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1. 2 209 2 8 Viewing 3D Image 2 211 2 8 1 Successive Display of Images a 2 212 2 8 1 1 Changing the Successive Display Speed a 2 213 2 8 2 ANIMA UON ai at Sg aan Bite ee nee i ede ee ea ee de 2 214 2 8 3 Building Stereo 3D Images arrsa 2 216 2 8 4 Building a 3D Image to be Viewed Through Color Red Green Eyeglasses2 218 2 9 Viewing Images Following the Progress of Time 2 220 2 9 1 Displaying Images Together 2 220 2 9 2 Displaying Images Successively a 2 220 2 10 Transferring Data to Another Application 2 223 2 10 1 Transferring Analysis Data to Another Application 2 223 2 10 2 Transferring the Plot Image of Analysis Data to Another Application 2 226 2 10 3 Transferring Image Data to Another Application microVoxel Paint etc 2 227 2 11 Entering Comment in Image 1 2 228 2 11 1 Writing Characters in Image a 2 228 CONTENTS a 2 13 1 Displaying the Image Intensity
2. a 1 22 1 2 3 Focusing on the Specimen I 1 24 1 2 3 1 Combination with BX a a s 1 24 1 2 3 2 Combination with AX e cece eeee serene teense taeeetaeeeeaaeetegeeeeeaeesenaees 1 26 1 2 3 3 Combination With IX sc ec en ected eh aii enn eee aaa a i oia 1 28 1 2 3 4 Combination of IXBP Bottom Port 1 29 1 2 4 Setting the LSM Light Path r 1 31 1 2 4 1 Combination with Upright Microscope BX 1 31 1 2 4 2 Combination with Inverted Microscope BX50W I 1 33 1 2 4 3 Combination with Upright Microscope AX 1 35 1 2 4 4 Combination with Upright Microscope AX70A a 1 37 1 2 4 5 Combination with Inverted Microscope IX a 1 39 1 2 4 6 Combination of IXBP Bottom Port 1 41 1 2 5 Selecting the Dyeing Method 1 43 1 2 6 Selecting the Filters a a 1 46 1 2 7 Selecting the ND Filters ccccccccceeeeceeeeeeeeeeeeee
3. UV0528 TF Y mage Const Y mage Comst Gaclastated mae E m r Pesan a q eae TIF Click the mouse right button ty Beg eae here The pop up menu appears ieee ows showing the file names that fU Te can be selected poet Select the file name to be ew fe processed ES te all views ca tile New Page Delete Page APARA _ 11 Drag Drop images on input basins to Fig 2 131 Function Panel Showing Pop up Menu 2 268 Page Appendix A List of Tools List of Tools Appendix A List of Tools Appendix A 1 List of Tools Appendix A 1 1 Toolbar The horizontal array of buttons at the bottom left of the panel form the toolbar The most frequently used FLUOVIEW functions are gathered here xl lAn lt Help gt button Displays on line help Allows the function and operation descriptions to be referenced while operating the application lt Full Screen gt button Displays the image full screen Allows only the image to be displayed by erasing all other image components such as the toolbar panels and status bar lt LUT gt button Allows the simulated colors to be changed or created originally by editing the LUT Look Up Table lt Annotate gt button Allows the analysis area to be specified or comment to be drawn in the image lt Experiment List gt button Allows the processing target image to be selected or deselected lt Print gt button Outputs the ima
4. Sc 6 Experiment Editor 4 XY 2 TIF APPLIED OPERATIONS Merger Extraction of Image Channels From the file list in the Experiments in Memory dialog box select the file name of the first image and drag it into the frame at the top left of the Experiment Editor group box The icon of the first image is displayed in the frame at the top left of the Experiment Editor group box TIP The mouse pointer turns into the image icon during dragging From the file list in the Experiments in Memory dialog box select the file name of the second image and drag it into the frame at the top left of the Experiment Editor group box The icon of the second image is displayed in the frame at the top left of the Experiment Editor group box NOTE when the image of the second selected image file is composed of multiple image slices the setting of the image slice range is ineffective even when the range is set Up to 3 channels are selected automatically beginning with the first image set in the Experiment Editor group box and connected to Merger by lines NOTE The lines of channels are colored as shown below Ch1 Navy blue Ch2 Light blue Ch3 Green Ch4 Yellow green Ch5 Orange Ch6 Red A TIP k When the mouse pointer is approached to the icon side end of a line connected to Merger the color of the line end turns into yellow Clicking the line in this condition switch
5. Laser Intensity Disabled C REX mode Bleach mode Make REX mask Fig 2 25 Lasers Sub panel Optics Among the laser lt Off gt buttons on the right of the Laser Intensity scales in the Lasers group box click the lt Off gt button of the laser you want to use in transmitted observation This should change the lt Off gt button to the lt Rdy gt button displayed in the pressed in condition Click the lt OK gt button in the FLUOVIEW dialog box Image acquisition starts Adjust the brightness with the PMT Offset and Gain LED sliders in the Ch group box in the Acquire panel See section 2 2 1 4 9 Adjusting the Image Brightness for details After acquiring images select lt Normal gt from the list displayed below the Surface XY Norm option button in the Scan Mode group box to set the scanning range to the original setting APPLIED OPERATIONS Image Acquisition 2 2 7 Image Acquisition of Rectangular Position at Desired Angle When the sample is not standing upright the image of a rectangular position can be acquired by changing the angle 1 Acquire an image in the XY observation mode See section 2 2 1 Image Acquisition with XY observation for operation procedure 2 Select lt Rect gt under the Surface XY option button in the Scan Mode group box in the Acquire panel A frame indicating the control range appears in the
6. Page 2 167 APPLIED OPERATIONS image Processing 2 5 1 4 DIC Correcting DIC Level Irregularities DIC level irregularities refers to uneven brightness of the image background which may be observed when a transmitted image is acquired in IDC observation The DIC level irregularities can be corrected to make the image easier to view 1 Display the Filters sub panel in the Process panel ie VOY Tile YhrocessY Analya Live Y SAMBDIC ORG y _ a Smoothing Filters Average Berem Lowpass Irregularities in brightness are observed across the diagonal line of the background DIC Image Filter lt DIC leveling gt button ae asas kf 13 Automatic correction of irregularities of the image background kg text box Enter the correction factor of the image background xe Zio 2 JEAFLOOViEWAIMAGESIDIC FROM Fig 2 73 Filters Sub panel 2 Enter the appropriate correction factor for the image background in the kg text box TIP The correction factor can be set between 0 1 and 1 in steps of 0 1 The standard factor is 1 If the effect is extreme try using 0 5 2 168 Page APPLIED OPERATIONS Image Processing Doo 3 Click the lt DIC leveling gt button The Level panel is newly created in the Display panel and shows an image after the correction of the DIC level irregularities of the background Te 1 evro Y
7. Page Down Moves the Z stage down Fine adjustment Page Down Moves the Z stage down Coarse adjustment Page Up Sets the Z stage to the Stop Z position Page Down Sets the Z stage to the Start Z position Insert Switches the Locked check box motor excitation status Delete Cancels the Start and Stop settings lt Set Zero gt Z motor The current position becomes 0 0 B 2 Page Appendix B List of Hot Keys Key Target Operation Piezo Returned to the 0 0 position Home Moves the stage till the Stop Z position Moves the stage till the Start Z position Transmitted illumination lamp ON OFF keys Key Target Operation Switches the transmitted illumination ON OFF lt Trans Lamp gt Target Operation Save the image in the Display panel being displayed as series images lt SAVE gt Image save key Panel select keys Main panel Target Operation Selects the Acquire panel Selects the File I O panel Selects the Tile panel Selects the Analysis panel Selects the Visualize panel Alt T Selects the Process panel Page B 3 Appendix B List of Hot Keys Acquire sub panels Key Target Operation Selects the Scan sub panel Sel
8. 2 119 2 3 3 Shredding IMAGES xccssccsssasseseeeveedcassndty dhenveea aratan aa eaen beia tanina atenat 2 119 2 3 4 Saving Comment Together with IMage 2 121 2 3 5 Checking the Image Information Acquisition Parameters 2 123 2 3 6 Saving the Image Information Observation Condition 2 127 2 3 7 Saving Reading the Region File cccccccceececeeeeeeeeeeeeeeeeeeeetneeeeeeees 2 130 2 3 7 1 Saving the Region File u 2 130 2 3 7 2 Reading the Region File u u u 2 132 2 4 Changing the Image Display Method 2 135 2 4 1 Displaying an Image in Simulated Colors 2 135 2 4 2 Editing the LUT Look Up Table aa 2 137 2 4 2 1 LUT Graph Editing According to Colors 2 137 CONTENTS 2 4 2 2 LUT Graph Editing by Gamma Correction 2 139 2 4 3 Switching the Displayed Channels Ch1 Ch5 2 140 2 4 4 Displaying Images of Multiple Channels Simultaneously Side By Side Views Over And Under Views Single View ccccceeeeeeeeeeeeeeeeeeeteeeeeeteees 2 141 2 4 4 1 Displaying Images Separately
9. 2 130 Page APPLIED OPERATIONS Saving Opening and Shredding Images 3 Select the image file name including the region to be saved in the Experiments in Memory dialog box and click the lt Comments gt button The Image Comments dialog box as shown below appears Image Comments C IMAGES X lt YZ 2 TIF Image Attributes Image Fields Comments Name x Y Ch Size 12 bit 800 600 5 2 lt Annotations ROIs gt button in the Export to File group box Saves the region into a file Lens Magnification 4x oF Record Changes Cancel Changes lt Annotations ROIs gt button in the Import from File group box Opens the file saving the region Fig 2 46 Image Comments dialog box 4 Select the lt Annotations ROIs gt button in the Export to File group box The Save Experiment As dialog box as shown below appears Look in ROIs y S l File name Roi Files of tupe FLUOVIEW ROI roi 7 Cancel Fig 2 47 Save Experiment As dialog box 5 Select the save destination drive or folder in the Look in drop down list 6 Select FLUOVIEW ROI roi in the Files of type drop down list e Page 2 131 APPLIED OPERATIONS Saving Opening and Shredding Images 7 Enter the file name into the File name text box and click the lt Save gt button NOTE when the text file having the same name as the entered name is already
10. 8 Enter the value of the emission wavelength into the WaveLength 1 text box in the Emission Wave Length group box e g 480 TIP photometry enter the value of the wavelength into the WaveLength 2 te l box too 9 Click the lt OK gt button to close the Edit Dye dialog box 10 Select the lt Save New Setup gt button to close the FLUOVIEW Setup dialog box 1 22 Page Maintenance of Major System Units Laser Scanning Microscope 2 Maintenance of Major System Units This section describes the maintenance of the Microscope 2 1 Laser Scanning Microscope According to IEC60825 Safety of Laser Product and IEC60825 EN60825 this product is a CLASS 3B laser product Therefore all of activities such as attachment or removal of the microscope modules listed below are defined as the service activities I In the United States of America this product is classified as a CLASS Tb laser product according to the CDRH laser safety regulation and attachment or removal of the microscope modules listed below is not permitted as part of user maintenance activities Such attachment and removal should be performed as part of service activities For detailed imformation refer to our service representatives 1 Transmitted lamp housing 2 Transmitted light fiber cable 3 Cube turret of reflected light fluorescence unit 4 Objective revolving nosepiece 6 7 5 Objectives Condense
11. Condenser Turret group box Sets up the name and color of the universal condenser Filter Turret group box Sets up the name and color of the filter turret for transmitted observation Save New Setup Quit w o Saving 1 14 Page Software Setup Setting the System Configuration The example of setting procedure in the Microscope Setup window Changing the Number of Buttons Section 1 3 1 1 Setting the Button Section 1 3 1 2 Editing the name of the buttons in the Mirror Unit Condenser Turret and Filter Turret group boxes Setting the Name Editing in the Nosepiece group box Setting the Name Setting the Objective Magnification Setting the condenser worked with Setting the magnification of the objective Setting the N A of the objective Setting the number of the confocal pinhole Setting the refractive Setting the Color Section 1 3 1 3 Setting the color of the button disengaged from the light path Section 1 3 1 3 1 Setting the color of the button engaged into the light path Section 1 3 1 3 2 Finishing the setup Section 1 3 2 Page 1 15 Software Setup Setting the System Configuration 1 3 2 1 Setting the Number of Buttons 1 Right click the mouse on the area outside of the buttons in the group box where the number of buttons to be displayed is changed The pop up menu as shown below appears Select Hole Count and select the number of the buttons to be displayed
12. FLUOVIEW FV500 can store the system setup information PMT Voltage Gain Offset etc on a per user basis To make this possible you have to register yourself as a user and log in personally when starting up the FV500 software Appendix H 1 User Registration Register yourself as a new user 1 Double click the FLUOVIEW Setup icon on the desktop When a dialog box as shown below appears display the Users panel at the front Coloring Tables Registration Microscope Z Stage N Scanning Unit FLUOVIEW Setup icon Software Hardware Lasers FLUOVIEW Users You are logged in as as Administrator Administrator a lt Add a user and Log in gt button Used to add a new user Add a user and Reset user to Login Factory Defaults Delete a user Quit w o Saving Save New Setup lt Reset user to Factory Defaults gt button lt Delete a user gt button Resets the system setups of the users selected in Deletes the user selected in the list box to the factory defaults the list box The Administrator and the user who log in now cannot be deleted 2 Click the lt Add a user and Log in gt button 3 The New FLUOVIEW User dialog box appears w New FLUOVIEW User User Name text box Enter the user name to be registered Page H 1 Appendix H USER REGISTRATION OF FV500 Logging into the FV500 4 Enter the user name in the User Name
13. TIP For the channel switching see section 2 4 3 Switching the Displayed 7 Channels h 4 Click the lt Animation gt button in the Save group box The Save Experiment As nimation dialog box appears as shown below lt Save Animation gt button Save in ja Images x cH File name XV2CH AM Save as type javi Uncompressed avi x Cancel Fig 2 34 Save Experiment As Dialog Box 5 When it is required to change the save destination drive or directory use the Save in drop down list 6 When it is required to change the saved file type use the Save as Type drop down list e Page 2 111 APPLIED OPERATIONS Saving Opening and Shredding Images q TIP Two saving methods can be selected AVI compressed Saves the AVI file being compressed 7 Enter the file name in the File Name text box 8 Click the lt Save gt button NOTE If a file with the same name as the entered file name already exists the dialog box appears to ask if you want to overwrite the existing file If you do not want to do so click the lt No gt button and enter another file name 2 3 1 5 File Types Available for Save The file type used for saving image in a file can be selected by the user See section 2 3 1 1 Saving Images As a Series for the operation method 1 Click the lt Experiment gt button in the Save group box 2 Select the file type from th
14. e Page 2 171 APPLIED OPERATIONS Image Processing 2 172 Page 6 Enclose the histogram section of interest using the scale in the Master View field 7 The magnified view of the region selected by the scale is shown in the Selected 8 View field Histogram of CIMAGES OL YMPUS TIF Master View 1445 0 Max Selected View The vertical chart axis represents the degree and the horizontal axis represents the mapped intensity values Black level White level 1689 Intensity Mapping Channel ere O4C Compute Histogram Fig 2 77 Histogram of the Selected Region When the image was acquired in the multi channel mode select the channel s to be subjected to LUT intensity mapping re assignment using the Channel option buttons Select the region to be subjected to mapping re assignment using the scale in the Selected View field While moving the scale confirm the change in contrast in the Display panel The intensity values in the selected region are mapped re assigned to intensity values from 0 to 255 and displayed APPLIED OPERATIONS Image Processing 2 5 3 Mathematical Operations Between Images Arithmetic or logical operations can be applied between two different images or between an image and a constant 2 5 3 1 Image Addition Addition of an image to an image constant to an image is possible as described below 1 Display the
15. Add Remove Programs The Add Remove Programs Properties dialog box as shown below will appear Add Remove Programs Properties 2 x Install Uninstall Windows NT Setup To install a new program from a floppy disk or CD ROM drive click Install 7 The following software can be automatically removed by w Windows To remove a program or to modify its installed components select it from the list and click Add Remove Internet Explorer 3 0 MGA NT PowerDesk 3 20 055 Visual Basic 5 0 Professional Edition Add Remave 3 Select the lt Install gt button The dialog box as shown below will appear Install Program From Floppy Disk or CD ROM Insert the product s first installation floppy disk or CD ROM and then click Next 4 When the Install Program From Floppy Disk or CD ROM dialog box appears select the lt Next gt button Page 1 3 Software Setup New Setup of the Software 5 When the Run Installation Program dialog box appears ensure that the lt CD ROM drive name gt SETUP EXE is displayed correctly and select the lt Finish gt button Run Installation Program If this is the correct installation program click Finish To start the automatic search again click Back To manually search for the installation program click Browse Command line for installation program lt Back Cancel 6 When the Choose Destination Location dialog box ap
16. Assign dyes manually check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set Icon The dyeing method is set Gain Offs NV FITC a JEn FEST Lc 800v 10 0x 7 Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Page 1 45 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 6 Selecting the Filters The excitation filter spectral filter and barrier filters are set automatically to the light path according to the dyeing method selected for the specimen To change filters see section 1 3 2 4 Configuring the Filters in this volume and follow instructions in the Optical System Configuration window The following table shows the possible combinations of the barrier and excitation filters Spectral Spectral Spectral Laser Combination Excitation Filter Barrier Filter 1 Barrier Filter 2 Barrier Filter 3 Barrier Filter 4 Filter 1 Filter 2 Filter 3 DM351 488 Fix DM488 568 Fix G DM488 543 Fix9 m 1 46 Page Spectral Filter 1 Laser Combination Excitation Filter DM351 488 DM488 543 DM488 543 633 Ar Kr HeNe R 488 568 633 DM351 488 DM488 568 DM488 568 633 Getting Started FLUOVIEW Outline of LSM Observation Procedures Spectral Spectral Barrier Filter 1 Barrier Filter 2 Barrier Filter 3 Barrier Filter 4 Filter 2 Filter 3
17. Format Point JAX Line Series40 General Marks Data Source Enables or disables the 3D inflection point display Sets the height of inflection points J Visible Width fa Selects whether the ends of 3D the inflection points are a4 aligned with the coordinate Inflate Margins axis when checked or the oF centers of the inflection Dark 3D Style Diagonal Cross points are aligned with the I coordinate axis when x ackground M Default Border unchecked 2 Enables or disables the dark shadowed 3D display of inflection points Sets the inflection point Resets the background color to background color the default color Sets the type of inflection points Sets whether or not the inflection point borders are displayed and sets the color width and tvoe General panel Used to set the general items Editing E Chart Series40 a Z Line Series40 Format Point General Marks Data Source Selects whether the labels are displayed on or below the chart Enables or disables the chart display inside the legend Horizontal Axis Top C Both Bottom General Show In Legend Sets the shape that the cursor changes when it is placed on the chart Usually leave this box unchecked Formats Values t 80 ttt Percents HHO HH Left C Both r Bight Selects whether the labels are
18. Recommended Confocal Name of the Objective Magnification UPLAPO 60XWPSF 60 2 k lt To change the details objective name pinhole No magnification of an objective gt Double click the objective for which you want to change the detailed information When the dialog box appears change the desired information items and select the lt OK gt button lt To add an objective to the list gt Double click Double Click here at the top of the list on the right When the dialog box appears set the detailed information on the new objective and select the lt OK gt button lt To add one of the objectives in the list on the left to the list on the right gt Place the mouse pointer on the objective to be added in the list on the left and drag it to the list on the right Software Setup Setting the System Configuration 6 Display the Fluorescence panel in the front position In this panel select the items to be included in the list of dyes used by the FLUOVIEW application Each user should set the dyes that the user wants to use with FLUOVIEW FLUOVIEW Setup Scanning Unit Soft Hardw Le Coloring Tables Registration Microscope ZStage Objectives Fluorescent Dyes Colors All Known Dyes Dyes on your Samples Calcium Crimson a Calcium Green 1 Calcium Green 2 List of all dyes Calcium Green 5N availab
19. UV Ar HeNe G HeN e R 351 488 543 633 DM488 543 DM488 543 633 UV Ar Kr HeNe R 351 488 568 633 BA400 470 BA400 470 Invaild If the equipment of anoter filter set for laser configuration is required please contact your local Olympus representative Page 1 47 1 48 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 7 Selecting the ND Filters e When the laser combiner is used 1 Display the Acquire panel In the Laser Intensity group box of the Lasers sub panel set the ND filter scale crosstalk and fading of each laser according to the levels of specimen brightness fluorescence Laser Intensity a a AOTF EE froy EJ roy S Laser Intensity group box Set the ND of the lasers in the scales The number of displayed lasers varies depending on the number of input channels REX Function Disabled C REX mode C Bleach mode Make REX mask _ SSSSSS gt Fig 1 11 Lasers Sub panel When the Green HeNe laser is used try ND of 50 by setting the Intensity scale in the Lasers group box to 50 With other lasers try ND of 10 first Page Getting Started FLUOVIEW Outline of LSM Observation Procedures One Point After the dyeing method has been set with the Dyes sub panel the ND filters can be set using the Ch group box in the upper part of the Acquire
20. Use the following procedure to change these filters 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Light Path Ej os HE lt SU Control gt button Click to display the filter and laser types to be set Combination with the laser combiner sets up the laser automatically Microscope Control Trans Scope SU Lamp Control Control Fig 1 21 Optics Sub panel 1 72 Page Laser Unit group box Shows the type of laser to be used With the laser combiner operation the laser type is set and displayed automatically When the lt On gt button is pressed in the laser is oscillating the beam When the lt Stby gt button is pressed in the laser is not oscillating When the Auto standby check box is checked the After text box appears below it When not using laser for a long time in order to suppress useless electric power consumption we recommend you making lt Stby gt mode The laser oscillation stops when the time shown in this box has elapsed after the end of laser scanning The laser oscillation stops when the time shown in this box has elapsed after the end of laser scanning The laser is suspended as standby mode using Ar or Kr laser or the laser oscillation stops using UV Ar laser When the time shown in this box has elapsed after the end of laser scanning Excitation DM and Beam splitter drop down lists Cha
21. 1 From the page tabs on the bottom right of the Acquire panel select the Z Stage sub panel Step Size 2 The number of steps has already been set and displayed in the Step Size text box This number can be changed using the lt gt or lt Y gt button in the Step Size text O 25um Ideal 1 1 box Step Size text box 3 TIP The number of steps in the Step Size text box has been calculated by the system so that the depth scale of the acquired image is identical to the The number of steps calculated by the system may be erroneous unless the XZ observation mode has been set previously Slices Slices text box TIP ie The number of acquired images shown in the Slices text box can also bei input from the keyboard After setting Start Z Z direction scan start position and Stop Z Z direction scanning stop position input the desired number of images in the Slices text box This automatically sets Step Size number of steps 5 Acquiring image 1 Click the lt XZ gt button in the Acquire panel The acquired image will be displayed in the Live panel Page 2 35 APPLIED OPERATIONS Image Acquisition pO 2 2 2 2 XT Observation Mode 2 36 The description in this section will be focused on the image acquisition operations in the XT observation mode that are not used in the XY observation modes which are the operations enclosed in Cc in the chart on the next pag
22. 4 Setthe interval time to 10 using the lt gt and lt Y gt buttons in the Interval text box Change the interval time corresponding to the specimen 5 Setthe number of scans to 30 using the lt gt and lt Y gt buttons in the N text box in the Time Series sub panel Change the number of scans corresponding to the specimen 6 Confirm that the time for image acquisition is set to 5 minutes or 300 seconds in the Total Time text box The time for image acquisition differs according to the interval and the number of scans 6 Setting the TIME COURSE Software optional Setting of the real time graph is required when you use the TIME COURSE software See the FV TIEMPO TIME COURSE software User s manual for details of the TIME COURSE software Page 2 97 APPLIED OPERATIONS Image Acquisition pO 7 Acquiring Data The experiment data before and after photobleaching of certain region can be obtained after performing an ordinary image acquisition in the first scan an image acquisition in the Bleach mode in the second scan and an ordinary image acquisition in the third scan When the REX mode is selected in the third scan the data of the region other than the specified region can not be obtained since the laser is irradiated only to the specified region However the REX mode is effective to prevent fading of the obtained data 1 Select the Disabled option button in the AOTF group
23. Getting Started FLUOVIEW Basic Operations Combination with IX 6 Filters 4 Transmitted light DIC slider FV5 DICT optional 1 Light path selector 7 Intermediate magnification knob 3 Analyzer FV5 ANI Page 1 7 Getting Started FLUOVIEW Basic Operations pO 1 Light path selector Select the light path between laser microscopic observation direct observation and photography observation e Refer to the following table and set the dial switch to the position corresponding to the required light path lt lt Light intensity of each light path gt gt h Side port LSM SLR port z 100 s Binocular section 100 2 Cube turret Select the fluorescence observation tube by rotating the turret e Engage the desired cube in the light path for visual fluorescence observation e For laser microscopy or for visual transmitted light observation rotate the turret to page tab CO This set the cube turret so that no cube is engaged 3 Analyzer FV5 ANI Polarizing plate for use in differential interference observation and polarized light observation e Set to the pushed in position to engage the FV5 ANI in the light path for visual transmitted light differential interference observation or transmitted polarized light observation e Set to the pulled out position to disengage the FV5 ANI for laser microscopy Engaging the FV5 ANI in the light path degrades image quality 4 Tran
24. Remove any disks from their drives and then click Finish to complete setup Page 1 5 Software Setup Setting the System Configuration 1 3 Setting the System Configuration If it is required to change the settings after having set up the software perform the following procedure 1 3 1 Overall Setting of FLUOVIEW 1 Double click the FLUOVIEW Setup on the desktop amp tl The FLUOVIEW User Login dialog box appears as shown below iw FLUOVIEW User Login FLUOVIEW Setup icon fe h hie US User Name a 2 Enter the user name in the User Name text box and click the lt OK gt button to log into FLUOVIEW FV500 Refer to Appendix H 2 Logging into the FV500 for details 3 The dialog as shown below appears for use in saving the system configuration First display the Microscope panel in the front position select the type of microscope and set the extra intermediate attachment magnification to 1 0 Software Hardware Lasers Uses J Scanning Unit Microscope Zstage Objectives Fluorescence Coloring Tables Registration Microscope O O r Configuration E CAX FV Review Station CH oo E C Fv Upgrade C px C P300 o F500 ic C 2CH C 3CH Extra Magnification 1 00 4CH TD Auto installed X Yes No Save New Setup Quit w o Saving Selecting BX51 52 or BX51 52WI and Automatic in the Microscope group box or BX61 62 or
25. Surface XY Horm 123 Hormal Fast Sequen C Line xT Cc Ler xr fC Pemt Size 800 by 600 B es UPLAPO 10x PPan I Az x1 Scan Speed Fast Slow 47s Sean 17s image _ Acquisition Settings Load Save Focus Mode O x2 Cx4 p Filter Scan Z Stage Time Normal EER z Series Optics Lasers CO Accumulate To Peck 4 sub panels These are used to set the information required for image acquisition Display Panel PTE 1 64 Page e Getting Started FLUOVIEW Online Help 2 From the panel page tabs shown on the bottom right of the Acquire panel select the Optics sub panel Light Path BI LSM Tv lt Scope Control gt button Displays useful information for the system setup Microscope Control Fig 1 18 Optics Sub panel Select the lt Scope Control gt button at the bottom of the panel The window as shown below will appear in case of a combination with the IX FE Microscope Configuration Fitters SW Vid Fig 1 19 Microscope Configuration Window Page Filters box This represents the filters N oe Analyzer box Represents the FV5 ANA analyzer optional Turret Cube box Represents the cube turret Observation group box LSM represents the laser scanning microscopy light path and BI repres
26. below appears x Set as Default Font Enter an annotation or pick one from the list click on the image to place it Close Fig 2 117 Active Overlays Dialog Box 3 In the drop down list inside the dialog box select x lt x hotspot value units gt to display the X coordinate position or Y hotspot value units gt to display the Y coordinate position 4 On the image place the mouse pointer on the position that you want to display the X coordinate position or the Y coordinate position and click the mouse 5 Click the lt Close gt button to close the Active Overlays dialog box g TIP E The displayed value indicates the distance from the origin when assuming Page 2 231 APPLIED OPERATIONS Entering Comment in Image 2 11 4 Drawing a Figure in Image IN lt Line gt button ea lt Poly Line gt button m lt Rectangular gt button is lt Circle gt button fo lt Polyregion gt button Q lt Free Region gt button lt Free Line gt button 2 232 Page This facility is used to draw figures in the image FLUOVIEW provides seven figure drawing modes Display the Display panel of the image in which you want to draw figures Select one of the following command buttons and draw a figure in the image using the mouse The operation methods of the command buttons are described below To draw a straight line On the image click t
27. lt Set end position gt button When successive display or frame by frame display is required set the image with which the display should end Assuming that the number of images is n n 1 is set when this button is not used lt Loop Bound switch gt button lt Display gt button Set the speed of successive display lt XYZ series gt button Displays one of multiple images by selecting it according to the multiple sections Z lt XYT series gt button Displays one of multiple images by selecting it according to the time T lt Bound gt button Every time successive display or frame by frame display in a direction ends at the start or end position the display is restarted in the opposite direction lt Display gt button Press to start display of n slices of image from the start position to the end position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame lt Rabbit gt button Press for successive display of image slices without pause lt Clock gt button Press for successive display of image slices at the time interval set in the Time Series button lt Turtle gt button Press for successive display of image slices at an interval of 0 8 sec lt Display gt button Press to start display of n slice of ima
28. 1 1 1 1 Display the Display panel of the image to be processed with Laplacian filtering Ss 2 When the image is composed of multiple image slices the range of image slices to lt Set start position gt be filtered can be specified using the lt Set start position gt and lt Set end position gt suton buttons above the image First display the image slice to start filtering using the lt Display gt button and click the lt Set start position gt button Then set the image slice lt Set s to end filtering in the same way as above u 3 When the image to be filtered was acquired in the multi channel mode filtering is applied only to the channels being displayed lt Display gt button Example When only the Ch1 image is displayed filtering is applied to the Ch1 aae x 7 image only lt Display channel switch gt buttons t 2D Laplacian X TIP k For the switching of channels see section 2 4 3 Switching the Display 4 Click the lt 2D Laplacian gt button A new Display panel having the page tab named Filter appears showing the filtered image s TIP gt During fitering the status bar shows the progress of processing 1 e Page 2 163 APPLIED OPERATIONS Image Processing File v0 Y Tile Yarocess Anatye Live xv2cH TIF Y Fitter 1 Filter mj D P XY CH 1ot 2 Smoothing Filters WW a Lowpass Median Sharpening Filters Highpass X Highpass Y
29. 7 Click the lt Extract gt button in the Experiment Editor group box A new Display panel showing Extract in the page tab appears and the image of the extracted channel s is displayed in the panel Acquire Y File vo Y Tie roced Open Save File Type Live Y xam Y xv2ur Y Extract 1 Extract xY Tift TIF Experiment Editor Extract CAMAGESWXY 1 TIF CAIMAGES XY 2 TIE E Comment Done xleieiZizcle Extract Fig 2 127 Extract Panel APPLIED OPERATIONS Changing the Chart Display Method 2 14 Changing the Chart Display Method When the processed analysis data chart in the Analyze panel is double clicked the Enhanced Profile Plot dialog box appears By clicking the lt Properties gt button the Editing dialog box can be displayed allowing the detailed chart settings and chart display method to be changed 2 14 1 Chart Panel Series panel This panel is used to change the type title or other settings of the displayed chart as well as to add or delete charts List of displayed charts k M D Series2 E a Series3 Move the positioning of the chart selected in the Series Title box Adds the chart selected in the Series Title box by specifying the format Deletes the chart selected in the Series Title box Re titles the chart selected in the Series Title box Copies the chart se
30. APPLIED OPERATIONS Image Acquisition 2 2 1 2 Setting the Filters The excitation filter spectral filter and barrier filters are set automatically to the light path according to the dyeing method selected for the specimen To change filters use the following procedure 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Optics sub panel Light Path BI LSH TY lt SU Control gt button Click to display the filter and laser types to be set Combination with the laser Trans Scope SU r combiner sets up the laser Lamp Control Control automatically Microscope Control Fig 2 6 Optics Sub panel e Page 2 15 APPLIED OPERATIONS Image Acquisition 2 Click the lt SU Control gt button at the bottom of the panel The window as shown below will appear Laser Unit group box Shows the type of laser to be used With the laser combiner operation the laser type is set and displayed automatically When the lt On gt button is pressed in the laser is oscillating the beam When the lt Stby gt button is pressed in the laser is not oscillating When the Auto standby check box is checked the After text box appears below it When not using laser for a long time in order to suppress useless electric power consumption we recommend you making lt Stby gt mode The laser oscillation stops when the time shown in this box has elapsed
31. After the key is set to ON it takes tens of sss seconds before the laser oscillation starts 2 3 HeNe Green Red laser 1 Turn the key to the I ON position meues emor After the key is set to ON it takes tens of seconds before the laser oscillation starts NOTE To stabilize the laser beam output we recommend leaving the system for warm up after turning the laser power ON The warm up period should exceed 10 minutes when the Error indicator laser system in use uses an Ar laser or exceed 30 minutes Power hour counter UV laser power supply when it uses a Kr laser or Green HeNe laser 2 4 UV Ar laser When using the system for the first time check that the remote cable is connected the POWER switch on the front panel of the chiller is set to O OFF its COOL DOWN CYCLE switch is set to I ON and the remote operation mode is set a I ON MAIN POWER Key switch Fig 1 3 Chiller Front Panel Output control From the second time and on use the following method 1 Set MAIN POWER of the laser power supply to ON and turn the key switch to the I ON position It takes about 30 seconds before the laser starts oscillation together with a trigger sound All error indicators turn off at this time e Page 1 19 Getting started FLUOVIEW Outline of LSM Observation Procedures 2 Make sure that the output control is set to the maximum position 3 Warm up for about 30 minu
32. Cancel Fig 2 121 Print Dialog Box 3 Select the connected printer name from the Name drop down list 4 If itis required to set the detailed data of the printer click the lt Properties gt button in the dialog box 5 Click the lt OK gt button of the dialog box NOTE It is required to install and select the printer driver before the above operation Refer to the Windows manuals for details For the printer operation procedures refer to the printer manuals 2 242 Page APPLIED OPERATIONS Image Output at Printer One Point The Print dialog box can also be displayed by mouse operation on the image 1 Display the image to be output at the printer at the front of the Display panel and click the right button of the mouse on the image 2 A pop up menu as shown below appears 3 Select Print from the menu FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Views Experiment Properties Page 2 243 APPLIED OPERATIONS Merger Extraction of Image Channels 2 13 Merger Extraction of Image Channels 2 13 1 Setting the Range of Multiple Image Slices When merging or extracting channels in images composed of multiple image slices such as time lapse images and images acquired by varying the cross sections it is possible to select only some of image slices as the target of channel merger or extraction This section descri
33. Enter the file name in the File Name text box Click the lt Save gt button NOTE if a file with the same name as the entered file name already exists a dialog box is displayed to ask if you want to overwrite the existing file If you do not want to overwrite it click the lt NO gt button and enter another file name One Point The Save Experiment As dialog box can also be displayed by a mouse operation 1 Display the image to be saved at the front of the Display panel and right click a point in the image 2 A pop up menu as shown below is displayed 3 Select Save Experiment from the menu FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Views Experiment Properties Page 2 105 APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 1 2 Saving a Display The displayed images can be hardcopied and saved in a disk This method is used when it is required to use a FLUOVIEW image in another application gt 1 Display the Display panel showing the image to be saved at the front lt Display gt button 2 When there is more than one image to be saved display the image to be saved using the lt Display gt buttons 3 Click the lt Display gt button in the Save group box The Save Display dialog box as shown below appears Save Display Save Area Selected View AH Vigas Selects All Views when you want to save Ti
34. In the Lasers group box of the Lasers sub panel set the ND filter scale of each laser according to the levels of specimen brightness fluorescence crosstalk and fading rr Jm 4 _ gt hoy EJ se gt Z Stage Laser Intensity group box Set the ND of the lasers in the scales The number of displayed lasers varies depending on the number of input channels AOTF REX Function Disabled C REX mode C Bleach mode Make REX mask When the HeNe Green laser is used try ND of 50 by setting the Intensity scale in totes Dyes Vine Sere Lasers the Lasers group box to 50 With other lasers try ND of 10 first One Point After the dyeing method has been set with the Dyes sub panel the ND filters can be set using the Ch group box in the upper part of the Acquire panel 1 In the upper part of the Acquire sub panel open the Ch group box for the ND to be changed Click the lt More gt button The field as shown below is displayed below the Ch group box Clicking this button allows fine C A Ar adjustment of the ND value Display of the optimum laser and ND value which are set Clicking this field allows the ND value automaticaly ae to the to be changed on a large scale A aaa The ND values which are usually used are displayed in green Display of the ND value set by clicking the lt gt and lt gt buttons or the field The set ND value can also
35. Page 1 40 Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 4 6 Combination of IXBP Bottom Port 1 From the index tabs on the bottom right of the Acquire panel select the Optics sub panel Light Path BI LSM Time Serie Microscope Control Trans Scope SU Lamp Control Control Fig 1 5 Optics Sub panel 2 Click the lt LSM gt button in the Light Path group box The lt LSM gt button looks pushed in to indicate that it is selected When scanning is started while the lt BI gt button is selected the LSM light path is selected automatically It is switched back to the visual observation automatically when scanning completes 3 Rotate the cube turret 2 of the vertical fluorescence illuminator to CO 4 Ifthe FV5 ANI analyzer 3 is engaged in the light path pull the lever out to disengage it 5 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider 4 from the light path When performing transmitted DIC observation or fluorescence transmitted light DIC simultaneous observation engage the FV5 DICT as well as the transmitted light DIC slider matching the objective in use in the light path by operating the condenser 5 With the fluorescence transmitted light DIC simultaneous observation the presence of the FV5 DICT in the light path may deteriorate the fluorescence image resolution somewhat 6 During transmi
36. Sections 2 2 1 1 amp 2 2 1 2 in OPERATION Set a lower scan speed Set the objective magnification Sogijgn 2 2 1 4 10 igi ERATION Section 2 2 1 4 1 in OPERATION If the image If image b does not Set the zoom ratio to 1X become Section 2 2 1 4 2 in OPERATION clean clean Set the channel to be acquired i Re adjust the image brightness Section 2 2 1 4 3 in OPERATION Section 2 2 1 4 9 in OPERATION Stop repeated scanning Section 2 2 1 4241 in OPERATION Set the observation mode Section 2 2 2 1 2 in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION Set the XY observation mode Section 2 2 1 4 5 in OPERATION Perform repeated scanning Section 2 2 1 4 6 in OPERATION Set the observation line Section 2 2 2 1 3 in OPERATION Adjust the Z position to observe the desired multiple sections Section 2 2 1 4 7 in OPERATION Set the numbers of Z direction steps and acquired image slices Set the observation range Section 2 2 2 1 4 in OPERATION Section 2 2 1 4 8 in OPERATION Acquire image Section 2 2 2 1 5 in OPERATION Adjust the image brightness Section 2 2 1 4 9 in OPERATION Save image Section 2 3 1 in OPERATION 2 32 Page APPLIED OPERATIONS Image Acquisition 1 Setting the Z direction scanning range While acquiring image move the Z stage according to the range of the multiple sections to
37. See section 2 10 Transferring Data to Another Application for 2 6 2 2 Intensity Distribution on a Planar Region Histogram The histogram on a region in an image can be displayed The histogram on a region in an image can be displayed The operation method is identical to displaying the intensity profile on a line See section 2 6 1 1 Intensity Values on a Line Line Profile Double click the Region Histogram window The Enhanced Histogram Plot windows appears as shown below lt Properties gt button Displays the Editing dialog box for use in detailed setting of the chart or change of the chart display See section 2 14 Changing the Chart Display Method for details lt Copy gt button Copies the plotted image in the clipboard lt Save gt button Saves the profile data in a file using an Excel compatible format lt Close gt button Quits the Enhanced Profile Plot window Grey Level and returns to the Analyze panel Fig 2 92 Enhanced Histogram Plot Window Line Specification e Page 2 193 APPLIED OPERATIONS Image Analysis 2 194 Page When a desired area is specified by dragging the left button of th When the right button of the mouse is dragged on the graph the grap ican be scrolled The magnification or scrolling of the graph can be canceled b dragging the left button of the mouse from the bottom left to the right When the mouse poin
38. Views Experiment Properties 2 144 Page 3 4 APPLIED OPERATIONS Changing the Image Display Method Select Views then select Add a view in the sub menu Annotate FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Add a view Remove last view Experiment Properties A view is added to the rightmost position on the Display panel Acquire File vo Y Tile roces Live xvz 2 11F hi XVZ 2 TIF P a p gt fr zm TE XYZCH File Type Titt TF P Files Name icon x g th pitit p w xy2chtit so i PEE xyztit Mm e Sc Se ha p Load Experiment Animation x lejel l 12 Fig 2 58 Display Panel After View Addition TIP Up to 6 views can be displayed at once UU Page 2 145 APPLIED OPERATIONS Changing the Image Display Method 2 4 5 2 Decreasing the Number of Divided Images 1 Display the Display panel of the image to be changed at the front i Acquire File o Tile Y2roces Live XVZ 2 TIF File Type Tif TIF XYZ 2 TIF XYZCH Files Name Icon x NY th pi tit 800 600 xy2ch tif 800 600 xyz tif fl 800 600 Load Save Experiment Experiment Animation xean Display Panel Fig 2 59 Display panel 2 Right click the imag
39. below The operation method is identical to Image Image Image Constant except for the following point See section 2 5 3 1 Image Addition e To multiply an image by an image slisala Click the lt Multiply 2 Images gt button in the Multi Image Operations group box A lt Multiply 2 Images gt N h buttotn new Display panel showing Image Image in the page tab appears displaying the image obtained by the multiplication operation To multiply an image by a constant Multiply Image with Click the lt Multiply Image with gt button in the Scalar Operations group box A new lt Multiply Image with gt button Display panel showing Image Const in the page tab appears displaying the image obtained by the multiplication operation 2 5 3 4 Image Division Division of an image by an image image by a constant is possible as described below The operation method is identical to Image Image Image Constant except for the following point See section 2 5 3 1 Image Addition 2 176 Page APPLIED OPERATIONS Image Processing Doo O To divide an image by an image Vivid 2 lays Click the lt Divide 2 Images gt button in the Multi Image Operations group box A lt Divide 2 Images gt button new Display panel showing Image Image in the page tab appears displaying the image obtained by the division operation To divide an image by a constant Divide Image by Click
40. change on a large scale oon YE G E Clicking the top or bottom arrow button allows fine 7 m adjustment of the value mance Dragging the square knob allows the value to vary ersan se irectly Acqulltion settings ea Pena wes F Sub panel k i A sub panel is provided for use uen in detailed setting or information O Accumulke Topes 4 display of a function Byes me Sere xeen Display Pan oe Fig 1 1 Panel and Major Functions Status bar Shows the description of the command being pointed by the mouse pointer During lengthy processing the status bar shows how far processing has progressed 1 14 Page Getting Started FLUOVIEW Basic Operations displayed in a different color from that of other windows Minimize button lt gt Click to turn the window into an icon Original size button lt Click to return the maxim size window to its original size Control menu box Clicking this box displays a control menu which contains the commands for use in controlling the window E Microscope Configuration C Fluorescence Homarski Optical Path Scroll bar BX Stop Z Go ea alle l pay Check box Elre Clicking this box enables or disables the indicated item The item is z Eeg enabled when the check box is checked x
41. operation procedure x When the images in both image files are composed of multiple image slices the overlaid image will have the same number of image slices as the file with the fewer image slices x Example 1 XYZ image XY image XY image i u The first image slice of the XYZ image is overlaid with the XY image u Example 2 XYZ image with 10 slices XYZ image with 5 slices XYZ image with 5 slices x The first five image slices are overlaid UU e Page 2 245 APPLIED OPERATIONS Merger Extraction of Image Channels pO 2 Click the lt Experiment List gt button in the toolbar at the bottom of the File I O panel The Experiments in Memory dialog box appears as shown below Experiments in Memory lt Experiment List gt button CAIMAGES XY 1 TIF CAIMAGESY 2 TIF comens pone Fig 2 122 Experiments in Memory Dialog Box 3 Click the lt ADV gt button in the Experiments in Memory dialog box The Experiment Editor group box appears as shown below lt ADV gt button Experiments in Memory AS y CAIMAGES lt Y 1 TIF CAMAGES XY 2 TIF E commens done Fig 2 123 Experiment Editor Group Box Displayed by lt ADV gt Button P A EAE EE IEEE AE E E sonny EA TE AAE E ns E E IEE K TIP The frame at the top left of the Experiment Editor group box shows th icon of the image file displayed at the front of the Display panel 2 246 Page
42. 2 In the Experiments in Memory dialog box select the file name of the image to check the image information and acquisition parameters and click the lt Comments gt button 3 Display the Image Attributes panel at the front The panel as shown below appears where the information on the selected image is shown CH IZ CH f Displays number of Z Shows the number of image aise y acquisition channels PXYIY Show the resolution values in the X and Y directions Image Comments C MMAGES CY 7 2 TIF NN Comments lI ntensi ty Image Attributes Shows the length of the data expressing the intensity value Size 12 bit 800 600 5 2 Z Shows the number of steps in the Z direction Resolution 1 0 0 5 0 5 0 3 0 0 X Resolution Y Resolution T Show the lengths per pixel in a m the X and Y directions Lens 40x IPP ee Lens Magnification Record Ch Displays objective Lens a Au wis Magnification when the image is acquired Cancel Changes Import from File AnnotationsROls Export to File AnnotationsiROIs Properties Fig 2 39 Image Attributes Panel e Page 2 123 APPLIED OPERATIONS Saving Opening and Shredding Images he X Size Y Size and Z Size text boxes can also be used to chang e lengths per image pixel or the number of steps in the Z direction Thes alues are used in the scale display and
43. 3 Setting the observation mode 1 In the Scan Mode group box in the Acquire panel select the Surface option button 2 In the Acquire panel select the XYZ observation mode option button 4 Acquiring image 1 Click the lt XYZ gt button in the Acquire panel The acquired image will be displayed in the Live panel While acquiring an image in the XYZ observation mode clicking the lt STOP SCAN gt button changes the buttons at the upper part of the Acquire panel as shown below The lt Resume gt button restarts image acquisition at the frame next to the frame where the acquisition is suspended STOP SCAN File vO Tile Y Proce lt Resume gt button 7A Resume Series Does lt Series Done gt button Restarts image acquisition at T Determines the acquired the frame next to the frame Vv v k Transmitted images Once this button is where the acquisition is a clicked it is not possible to suspended append an image Page 2 49 APPLIED OPERATIONS Image Acquisition 5 Appending image An additional image can be appended to an image acquired in the XYZ observation mode Immediately after acquisition of an image in the XYZ observation mode the buttons at the upper part of the Acquire panel changes as shown below lt Append Next gt button x C N File i Tile Proog lt Series Done gt button Acquires another image and Determines the acquired appends it to the image Ap
44. BX61 62WI in the Microscope group box shows the Microscope Setup dialog box to edit the BX operation panel BX Control Panel For details see section 1 3 1 Setting the BX Control Panel 1 6 Page Software Setup Setting the System Configuration 4 Display the Z Stage panel in the front position and check and set the presence of motor controller positioning of the motor controller backlash and moving amount of the Z motor a Sue ane Set the backlash of motor operation Hasa e A caomagfanies in the Z Stage sub panel Z Stage Set the backlash during Is there a Z Stage Motor installed lt Yes C No scanning involving the Z stage Motor on which side of microscope Left x Right Ae Oh j Set the range where the stage Backlash correction during roaming 0 0 um can move during the Z motor oneration Set the moving amount assigned to the lt Z stage coarse adjustment gt buttons Backlash correction during z series 5 0 um Maximum range for z series 10 mm Fast Z stage increment 10 um Slow Z stage increment 0 1 um Set the moving amount assigned to the lt Z stage fine adjustment gt buttons Save New Setup Quit w o Saving i The Maximum range for Z series mm text box accepts a setting value lt TP were between 1 0 and 3 0 mm The values in the Start Z and Stop Z text boxes in the Z Stage sub panel are set according to the value set there f Fo
45. Di lt ge gt UPlan Apo 100X 0111 lt o gt oo Pls feo aaee Skat lt 01 Plan keia Plan Ao ax entered and the previous screen will return lt 013 gt Plan Apo GK Oil lt B14 gt Plan Aoo 100X Dil lt 015 gt Plan Apo 200x 0il 5 to enter the data of other objectives repeat the above procedure Page D 3 Appendix D U MCB Setting Objective Data Input OB The following table shows the objectives Objective PLAPO 40X PLAPO 60XO PLAPO 100XO PLAPO 40XWLSM PLAPO 60XWLSM PLAPO 60XOLSM UPLAPO 10X UPLAPO 20X UPLAPO 20XO UPLAPO 40X UPLAPO 40XOI UPLAPO 60X UPLAPO 60XW UPLAPO 60XWPSF UPLAPO 100XOI UPLFL10X UPLFL20X UPLFL40X UPLFL60XOI UPLFL100XO UPLFL100XOI UMPLFL10XW UMPLFL20XW LUMPLFL40XW LUMPLFL60XW D 4 Page Appendix D U MCB Setting Transmitted light DIC prism Setting UCD Appendix D 3 Transmitted light DIC prism Setting UCD Please register the DIC prism is suitable for a objective NOTE in the case that you do not have the suitable prism for a objective lens please register the prism temporarily NOTE Leave at least one position NONE for use TIP 11 optical element mounting hole on the INIT input screen Fig D 9 microscopic observation using the FV500 system 1 To display the UCD DATA SET screen Fig D 10 press UCD 1 2 When the TURRET number 2 to be entered is
46. For detailed microscope operation procedures refer to the instruction manuals of your microscope Combination with BX51 or BX61 2 Cube display window 1 Light path 4 Transmitted light DIC selector button FV5 DICTS WI DICTH with BX61WI optional 3 Analyzer U MDICT3 6 Universal condenser 5 Filters LBD ND6 ND25 7 Hand switch When using BX61WiI U HSTR2 LBD U FH is optionally FFR available with BX61 This figure shows the case of BX age 1 1 Getting Started FLUOVIEW Basic Operations pO 1 Light path selector button Select the light path between the visual observation and laser microscopy e Select the lt aWq gt button for visual observation e Selectthe lt 1 gt button for TV or photomicrography e Selectthe lt 2 gt button for laser microscopy 2 Cube turret Select the fluorescence observation tube by rotating the turret e Engage the desired cube in the light path for visual fluorescence observation For laser microscopy or visual transmitted light observation rotate the turret to page tab CS This sets the cube turret so that no cube is engaged 3 Analyzer U MDICT3 Polarizing plate for use in differential interference observation and polarized light observation Rotate the turret to engage the U MDICT3 in the light path for visual transmitted light differential interference observation or transmitted polarized light observation e
47. OPERATION Set a lower scan speed Section 2 2 1 4 10 in OPERATION Set the objective magnification Section 2 2 1 4 1 in OPERATION Set the zoom ratio to 1X If the image If the image Section 2 2 1 4 2 in OPERATION becores does not become clean clean Set the channel to be acquired Section 2 2 1 4 3 in DPERATION Re adjust the image brightness Set the highest scan speed Section 2 2 1 4 9 in OPERATION Section 2 2 1 4 4 in DPERATION Set the XY observation mode Section 2 2 1 4 5 in OPERATION S pp repeated SPnning Section 2 2 1 4 11 in OPERATION Perform repeated scanning Section 2 2 1 4 6 in OPERATION Acqiigne image Section 2 2 1 5 in OPERATION Adjust the Z position to observe the desired multiple sections Save the image Section 2 3 1 in OPERATION Section 2 2 1 4 7 in OPERATION Section inf Set the observation range Section 2 2 1 4 8 in OPERATION Adjust the image brightness Section 2 2 1 4 9 in OPERATION 2 8 Page APPLIED OPERATIONS Image Acquisition 2 2 1 1 Configuring the Microscope Set the light path so that the image can be observed through the microscope 1 Display the Acquire panel lt Focus gt button none putton The repetition images can be acquired at a high speed Use this button to find an optimum image Acquires an image in the currently selected observation mode and display the image in t
48. Page When displaying images acquired in a multi channel mode select the display method from the Display Type drop down list Click the lt New Page gt button A new Display panel appears showing the two images one above the other The image of the file displayed in the frame at the top left of the Tile panel is displayed on the upper part of the Display panel and that of the file displayed in the frame at the top right is displayed on the lower part NOTE Use the lt Retile gt button when it is required to re arrange the images in the currently displayed Display panel Acquire File YOY Tile Yeroces Live Wi XYZ TIF iC TRATF Y X Z Tiled 1 XYZ TIF TRA TIF Columns Rows Tia f Experiment x a00 x 600 v 4 800 Y 600 CH FZ Animate all views Tile Over z Y Self Display Type Side by Sid gt Single View gt Retile New Page Delete Page xalla Tooo Fig 2 68 Panel Displaying Two Different Images Together APPLIED OPERATIONS Changing the Image Display Method 2 4 8 Displaying an Image in Full Screen Only the image itself can be displayed to fill the screen by erasing all other display components such as the toolbar panel and status bar This feature is useful for taking pictures using an analog printer for creation of a slide 1 Display the Display panel of the image to be displayed 2 Click the
49. Sec 1 2 7 Set the observation condition Set the objective magnification Sec 1 2 8 1 Set the zoom ratio to 1X Sec 1 2 8 2 Set the channels Sec 1 2 8 3 Select the light path for 100 binocular tube and focus on the specimen Section 1 2 3 Set the highest scan speed Select the LSM light path Sec 1 2 8 4 Section 1 2 4 Select the XY observation mode Sec 1 2 8 5 Perform repeated scanning Set the dyeing method Sec 1 2 8 6 Sec 1 2 5 J asassssssssllasssssssasassssswasanmanmawnaana If noimage is displayed p eldsip jou s s eeu oy J When the combination using the laser i Image is combiner is used set the optimum Adjust PMT Voltage displayed in the Sec 2 1 1 4 9 ND filters for the laser Section 1 2 7 Live panel of If an image the soft x bhon O is displayed Set the multiple sections to be observed Sec 1 2 8 7 Set the area to be observed Sec 1 2 8 8 Set a lower scan speed Section 1 2 8 9 Stop repeated scanning Sec 1 2 8 10 Acquire an image Sec 1 2 9 Save the image Sec 1 2 10 Exit from the FLUOVIEW software Sec 1 2 11 Turn the system power OFF Sec 1 2 12 1 16 Page e Getting Started FLUOVIEW Outline of LSM Observation Procedures e Transmitted light observation procedure Start the system e Turn the system power ON Sec 1 2 1 e Start the FLUOVIEW software Change the ND filter with a high
50. Then exit from windows and reboot the computer 19 The power supply of The fuse has run out Contact your local Olympus Power Unit FV5 PSU representative for assistance is not turned on then replace the huse 20 The current limit The laser is in standby indicator on the UV Ar condition laser power supply is blinking the Laser Unit group box in the Optical System Configuration window to restart laser oscillation 21 The error indicator on Consult your local Olympus the UV Ar laser power representative supply will not turn off 22 Bios some check error The battery maintains Bios Contact your local Olympus message appears at the set value has run out representative for assistance moment of starting up to replace the battery the computer Bios has broken Consult Olympus Page 1 5 OLYMPUS OLYMPUS OPTICAL CO LTD 2 43 2 Hatagaya Shibuya ku Tokyo Japan OLYMPUS OPTICAL CO EUROPA GMBH Postfach 10 49 08 20034 Hamburg Germany OLYMPUS AMERICA INC 2 Corporate Center Drive Melville NY 11747 3157 U S A OLYMPUS SINGAPORE PTE LTD 491B River Valley Road 12 01 04 Valley Point Office Tower Singapore 248373 OLYMPUS OPTICAL CO U K LTD 2 8 Honduras Street London EC1Y OTX United Kingdom OLYMPUS AUSTRALIA PTY LTD 104 Ferntree Gully Road Oakleigh Victoria 3166 Australia This publication is printed on recycled paper Printed in Japan 2001 05
51. Using the scale in the Threshold group set the threshold value for the intensity values used in operation The intensity data above the threshold values set here will be used in the operation Page 2 199 APPLIED OPERATIONS Image Analysis pO 14 Click the lt Begin Analysis gt button The mean value of the specified regions will be displayed graphically in the Mean Intensity box NOTE The colors of the chart lines corresponding to the colors assigned to the regions NOTE When the image was acquired in the multi channel mode the channel number is displayed to the right of each chart line heo Y Tile process an ya Live 16 XYZ 2 TIF Y MERGE TIF 3 MERGE TIF Em iL XYCH Single Series Intensity vs X Single Slice Integration x Threshold chi o Ch2 0 ch3 LI Mean Intensity 2203 O lt ili Integrated Intensity 2 0234e 05 0 xaea CIMAGES MERGE TIF Fig 2 94 Panel After Analysis 15 Double click the Mean Intensity box The Average Intensity Trace window appears as shown below 2 200 Page lt Properties gt button Displays the Editing dialog box for use in detailed setting of the chart or change of the chart display See section 2 14 Changing the Chart Display Method for details lt Copy gt button Copies the plotted image in the clipboard lt Save g
52. buttons to let them 2 disappear lt Channel 2 gt button Acquire File vo Y Tie roces 3 lt XYZ 2 TIF lt Channel 3 gt button cm so XYZCB 2ot2 Live xvz 2 TIF ii File Type 4 Titt TIF lt Channel 4 gt button th pi tit m 800 600 xy2chtif 800 600 5 race ae 800 600 lt Channel 5 gt button aq ci Images from multiple channels can be displayed either by merging them or placing them side by side It is also possible to display the image of only one of these channels Use the buttons displayed at the top of the Display panel and those on the bottom right which are displayed when the corresponding image is clicked For the display of the image of only one channel see section 2 4 3 Switching the Displayed Channels e Page 2 141 APPLIED OPERATIONS Changing the Image Display Method 2 4 4 1 Displaying Images Separately Per Channel Side By Side Views Over And Under Views 1 Display the Display panel of one of the images to be displayed side by side at the front E 2 Click the lt Display switching gt button at the top of the Display panel The list of lt Display switching gt button buttons as shown below appears 3 From the displayed list of buttons click one of the lt Side by side Views gt button or lt Side oe lt Over and under views gt button The icon shown in the lt Di
53. gt Fine Save as type fasci text txt 7 Cancel Fig 2 44 Save As ASCII Text dialog box 7 In order to change the save destination drive or directory use the Save in drop down list 8 Confirm that ASCII text txt is selected in the Save as type drop down list 9 Enter a file name into the File Name text box 10 Click the lt Save gt button NOTE When the text file having the same name as the entered name is already exists the dialog box appears to ask you to overwrite the existing file or not When overwriting is not required click the lt NO gt button and enter another name to save the file When the image to be saved was acquired through multiple channels the observation conditions for all channels are saved Page 2 129 APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 7 Saving Reading the Region File The information on the shape position and color of certain region can be saved in a file and they can also be read 2 3 7 1 Saving the Region File 1 Display the Display panel of the image including the region to be saved at the front position 2 Select the lt Experiment List gt button in the tool bar at the bottom of the File 1 0 panel The Experiments in Memory dialog box as shown below appears lt Experiment List gt button Experiments in Memory 2 Comments pone Fig 2 45 Experiments in Memory dialog box
54. in the sub menu Select Default for ordinary use Hole Count Nothing Selected Hole Color 1 2 3 4 5 6 7 v 8 Default 1 3 2 2 Setting the Name 1 Editing the name of the buttons in the Mirror Unit Condenser Turret and Filter Turret group boxes 1 In the Mirror Unit Condenser Turret or Filter Turret group box right click the mouse on the button whose name is to be edited The pop up menu as shown Edit Color 2 Select Edit The Edit dialog box as shown below appears below appears ict OK Cancel 3 Enter a name into the text box and click the lt OK gt button 2 Editing in the Nosepiece group box 1 Inthe Nosepiece group box right click the mouse on the button whose name is to be edited The pop up menu as shown below appears Edit Color 1 16 Page Software Setup Setting the System Configuration 2 Select Edit The Edit dialog box as shown below appears Recommended Confocal Pinhole text box Enter the number of the confocal pinhole with keyboard Name of the Objective drop down list Select the name of the Name ofthe Objective objective in the list or enter the name with kevboard Name of the Linked Condenser drop down list S lact the condenser Magnification text box worked with Enter the magnification of N A text box n text box the objective with Enter N A of the Enter refractive index with k
55. lt Copy gt button Copies the plot image to the clipboard pass Copy Save lt Close gt button Quits the Enhanced Profile Plot LAm window and returns to the Analyze Position um panel Fig 2 113 Enhanced Profile Plot Window 2 226 Page APPLIED OPERATIONS transferring Data to Another Application Doo O 2 Click the lt Copy gt button to copy the plot image to the clipboard 3 Exit from FLUOVIEW or display the Start menu by pressing the Windows key or the Ctrl Esc keys 4 Select Programs Accessories and issue the Paint command 5 From the Edit menu of Paint select the Paste command and paste the plot image which has been copied to the clipboard in step 3 lt TIP For detailed operation procedures of Paint refer to the help provided by 2 10 3 Transferring Image Data to Another Application microVoxel Paint etc To transfer image data to another application the image data should be saved in a file and the file should be transferred 1 Save an image using one of the formats that can be handled by the destination application See sections 2 3 Saving Opening and Shredding Images and 2 3 1 1 Saving Images As a Series for the image saving procedure 2 Exit from FLUOVIEW or display the Start menu by pressing the Windows key or the Ctrl Esc keys NOTE To transfer data to microVoxel it
56. lt Display gt button Press to start display of n slices of image from the end position to the start position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame 2 212 Page APPLIED OPERATIONS viewing 3D Image pO 3 Click the lt Set start position gt button If the start position is not set image No 0 becomes the start image automatically 4 Display the image slice to end the successive display by using the lt Frame advance gt button at the top of the Display panel 5 Click the lt Set end position gt button If the end position is not set image No n 1 assuming that the number of images is n becomes the start image automatically 6 Click the lt Successive display gt button The images will be displayed successively from the start position to the end position Click the lt Stop gt button to stop the successive display 2 8 1 1 Changing the Successive Display Speed The buttons on the top of the Display panel can be used to vary the speed of successive display of multiple image slices 1 Display the Display panel of the image to be subjected to successive display speed change 2 The buttons as shown below are displayed at the top of the Display panel Usually the lt Rabbit gt button is displayed Clicking it displ
57. start 0 is set when this button is not used lt Set end position gt button When successive display or frame by frame display is required set the image with which the display should end Assuming that the number of images is n n 1 is set when this button is not used lt Loop Bound switch gt button lt Display speed switch gt button Set the speed of successive display lt XYZ series gt button Displays one of multiple images by selecting it according to the multiple sections Z lt XYT series gt button Displays one of multiple images by selecting it according to the time T lt Bound gt button Every time successive display or frame by frame display in a direction ends at the start or end position the display is restarted in the opposite direction lt Display gt button Press to start display of n slices of image from the start position to the end position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame lt Rabbit gt button Press for successive display of image slices without pause lt Clock gt button Press for successive display of image slices at the time interval set in the Time Series sub panel lt Turtle gt button Press for successive display of image slices at an interval of 0 8
58. then select lt Clip gt in the Scan Mode group box and reselect the scanning range 2 70 Page APPLIED OPERATIONS image Acquisition Doo 2 2 5 High Speed Image Acquisition An image can be acquired in 0 25 second This image acquisition mode is valid under the following condition e Max 2 channels e Image size 512 x 512 pixels e Zoom ratio of 2X or more with setting in 2X steps 1 Check one or both of the Ch check boxes in the Ch group box in the Acquire panel 2 Select the Surface XY None option button in the Scan Mode group box in the Acquire panel Then select lt Fast gt in the list shown below Scan Mode Surface XY Horm e E Hormal Fast C Line XT Depth XZ C Point Size snz by 512 7 512 by 512 is set automatically in the Size drop down list in the Scan Mode group box X2 is set and shown in gray out display automatically in the Zoom scale in the Pan group box 3 To change the zoom ratio use the Zoom scale in the Pan group box xY Repeat 4 Select any button to acquire image such as the lt XY Repeat gt button lt XY Repeat gt button a Wa S E a umpu paskisqa qaa sisa sss s TIP ee ae High speed image acquisition is available in the XY XYT XT observations lt Once gt button ee 5 After acquiring images select lt Normal gt from the list displayed below the Surface XY Norm option button i
59. 1 TROUBLESHOOTING GUIDE i Phenomenon Manual Ref Pages 1 Fluorescence image The fluorescent dye and Select a laser suitable for the 1 2 7 OPERATION cannot be observed excitation wavelength does fluorescent dye method INSTRUCTIONS not match Change the ND filters by reading 1 2 7 Selecting the ND Filters in OPERATION INSTRUCTIONS The fluorescent dye does not Select the barrier filter 1 2 6 amp 1 3 2 4 match the excitation dichroic matching the fluorescent dye OPERATION mirror dichroic mirror and or method INSTRUCTIONS barrier filter See 1 3 2 3 Configuring the Filters in OPERATION INSTRUCTIONS and follow instructions of the Optical System Configuration window Focusing is not correct Adjust focusing 1 2 3 OPERATION INSTRUCTIONS The photomultiplier tube Increase the photomultiplier 2 2 1 4 9 voltage of the detector is low tube voltage using the PMT OPERATION scale in the Acquire panel INSTRUCTIONS The OFFSET value is too Decrease OFFSET to an 2 2 1 4 9 high optimum value OPERATION INSTRUCTIONS The photomultiplier tube of Check the Ch1 Ch2 1 2 8 3 amp 2 2 1 4 3 the channel set with the Ch3 Ch4 or Ch5 check OPERATION detection mode setting slider box in the Acquire panel INSTRUCTIONS is not set 2 Transmitted image cannot be observed The transmittance detection In the Acquire panel check 2 2 3 3 channel is not selected the chec
60. 2 230 2 11 3 Displaying the X coordinate Y coordinate of the Image 2 231 2 11 4 Drawing a Figure in Image 2 232 2 11 5 Drawing a Scale in IMage ccc eeeceeeeeeeceeeeeeeeeeeeeeeeeeeeeeeeeeneeeeeeeeeeteees 2 233 2 11 6 Drawing an Arrow in Image r ennnen 2 235 2 11 7 Drawing Color Bars in Image I 2 236 2 11 8 Deleting Comment n 2 237 2 11 9 Moving Comment a n 2 237 2 11 10 Changing the Comment Size a 2 239 2 11 11 Changing the Comment Color a 2 240 2 11 12 Changing the Comment Font a 2 240 2 12 Image Output at Printer 1 2 242 2 13 Merger Extraction of Image Channels 2 244 2 13 1 Setting the Range of Multiple Image Slices 2 244 2 13 2 Merging Image Channerls ars 2 245 2 13 3 Extracting Channels from Image ccceeeseeceeeeeeeeeeeneeeeeeeeeeennseeeees 2 249 2 14 Changing the Chart Display Method 2 253 2 14 41 Ch rt Panel s u yuya umma steel eH OAR A eGR 2 253 2 14 2
61. 20 34 1995 Ch Number of image channels An Number of images created when constructing a stereo image using the Visualize function Stx 2 is displayed when stereo images stereo 3D image or stereo image to be viewed through color eyeglasses are constructed and saved using the Visualize function Size Image file sizes Date Image saving dates User Comment Part of the comment saved with each image Page 2 103 APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 1 Saving Images Either a series of images or single image being displayed can be saved 2 3 1 1 Saving Images As a Series The displayed images can be saved in a disk as a series image file 1 Display the Display panel showing the image to be saved at the front T gt 2 When there is more than one image to be saved the lt Set start position gt and lt Set Ea lt Set start position gt button end position gt buttons will be displayed on the upper part of the image Display the lt Set stop position gt button image with which the saving should start using the lt Display gt buttons then click the ay lt Set start position gt button Also set the save end position in the similar way 3 When the images to be saved are acquired from more than one channel select lt Display gt button 4 2 3 4 5 whether saving images from more than one channel simultaneously or only a single lt
62. 4 Click the lt Rectangular gt button in this displayed buttons 2 108 Page APPLIED OPERATIONS Saving Opening and Shredding Images Specify the area to be saved in the image in the Display panel _ The area is displayed on the image with the handles a I I on its frame h d The area becomes the save target while these L H d handles are displayed k Handle 6 Click the lt Annotate gt button so that the list of buttons disappear 7 Click the lt Experiment gt button in the Save group box The Save Experiment As dialog box appears Save Experiment As HE Save in E Images gt al e j 86 3d1 tif E 86xt1 tif E 86xpt tif E 86 xy22 2 ti a 86 3d2 tif 86xt2 tif E 86xyt2 tif 86xyz3 tif fa 86 3d3 tif E 86 t3 tif E 86xyt3 tif E 86xyz3 2 ti E 86 3Dst1 tif 86xy1 tif 86sxyz1 tif B6xyzt1 tif fa 86 3Dst2 tif E 86xy2 tif E 86xyz1 2 tif E 86xyzt2 tif E 86 3dst3 tif E 86 y3 tif E 86xyz2 tif 86xyzt3 tif lt gt File name fxV2CH TIF Save as type FLUOVIEW MultiT if tif x Cancel Fig 2 32 Save Experiment As Dialog Box 8 Use the Save In drop down list if you want to change the save destination drive and directory 9 Select FLUOVIEW Multi Tiff in the Save as Type drop down list 2 TIP f It is not possible to save only the specified area of image in the Single TIF f 10 Enter the file name in the File Name text box 11 Click
63. 70 2 2 5 High Speed Image Acquisition raras 2 71 2 2 6 Acquiring Finer Image Sequential Scan 2 72 2 2 7 Image Acquisition of Rectangular Position at Desired Angle 2 75 2 2 8 Image Acquisition of a Line at Desired Angle 2 77 2 2 9 Display the change of image intensity Point Scan 2 78 2 2 10 Image Acquisition on Desired Line XZ XT or XZT Observation 2 83 2 2 11 Image Acquisition in the Laser Excitation Mode 2 86 2 2 11 1 Making REX Mask File u 2 87 2 2 11 2 Example of FRAP experiment 2 92 2 3 Saving Opening and Shredding Images 2 101 2 3 1 Saving Images ua nun ee l aaa a aaa aaa eae 2 104 2 3 1 1 Saving Images AS a Series teier 2 104 2 3 1 2 Saving a Display m W ee ene Adee 2 106 2 3 1 3 Saving Specified Area Of Image u 2 108 2 3 1 4 Saving Animation Images 2 111 2 3 1 5 File Types Available for Save 2 112 2 3 2 Opening Previously Saved Images
64. A Diameters The pinhole diameters are set automatically according to the selected dyeing method Use the following procedure to change the pinhole diameters 1 Display the Acquire panel P Fv500 Acquire File O tive XY Repeat Once Fuci V Transmitted PMT Gain Offset 320v x4 0 0 xY OXYT Oxyz OXYZT r Scan Mode Surface XY Horm Ch group box Sets whether the image of each channel is to be acquired or not the PMT voltage Gain and Offset Right clicking the mouse inside this group box displays the Ch group boxes of all channels 1 23 Hormal Fast Sequen C Line xT Cc Ler XT C Pemt Size soo by 600 gt upjecuue UPLAPO 10X Pan Zoom gt Ja C Scan Speed j Z Fast Slow 17s Scan 1 75 Image r Acquisition Settings Load Save r Focus Mode Display Panel 1 74 Page Getting Started FLUOVIEW Online Help 2 Right click the mouse inside the Ch group box to display the Ch group boxes of all channels Ch1 Ch2 Ch3 Ch4 and Ch5 group boxes Select the channels from which z V DAPI v FITC you want to acquire images atti can oast Offset 5 PMT Gain and Offset m K os iV os I Transmitted PMT Gain Offset PMT Gain Offset j oi Offset 800v sal 800v 10 0 Z 485v 43x 0 LED sliders Set these values
65. After image acquisition select lt Normal gt under the Surface XY option button in the Scan Mode group box in the Acquire panel to return from the image acquisition mode 2 82 Page APPLIED OPERATIONS Image Acquisition 2 2 10 Image Acquisition on Desired Line XZ XT or XZT Observation When the sample is in curved form the image can be acquired by drawing a desired line on it 1 Acquire an image in the XY observation mode For detailed operation see section 2 2 1 Image Acquisition with XY observation 2 Select Line XT option button in the Scan Mode group box in the Acquire panel or lt Free gt under the Depth XZ option button A line indicating the scanning range appears in the Live panel lt Free gt under the Line XT option button is selected in the following Fig Scan Mode Surface XY Horm ft Line xT s A Horrnal Slant Free Depth XZ C Point Size soo by T gt TIP Aline can be specified for one display UU ky eee 3 Move the curved line to the area you want to observe To move the curve place the mouse pointer in the box and drag the mouse Page 2 83 APPLIED OPERATIONS Image Acquisition fe j 2 84 Page Change the curved line shape Place the mouse pointer within the box and right click to display the pop up menu Then select lt Edit gt apy Move size Edit Properties The cross shaped handles appear around th
66. Calcium Green 1 Calcium Green 2 Calcium Green 5N Calcium Orange Calcium Orange 5N Fluo 3 gt Dio gt and drop them here to identify those Dyes you actually use Double click to edit Save New Setup Quit w o Saving 4 Double click the Double click here to make a new Dye in the Dyes on your Samples list m Name of the Dye Laser Type D 7 The Edit Dye dialog box as shown below appears Excitation Wave Length 4 r Emission Wave Length Excitation Wave Length group box Enter the excitation wavelength In case of the 2 wavelength and 1 photometry enter the value into the WaveLength2 text WaveLength 1 WaveLength 2 box Emission Wave Length group box WaveLength 1 Enter the emission wavelength In case of the 1 wavelength and 2 photometry enter the value into the WaveLength2 text box too WaveLength 2 5 Enter the name of the dyeing method into the Name of the Dye text box e g CFP 6 Select the laser to use using the Laser Type drop down list e g HeCd TIP As the laser to use helium cadmium laser should be set in advance When Heca is not displayed check the HeCd check box in the Laser Page 1 21 Software Setup Adding the dyeing method 7 Enter the value of the excitation wavelength into the WaveLength 1 text box in the Excitation Wave Length group box e g 442
67. Channels Experimenta in Memory 4 From the file list in the Experiments in Memory dialog box select the file name of Experiment Editor XY 1 TIF the image from which to extract channels and drag it into the frame at the top left of the Experiment Editor group box The icon of the image is displayed in the frame at the top left of the Experiment Editor group box CAMAGESVOY2 TIF 5 The channels of the image set in the Experiment Editor group box are connected to Extract by lines The lines of channels are colored as shown below Ch1 Navy blue Ch2 Light blueCh3 Green Ch4 Yellow green Ch5 Orange Ch6 Red When the mouse pointer is approached to the icon side end of a line connected to Extract the color of the line end turns into yellow Clicking the line in this condition switches the channel between the selected and deselected status alternately The lines connected to Extract indicate the selected channels Experiment Editor eo EA Clicking a channel line in this area switches the Aail channel between the selected and deselected T status XYCH Page 2 251 APPLIED OPERATIONS Merger Extraction of Image Channels Experiment Editor XY 1 TIF Oat 2 252 Page 6 Among the channel lines connected to Extract deselect the unnecessary channels as described in the TIP on the previous page so that only the necessary channels are selected
68. Data Type Choose the file type that best describes your data Delimited Characters such as commas or tabs separate each field Excel 4 0 standard C Fixed Width Fields are aligned in columns with spaces between each field Start Import at Row 1 Fie Origin Windows ANSI Preview of file C IMAGES OGAWA XLS a ine Intensity Profile 112 000000 to 2176 000000 osition um NolPositionl rH Ps ss 000000 Fig Appendix G 1 Dialog Box Displayed When File is Opened with Excel 1 3 4 Click the lt Next gt button When the dialog box as shown below appears check the Tab check box in the Delimiters check box then select none from the Text Qualifier drop down list Text Import Wizard Step 2 of 3 This screen lets you set the delimiters your data contains You can see how your text is affected in the preview below p Delimiters Treat consecutive delimiters as one IX Tab Semicolon Comma Space Other Text Qualifier none Data Preview ine Intensity Profile 112 000000 to 2176 000000 osition um No 0 kk Fig Appendix G 2 Dialog Box Displayed When File is Opened with Excel 2 3 Page G 1 Appendix G Converting Analysis Data into a Chart Using EXCEL 5 Click the lt Next gt button When the dialog box as shown below appears click the General option button in the Columns Data Format group box then click the l
69. Display Method Click the lt Experiment List gt button in the toolbar at the bottom of the Tile panel The Experiments in Memory dialog box appears as shown below Experiments in Memory CAIMAGEYS YZ TIF CAIMAGE TR 1 TIF is C ZARA Fig 2 67 Experiments in Memory Dialog Box From the Experiments in Memory dialog box select the file name of the second image to be displayed and drag it into the frame at the top right of the Tile panel The icon of the second image is displayed in the frame at the top left of the Tile panel and the acquisition parameters used in the image acquisition are displayed in the Experiment panel TIP _ The mouse pointer turns into the image icon during dragging Click the lt Done gt button in the Experiments in Memory dialog box to close it Set the number of images to be displayed together by using the lt gt and lt Y gt buttons in the Columns and Rows text boxes How the images will be arranged can be confirmed in the gray box at the upper part of the Tiling group box When there are multiple images to be displayed select the following items in the Tile Over drop down list e Self The same images as the image being displayed will be displayed e Z Images are displayed according to change in multiple sections e T Images are displayed according to change in time Page 2 157 APPLIED OPERATIONS Changing the Image Display Method 2 158
70. Dsub15pin UMCB Dsub9pin combination LYN O CO with AX only 11 Dsub15pi rT BX UCB q i FV5 PSU combination with BX51 61 only i U PS q POWER combination anc i SUPPLY L with AX only Li FV5 SU LI LI BQ Square 3 pin SCAN UNIT i SX Dsub25pin Circular 6 pin FV5 zZM 8 2 9m bare ee Z MOTOR Dsub25pin 1 2 9m 1 2 9m LITRANSMITTEL _ HeNe GTASER 1 8m Square 2 pin POWER SUPPLY q UNIT ee oneal Tam 10 H POWER SUPPLY _ 1 2 9m E Square 2 pin UNIT ar sub25pin DsubtSpin mo L D ae 2 ia UV Ar LASER FV5 LUUV E p U POWER Dgub9pin 1 s CHILLER tH supply s REMOTE SW POWER SUPPLY q FV5 LDUIR Dsub25pmi Circular 8 pin FV5 COMB I q 3 Circular S7PIOT lar LASER POWER 5 5m PAROM fag Se ears TRANSMIT j 2am y SUPPLY UNIT TED LIGHT fPsub25pin sube pin i 4 2 9m BNC DETECTOR lt r LASER POWER 55m 2 t r SUPPLY UNIT Circular 37 pin 3 im E Circular 37 pin LASER COMBINER Square 3 pin IILI KrLASER Duct 2 2m SIROCCO FAN U RFL T 8Mq BH2 RFL T3 2 2 9m HeCd LASER J Reflected Square Z pn POWER SUPPLY DH FV5 LUHECD L I LJ Fluorescence Light Power Supply Wnit Fea as a oN Si er ay eee eer e a eo eee e eS a he eee es ed 1 6 Page SYSTEM OVERVIEW System Configuration The block diagram in AOT
71. Editing Enables or disables the label display Series General Axis Titles Legend Panel Paging M Show Axis cales Title Labels Ticks EZtion Axis Not used with the present application IV Visible JV Label On Axis Multiline _I Round First Enables or disables the scale display according to the maximum and minimum values in the chart Bottom C Depth fals 3D Size 0 Min Separation 10 Angle fo Style Auto C Value V Visible None C Mark Sets the distance between labels Ticks sub panel Used to set the coordinate axis line Sets the type color and width of the coordinate axis Sets the type color and width of the scale line on the label side of the coordinate axis Axis Border Sets the type color and width of the scale line on the iio chart side of the coordinate axis Bottom Show Axis Grid Border Centered Ticks Len f4 IV At Labels Only Inner C Depth Sets the length of the scale line from the coordinate axis to the chart side V Visible Sets the type color and width of the scale line between labels Sets the length from the coordinate axis of the scale line between labels to that on the label side 2 256 Page Enables or disables the label display on a point of crossing with another axis Sets the label character font Sets the distance from the title to the lab
72. FLUOVIEW Outline of LSM Observation Procedures v TIP The focus mode makes it possible to increase the line skipped scan speed f r i From the page tabs on the bottom right of the Acquire panel select the U Scan sub panel i Select either option button in the Focus Mode group box r Acquisition Settings Load Save i m I Focus Mode group box Focus Mode X2 option button 4 Acquires image at twice the X2 C x4 highest speed i X4 option button m Filter i Acquires image at 4 times the Hormal highest speed 2 i Increasing the number of Kalman 8 divided images in the Display panel line skipped scan at 4 Accumulate To Peak Sl i times Focus cannot be done NOTE The Focus function reduces the scanning time by line skipped scan As a result the acquired images become coarse 1 2 8 5 Setting the XY Observation Mode 1 In the Scan Mode group box in the Acquire panel select the Surface option button 2 In the Scan Mode group box in the Acquire panel select 800 by 600 from the Size drop down list 3 In the Acquire panel select the XY observation mode option button Page 1 51 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 8 6 Repeated Scanning Operation 1 Select the lt XY Repeat gt button The acquired image will be displayed in the Live XY Repeat panel pure
73. File gt button Description The name of a panel dialog box list box or check box is enclosed inside square brackets The name of a button in a panel or dialog box is enclosed inside angled brackets lt gt lt gt Notation of Mouse Operations Notation clicking double clicking dragging Description Action of pressing then immediately releasing the mouse button Action of clicking the mouse button twice in quick succession Action of moving the mouse while holding down the mouse button then releasing the mouse button at the desired destination Note In this manual clicking double clicking and dragging involves pressing the left button of the mouse unless otherwise specified Page 3 NOTATIONS IN THIS MANUAL lt Notation of key operations Notation Description The name of a key is enclosed inside _ J Alt F1 The positive sign expresses the combination of more than one key operation For example refers to pressing the key while holding the key down Direction keys Generic names given to the eS J and keys lt Notation of system specific terms Notation Description XY observation Refers to observation with XY scanning Other observations The same principle also applies to other observations such as XZ Xt XYZ XYt and XYZt Note that some of the panels and dialog boxes shown in this manual are not the precise reproductions of the originals Som
74. For laser microscope always disengage the U MDICT3 Engaging the U MDICTS in the light path degrades the image quality 4 Transmitted light DIC FV5 DICTS WI DICTHRA when using BX61W1 optional This is the prism for use in differential interference observation Engage the transmitted light DIC in the light path for laser differential interference observation or visual transmitted light differential interference observation Leaving the transmitted light DIC engaged during laser fluorescence observation will degrade the resolution somewhat We recommend disengaging the transmitted light DIC from the light path when simple laser fluorescence observation is required 1 2 Page Getting Started FLUOVIEW Basic Operations NOTE With transmitted light differential interference observation using an immersion objective set the microscope s field diaphragm so that it circumscribes the field of view Otherwise the contrast may degrade This applies to both visual observation and laser differential interference observation 5 Filters These filters are used to adjust the transmitted light e Be sure to disengage any filter from the light path for transmitted observation using lasers Leaving a filter engaged in the light path will degrade the image quality When you perform transmitted observation using laser with BX61WI use the filter knob to disengage the LBD from the light path and engage the FR Frost into th
75. Per Channel Side By Side Views Over And Under 7 Oe ean a aC ever aT ana haatun nee ere eee Tete 2 142 2 4 4 2 Displaying Merged Image of Multiple Channels Single View 2 143 2 4 5 Changing the Number of Divided Images 2 144 2 4 5 1 Increasing the Number of Divided Images 2 144 2 4 5 2 Decreasing the Number of Divided Images a 2 146 2 4 6 Switching the Display Method of Multiple Images 2 148 2 4 7 Displaying Multiple Image Slices Together 2 149 2 4 7 1 Displaying Multiple Images Per Channel a 2 150 2 4 7 2 Displaying Images of Two Channels Together 2 152 2 4 7 3 Displaying Time Lapse Images a 2 153 2 4 7 4 Displaying Multiple Multiple sections Images 2 154 2 4 7 5 Displaying Same Images in Different Display Methods 2 154 2 4 7 6 Re arranging Images Using the Same Display Method 2 156 2 4 7 7 Displaying Different Images Together 2 156 2 4 8 Displaying an Image in Full Screen 7 2
76. Sel Stat Z Ep Step Size Slices Title bar Shows the title of the window The title OSD A ae bar of a window that is active is Group box The group box groups functions with specific meanings and encloses them ina frame Option buttons This is a group of multiple items among which only one can be selected Clicking one of the round buttons selects the corresponding item The option button of the selected item is displayed with a black dot in the center of it Information Shows information on the operations and meanings of functions The scroll bar is displayed when there are too many data items to be displayed in a field at once and is used to display the data items outside the field Clicking a point in the scroll area allows data items to be scrolled in large steps Clicking the top or bottom arrow button allows fine scrolling of the data items Dragging the square knob allows direct scrolling List box Shows the list of available items for selection All items in the list can be displayed by scrolling To select an item in the list double click or drag the item Page 1 15 Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 Outline of LSM Observation Procedures e Fluorescence observation procedure Start the system e Turn the system power ON Sec 1 2 1 e Start the FLUOVIEW software Sec 1 2 2 Change the ND filter with a filter with higher transmittance
77. Series Pa el u uuu ununwan yas aaa aussi Rau 2 261 2 15 Pop up Menus l 2 264 Appendix A List of Tools A 1 Appendix A 1 List of Tools u A 1 Appendix A 1 1 Toolbar U A 1 Appendix A 1 2 Tools at the Top of Display Panel A 2 Appendix B List of Hot Keys B 1 Appendix C Glossary C 1 Appendix D U MCB Setting D 1 CONTENTS pO Appendix D 1 Mirror Cube Data Input CUBE D 2 Appendix D 2 Objective Data Input OB D 3 Appendix D 3 Transmitted light DIC prism Setting UCD D 5 Appendix D 4 Setting PC Communications D 7 Appendix E User Registration E 1 Appendix F Formatting of Magnetic Optical Disk F 1 Appendix G Converting Analysis Data into a Chart Using EXCEL G 1 Appendix H USER REGISTRATION OF FV500 H 1 Appendix H 1 User Registration H 1 Appendix H 2 Logging into the FV500 H 2 Appendix H 3 Deleting a User H 3 Appendix Change of Default Folder for File I O Panell 1 Appendix J List of Functions in the Active Overlays D
78. TIF a XYZCH Single Measurement Results Line Length 20 488 um x 42um Y 15 8 um Statistics Total 44577 25353 Average 564 27 320 92 Std Dev 473 07 379 06 Intensity Profile o FullScreen Display Paste Print Channel 2 Save Display Region Histogram Save Experiment ote UCU Select All Overlays ViewProcessor Views Experiment Properties x aje zJ 2 Fig 2 130 Display Panel Showing Pop up Menu FullScreen Display Displays the image full screen Paste Pasts the comment copied on the image Print Outputs the image at the printer Save Display Saves the image as a single image Save Experiment Select All Overlays ViewProcessor gt Views Saves the image as a series of images Selects all the comments overlaid on the image Edits the image LUT Sets the number of image divisions when displaying more than one image Experiment Properties Edits the image comment Page 2 267 APPLIED OPERATIONS Pop up Menus Pop up menu of the Experiments in Memory dialog box When the right button of mouse is clicked in the frame for opening an image file in the File I O Tile or Process panel a pop up menu appears This makes it possible to select the file to be opened when performing tile display inter image operations image channel merger extraction etc Tie Y Process Analyze
79. The operation method is identical to the method for displaying time lapse images except for the following point See section 2 4 7 3 Displaying Time Lapse Images Select Z from the Tile Over drop down list _ Fiero Y me acess _ XYZ_2 TIF XYZ 2 Tied on ECE Der jo XYZCH Tiling Columns Rows 714 P Experiment Y 600 Z 4 Animate all views Tile Over Display Type Reie New Page Delete Page Drag Drop images on input basins Fig 2 65 Panel Displaying Different Multiple sections Images Together 2 4 7 5 Displaying Same Images in Different Display Methods 2 154 Page Images composed of multiple image slices can be displayed in more than one display methods together 1 Display the Display panel of one of the images image composed of multiple slices to be displayed together The icon of the image is displayed in the frame at the top left of the Tile panel and the acquisition parameters used in image acquisition are displayed in the Experiment panel 2 Set the number of images to be displayed together by using the lt gt or lt Y gt buttons in the Columns and Rows text boxes How the images will be arranged can be confirmed in the gray box at the upper part of the Tiling group box 3 Select Z or T from the Tile Over drop down list APPLIED OPERATIONS Changing the Image Display Met
80. Tie Y rocess Anat Y__SAMBDIC ORG Y tevet The background irregularities are solved Logical Histogram Filters Display zoom 100 1 Fig 2 74 Level Panel After DIC Level Correction 4 If the DIC level irregularities cannot be reduced repeat steps 1 to 3 above by varying the correction factor e Page 2 169 APPLIED OPERATIONS Image Processing 2 5 2 Contrast Conversion The LUT intensity can be mapped re assigned while observing a histogram Mapping re assignment results in changing the image contrast An image acquired by observation contains intensity information in values from 0 to 4095 but the intensity information used in actual display takes values from 0 to 255 by assigning the original values from 0 to 4095 to values from 0 to 255 usually This facility between 0 and 4095 and mapping this section to values between 0 and 255 lt oe 0 255 LJ 0 1 Display the Process panel at the front 2 Display the Histogram sub panel of the Process panel at the front Acquire File o Y Tile 2roce Sets the image to be subjected to OLYMPUS TIF contrast change The icon of the image is Tio displayed here g XY 2 5 5 CH Live ovvmpus niF et gt Ulead oe Histogram of SFLUOVIEWIMAGES SAMPLE1 TI Master View 2011 A a Min 0 Max 4095 Selected View Channel option button Select the channel to be
81. To display the line images use the Visualize panel Display the Visualize panel e Page 2 209 APPLIED OPERATIONS Building an Image from a Different Viewpoint 1 Display the Display panel of the XYZ multiple sections image Make Multi Plane view 2 Click the lt Make Multi Plane View gt option button in the Rendered View panel lt Make grey View gt The 3D panel is created to start the line image building utton Line image display in XZ direction Display the image of the line specified on the XY direction image Begin Visualize Se Rn 2 eats SS Image display in XY direction C Extended Focus View The line image to be displayed Atina view in XZ or YZ direction can be 7 moved by dragging the red line oe XZ or brown line YZ on the gt gt XY direction image west C Initial view Sequence views Rotation Angle Increment AX TT AY 54 az of Standard Views Default Number of views TTE Current View XY view 22288 um Sice 5 Line image display in YZ direction intersection X 243 Y 256 Z5 Display the image of the line specified on the XY direction imaqe x je e i 2 Display Panel Fig 2 102 Panel showing the line images to be viewed in Z direction Upper row shows the current slice and the steps in Z direction acquiring the image z the number of current steps Slice
82. box and the Y axis label in the Value Y text box G 4 Page Appendix G Converting Analysis Data into a Chart Using EXCEL 17 Click the lt Finish gt button A chart will be drawn on the sheet Microsoft Excel OLYMPUS XLS File Edit View Insert Format Tools Data Window Help Hew Sl PPD ESEE s eis 2s oy ay Chart 1 pa ne B q D E F G H l J K L M N 0 E2 73 000000 to 1658 000000 I3 Position um 0 000000 to 20 500000 W no Position 0 0 73 6 1 0 25 104 El 2 0 5 130 3 3 0 75 108 f 1 feu 4 1 83 Enhanced Profile Plot 5 1 25 121 1 6 15 100 42 7 175 110 1m 13 8 2 30 1600 a4 9 225 87 15 10 25 119 1400 46 11 275 124 47 12 3 88 1200 48 13 325 234 gt 14 35 492 g 00 20 15 375 1356 e 21 16 4 1658 22 17 425 1635 600 23 18 45 1450 24 19 475 1085 400 25 20 5 938 26 21 5 25 775 200 27 22 55 653 28 23 575 622 sR E i A SRE AAAS EA Si o Ea 29 24 6 609 SS o ku p tu S OS I Oe Oe SONS eR 526 Position um 32 2 6 75 481 4 33 28 7 471 34 23 7 25 481 35 30 75 501 36 31 775 481 z HA Olympus Eo Double click chart to edit ES ae Lr as s E Fig Appendix G 9 Sheet with Inserted Chart pe Sees I iWhen the mouse is clicked on the chart black handles will appear around the chart The f
83. box in the Lasers sub panel AOTF REX Function Disabled C REX mode Bleach mode Make REX mesk XYT 2 Click the lt XYT gt button to start image acquisition lt XYT gt button Disabled option button 3 Select the Bleach mode option button in the AOTF group box in the Lasers sub panel AOTF REX Function REX _Live 0 l C Disabled Bleach mode option button REX mode Bleach mode Make REX mesk xY NOTE Select the Bleach mode option button within 10 seconds before the next scanning is started NOTE in the TIME COURSE software optional when the REX mask file in the REX or Bleach mode is changed while image scanning is performed obtaining the real time graph the laser is irradiated while the pop up menu is displayed however the real time graph is not plotted Please be aware this before using 2 98 Page 4 APPLIED OPERATIONS Image Acquisition if The laser of strong intensity is irradiated to the specified region Start the second scan in 10 seconds after the first scan is performed Beauly Y TE Y 1 tive Y STOP SCAN F Offset I F mito rj PMT Gain a 690v 10x YJ xY_ xYI C xyz OXZI Mode G Surface XY Clip EA 140 25 Normal Clip Sq Clip C Line 100 90 Scan Speed m Tall mr KA f m E gt or Em on H Propertie 4000 Ae Function RE
84. close the dialog box e Page 1 69 Getting started FLUOVIEW Online Help 1 3 2 3 Configuring the Microscope AX70A When using the combination with the AX70A the microscope and scan unit can be configured from the FLUOVIEW software Use the following procedure for this 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Optics sub panel Light Path x SE e lt Scope Control gt button Click to start the system setup Microscope Control Trans Scope SU Lamp Control Control Fig 1 20 Optics Sub panel 1 70 Page Getting Started FLUOVIEW Online Help 2 Click the lt Scope Control gt button at the bottom of the panel The window as shown below will appear LVT group box Cube group box Switches the light path Select the excitation filter from the drop down list Click lt BI gt button for visual observation or The selection from the drop down list is possible when lt TV gt button for TV observation the lt Reflected Fluorescence Transmitted Differential It is not necessary to change the setting Interference gt or lt Reflected Fluorescence gt button is when performing LSM observation selected in the Observation mode group box Observation mode group box Changes the microscopy mode Observation by combining more than one microscopy technique is also available The lt FL gt button performs reflected light obser
85. date at which the image was captured can be displayed Syntax lt Date gt Appendix J 3 4 Time of Image Capturing The time of the day at which image was captured can be displayed Syntax lt Time gt Appendix J 3 5 Image File Name The image file name can be displayed Syntax lt Name gt Page J 5 MAINTENANCE On This Volume This volume describes the user maintenance procedures of the FLUOVIEW FV500 system Please read this volume so that you can use the system for an extended period of time CONTENTS pO 1 Software Setup 1 1 1 1 Re setup or Updating of the Software 1 1 1 2 New Setup of the Software 1 3 1 3 Setting the System Configuration 1 1 6 1 3 1 Overall Setting of FLUOVIEW a ssssssssssssss 1 6 1 3 2 Setting the BX Control Panel 1 14 1 4 Adding the dyeing method 1 20 2 Maintenance of Major System Units 2 1 2 1 Laser Scanning Microscope s 1 2 1 Dee UV Ar EAS CM suu uuu silus A ATT 2 2 2 2 1 Auto Alignment Operation Remote Switch 2 2 2 2 2 Checking Refilling and Replacin
86. detector but the images will not be Light detector confocal images because there is no confocal aperture in front of the detector The transmitted light image can be used by combination with a confocal fluorescence image to observe fluorescent localization Page 1 1 SYSTEM OVERVIEW Features of FLUOVIEW FV500 1 2 Features of FLUOVIEW FV500 1 The detector has 12 bit resolution and is capable of detecting very small changes in fluorescence inside cells 2 The system has high resolution of 2048 x 2048 pixels The output video signal is non interlaced for clear images without flickering 3 The transmitted light is detected by a photomultiplier for bright sharp transmitted images 4 The auto gain adjustment feature eliminates the need for complicated gain adjustment operation 5 A total of 5 image channels including up to 4 fluorescence images and a transmitted image can be acquired simultaneously 6 The scan unit can be used in common with an upright or inverted microscope 7 The confocal aperture is continuously variable and the optimum confocal aperture for each objective is set automatically 8 High speed scanning acquires 4 images per second 512 x 512 pixels 9 Sequential scan for acquiring images without crossover 10 In addition to raster scanning scanning modes such as vector scanning and oblique scanning are provided to meet a wide range of applications 11 The dichroic mirror for excita
87. displayed to the left or right of the chart Sets the display formats when the label display is set to the numerical value display or percent display DateTime Usually leave this box unchecked 2 262 Page APPLIED OPERATIONS Changing the Chart Display Method Marks sub panel Enables or disables the item display in the chart Sets the background color of the item display area Sets the item character font Sets the color width and type of the outer frame of the item display area Sets the color width type and length of the arrows indicating the item display area Used to set the items to be displayed along the coordinate points Editing Back Color IV Transparent Font Color Length jo Data Source sub panel Percent Label Label and Percent Label and Value Legend Percent Total Label amp Percent Total X Value Enables or disables the transparent background display Enables or disables the item display of lines outside the coordinate axis Sets the contents of the displayed item Do not change the settings here but leave them in the initial condition Page 2 263 APPLIED OPERATIONS Pop up Menus 2 15 Pop up Menus Selection of function panels and display panels and other frequently used FLUOVIEW functions full screen display printer output image save LUT setting comment setting can
88. each user shall log into it in AE different ways See Appendix H User Registration of FV500 in this i Volume f PEE E PE AE ALEA DOET ALAA T ATTN TEE EA AE D EA E POE E E E IE A PT A A EA EEEE OT EE E EA STAA R TIP it takes 20 to 30 seconds to start up software since double clicking the FLUOVIEW icon 1 22 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures 3 The following window appears when the FLUOVIEW software starts ne 2 s o Gx OxT Oxz OXZ H g Li Pan Zoom xt TID ScnSped SS Fast y slow 78 Seen 17s image p Acquisition Settings Load Save Focus Mode UPLAPO 10x z LASER SCANNING MICROSCOPE FLUOVIEW Fig 1 3 Window at Start up e Page 1 23 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 3 Focusing on the Specimen 1 2 3 1 Combination with BX 1 From the page tabs on the bottom right of the Acquire panel select the Optics sub panel Light Path Microscope Control Trans Scope SU Lamp Control Control Fig 1 4 Optics Sub panel 2 Select the lt BI gt button in the Light Path group box The lt BI gt button looks pushed in to indicate that it is selected 3 Push the cube button 3 of the hand switch 2 to engage the optimum cube for specimen dye In the cube display window 1 the
89. hard copy unit and transmitted light unit when the transmitted light detector is used Turning the monitor ON Refer to the instruction manual of your monitor As this unit should be connected to the power outlet unit leave its power switch in the ON position Turning the microscope ON Refer to the instruction manual of the microscope combined with the system As this unit should be connected to the power outlet unit leave its power switch in the ON position Turning the transmitted light unit when the transmitted light detector is used Page 1 21 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 2 Starting the Software Before starting up this software wait for more than 2 minutes after NOTE turning the microscope and power supply unit ON s TIP When executing a function that does not need image acquisition such a analysis of image data previously saved in a file it is enough to turn on NOTE Before proceeding to the following wait for more than 2 minutes after turning the microscope and power supply unit ON NOTE When using the AX70A and U MCB start the software while the MAIN screen of U MCB is displayed 1 Input the user name and password and log onto Windows NT NOTE Input the user name authorized as an Administrator Nig 2 Double click the FLUOVIEW icon on the desktop FLOOVIEY esa FLUOVIEW icon TIP When more than one user uses the FV500
90. image in the XY observation mode For the detailed operation method see section 2 2 1 Image Acquisition in XY Observation Mode 2 Select the Surface XY Norm option button in the Scan Mode group box in the Acquire panel then select lt Clip gt from the list displayed below it A frame indicating the scanning range will be displayed in the Live panel Scan Mode tf Surface XY Horm mi Normal Clip C Line XT C Depth XZ C Point Size 00 by 300 Clip Box 3 Move the frame around the range to be observed The frame can be moved by placing the mouse pointer inside it and dragging 4 Change the frame size To change the frame size click a point inside the frame with the mouse pointer When square handles are displayed on the frame edges place the mouse pointer on one of them and drag it 5 Acquire images by clicking the lt XYZ gt lt XYT gt or lt XYZT gt button 6 After acquiring images select lt Normal gt from the list displayed below the Surface XY Norm option button in the Scan Mode group box to set the scanning range to the original setting It is not permitted to change the scanning range after having acquired NOTE images If you want to change the scanning range again select lt Normal gt from the list displayed below the Surface XY Norm option button in the Scan Mode group box to set the scanning ragne to the original setting acquire an image in the XY observation mode
91. in the X direction from the top to bottom The image shown after image acquisition is the same a width as that x in the X direction specified in the Live panel Same as that in the XT 8 After image acquisition select lt Normal gt under the Surface XY option button in the Scan Mode group box in the Acquire panel to return from the image acquisition mode Page 2 85 APPLIED OPERATIONS Image Acquisition 2 2 11 Image Acquisition in the Laser Excitation Mode When you use the FV system with AOTF FV5 COMBA the function described in this section is available In the FV system with AOTF it is possible to cut excitation of laser except the region where scanning is performed Moreover it is also possible to set up a region of laser excitation to excite laser only to the target part u i represents the region of laser excitation in this section _TIP REX represen ts the region of laser excitation in this section U l Three kinds of the laser excitation mode Disable REX and Bleach are selectable according to your purpose And each mode can be switched in the XYT observation In use of the REX or Bleach mode a setup of REX mask file is required Disable mode An ordinary mode to acquire an image of specified size REX mode The mode which sets the REX mask file to excite laser only to the target part and acquire image The same laser setup as Disable mode is applied Bleach mode The
92. lt Full Screen gt button in the toolbar at the bottom left of the screen lt Full Screen gt button TIP When the mouse left button is clicked while the image is displayed full screen the buttons which are usually displayed on the top of the Display panel lt Display gt buttons etc and those usually displayed on the bottom Tight lt Display channel switch gt button can be displayed in the image Fig 2 69 Panel Showing the lt Display channel switch gt lt Display gt and Other Buttons 3 Click the mouse right button on the image to cancel the full screen display e Page 2 159 APPLIED OPERATIONS Changing the Image Display Method One Point The image can also be displayed full screen by the mouse operation on the image Display the Display panel of the image to be displayed full screen and click the mouse right button on the image A pop up menu as shown below appears Select FullScreen Display from the FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Views Experiment Properties 2 160 Page APPLIED OPERATIONS Changing the Image Display Method 2 4 9 Magnifying Reducing an Image The i Disp 1 2 mage can be magnified or reduced using the buttons displayed at the top of the lay panel Magnification or reduction up to 3 1 or 1 3 the original image is possible Display the Display panel of the image to be magn
93. maaek record euler selection or the mode allowing to be displayed by displaying multiple single entry of comment Display panels together C Uses next color in sequence Tab Display Number of tabs on Function Set the number of Function ae nee he panels that can be displayed at a Display Panel visible at once time Set the number of Display panels that can be displayed at a time Save New Setup Quit w o Saving Coloring for new overlays group box Set the color of the comment to be overlaid in image in the Display panel 1 10 Page Software Setup Setting the System Configuration 9 Display the Hardware panel in the front position In this panel select Enabled Disabled to enable disable the automatic black level adjustment enter the values for delay timing for TD unit and enter the maximum duration for continuous bi directional scanning when setting fast scan mode Delay Timing for TD Unit group box This data should usually be left in the default values and does not have to be changed Delay shutter changes sec text box Enter the opening closing wait time of the shutter in the TD unit in seconds Delay for bulb changes sec text box Enter the ON OFF wait time of the lamp in the TD unit in seconds FLUOVIEW Setup Microscope Software Objectives N Fiforescence 3 Coloring Tables Registration Lasers Users Scanning Unit Delay Timing for TDU
94. margin between the chart display area and legend display area Sets the type color and width of the lines dividing the legend items Enables or disables the inversion of the display order of legend items Sets the color and width of the shadow area of the shadowed display of legend area Panel sub panel Used to set the chart display panel Set the format of he borders wawa wasa Chart Series of the chart display area Bevel Inner sets the inner borders of the chart display area and Bevel Outer sets the outer borders of the chart Bevellnner p Bevel Outer display area None C None Panel Color Enables or disables the Pitman lu display outside the outer 3 frame of the chart display Raised Raised Area Sets the background color of Series General Axis Titles Legend Panel Paging Walls 3D te chartidisplay area Sets the width between the inner and outer borders ie idth tadient F visible Statt Color Sets whether or not the chart yle display area background is Stretch Bl End Color displayed with gradations Tile and sets the colors and type C Center Direction Top Bottom x of the background Sets the width of the inner and outer borders Specifies the background image of the chart display area sets whether it is displayed inside the chart or in the entire chart display area and sets its display position 2 258 Page APP
95. method in the Ch1 Ch2 Ch3 Ch4 group box is check marked to indicate that the corresponding channel is ready for image acquisition Ch3 check box Ch2 check box Ch4 check box DAPI M FITC Transmitted a Offset PMT Gain Offset L Gain Offset Ara Gain Offset PMT Gain Offset Ch1 check box Ili j i z I lil II I III 800v 10 0x Y 800v 10 0x 7 800v 40 0x Si 800v 10 0x Y 186v 43x 0 e Set the brightness of the transmitted image Please use PMT Offset and Gain LED sliders in Ch5 group box to adjust the brightness of the transmitted image For details see section 2 2 1 4 9 Adjusting the Image Brightness in the OPERATION volume When observing a fluorescence image s simultaneously also set the brightness of the fluorescence image s Adjust the brightness of the image of the transmitted image acquisition channel s by using the PMT Offset and Gain LED sliders in its group box in the Acquire panel For details see section 2 2 1 4 9 Adjusting the Image Brightness in the OPERATION volume Page 2 69 APPLIED OPERATIONS Image Acquisition 2 2 4 Acquiring Multiple Images XYZ XYT or XYZT Observation Acquisition of a large number of image slices takes a very long time In such a case it is possible to acquire the images of only the required areas in a shorter time by narrowing the observation range and scanning range 1 Acquire an
96. of MO disk The disk is not loaded in the Insert the disk properly Instruction manual drive of MO disk The disk is write protected Release the write protection Instruction manual of MO disk The disk is not formatted Format the disk before use Appendix F OPERATION INSTRUCTIONS The available space in disk Increase the available space is insufficient in disk by deleting Appendix F unnecessary files or use OPERATION another disk be sure to INSTRUCTIONS format a new disk 16 The image cannot be output at the printer The printer is not recognized Ensure that the printer is ON Instruction manual of printer The printer is not recognized Check the cable connection Instruction manual of printer The printer is out of paper Supply paper Instruction manual of printer 17 The FLUOVIEW The FLUOVIEW software Press Alt Tab on 1 2 2 OPERATION software cannot be has already been started the keyboard to switch to INSTRUCTIONS started FLUOVIEW Another application is Exit from the running running software before starting the FLUOVIEW software 1 4 Page M TROUBLESHOOTING GUIDE TT O Phenomenon Manual Ref Pages 18 The mouse pointer in The software is Press Ctrl Alt 1 2 1 amp 1 2 11 the screen cannot be _ malfunctioning Delete on the keyboard OPERATION moved by moving the mouse following the displayed messages
97. of a channel Ch1 Ch2 Ch3 Ch4 The subsequent description deals with the differences in operation between the monochrome dyeing and dual fluorochrome dyeing e Set the dyeing method 1 From the page tabs on the bottom right of the Acquire panel select the Dyes sub panel r Selected Dyes Assign dyes manually check box Place the pointer on the Checking this enables the manual setting Dragging the dyeing method in the list directly to the Ch group box assigns the dye to the desired channel Assign dyes manually icon displayed in the Selected Dyes and the dyeing method is shown in the pop up display Available Dyes Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method lt Prev gt button Sets the dyeing method which was set last time by clicking the lt Apply gt button lt Apply gt button Applies the dyeing method dragged in the Selected Dyes group box to the Ch group in the Acquire panel Rhodamine Phalloidin Texas Red JE sssi Fig 2 20 Dyes Sub panel l lt Clear gt button Apply Clear the set dyeing method 2 Select the specimen dyeing method in the Available Dyes list box in the Selected Dyes group box and drag the selected method into the field immediately above the list box 3 Click the lt Apply gt button to apply the se
98. of the file or directory F File Group of information which is named and saved in a disk e Gain This function brightens the image by the ratio set at the time of image acquisition Use Gain when a bright image cannot be obtained even by setting PMT Voltage to 800 V Group Refers to the applications registered in the program manager When a group is opened on the program manager more than one icon is displayed in the window When the group is closed the group becomes an icon of the window m Icon An icon is a small figure with characters below it The icon indicates the status in which the window is closed or minimized Iconize This refers to turn a window into an icon display by using the iconize button of the Iconize command in the control menu The application continues to run even after it has been turned into an icon Selecting an icon returns it to the active application Intensity Brightness of each pixel in an image Items displayed in pale color The menu commands and buttons which cannot be used are displayed in less visible way i e in a pale color or gray k Keyboard input Action of inputting an alphanumeric character from the keyboard of the computer LUT LookUpTable The image acquired by observation input has 12 bits of brightness data per pixel Meanwhile the brightness data which can actually be displayed output consists of 8 bits per pixel for each of R G and B T
99. overwrite it click the lt NO gt button and enter another file name One Point The Save Display dialog box can also be displayed by a mouse operation Display the image to be saved at the front of the Display panel and right click a point in the image A pop up menu as shown below is displayed Select Save Display from the menu FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Views Experiment Properties Page 2 107 APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 1 3 Saving Specified Area of Image Only the specified part of an image can be saved as an image in a disk 1 Display the Display panel of the image containing the part to be saved in the front 2 When the image was acquired through multiple channels you can select whether the image slices for multiple channels are saved together or the image slice of only 1 channel is saved Use the lt display switching gt button for the selection The image will be saved in the channel condition as displayed es TIP k For the channel switching see section 2 4 3 Switching the Displaye f 3 Specify a desired area in the image This operation is not necessary if an area has lt Annotate gt button already been specified Click the lt Annotate gt button in the toolbar at the bottom of the Acquire panel A list of buttons as shown below will appear A oS ee K Ga Z
100. panel 1 In the upper part of the Acquire sub panel open the Ch group box for the ND to be changed Click the lt More gt button The field as shown below is displayed below the Ch group box C A Ar Clicking this button allows fine adjustment of the ND value Display of the optimum laser and ND value which are set ay a automatically according to s Clicking this field allows the the selected dyeing method np value to exchanged On arge scale The ND values which are usually used are displayed in green S Display of the ND value set by clicking the lt gt and lt gt buttons or the field The set ND value can also be changed by entering its value directly from the keyboard 3 Vary the ND value using the Laser LED slider Each click of the laser ND lt gt or lt gt varies the laser ND value by 1 Each click of the laser ND slider varies the laser ND value by 5 1 2 8 Setting the Observation Condition 1 2 8 1 Setting the Objective Magnification From the drop down list on the center of the Acquire panel select the objective being used with the microscope UPLAPOAKO ee NOTE With a combination using a microscope other than the AX70A the measurement results will be inaccurate if the objective magnification set here does not match the actual magnification of the objective in use i TIP if you change the objective click the lt Apply gt button in the Dy
101. panel at the front Sets the image to be subjected to operation The icon of the image is displayed here lt Image AND Image gt button Executes an AND operation lt Image OR Image gt button Executes an OR operation lt Image XOR Image gt button Executes an exclusive OR operation Acquire File vO Y Tile 2roce Live Y wwam Y Fitter j Sets the second image a of the operation The m m icon of the image is N xY displayed here Image OR Image NOT Image J AND Image J Image R Image xisi Fig 2 83 Logical Sub panel 2 Click the lt Experiment List gt button in the toolbar at the bottom of the Process panel The Experiments in Memory dialog lt Experiment List gt button box appears as shown below Experiments in Memory Fig 2 84 Experiments in Memory Dialog Box Page 2 179 APPLIED OPERATIONS Image Processing 3 From the Experiments in Memory dialog box select the file name of the first image and drag it to the frame at the top left of the Process panel The icon of the image is displayed in the frame at the top left of the Process panel Scalfr Operations cflar Value Add to Image e LYS peseeeensneneenseensnecneneceeeeceeseneesennesnenecennenecnessesenaeaesusnecneseceeeeceeseeseeaeaenesneneeeeeseeessesesaeeeeneeeneneeeeeseseesessSueeeeeeeeeneeeeeeeaeseeesSeeneeeeeeeeseeeeeeeeaeseSeeQeeneeeeneeeeeeeey F H TIP k Before the dra
102. path 6 Filters These filters are used to adjust transmitted light e For transmitted observation using laser disengage the LBD filter from the light path and engage the FR frost filter in the light path by operating the filter levers If the FR filter is disengaged from the light path the image may suffer from stripe interference 7 Intermediate magnification knob Select the intermediate magnification from 1X or 1 5X e Always set to 1X for using the microscope as a LSM It cannot be used as a LSM if this knob is set to 1 5X Page 1 9 Getting Started FLUOVIEW Basic Operations Combination of IXBP Bottom port 6 Filters 4 Transmitted light DIC slider FV5 DICT optional 1 Light path selector 2 Cube turret 3 Analyzer FV5 ANI 1 Light path selector knob Switch the light path between laser microscope observation visual observation and side port e Set the knob to the position for the desired light path by referring to the following table Light ratios of light paths nob Position Indication q el SM Side port Binocular tube or LSM 100 1 10 Page Getting Started FLUOVIEW Basic Operations 2 Cube turret Switch the cubes for fluorescence observation by rotating this turret For visual fluorescence observation engage the specified cube in the light path For laser microscopy or for visual transmitted light observation rotate the
103. processing is useful for making structures clear or extract the edges The filter format is as shown below 1 0 1 1 0 1 1 0 1 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter Highpass Y Click the lt Highpass Y gt button lt Highpass Y gt button e Page 2 165 APPLIED OPERATIONS Image Processing s 5 Prewitt filter This filter enhances the contours of image grains in a similar way to the Sobel filter but more strongly than it It has two filter formats X and Y as shown below The format providing the larger value after filtering is used Y 2 2 2 2 0 2 0 0 0 2 0 2 2 2 2 2 0 2 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter e Click the lt Prewitt gt button lt Prewitt gt button 2 5 1 2 Noise Reduction When random noise interferes with an image its irregularity increases and it become harder to see Such noise can be reduced by means of filtering Three kinds of filters are available for noise reduction 1 Averaging filter The averaging filter is used to eliminate details in image or reduce noise However as it makes everything in an image smooth it also makes the edge sections dull which sometimes result in the image resolution deterioration The filter format is as shown below 1 1 1 1 1 1 X 1 9 1 1 1 T
104. provided by software With the panel system a software function can be executed easily by selecting the panel page tab of the function to be executed just like when using a system notebook or file folder The FLUOVIEW software is organized by two kinds of panels the function panels and display panel The function panels include the Acquire File I O Tile Process Analyze and Visualize panels The display panel shows either the Live panel or the panel image loaded from a file filename panel Function panel Display panel Fite vo Y Tie YrocessYanay W XYZ 2 TIF XYZCH File Type Tift S TIF X Files Name y a momatif fitc pitit j 600 ida011 tit 600 ptk2 tit 600 shimal tit 600 tr pistit u40fpz2ctif Se DATA x Load Save Experiment Experiment Display Animation Display Panel o NOTE In this manual the function panels are referred to simply using their page tabs Namely the File I O panel of function panel is referred to simply as the File I O panel Page 1 9 SYSTEM OVERVIEW Software Functional Configuration 1 5 2 Panel Structure of the Software This software cannot show the indexes of all function panels at a glance but uses scrolling to display the desired page tab Please use the following list of the panels for reference in scrolling Acquire Sets up the image acquisition and executes actual acq
105. stage coarse adjustment gt buttons Displaces the Z stage on a large scale lt Z stage fine adjustment gt buttons Displaces the Z stage on a fine scale Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Step Size text box Set the number of steps using the lt a gt or lt v gt button This number can also be input directly from the keyboard tep Sits i um R ded st i ecommended step size O 25um ideal 4 4 Shows the number of steps calculated by the system so that the scale of depth LA in the Z direction of the acquired image is identical to the scale of the plane in the X and Y directions Slices text box Shows the number of images acquired This number can also be input directly from the keyboard Fig 2 16 Z Stage Sub panel Moves to the set scanning stop position Use this button to check the scanning stop position lt Set gt button Sets the current stage position as the scanning stop position of the range of the observed cross section Z direction scanning range Current Pos text box Shows the current position of the stage lt Set Zero gt button Sets the current stage position as the home position Pressing this button also clears the Stop Z and Start Z values lt Set gt button Sets the current stage position as the scanning start position of the range of the observed
106. subjected to LUT intensity mapping re assignment a Black level 0 White lever 4095 Intensity Mapping Channel e2ze4 lt Compute Histogram gt button SES om le Histogram Displays the histogram eS Xp leer al Fig 2 75 Histogram Sub panel 3 Display the Display panel of the image you want to change the contrast The icon of the image is displayed in the frame at the top left of the Process panel 2 170 Page APPLIED OPERATIONS Image Processing Doo O 1 2 3 4 5 4 When the image was acquired in the multi channel mode select whether the LUT lt Dispiay aba switch gt intensity mapping re assignment is applied to multiple channels simultaneously or to a single channel To select the target channel s use the lt Display channel switch gt buttons The histogram of the selected channel s is displayed Example When only the Ch1 image is displayed the histogram of Ch1 is displayed and mapping of only the Ch1 image is possible ss TIP 4 For the switching of channels see section 2 4 3 Switching the Display Channels 5 Click the lt Compute Histogram gt button A histogram appears as shown below Histogram of CHMAGESOLYMPUS TIF Master View 2011 Min 0 Max 4095 Selected View Kif m3 Jwi Black ievel 0 wwhitelevel 4095 1 Intensity Mapping Channel es ES Compute Histogram Fig 2 76 Histogram
107. text box and click the lt OK gt button The newly set user name is added in the list box FLUOVIEW Setup Microscope Z Stage Objectives i Fluorescence Coloring Tables Registration Software Hardware Lasers Scanning Unit The registered user name is shown here FLUOVIEW Users You are loggedinas a Administrator as r The user name is added Add a user and Reset user to Log in Factory Defaults Delete a user Save New Setup Quit w o Saving 5 To register other users repeat steps 3 to 5 for each one 6 Click the lt Save New Setup gt button or lt Quit w o Saving gt button to close the dialog box TIP The Administrator is the user name which saves the factory defaults of the Appendix H 2 Logging into the FV500 After the user name has been registered and the FV500 started a dialog box for entering the user name appears Enter the user name to log in the FV500 Nig 1 Double click the FLUOVIEW icon on the desktop The dialog box for entering the user name appears as shown below FLUOVIEW FLUOVIEW icon OLYM P us FLUOVIEW User kau Copyright 2000 Olympus Optical Co Ltd H 2 Page Appendix H USER REGISTRATION OF FV500 peleting a User 2 Enter the user name in the User Name text box and click the lt OK gt button 3 The FLUOVIEW software starts lt TIP lf no user name has been registered the FLUOVIEW User Logi
108. the Scan Mode group box in the Acquire panel to return from the image acquisition mode 2 2 9 Display the change of image intensity Point Scan The change of fluorescence intensity according to time lapse can be displayed graphically by irradiating the laser to certain point on the image If you have FLUOVIEW TIME COURSE software image acquisition can be started with buttons keys or input of external trigger signals 1 Acquire an image in the XY observation mode For the operation procedure see section 2 2 1 Image Acquisition with XY observation 2 Select lt Point gt under the Point option button in the Scan Mode group box in the Acquire panel Scan Mode C Surface XY Horm C Line XT Depth XZ Point PEBE s Shows the X Y coordinates on the Point image Size x 128 128 2 78 Page APPLIED OPERATIONS Image Acquisition The graph window showing the cross cursor for scanning and the intensity values appears in the Live panel Cross cursor 6560 32w gt PeT Scan Mode C Surface xY Norm C Line T c h Point pg Era Point Size 199 299 i Objective UPLAPO 20x Pan Zoom 5 4 Jal gt v f l 129 95 ail 1 Scan Speed Fast Siow 5 8698 Acquisition Settings Load Save i 9 E Display zoom 100 1 x2 Oxa Filter Normal C Kalman 4 C Accumul
109. the TIME COURSE software optional See the FV TIEMPO TIME COURSE software User s manual for details of the TIME COURSE software Page 2 93 APPLIED OPERATIONS Image Acquisition pO 4 Selecting REX Mask File and Setting the Laser ON OFF Set the REX mask file in the REX mode and Bleach mode and the laser ON OFF respectively 1 Select the REX mode option button in the AOTF group box in the Lasers sub panel Disabled REX mode le Bleach mode Make REX mR 2 A frame to specify the REX mask file appears on the right side of the REX mode REX mode option button option button Right click the mouse inside the frame to display the pop up menu as shown below Select the image to be masked in the menu The REX mask file displayed REX_Live in the Display panel is pop REX_Livett0 up displayed p TIP I In order to use the already saved file as a REX mask file open the imag l f beforehand The opening method of the REX mask file is completely the same as that an image For details see section 2 3 2 Opening Previously Save Images in this manual 3 The icon of the selected REX mask file is displayed inside the frame REX Function REX Live C Disabled The icon of the selected REX mode REX mask file is displayed C Bleach mode inside the frame And the file name and the xY observation mode are also MOKERS mesk displayed above and unde
110. the frame next to the frame ir Vv Viiv Transmitted images Once this button is where the acquisition is clicked it is not possible to suspended append an image Page 2 37 APPLIED OPERATIONS Image Acquisition pO 2 2 2 3 XZT Observation Mode The description in this section will be focused on the image acquisition operations in the EG XZT observation mode that are not used in the XY observation modes which are the operations enclosed in in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections 2 38 Page APPLIED OPERATIONS Image Acquisition Doo O Set the dyeing method Section 2 2 1 4 in OPERATION Set the range of the multiple sections to be observed the Z direction scanning range Configure the microscope and scan unit Section 2 2 2 3 1 in OPERATION Sections 2 2 1 1 amp 2 2 1 2 in OPERATION Set a lower scan speed Set the objective magnification Segja 2 2 1 4 10 ing RERATION Section 2 2 1 4 1 in OPERATION If the image If the image dodana Set the zoom ratio to 1X becomes b Bae Section 2 2 1 4 2 in OPERATION clean wA oe ine chant a be acquired Re adjust the image brightness Section 2 2 1 Bag OPERATIOND Section 2 2 1 4 9 in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION Stop re
111. the number of current slice Lower row shows the current X and Y coordinates and Z position on the XY direction image X current X position Y current Y position Z current Z position CG TIP E you change the slice to be displayed with the lt Display gt button Slice an lt Display gt button Z aren t effected Slice and Z show current slice and Z position according to moving the re r brown line indicating XZ or YZ direction on the XY direction imag s ETE Dost ES CY Saa asn 2 210 Page APPLIED OPERATIONS Viewing 3D Image 2 8 Viewing 3D Image Use the Visualize panel to view an image three First display the Visualize panel dimensionally Display panel Begin Visualize button Starts building the images for 3D display Method group box Brightest option button Live XYZ 2 TIF Shows the image The file name of the image is shown in the page tab of the panel Rendered View group box Extended Focus View IEEE YD Builds the image by accumulating the intensity value Stereo Pair check box To be checked when building a pair of stereo 3D images or a 3D image Rendered View Extended Focus View C Arbitrary View to be viewed through color WERS red green eyeglasses Brightest Sum option button Bene Builds the image by adding the intensity values Initial view option button Sets the angle at whi
112. turret so that the index indicates CO This sets the cube turret to an idle position without cube 3 Analyzer FV5 ANI Polarizer for use in DIC or polarized light observation For visual transmitted DIC observation or transmitted polarized light observation push in the lever to engage the analyzer in the light path For laser microscopy be sure to disengage the analyzer from the light path by pulling the lever out High quality image cannot be obtained if the analyzer is left engaged in the light path 4 Transmitted light DIC slider FV5 DICT optional Prism for use in DIC observation For laser DIC observation or visual transmitted light DIC observation engage If fluorescence observation using laser is performed with the FV5 DICT engaged in the light path the resolution may deteriorate somewhat It is recommended to disengage the FV5 DICT from the light path when performing only the fluorescence observation using laser NOTE With transmitted DIC observation using a water immersion objective reduce the field iris diaphragm of the microscope until it circumscribes the field of view Otherwise the contrast may deteriorate This applies to both visual and laser DIC observations Page 1 11 Getting Started FLUOVIEW Basic Operations pO 5 Condenser and polarizer Condenser for transmitted light The rotary turret for the transmitted light DIC slider and the polarizer for DIC observation are a
113. which have been used up to Ver 1 0 FITC etc are supplied with this version in the same names as before If you want to use such files change their settings here iw Edit LUT x Button label LUT file FITC C fluoview data FITC LUT 8 Display the Software panel in the front position In this panel select the initial magnification status when images are to be displayed in Display panels This panel is also used to set the mode of comment overlay in image and to set the numbers of Function and Display panels that can be displayed together Default Image Zoom in Single View group box Select the initial status of display magnification when a single image is to be displayed in a single Display panel FLUO EW Setup MicrQ scope Z Stage Objectives J Fluorescence i Coloring Tables Registration Hardware Lasers Users 3 Scanning Unit Default Image Zoom in Single View Overlay creation Overlay creation group box Auto Requires click on tool for each Default Image Zoom in Multiple View group box De When comment is to be overlaid in image select either the mode allowing repeated entries of 11 Repeats with each click on image Select th e in iti al statu s of d is la efault Image Zoom in Multiple View Coloring for new overlays are ila P y Auto Uses default color yellow comment per Annotation tool magnification when multiple images are O14 o
114. 00 system with AOTF FV5 COMBA 1 From the page tabs on the bottom right of the Acquire panel select the Scan sub panel r Acquisition Settings Load Save 5 r Focus Mode Filter group box X2 x4 Select the accumulation mode Na Filter Accumulate To Peak are availabe tits C Kalman C Accumulate To Peak a Fig 2 12 Scan sub panel 2 In the Filter group box select the Kalman option button Page 2 29 APPLIED OPERATIONS Image Acquisition pO 3 Enter the accumulation count in the text box and select the Line Kalman option button displayed r Acquisition Settings Load Save Scan Focus Mode x2 C x4 m Filter O Hormal Kalman Mode Line Kalman option Frame Kalman button Line Kalman Enter the accumulation count I TIP The accumulation count can be set up a maximum of 63 times When 0 is set as the number of times of accumulation an ordinary image 4 Click the lt Once gt button in the Acquire panel The acquired image will be displayed in the Live panel And the accumulated image can be displayed in the Display panel without being displayed in full The difference between the Frame and Line modes of Kalman Accumulation In the frame mode an image is created by accumulation after scanning is performed per image at specified times Meanwhile in the line mode scanning is performed per lin
115. 1 Set the number of scans using the lt gt or lt Y gt button in the N text box in the Time Series sub panel 6 Acquiring image 1 Click the lt XYZT gt button in the Acquire panel The acquired image will be displayed in the Live panel When the built in power supply for transmitted light illumination is used for a long period in the XYZT mode the metallic parts may be expanded by heat causing the focusing to be deviated To acquire precise image data it is recommended to turn off the power of the transmitted light While acquiring an image in the XYZT observation mode clicking the lt STOP SCAN gt button changes the buttons at the upper part of the Acquire panel as shown below The lt Resume gt button restarts image acquisition at the frame next to the frame where the acquisition is suspended STOP SCAN Weg Fite vO Tile Proce _ lt Resume gt button Resume Series Dome lt lt Series Done gt button Restarts image acquisition at Determines the acquired the frame next to the frame VV Vv Vii Transmitted images Once this button is where the acquisition is i clicked it is not possible to suspended append an image Page 2 59 APPLIED OPERATIONS Image Acquisition 2 2 3 Differences in Image Acquisition Method Between Fluorescent and Transmitted Images 2 2 3 1 Monochrome Image The wavelength obtained by monochrome dyeing can be acquired and observed as an image
116. 1 3 Setting the ND Filters u u 2 17 2 2 1 4 Setting the Observation Condition 2 18 2 2 1 5 Acquiring Image a 2 26 2 2 1 6 Acquiring Image in Accumulation Mode 2 27 2 2 1 7 Saving the Acquired Image in File _ 2 30 2 2 2 Image Acquisition in Other Observation Modes 2 31 2 2 2 1 XZ Observation Mode sassa 2 31 2 2 2 2 XT Observation Mode n seeaeeeeaeeeea 2 36 2 2 2 3 XZT Observation Mode u u cs erties need hase ee 2 38 2 2 2 4 XYZ Observation Mode sassa 2 45 2 2 2 5 XYT Observation Mode uu L L SISI T us 2 51 CONTENTS 2 2 2 6 XYZT Observation MOde cccccccccccccesecceceeeeeeeeeceeeeeeeeeeseeaeeseeeeeeuaegeeeeutenees 2 54 2 2 3 Differences in Image Acquisition Method Between Fluorescent and Transmitted IMA JES miie a O Da a a E aiala 2 60 2 2 3 1 Monochrome Image u u 2 60 2 2 3 2 Dual Fluorochrome Image 2 63 2 2 3 3 Transmitted IMage u u U LL SQ S usu naihain E hoiii 2 66 2 2 4 Acquiring Multiple Images XYZ XYT or XYZT Observation 2
117. 11 7 2 11 8 and 2 11 9 in section 7 Click the lt Annotate gt button so that the list of buttons disappears 8 Click the lt Begin Analysis gt button The bird s eye view of the specified region will lt Annotate gt button be displayed in the Intensity Profile box of the Analyze panel The Measurement Results box in the Single sub panel shows the measurement results such as the area Area horizontal and vertical lengths X Y Z or T and perimeter length Perimeter of the region and the statistic result of each channel such as the total Total average Average and standard deviation Std Dev Page 2 189 APPLIED OPERATIONS Image Analysis Shows Measurement results including Perimeter Area XY Z or T Statistical channel data including Total Average Std Dev 2 190 Page merae 443 21 269 51 9 When the image was acquired in the multi channel mode the channel s to be subjected to the bird s eye view display can be selected using the Channel option buttons jevo Y Tile YY resesey analya E XYCH Y MERGETIF Measurement Resuits Perimeter 142 um extent 36 6 um Y extent 34 8 um Area 1259 4 um 2 Statistics Total 1 3955e 007 8 4858e 006 Intensity Profile Channel 1 O O 3 Region Histogram 0 Channel 1 2 3 C IMAGES MERGE TIF Fig 2 89 Panel After Analysis Region Specifi
118. 159 2 4 9 Magnifying Reducing an Image a ar 2 161 2 5 Image Processing l 2 162 2551 F It riRgua s u anu ma na mu h nais ma kasta eno aa 2 162 2 5 1 1 Contour Enhancement a 2 163 2 5 1 2 Noise REGUCHON sic sete Sere ch unai a a aetna A ade tines 2 166 2 5 1 3 Image Sharpening a 2 167 2 5 1 4 DIC Correcting DIC Level Irregularities 2 168 2 5 2 Contrast Conversion a 2 170 2 5 3 Mathematical Operations Between Images 2 173 2 5 3 1 Image Additional alee Aun ieee 2 173 2 5 3 2 Image Subtractlib uuu noroi te eset days ss upah Qaqa aku 2 176 2 5 3 3 Image Multiplication a 2 176 2 5 3 4 Image DIVISION u u kde AL aa i hala q lai 2 176 2 5 3 5 NOT IMAGES tice t L L a u u S l u aun us 2 177 2 5 3 6 Image AND Image u 2 179 CONTENTS pO 2 5 3 7 Image OR Image uha ulus u a aha uY 2 181 2 5 3 8 Image XOR MAJE u u aiaeeiiee aa ae a usasapa 2 182 2 6 Image Analysis uuu uuu uussssssssssasassasa sasa asq asqsassskssskasa 2 183 2 6 1 Checking the Intensity of a Specific Part 2 184 2 6 1 1 Intensity Values on a Line
119. 2 49 Image Comments dialog box 4 Select the lt Annotations ROIs gt button in the Import from File group box The Save Experiment As dialog box as shown below appears Lookin ino x cH File name Roi Files of type FLU OVIEW ROI roi x Cancel Fig 2 50 Save Experiment As dialog box 5 Select the drive or folder where the file to be read is saved in the Look in drop down list 6 In the list below the Look in drop down list double click the folder or sub folder where the data to be read is saved to open 7 Select FLUOVIEW ROI roi in the Files of type drop down list e Page 2 133 APPLIED OPERATIONS Saving Opening and Shredding Images 8 Select the file to be read and click the lt Open gt button hen the image size of the file including the region to be read and that o e specified file differ the Careful dialog box as shown below appears Careful The ROls you requested were created on a different sized data set Sure you want to load them here No licking the lt OK gt button reads the file in different image size wherea licking the lt No gt button cancels reading the region file hen the region file is read in different image size the upper left of th iimage is assumed as reference 2 134 Page e APPLIED OPERATIONS Changing the Image Display Method 2 4 Changing the Image Display Method The method of displaying a
120. 2D Laplacian Sobel Prewitt Sharpen DIC Image Filter DIC leveling kg EF Fig 2 72 Panel Displaying the Laplacian Filtered Image 2 Sobel filter This filter enhances the contours of the image grains If the image contains noise the noise is also enhanced It has two filter formats X and Y as shown below The format providing the larger value after filtering is used 1 2 1 1 0 1 0 0 0 2 0 2 1 2 1 1 0 1 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter Sobel Click the lt Sobel gt button lt Sobel gt button 2 164 Page APPLIED OPERATIONS Image Processing I 3 High pass X filter The HIGH PASS X filter passes the high frequency structures in the X direction of image In this way it can extract details by detecting positions with large variation This processing is useful for making structures clear or extract the edges The filter format is as shown below 1 1 1 0 0 0 1 1 1 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter Highpass X Click the lt Highpass X gt button lt Highpass X gt button 4 Highpass Y filter The HIGH PASS Y filter passes the high frequency structures in the Y direction of image In this way it can extract details by detecting positions with large variation This
121. Acquisition Doo 3 Click the lt Apply gt button to apply the selected dyeing method to the Ch group box on the upper part of the Acquire panel TIP When the dyeing method is selected from the Available Dyes list box and ay aa the lt Apply gt button is clicked a channel for acquiring fluorescence is seti automatically according to the changed filter And the dyeing method is shown in the Ch group box The Confocal Aperture value is also set automatically according to the TIP At you change the objective click the lt Apply gt button in the Dyes sub panel The Conforcal Aperture value is set appropriately U U e Page 2 11 APPLIED OPERATIONS Image Acquisition One Point The Assign dyes manually check box can also be used to set the dyeing method to the desired channel 1 Check the Assign dyes manually check box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box To assign dyes Drag and drop channel either flo or from the dyed i Assign dyes manually feet check box rims 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set a oe I The dyeing method is set A cain orrs con W N FITC aa JZ EST 0 2 800v 10 0x Y Dragging the icon to the out of the Ch check box field cancels the setting of the
122. Blinking which indicates the area where a character can be input When a keyboard key is pressed the character is entered in the position of the cursor p Dialog box Some functions require fine settings so that they can be executed and some functions require the confirmation of settings before being executed The dialog box is a sub window displayed in such cases Directory Hierarchical classification of the space inside a disk so that the files can be arranged in a significant manner according to their categories Disk drive The storage device storing the files Some disk drives such as the hard disk drive and floppy disk drive are capable of both input and output and some such as the CD ROM drive is designed for read only Page C 1 Appendix C Glossary Dot Pixel Double click Action of pressing and releasing the mouse button quickly twice without moving the moue Drag Action of placing the mouse pointer on the target function pressing the mouse button moving the mouse while keeping the mouse button pressed and releasing the mouse button at the destination position Drive Disk drive Drive name Character such as A and C assigned to each drive Disk drive E Extended focus View of an XYZ image obtained by projection in the Z direction Extension Up to 3 characters after a period which are attached at the end of a file name The extension usually represents the type
123. Display channel switch gt image from a single channel buttons Use the lt Display channel switch gt buttons for this selection The images will be saved according to the conditions of the selected channels Example When only the image of Channel 1 is displayed only the image of Channel 1 will be saved TIP or the channel switching see section 2 4 3 Switching the Displayed i Pannes ees reese ceca snipers carers seer nese mnetinem eri resi aroma aa 4 Click the lt Experiment gt button in the Save group box The Save Experiment As dialog box as shown below appears Save Experiment As Save in ja Images v cl 86 3d1 tif 86xt1 tif B6xyt1 tif 86xy22 2 ti fa 86 3d2 tif E 86xt2 tif E 86xyt2 tif fa 86xyz3 tif 86 3d3 tif E 86 t3 tif E 86xyt3 tif E 86xy23 2 ti 86 3Dstt tif E 86xy1 tif 8Bsyz1 tif 8Bxyztl tif fa 86 3Dst2 tif E 86xy2 tif E 86syz1 2 ti E SBxyzt2 tif 86 3dst3 tif E 86xy3 tif E 86xyz2 tif 86xyz2t3 tif gt File name lt 72CH TIF Save as type FLUOVIEW Multi ti v Gaol Fig 2 29 Save Experiment As Dialog Box 2 104 Page APPLIED OPERATIONS Saving Opening and Shredding Images When it is required to change the save destination drive or directory use the Save in drop down list When it is required to change the saved file type use the Save as Type drop down list See section 2 3 1 5 File Types Available for Save for details
124. Display gt button at the top of the Display panel 4 Click the lt Set start position gt button If the start position is not set image No 0 becomes the start image automatically 5 Display the image slice to end the range at the front using the lt Display gt button 6 Click the lt Display gt button If the end position is not set image No n 1 assuming the number of image slices is n becomes the start image automatically 2 13 2 Merging Image Channels For instance when a specimen is imaged with two scans for 2 channel fluorescence observation and 1 channel transmitted light observation the transmitted light image can be merged to the fluorescence images to create a 3 channel image Note that the display gradation of the images obtained by overlaying 3 channels may be poor than original 1 Open the two files to be used in creating a new image The image files used in overlay are subjected to the following NOTE a restrictions The sizes of the images in the two image files should be identical The number of data bits in the two image files should be identical For example it is not possible to overlay a Fluoview Multi Tiff file with a Single TIF S 8 bit file TIP When the image in one of the image files is composed of multiple image slices it is possible to use only some of the slices by setting a slice range See section 2 13 1 Setting the Range of Multiple Image Slices for the
125. Displays the images by arranging them in the currently displayed Display panel Delete Page button Deletes the Display panel being displayed x 8le Z e5 2 Fig 2 61 Tile panel Fr e Page 2 149 APPLIED OPERATIONS Changing the Image Display Method 2 4 7 1 Displaying Multiple Images Per Channel 1 Display the Display panel of one the images which are to be displayed together The icon of the image is displayed in the frame at the top left of the Tile panel and the acquisition parameters used in image acquisition are displayed in the Experiment panel 2 Set the number of images to be displayed together by using the lt gt and lt Y gt buttons in the Columns and Rows text boxes How the images will be arranged can be confirmed in the gray box at the upper part of the Tiling group box 3 When there are multiple images to be displayed select the following items in the Tile Over drop down list e Self The same images as the image being displayed will be displayed e Z Images are displayed according to change in multiple sections e T Images are displayed according to change in time 4 Select the display method from the Display Type drop down list 5 Click the lt New Page gt button A new Display panel appears showing the images displayed per channel NOTE Use the lt Retile gt button when it is required to re arrange the images in the currently displayed Display pa
126. Drive drop down list Click lt gt to display the existing drives and select one of them lt Experiment gt button Click to open the selected image file AN I Open Pi L XY Ch 1ot2 File Type Tit THF Files sampiet TIF m 800 600 sample2 TIF l 160 120 testa 1040 682 4 ac Gyrivoview lt Display Animation Fig 2 28 File I O Panel Display panel Displays the image The image file name is shown in the panel page tab Directory list box Shows the directories in the hierarchical structure Double click the required directory If the required directory is not shown double click the upper level of the current directory or the root directory lt Experiment gt button Saves the image displayed in the Display panel together with its file name lt Display gt button Saves the image displayed in the Display panel as if a hardcopyed image lt Animation gt button Saves the animation image displayed in the Display panel Page 2 101 APPLIED OPERATIONS Saving Opening and Shredding Images lt Image Icons gt Images are represented by icons which can also Image Icon Significance identify the observation modes used when XZ observation acquiring them The icon of the selected image image in the XZ observation 2 channel mode Displ l is displayed in the f t th Display panel is di
127. Experiments in Memory lt Shredder gt button 4 Click the lt Done gt button in the Experiments in Memory dialog bo to close 2 120 Page e APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 4 Saving Comment Together with Image 1 Click the lt Experiment List gt button in the toolbar at the bottom of the File I O panel lt Experiment List gt button The Experiments in Memory dialog box appears as shown below Experiments in Memory 3 CAIMAGE SY 2 2 TIF CAIMAGEYSY Z Fig 2 36 Experiments in Memory Dialog Box In the Experiments in Memory dialog box select the file name of the image to be saved with comment and click the lt Comments gt button The Image Comments dialog box appears as shown below Image Comments C IMAGES XYZ 2 TIF Image Fields Ee OLYMPUS FLUOVIEW Save Comments To Image J Restore Comments J Import frorn File Export to File l AnnotationsROls AnnotationsROls Properties Fig 2 37 Image Comments Dialog Box Display the Comments panel at the front Page 2 121 APPLIED OPERATIONS Saving Opening and Shredding Images 4 Enter comment from the keyboard NOTE During modification of previously entered comment if it is required to restore the originally entered comment click the lt Restore Comments gt button However once the lt Save Comments To Image gt button is pressed the original comme
128. F FV COMBA use 6 2 9m MONITOR q 11 50pin 5 2 9m FV5 LCU BX UCB FV5 PSU combination with BX51 61 onl U PS q combination with AX onl BNC SUPPLY UNUT x2 1 2 9m Dsub25pin MICRO FV5 SU ae SCOPE Circular 8 pin Sy SCAN UNIT ORN Square a 3 pin oo im M IDsub25pin causay 8 2 9m 4 2 9 4 2 9m TRS 4 2 9m ode kam 3 3m y Dsub25pin TED LIGHT i Dsub25pin 2 2 9m BNC DETECTOR Circular H SUPPLY UNIT wee 37 pin 3 _ HeNe GTASER 4 9m im s Square 2 pin POWER SUPPLY q i Circular 8 pin UNIT FV5 COMBA E Circular 37 pin eNe 1 8 40 H POWER SUPPLY 1 2 9m Square 2 pin UNIT pese sub25pin L u Essl 2 UV Ar LASER FV5 LUUV U POWER Dsub9pin SUPPLY m E i 1 Q CHILLER 2 29m _ BNC iRiaseR tt POWER SUPPLY g FV5 LDUIR Dsub25pin Fluorescence omo 2 2 9m HeCd LASER r Square Zpn POWER SUPPLY pozem FV5 LUHECD Light Power Le Lt Supply Unit dese sea in eee eee ee a ts Me tulatauat a a a E coie LASER COMBINER with AOTF Square 3 pin Circular 7 pi 4 ummm kr LASER Duct 2 2m sIROCCO FAN U RFL T BH2 RFL T3 Reflected Page 1 7 SYSTEM OVERVIEW System Configuration For connection to FV5 PSU PC SYSTEM 50 pin For connection to PC PC SYSTEM 50 pin Outputs for laser in
129. Green X Blue box they are reset to the default ma LUT Save LUT values 0 with the Low scale i i Standard Color LUTs 4095 or 255 with the High i i Gray Red Green Blue scale and 1 0 with the Gamma Hi Lo Fall Spec1 Spec2 Intensity Mapping n Gamma 6 The inclination of the graph in the Color Enw TI ma High 4095 4 si Chi C Ch2 OCh3 C Ch4 C ChE IV Apply to all views selected LUT will immediately be applied DK i to the image in the Display panel LUT Tool group box can be changed by dragging an end of the graph line The 7 fiti ired t the edited LUT i MS OA une o RANS E EENS ma Fig 2 52 LUT Intensity Graph file click the lt Save LUT gt button in the Display Color LUT Tool group box e Page 2 137 APPLIED OPERATIONS Changing the Image Display Method 8 Click the lt Graph display gt button at the top right of the Color Tool dialog box again 9 Finally click the lt OK gt button to exit from the LUT editing One Point The Color Tool dialog box can also be displayed by a mouse operation Display the image to be colored at the front of the Display panel and right click a point in the image A pop up menu as shown below is displayed Select View Processor from the menu then select Intensity Scaling from the displayed sub menu Annotate FullScreen Display Paste Print Save Display Save Experiment Sele
130. IONS Changing the Image Display Method 2 4 7 3 Displaying Time Lapse Images Multiple images acquired over time can be displayed side by side for simultaneous view 1 Display the Display panel of one of the time lapse images to be displayed together 2 Set the number of images to be displayed together by using the lt gt and lt Y gt buttons in the Columns and Rows text boxes How the images will be arranged can be confirmed in the gray box at the upper part of the Tiling group box 3 Select T from the Tile Over drop down list 4 When the time lapse images were acquired in a multi channel mode select the display method from the Display Type drop down list 5 Click the lt New Page gt button A new Display panel appears showing the images displayed per channel NOTE Use the lt Retile gt button when it is required to re arrange the images in the currently displayed Display panel Eero Y me XYTA Tiled Tiling io XYZCH T Display Tile Over Type Single View Retile New Page xian Drag Drop images on input basins Delete Page Fig 2 64 Panel Showing Time Lapse Images Together Page 2 153 APPLIED OPERATIONS Changing the Image Display Method 2 4 7 4 Displaying Multiple Multiple sections Images Images acquired from different multiple sections can be displayed together for simultaneous view
131. LIED OPERATIONS Changing the Chart Display Method Paging sub panel Used to paginate a chart for detailed viewing Chart Series Series General Axis Titles Legend Panel Paging walls J Sets the number of horizontal axis points displaved per paqe Enables or disables the change in scaling in the last paqe Sets the current page number I Scale Last Page Points per Page and total number of pages Current Page Totak 1 1 M to th t Moves to the first page oves to the next page 44 First Previous Next gt Last pp Moves to the previous page Moves to the last page Close Walls sub panel Used to set the background of the axis of XYZ or XYt observation chart Chart Series i i il ji Wall Enables or disables the chart Series General Axis Titles Legend Panel Paging Walls 30 axis background display Ss Visible Walls Left Wall Bottom Wall Back Wall Background m Border Changes color of Left Bottom and Back wall lt Background gt button Sets the background color lt Border gt button Sets whether or not the background frame is displayed and sets its color width and type lt Pattern gt button Sets whether or not the background pattern is displayed and sets it color width and type Transparent check box Enables or disables the transparent background display Size text box Sets
132. Line Profile 2 184 2 6 1 2 Intensity Values on a Planar Region Bird s Eye View 2 187 2 6 2 Checking the Intensity Distribution of a Specific Part 2 192 2 6 2 1 Intensity Distribution on a Line Histogram 2 192 2 6 2 2 Intensity Distribution on a Planar Region Histogram 2 193 2 6 3 M asuring a Parl u u u s Nu Sauna asnaq a ua u uY 2 195 2 6 3 1 Length Measurement u 2 195 2 6 3 2 Area Measuremernt 1 u 2 195 2 6 3 3 Measuring the Change in Mean Value of Intensity 2 196 2 6 3 4 Measuring the Change in Integrated Intensity 2 202 2 7 Building an Image from a Different Viewpoint 2 204 2 7 1 Building Extended Focus Image from XYZ Image 2 204 2 7 1 1 Display Switching to Built Image 2 204 2 7 1 2 Turning Built Image into Single Image 2 207 2 7 1 3 Turning Built Image into time series image a 2 209 2 7 2 Building line images to be viewed in Z direction
133. Live panel Scan Mode Surface XY Horm Normal Rect C Line XT C Depth xz C Point Size 00 by 50 Rot Box z 3 Move the frame to the area to be observed To move the frame place the mouse pointer inside it and drag the mouse 4 Change the frame size Place the mouse pointer inside the frame and click to display square handles around the frame Then place the mouse pointer on one of these handles and drag it to change the frame size Handle N Page 2 75 APPLIED OPERATIONS Image Acquisition pO 5 Change the inclination angle of the frame Click the mouse right button inside the frame to display the pop up menu and select Rotate in it Beja Move Size Edit Rotate Properties 6 The inclination angle of the frame can be varied according to the mouse pointer movement Place the mouse pointer on an inclining position and click the mouse left button to fix the frame inclination angle 7 Acquire the image with the buttons such as the lt XYZ gt lt XYT gt lt XYZT gt buttons 8 After acquiring the image select lt Normal gt under the Surface XY option button in the Scan Mode group box in the Acquire panel to return from the image acquisition mode 2 76 Page APPLIED OPERATIONS Image Acquisition 2 2 8 Image Acquisition of a Line at Desired Angle When the sample is not standing upright the image of a line can be acquired by changing th
134. MEJp ZYD LUD u uAA 9lduiexg sobew uolipuoo peAe dsip JUSWIWOD ebew p pu x unes sebew pebiew Bunes apis Aq apis Bunes J pun jauueyo e unes sebeuw Buippeiys pue Bulusdo 65uAeS GNO LV 3dO q3lTddv panes eq ueg sebewi uo UMeJp yUsWWOD 0 SON sbeuw p ebjopoqe 0 ON Bew Jn1 06J poqe S WEU lJ PACS s BEeuul u Jo I e Bune nuunooe Aq p uiezqo shew y S BI JSe y u Beuu oul L pue Q SON s ew Bune nuinooe A p uiezqo ew y S 10 S ON l 0 ON ew s 10 S 0 ON l 3 CHI y uo sebeu p I N pue sobeul apis q pis uonipuoos p ejds p sepun j uueyo e Bunes s sjauueyo p ejds p y Jo uonipuo5 y uo Bulpuedep BIQeWeA Je Sally pue sjauueyo pares y u m q diysuoljejas ou ON Eyo ZuO LUO sly u6j poqe 0 ON 4uO Zyo yo y OUByepoqe SOWEU IJ BACS pedes ae soebeul g uO pue zu yo bulbiew Aq p ule qo sobeul y Lee suo ng yoyms j uueyo Aeldsiq p eldsip aie yD pue ZUD LUO u uA 9lduuexg sebew pebiew Bunes uonnq YoMS Sees Z ebew p pu x unes guo dV LLAUByepoge 149 Hyr 6AUByepoge 49 Hr gaybjapoqe 149 Ji g9Au6J poqe guo Ji SAu6J poqe LUD sr eAUByepoge guo Ji ZAu61J poqe kuo J 0Au6J poqe S WELU lJ S AES UO pue zu Lyo JO yoga wou panes aie sobeul y lean E s
135. Math sub panel of the Process panel Sets the second image of the operation The icon of the image is displayed here This image needs not be set in operations between image and constant Y xv I Filter Sets the first image of the operation The icon of the image is displayed here Scalar Operations group box Provides the command buttons for use in operations between image and Scalar Operations constant Scalar Value Add to Image Subtract from Image Filters J Math Multiply Image with Divide Image by Multi Image Operations group Log base 10 5 box a Provides the command buttons for l Bas Image Operations use in operations between two Add 2 Images different images Subtract 2 Images Logical Multiply 2 Images Divide 2 Images Fig 2 78 Math Sub panel e Page 2 173 APPLIED OPERATIONS Image Processing Click the lt Experiment List gt button in the toolbar at the bottom of the Process panel 2 The Experiments in Memory dialog box appears as shown below lt Experiment List gt Experiments in Memory button HES ilter is H C En dii Fig 2 79 Experiments in Memory Dialog Box 3 From the Experiments in Memory dialog box select the file name of the first image and drag it to the frame at the top left of the Process panel The icon of the image is displayed in the frame at the top left of the Proces
136. OLYMPUS User s Manual FLUOVIEW FVS00 CONFOCAL LASER SCANNING BIOLOGICAL MICROSCOPE Ver 3 3 This user s manual is for the software to be run on Olympus FLUOVIEW FV500 Confocal Laser Scanning Biological Microscope To ensure safety obtain optimum performance and familiarize yourself fully with this product we recommend that you study this manual thoroughly before operation This user s manual is composed of two volumes including SYSTEM OVERVIEW OPERATION INSTRUCTIONS MAINTENANCE and TROUBLESHOOTING Together with this manual please also read the User s manual FLUOVIEW FV500 and the instruction manual of the microscope in order to understand overall operation methods To ensure the safety operation of laser system we recommend you to study the manual of each laser and the light source equipment besides this manual Retain this manual in an easily accessible place near a system for future reference A CAUTION 1 Reproduction copying or duplication of a part or all of this software and manual is prohibited 2 The information described in this manual may be subject to change without notice Registered Trademarks Microsoft Microsoft Windows Excel for Windows are registered trademarks of Microsoft Corporation Other brand names and product names are trademarks or registered trademarks of their respective owners Page FLUOVIEW MANUAL CONFIGURATION FLUOVIEW MANUAL CONFIG
137. Optics Sub panel 3 Click the lt BI gt button in the Light Path group box The lt BI gt button looks pushed in to indicate that it is selected 4 Rotate the cube turret 2 to engage the cube matching the specimen dye in the light path k rr lt e Page 1 29 Getting started FLUOVIEW Outline of LSM Observation Procedures 5 Look into the eyepiece and bring the specimen into focus Be sure to adjust the eyepiece diopter correctly refer to the instruction manual for the IX50 70 When the Z motor is in use clear the check mark in the Locked check box in the Z Stage sub panel in the Acquire panel see section 2 2 1 4 7 of this volume before bringing the specimen into focus using the focus adjustment knobs of the microscope If the fine focus adjustment knob of the microscope is rotated while the Locked check box is checked the Z motor may be destroyed NOTE The specimen may float during immersed observation In this case attach the stage clip U SCL to the microscope p 0 lt 2 1 30 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 4 Setting the LSM Light Path 1 2 4 1 Combination with Upright Microscope BX 1 From the page tabs on the bottom right of the Acquire panel select the Optics sub panel Light Path BI LSM T Microscope Control Trans Scope SU Lamp Control Co
138. PERATION INSTRUCTIONS 11 The position The Z position fluctuates Make the microscope s Instruction manual reproduction of the Z because the coarse coarse movement knob of microscope motor is poor movement knob of heavier by turning the microscope is too light microscope s rotation tension adjustment ring Page 1 3 TROUBLESHOOTING GUIDE i Phenomenon Manual Ref Pages The Acquire panel The power unit is not Check the connection 1 2 1 OPERATION cannot be displayed recognized between the computer and INSTRUCTIONS power unit Turn the power unit ON If the combination with the Check the connections of the 1 2 1 OPERATION AX70A is used the PC and microscope INSTRUCTIONS microscope is not recognized 13 The scale of the Z The Z stage Z revolving Check the Locked check 2 2 1 4 7 Stage sub panel in the nosepiece motor is not box in the Z Stage sub OPERATION Acquire panel cannot excited panel INSTRUCTIONS be moved _ N Turn the microscope on 14 The fine movement The Z stage Z revolving Clear the Locked check box 2 2 1 4 7 knob of microscope nosepiece motor is excited in the Z Stage sub panel JOPERATION cannot be turned or is INSTRUCTIONS not smooth 15 The image cannot be The disk drive is not ON Ensure that the MO disk Instruction manual saved in the disk drive is ON of MO disk The MO disk is not Check the cable connection Instruction manual recognized
139. PERATIONS Building an Image from a Different Viewpoint 2 7 Building an Image from a Different Viewpoint 2 7 1 Building Extended Focus Image from XYZ Image 2 7 1 1 Display Switching to Built Image gt lt Display gt button 2 204 Page An extended focus image can be built from XYZ multiple sections images and the display can be switched to show the built image Display the Display panel of the XYZ multiple sections image The following button is displayed at the top of the Display panel Usually only the lt XYZ series gt button is displayed When it is clicked a list of buttons appears as shown below Fa lt XYZ series gt button Displays only one of multiple sections image slices lt Extend gt button Displays the extend image Click the lt Extend gt button Click the lt Display gt button at the top of the Display panel repeatedly to build the extended focus image The extended focus image can also be displayed by clicking and holding the lt display gt button for successive display APPLIED OPERATIONS Building an Image from a Different Viewpoint Image No 2 Image No 3 Fig 2 97 Four Images Used in Building Extended Focus Image File V0 Y Tie Y rocess Analyz XYZ 2 TIF XYzCH File Type Tiff TIF X Files y la 600 Name a moma tif fte pi tit 600 600 600 600 ida011 tit Ptk2
140. PLIED OPERATIONS Image Acquisition 2 Setting the dyeing method 1 From the page tabs on the bottom right of the Acquire panel select the Dyes sub panel Assign dyes manually check box _Selected Dyes Checking this enables the manual setting Dragging the dyeing method in the list directly to the Ch group box assigns the dye to the desired channel Place the pointer on the icon displayed in the Selected Dyes and the dyeing method is shown Assign dyes manually in the pop up display Available Dyes list box Available Dyes Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method Time Serie lt Apply gt button Applies the dyeing method dragged in the Selected lt Prev gt button Sets the dyeing method which Rhodamine Phalloidin t last time by clicking th was set last time by clicking the Texas Red Dyes group box to the lt gt Apply gt button Ch group in the Acquire Prev Clear Apply panel lt Clear gt button Clear the set dyeing method Fig 2 2 Dyes Sub panel 2 Select the specimen dyeing method by dragging desired dye names in the Available Dyes list box in the Selected Dyes group box to the field immediately above the list box Selected Dyes s Zh Assign dyes manbally Available Dyes Fig 2 3 Dyes Sub panel 2 10 Page APPLIED OPERATIONS Image
141. REX mask file other than that of the REX mode can be set And each laser setup can also be obtained apart from the Disable REX mode Laser Intensity ger I zl Sets up the laser intensity B i Dyes A AOTF REX Function Disabled REX mode C Bleach mode Make REX mask Optics Switches the laser excitation modes A Lasers 2 86 Page APPLIED OPERATIONS Image Acquisition NOTE Jin the REX and Bleach mode image acquisition in the Focus mode fast scan and image acquisition in the Line mode Normal Slant and Free can not be performed NOTE The specimen in the images carried in this section is not for FRAP but for the user s manual 2 2 11 1 Making REX Mask File 1 Display the Live panel at the front position Bequi Eile voYY Die Yewa f tie Y Feche xw Repeat Once E m IM Mto PMT Cair exw CXT Oxyz oxn Mode Surface XY Clip ETE Lobes Wine sere i Dpi xaea Satan 2 Select the lt Annotate gt button in the tool bar at the bottom left of the screen lt Annotate gt button Page 2 87 APPLIED OPERATIONS Image Acquisition 3 Inthe list of buttons displayed as shown below select the lt Rectangular gt lt Circle gt lt Polyreg
142. Sets the coordinate axis display position with respect to the chart Sets the coordinate axis display range with reference to the start point Sets the coordinate axis display range with reference to the end point Sets the type color and width of the title frame Sets the title display position Sets the pattern and color of the title background Sets the title background color Page 2 257 APPLIED OPERATIONS Changing the Chart Display Method Legend sub panel Sets the items to be displayed in the legend Usually set Automatic Used to set the chart legend display Enables or disables the legend UR display hart Series SXes General Axis Titles Legend Panel Paging Walls Sets the legend display style This setting is effective when the numerical value display is selected in the Legend Style pull down menu Sets the legend background color Visible Legend Style Automatic Sets the legend character font Back Color Text Style Left Value eis Resize Chart Top Pos 10 Sets whether or not the chart is 2 Inverted 112 resized according to the legend Color with display Fine adjusts the positioning of the legend display from the top edge Sets the width of the lines Sets the type color and width Right C Bottom Shadow displayed in the legend of the legend frame line column Margin 0 Sets the legend display position and the
143. TIP eg nie Refer to Appendix J List of Functions in the Active Overlays Dialog Bo for other functions of the Active Overlays dialog box 2 11 2 Displaying the Image Intensity The intensity of any pixel of an image can be displayed without using the Analyze panel 1 Display the Display panel of the image that you want to display the intensity at the front 2 In the list of buttons displayed click the lt Text gt button The dialog box as shown below appears lt Text gt button Active Overlays x Set as Default Font Enter an annotation or pick one from the list click on the image to place it Close Fig 2 116 Active Overlays Dialog Box In the drop down list inside the dialog box select lt intensity hotspot value gt On the image place the mouse pointer on the position that you want to display its intensity and click the mouse 5 Click the lt Close gt button to close the Active Overlays dialog box 2 230 Page APPLIED OPERATIONS Entering Comment in Image 2 11 3 Displaying the X coordinate Y coordinate of the Image The X coordinate position or the Y coordinate position of any pixel of an image can be displayed Display the Display panel of the image that you want to display the X coordinate position or the Y coordinate position at the front lt Text gt button 2 In the list of buttons displayed click the lt Text gt button The dialog box as shown
144. URATION The FLUOVIEW system uses two manuals including this User s Manual and the on screen manual built into the software Online Help The User s Manual is composed of the five following volumes and subject matter e SAFETY GUIDE Anoter manual Describes notes and cautions on using the FLUOVIEW system and on types of warning labels e SPECIFICATIONS Another manual Describes the specifications of the FLUOVIEW system e SYSTEM OVERVIEW This manual Describes the outline of the FLUOVIEW system e OPERATION INSTRUCTIONS This manual Describes the operation procedures of the FLUOVIEW system for example methods for image acquisition and various image processing e MAINTENANCE This manual Describes maintenance of the FLUOVIEW system e TROUBLESHOOTING This manual Describes countermeasures in case trouble occurs For Online Help please see 1 3 Online Help in OPERATION INSTRUCTIONS of this manual 2 Page NOTATIONS IN THIS MANUAL NOTATIONS IN THIS MANUAL This manual complies with the following notations lt Notation of Caution Notes and Tips Notation A NOTE TIP 3 Description Caution to prevent injuries to the user or damage to the product including surrounding objects Note for the user Hint or one point advice for user reference lt Notation of panel Command Buttons and Dialog Boxes Notation Acquire panel lt OK gt button lt Open
145. With types of dyeing of the specimen to observe the Barrier Filters will be set automatically to the optical path To change the Barrier Filter types see section 1 3 2 4 Configuring the Filters and change by Optical system Configuration window Turn off the transmitted light bulb of the microscope 1 From the page tabs on the bottom right of the Acquire panel select the Optics sub panel EU a Path lt Trans Lamp gt button Turns off the transmitted light bulb of the Microscope Control microscope 7 The lamp lights when the button is in ans Sapo Lamp Control Control the pressed status Fig 2 23 Optics Sub panel 2 Click the lt Trans Lamp gt button to turn the lamp off set the button to the non pressed status In the Acquire panel make sure that the Transmitted check box in the Ch5 group box is check marked to indicate that Ch3 is ready for image acquisition Ch5 check box DAPI E G iv y PMT Gain Offset PMT Gain Offset PMT Gain Offset Big Gain Offset PMT Gain Offset x x 0 x 0 x 0 x x 0 x 800v 10 0x soov 10 0x Z soov 10 0 J soov 10 0x Z 486v 43x o 2 68 Page APPLIED OPERATIONS Image Acquisition Doo When observing a fluorescence image simultaneously set the required channel ready for acquisition of fluorescence image Make sure that the check box showing the dyeing
146. X_Live 0 lz smo come g Jt be x 20 2 20 400 600 s00 1000 1 0 ta ye 1800 2000 2200 2400 2600 2800 2 Sey 5 Select the Disabled or REX mode option button in the AOTF group box in the Lasers sub panel r AOTF REX Function Disabled C REX mode C Bleach mode Make REX mask The Disabled option button is selected NOTE Select the the Disabled or REX mode option button within 10 seconds before the next scanning is started NOTE in the TIME COURSE software optional when the REX mask file in the REX or Bleach mode is changed while image scanning is performed obtaining the real time graph the laser is irradiated while the pop up menu is displayed however the real time graph is not plotted Please be aware this before using Page 2 99 APPLIED OPERATIONS Image Acquisition 6 Start the third scan in 10 seconds after the second scan is performed Bea EON Te Y Pees tive Y EN STOP SCAN r F F mito PMT Gain Offset E The image of the specimen is fading only where the Am Oxz cxar le Surface XY Clip laser is irradiated al 233 Normal Clip Sq Cip Scan Speed fast h stow O57s Scan 0 57s image D J sr J j as mu Te Ao REX Function as Disabled 2500 C REX mode 2000 C Bleach mode 1 500 j H i 1 000 1 r H Make REX mask atl ah ar arena py See gt ae Dye Capea aap Oe anes Lest ames i
147. Y Tie Y2rocess Anatz v Y xvecH tiF lt Channel 2 gt button AT B File Type Titt TIF lt Channel 3 gt button Files Name con x Yo a brfz1s2atif g s e brz1s2c1 tif g brz1s2cr tif gg lt Channel 4 gt button tke tit E test1 tif i 640 480 All F lestxyz tif Si utu011 tit 1024 768 lt Channel 5 gt button c r Load r Save Experiment Experiment Display Animation xelena Display Panel Fig 2 56 Panel Displayed Merged Image of Two Channels e Page 2 143 APPLIED OPERATIONS Changing the Image Display Method 2 4 5 Changing the Number of Divided Images The number of images viewed simultaneously can be changed 2 4 5 1 Increasing the Number of Divided Images 1 Display the Display panel of the image to be changed at the front Acquire Fiero Tile 2roces_ Live x z 2 11F XYZ 2 TIF fur p om s fJ XY2CH File Type Any image x Files Name Icon x E th pitif 800 600 xy2ch tif K 800 600 xyz tif f em eso Sc Ej Load Save Experiment Animation erae Display Panel xiaj Fig 2 57 Display panel 2 Right click the image A pop up menu as shown below appears FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor
148. Z position to observe the desired cross section Section 2 2 1 4 7 in OPERATION Set the observation range Section 2 2 1 4 8 in OPERATION Adjust the image brightness Section 2 2 1 4 9 in OPERATION APPLIED OPERATIONS Image Acquisition Set the range of the cross section to be observed the Z direction scanning range Section 2 2 2 6 1 in OPERATION Set the numbers of Z direction steps and acquired image slices Section 2 2 2 6 2 in OPERATION Set a lower scan speed Section 2 2 1 4 10 in OPERATION If the image If thee b Ss does not MO become clean Re adjust the image brightness Section 2 2 1 4 9 in OPERATION Stop repeated scanning Section 2 2 1 4 11 in OPERATION Set the observation mode Section 2 2 2 6 3 in OPERATION Set the interval time Section 2 2 2 6 4 in OPERATION Set the number of scans Section 2 2 2 6 5 in OPERATION Acquire image Section 2 2 2 6 6 in OPERATION Save image Section 2 3 1 in OPERATION Page 2 55 APPLIED OPERATIONS Image Acquisition 1 Setting the Z direction scanning range While acquiring image move the Z stage according to the range of the multiple sections to be observed Z direction scanning range From the panel page tabs shown on the bottom right of the Acquire panel select the Z Stage sub panel lt Go gt button Moves to the set scanning stop position Stop Z text box Use this button to che
149. a X2 option button Acquires image at twice the r Filter highest speed Hormal A amp X4 option button Fa Acquires image at 4 times Sh tent 8 E9 the highest speed Accumulate To Peak EJ Sl Increasing the number of divided images in the Display panel line skipped scan at 4 times Focus cannot be done he focus mode is enabled when acquiring images using the lt Focus gt NOTE The Focus function reduces the scanning time by line skipped scan As a result the acquired images become coarse 2 20 Page APPLIED OPERATIONS Image Acquisition 5 Setting the XY Observation Mode 1 In the Scan Mode group box in the Acquire panel select the Surface option button 2 In the Acquire panel select the XY observation mode option button 6 Repeated Scanning Operation xY Repeat 1 Select the lt XY Repeat gt button The acquired image will be displayed in the Live panel lt XY Repeat gt button i Focus TIP Use the lt FOCUS gt button to acquire image at an even higher speed If the lt Focus gt button specimen is already being scanned stop scanning with the lt STOP SCAN gt button before selecting the lt XY Repeat gt button l The Focus function reduces the scanning time by line skipped scan As a f result the acquired images become coarse i 7 Setting the Multiple sections to be Observed While acquiring image move the Z stage to select the multiple s
150. a Type that best describes your data Original Data Type Choose the file type that best describes your data Delimited Characters such as commas or tabs separate each field Excel 4 0 standard C Fixed Width Fields are aligned in columns with spaces between each field Start Import at Row 1 Fie Origin Windows ANSI Preview of file C IMAGES OGAWA XLS 1 Line Intensity Profile 2 112 000000 to 2176 000000 3 Position um NolPositionl E ean ea Fig 2 110 Dialog Box When the File is Opened by Excel 1 3 2 224 Page APPLIED OPERATIONS transferring Data to Another Application 7 Click the lt Next gt button When the dialog as shown below appears check the Tab check box in the Delimiters group box then select none from the Text Qualifier drop down list Text Import Wizard Step 2 of 3 This screen lets you set the delimiters your data contains You can see how your text is affected in the preview below p Delimiters Treat consecutive delimiters as one IX Tab Semicolon Comma I Space Othe Text Qualifier none E Data Preview ine Intensity Profile 112 000000 to 2176 000000 osition um N o 0 EEEE Fig 2 111 Dialog Box When the File is Opened by Excel 2 3 8 Click the lt Next gt button When the dialog as shown below appears select the General option button in the Column Data Format group box then clic
151. aeeeeeeeeesenceeeeeeeeeteeeees 1 48 1 2 8 Setting the Observation Condition 1 49 1 2 8 1 Setting the Objective Magnification 1 49 1 2 8 2 Setting the ZOOM Ratio to 1X Y 1 50 1 2 8 3 Setting the Channels 1 50 1 2 8 4 Setting the Highest Scan Speed 1 50 1 2 8 5 Setting the XY Observation Mode 1 51 1 2 8 6 Repeated Scanning Operation 1 52 1 2 8 7 Setting the Cross section to be Observed 1 52 1 2 8 8 Setting the Area to be Observed a 1 53 1 2 8 9 Setting a Lower Scan Speed a 1 54 CONTENTS 1 2 8 10 Stopping Repeated Scanning cccccesscecccceceeeeeeeeeenneeeeeeeeeeeeessennseeeeeeeees 1 54 1 2 9 ACQUIFING IMAGE ccccccceeeeeeeeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeteeenenenenetenens 1 55 122 10 SAVING IMAGE u uuu n unupa ne aeaea eraka ATena araa 1 55 1 2 11 Exiting from the Software a r rarrsssssssssa 1 57 1 2 12 Turning Power Off ieee ae a
152. after the end of laser scanning The laser oscillation stops when the time shown in this box has elapsed after the end of laser scanning The laser is suspended as standby mode using Ar or Kr laser or the laser oscillation stops using UV Ar laser When the time shown in this box has elapsed after the end of laser scanning Excitation DM and Beam splitter drop down lists Change the excitation filter and beam splitter of each channel Barrier Filter drop down list Changes the barrier filter of each channel TD Unit group box Shows the transmitted light detection Dyes Shows the dyeing method set for each channel Z Resolution Shows the Z resolution set for each channel 3 Optical System Configuration s Laser Unit jn e usss ron sity Auto standby N Op eee i NH E si Auto standby DMs amp Filters in SU Excitation DM Beam Splitter B5278 SDM630 x a Confocal Aperture C A Perrier Filter TD Unit Resolution Close Fig 2 7 Optical System Configuration Window To change the displayed filter types use the Excitation DM Beam splitter and or Barrier Filter drop down lists 4 After completing the setup click the lt Close gt button 2 16 Page APPLIED OPERATIONS Image Acquisition 2 2 1 3 Setting the ND Filters e When the laser combiner is used 1 Display the Acquire panel
153. ag acquisition NOTE When the scan speed is decreased during fluorescence observation the saturation of fluorescence may darken the image of certain types of specimens In this case increase the scan speed and increase the PMT Voltage or use accumulation in scanning Scan Speed group box Set the scan speed by clicking a Sean Speed point on the scale line Festi ow 3 2s Scan 3 25 Image 1 2 8 10 Stopping Repeated Scanning After the brightness and gain have been adjusted select the lt STOP SCAN gt button in the Acquire panel to stop scanning temporarily STOP SCAN 1 54 Page e Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 9 Acquiring Image lt Once gt button Select the lt Once gt button The acquired image will be displayed in the Live panel Fig 1 14 Image Acquired in the Live Panel 1 2 10 Saving Image Display the File I O pane gt T XYCh 1o062 sample TIF 800 600 sempie2 TF Q 160 120 testi tf Bg e n gt TZ Sy FLUOVIEW OES Se z Load Save Experiment Experiment A f File Selection Page 1 55 Getting started FLUOVIEW Outline of LSM Observation Procedures 2 s lt Display channel switch gt buttons 1 56 Page When saving images acquired with mo
154. al interference observation or simultaneous fluorescence transmitted light differential interference observation is required engage the FV5 DICT and the optimum transmitted light DIC slider for the objective in the light path by operating the universal condenser 6 6 For transmitted light observation disengage the LBD filter 5 from the light path and engage the FR frost filter in the light path by operating the filter levers If the FR filter is disengaged from the light path the image may suffer from stripe interference Page 1 33 Getting started FLUOVIEW Outline of LSM Observation Procedures 3 Analyzer FV5 ANU optional 2 Cube turret 4 Transmitted light DIC slider FV5 DICT optional 6 Universal condenser 5 Filters LBD ND6 ND25 1 34 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 4 3 Combination with Upright Microscope AX 1 From the page tabs on the bottom right of the Acquire panel select the Optics sub panel D e Light Path Microscope Control Trans Scope SU Lamp Control Control Fig 1 8 Optics Sub panel 2 Select the lt LSM gt button in the Light Path group box The lt LSM gt button looks pushed in to indicate that it is selected When scanning is started while the lt BI gt button is selected the LSM light path is selected automatically It is switched back to the visual observation aut
155. and the graph showing the fluorescence intensity also appears in the graph window APPLIED OPERATIONS Image Acquisition Doo TIP E The image shown in the Live panel after image acquisition is the image that acquired at the coordinates specified in the Live panel and arranged in the X direction from the top left to the bottom right x The image shown after image acquisition is the same a width as that Lin the X direction specified in the Live panel U U UU UU Display zoom 100 t 0s 1 2 TIP s The size of the graph window can be modified by double clicking the 7 TIP When a certain area is specified by dragging the mouse from the top left to the bottom right on the graph while pressing down its left button l the specified area can be magnified mmm TIP X When the right button of the mouse is dragged on the graph the graph can be scrolled TIP The magnification or scrolling of the graph can be canceled by dragging the left button of the mouse on the magnified graph from the f bottom left to the top right the top right to the bottom left or the bottom Page 2 81 APPLIED OPERATIONS Image Acquisition s oct TIP i When click the mouse on the X or Y Axis on the graph window th Editing dialog box appears and the graph parameters or the grap display method can be modified U UU 8
156. ans Scope SU Lamp Control Control Fig 1 9 Optics Sub panel 2 Select the lt LSM gt button in the Light Path group box The lt LSM gt button looks pushed in to indicate that it is selected When scanning is started while the lt BI gt button is selected the LSM light path is selected automatically It is switched back to the visual observation automatically when scanning completes 3 When the FV5 ANA analyzer 1 is in use disengage it from the light path by setting the switch to the pulled out position 4 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider from the light path by pulling it out When the fluorescence transmitted light simultaneous interference observation is required the fluorescence resolution may be deteriorated slightly due to the engagement of the FC5 DICT in the light path or ce seeveneeneneeneneaneneaeensnsansueneseeseneeneneaeeneaesnenesesusnesueneneeneneaeeneaesuenenesnsnesneneneeneneeneneaesneneneeneseeneneneeneneeneneaeseneseeseseeneneeeeneeeeneneneseeseseeeneenenesneneneeneneseseenesee ean z H g TIP The cube turret is set automatically to con The optimum transmitted light DIC element for the objective in use is Page 1 37 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 Analyzer 2 Transmitted DIC slider FV5 DICT 1 38 Page Getting Started FLUOVIEW Outline of LSM Observation Pr
157. appears lt Exit gt button gt xt bu L Shut Down FLUOVIEW F Are you sure you want to Close all experiments and log in as a different user 2 To exit from this software select the Shut Down FLUOVIEW option button and click the lt OK gt button Then this software finishes 3 To logout from this software select the Close all experiments and log in as a different user option button and click the lt Yes gt button The dialog box asking whether to save the observation data or not appears If you want to save the data click the lt Yes gt button or the lt No gt button if saving is unnecessary It returns to the FLUOVIEW login screen 4 To shut down Windows select the Windows lt Start gt button to display the Start menu and select the Shut Down command from the menu 5 When the Shut Down Windows dialog box appears select the Shut down the Computer option box and select the lt Yes gt button Wait until message It is now safe to turn off your Computer is displayed e Page 1 57 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 12 Turning Power Off 1 Turn off the power supply to the units reflected light power supply switched power outlet unit 2 Turn the laser power OFF e Ar laser Turn the key to the OFF position then set the power switch to OFF For details refer to the instruction manual of your laser unit e Kr laser Turn the ke
158. ares y uy pue zuo yo pupa w pue Zyd LUD Jo yore CUD WO eq ued u m q diusuone i oy Aq p uiezqo soBbewi y wo pares ase sobeul y Ajuo pares ase sobeul cae efef LH ser suo ng yoyms jauueyo eldsiq suo ng yoyms j uueyo Aeldsiq suoynq apis Aq apis pea e sebewi uolinq uols s u s 1 7 padeidsip ee y9 pue j gy9 Ajuo u y mM 9lduiexg uo UMEJp ZUD LUD uayM 9lduuexg sebeul uonipuoo peAe dsip JUSWIWOD ebew p pu x unes sebew pebiew Bunes apis Aq apis Bunes J pun jauueyo e unes sebew Buipp ius pue Buiuedo unes SNOILYN AdO a3llddY APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 2 Opening Previously Saved Images Image files saved in the disk can be opened as follows 1 If the image file name that you want to open is not displayed in the Files list box change the drive and or directory to those containing the desired file using the Drive drop down list and or Directory list box 2 From the File Type drop down list select the file type of the files to be listed in the Files list box 3 Click the lt Experiment gt button in the Load group box TIP Other methods are also available for opening a file a Perform the same operations as steps 1 and 2 above before the following Place the mouse pointer on the desired image file name in the Files f list box and drag the file name to the Open frame on the upper part of the File I O pane
159. ate To Pesk 4 xla FC 2 ae Live F Graph window 3 Move the cross cursor to the area you want to observe To move the cross cursor place the mouse pointer on it and drag the mouse Scan the center of the cross cursor Tet lt Cross cursor 4 Set the scan speed for image acquisition Set the speed in the Scan speed group box of the Acquire panel Scan Speed Fast Slow 0 5s Sean 0 55 Image Page 2 79 APPLIED OPERATIONS Image Acquisition pO 2 80 Page TIP The speed recommended for point scan are as follows 2Qus The scroll bar indicates Fast position It is useful for th specimen changes rapidly 10us 100us The scroll bar indicate Slow position It is useful for the specimen changes slowly _ i Set the time for measurement From the page tabs on the bottom right of the Acuire panel select the Time Series sub panel Set the time for measurement with the lt gt or lt Y gt button in the Scanning text box Scanning text box Sean ime Serie 1 10 000 Dyes 0 0 sec Set the starting method for image acquisition If you have FLUOVIEW TIME COURSE software For setting procedure see FLUOVIEW TIME COURSE software user s manual Click the lt Pt T gt button to acquire the image The image at the coordinates set in the Live panel appears in the image display area
160. ays a list of buttons as shown below Click one of the buttons to select the display speed lt Rabbit gt button For successive display without pause lt Clock gt button Press for successive display of image slices at the time interval set in the Time Series sub panel lt Turtle gt button Press for successive display of image slices at an interval of 0 8 sec 3 The display speed provided by the lt Rabbit gt or lt Turtle gt button can be varied by clicking the mouse right button on each button The Animation speed dialog box appears when the button is right clicked max Cancel e Page 2 213 APPLIED OPERATIONS viewing 3D Image pO 4 Select the option button of the speed to be varied 5 Set the desired display speed in the scale on the right 6 Click the lt OK gt button to close the Animation speed dialog box 2 8 2 Animation 1 2 3 s lt Display channel switch gt 2 214 buttons Images composed of multiple image slices acquired by varying the multiple sections XYZ observation XYZT observation can be built into animation image which can be displayed in 3D by rotating images 1 Display the Display panel of the image composed of multiple image slices 2 When the images were acquired in the multi channel mode select whether animation is built from images of more than one channel or from an image of only one channel To select the target chan
161. be changed by entering its value directly from the keyboard 3 Vary the ND value using the Laser LED slider Each click of the laser ND lt gt or lt gt varies the laser ND value by 1 Each click of the laser ND slider varies the laser ND value by 5 Page 2 17 APPLIED OPERATIONS Image Acquisition 2 2 1 4 Setting the Observation Condition 1 Setting the Objective Magnification 1 From the drop down list on the center of the Acquire panel select the objective being used with the microscope UPa ao e NOTE With a combination using a microscope other than the AX70A the measurement results will be inaccurate if the objective magnification set here does not match the actual magnification of the objective in use TIP f you change the objective click the lt Apply gt button in the Dyes su panel The Conforcal Aperture value is set appropriately 2 Setting the Zoom Ratio to 1X 1 Use the Zoom scale in the Acquire panel to set the zoom ratio to X1 Pan Zoom x x Fig 2 8 Pan Zoom Group Box Using the UV Ar laser set the zoom ratio to X2 2 18 Page APPLIED OPERATIONS Image Acquisition bs 3 Setting the Channels 1 In the Channel 1 group box check the check box showing the applicable dyeing method to make the image acquisition ready 2 In the Channel 2 group box check the check box showing the applicable dyeing method to ready t
162. be controlled by clicking the right button of the mouse without selecting specific page tabs or buttons Pop up menu of function panel When the right button of mouse is clicked on the page tab of a function panel a pop up menu appears to allow selection of the function panel to be displayed at the front Click the mouse right button here Select the function panel to be displayed at the front from this pop up menu xy OXT OXZ C xYzT Scan Mode Surface XY Horm Normal Fast C Lie XT f Depth xz fC Point Size 800 by 600 x p voecuwve _______________ UPLAPO 10x I r Pan Zoom ag et 1 Scan Speed U rt Stow A7e7Scen 17s image Acquisition Settings mw Load Save FocusMode x2 Ox4 Filter Normal C Kalman C Accumulate To Peak xlali Display Panel 2 264 Page APPLIED OPERATIONS Pop up Menus Pop up menu of display panel When the right button of mouse is clicked on the page tab of a Display panel a pop up menu appears to allow selection of the Display panel to be displayed at the front Click the mouse right button here P g a KYZZTE lie Image Const l F that p Ost mikra am eee Image Canst eo XYZCH Select the display panel to be displayed at the front from this pop up menu Scalar Operations Scalar Value Add tolmage _Subtract from Image Multiply I
163. be observed i e the Z direction scanning range From the panel page tabs shown on the bottom right of the Acquire panel select the Z Stage sub panel Stop Z text box Shows the scan stop position in the range of the observed cross section Z direction scanning range lt Z stage coarse adjustment gt buttons Displaces the Z stage on a large scale lt Z stage fine adjustment gt buttons Displaces the Z stage on a fine scale Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Step Size text box Set the number of steps using the lt gt or lt v gt button This number can also be input directly from the keyboard Recommended step size Shows the number of steps calculated by the system so that the scale of depth in the Z direction of the acquired image is identical to the scale of the plane in the X and Y directions Slices text box Shows the number of images acquired This number can also be input directly from the keyboard _ 25um ideali lt Go gt button Moves to the set scanning stop position Use this button to check the scanning stop position lt Set gt button Sets the current stage position as the scanning stop position of the range of the observed cross section Z direction scanning range r Z Series Current Pos text box Shows the current position of the stage lt Set Ze
164. bes how to set the range of target image slices 1 Display the Display panel of the images composed of multiple image slices 2 The buttons as shown below are displayed at the top of the Display panel To switch to the image slice of another cross section click the lt Z T series switch gt button then click the lt XYZ series gt button in the displayed list of buttons To switch to the image slice of another instant in the elapsed time click the lt Z T series switch gt button then click the lt XYT series gt button in the displayed list of buttons In these operations the icon in the lt Z T series switch gt button changes to the icon of the selected button lt Loop gt button lt Z T series switch gt button Successive display or frame by frame display is repeated in the same direction lt Loop Bound switch gt button lt Set start position gt button When successive display or frame by frame display is required set the image with which the display should lt Display speed switch gt button start 0 is set when this button is not used Set the speed of successive display lt Set end position gt button When successive display or frame by frame display is required set the image with which the display should end Assuming that the number of images is n n 1 is set when this button is not used lt XYZ series gt button Displays one of multiple images by selecting it according to the mul
165. bservation will degrade resolution somewhat We recommend disengaging the FV5 DICT from the light path when simple laser fluorescence observation is required NOTE With transmitted light differential interference observation using an immersion objective set the microscope s field diaphragm so that it circumscribes the field of view Otherwise the contrast may degrade This applies to both visual observation and laser differential interference observation 5 Filters These filters are used to adjust transmitted light e Be sure to disengage any filter from the light path for transmitted observation using lasers Leaving a filter engaged in the light path will degrade the image quality Page 1 5 Getting Started FLUOVIEW Basic Operations pO 6 Universal condenser U UCDB Condenser for transmitted lighting In addition the rotary turret for the transmitted light DIC prism and the polarizing plate for differential interference observation polarizer are also provided e To perform differential interference observation engage the transmitted light DIC prism matching the objective in use in the light path For both visual observation and laser differential interference observation e To perform visual differential interference observation or laser differential interference observation engage the polarizing plate in the light path 7 Hand switch U HSTR Switches the objectives and cubes 1 6 Page
166. cation APPLIED OPERATIONS Image Analysis 10 Double click the Intensity Profile button The Intensity Map window appears as Angle scale shown below Sets the angle in the horizontal direction The result can be confirmed with the small bird s eye view in the frame on the Tilt scale Sets the angle in the vertical direction The result can be confirmed with the small bird s eye view in the frame on the top left lt Plot gt button Displays the bird s eye view with the angles set above iw Intensity Map lt Spin gt button Spins the bird s eye view by one turn The spinning starts from the front lt Copy gt button X um 43 to 81 75 Copies the plotted image in the Gre 3863 clipboard lt Save gt button Saves the profile data in a file using an Excel compatible format lt Close gt button 3 N TIP 3 The displayed data can be utilized in other applications Quits the Enhanced Profile Plot window and returns to the Analyze See section 2 10 Transferring Data to Another Page 2 191 APPLIED OPERATIONS Image Analysis 2 6 2 Checking the Intensity Distribution of a Specific Part 2 6 2 1 Intensity Distribution on a Line Histogram The histogram on a line in an image can be displayed The histogram is displayed in the Region Histogram box in the Single sub panel The operation method is identical to displaying the intensity profile on a line See s
167. ch the rotation should start The angle itself can be me se ays set using the Initial Rotation Angle scale immediately below the option button Sequence views option button Sets the rotation angle per rotation step The angle itself can be set using the Rotation Angle Increment scale immediately below the option button Display Panel aa option button Builds the extended focus image Arbitrary View option button Builds the image for 3D display Initial Rotation Angle Rotation Angle Increment scales Set the angles Standard Views drop down list Selects the rotation direction It is possible to set an arbitrary direction Number of views scale Sets the number of views displayed during rotation iy zoom 100 z Qum Channel 1 2 Fig 2 103 Visualize Panel and orientation Sub panel Threshold range Low High scale Sets the range of intensity values to be used in image building Depth Weight scale Sets the weighting in the depth direction By increasing this setting it is possible to provide the image with a perspective by darkening the far objects and brightening near objects Stereo Factor text box Sets the deviation between the left and right eyes when building a pair of stereo 3D images or a 3D image to be viewed through color red green eyeglasses Z stretch Factor text box Provides each multiple sections image with a feeling of thic
168. ch uses confocal optics for high resolution high contrast and drastically improved resolution in the light axis direction It offers researchers features for sectioning 3D construction and time series observation as well as a variety of image processing and analysis functions This section describes the principle and characteristics of the system and the functional configuration of software for use with the system 1 1 Principle A laser scanning microscope converges a laser beam into a very small spot using an objective and scans the specimen in the X Y directions It then detects the fluorescence and transmitted light from the specimen using light detector and outputs the image of the specimen on an image monitor The confocal optics place a confocal Light detector Se Confocal aperture aperture at a position which is optically conjugate with the focusing position i e confocal plane to eliminate light from parts other than those for the focusing position As a result the part corresponding to the eliminated light is darkened in the image making it possible to slice a thick tissue specimen optically On the other hand with ordinary optical microscopes light from a part other Laser than the focusing position overlaps with IF boaa Objective the image forming light on the focusing RH position so the overall image tends to be vague Specimen Transmitted light images can be obtained by use of a transmitted light
169. ck the scanning Shows the scan stop position in the range of the observed cross section Z direction scanning range lt Z stage coarse adjustment gt buttons Displaces the Z stage on a large scale lt Z stage fine adjustment gt buttons Displaces the Z stage on a fine scale Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Step Size text box Set the number of steps using the lt 4 gt gt f or lt wv gt button This number can also Step Size be input directly from the keyboard 0 1um Recommended step size 0 25 um cZ Series Shows the number of steps calculated by the system so that the scale of depth in the Z direction of the acquired image is identical to the scale of the plane in the X and Y directions a Slices text box Shows the number of images acquired This number can also be input directly from the keyboard Fig 2 18 Z Stage Sub panel stop position lt Set gt button Sets the current stage position as the scanning stop position of the range of the observed cross section Z direction scanning range Current Pos text box Shows the current position of the stage lt Set Zero gt button Sets the current stage position as the home position Pressing this button also clears the Stop Z and Start Z values lt Set gt button Sets the curr
170. copy Select the lt q gt button for visual observation e Select the lt 1 gt button for TV or photomicrography e Select the lt 2 gt button for laser microscopy 2 Universal vertical illuminator mirror cube housing The turret for the cube for reflected light observation can be switched with the hand switch for transmitted reflected light 7 Engage the desired cube in the light path for visual fluorescence observation e For laser microscopy or visual transmitted light observation press the BF cube switch button of the hand switch 7 This set the cube turret so that no cube is engaged 1 4 Page Getting Started FLUOVIEW Basic Operations 3 Analyzer FV5 ANA Polarizing plate for use in differential interference observation and polarized light observation e Set to the pushed in position to engage the FV5 ANA in the light path for visual transmitted light differential interference observation or transmitted polarized light observation e For laser microscope set to the pulled out position to disengage the FV5 ANA from the light path Engaging the FV5 ANA in the light path degrades image quality 4 Transmitted light DIC slider FV5 DICT This is a prism for use in differential interference observation e Engage the FV5 DICT in the light path for laser differential interference observation or visual transmitted light differential interference observation Leaving the FV5 DICT engaged during laser fluorescence o
171. cross section Z direction scanning range lt Go gt button Moves to the set scan start position Use this button to check the scan start position Locked check box Enables the Z Motor operation The moving amount assigned to the lt Z stage fine adjustment gt and lt stage coarse adjustment gt buttons can be changed x See section 1 3 in MAINTENANCE Setting the System Configuration for 1 Check the Locked check box in the Z Stage sub panel Do not turn the fine focus adjustment knob while the Locked check box is checked for this may damage the Z motor 2 47 Page APPLIED OPERATIONS Image Acquisition pO 2 While observing the image in the Live panel locate the upper edge of the range to be observed by moving down the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the bottom edge of the range to be observed by moving down the revolving nosepiece using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons 3 When the upper edge position is located click the lt Set gt button The Start Z text box will show the scan start position of the range of the multiple sections to be observed Z direction scanning range 4 While observing the image in the Live panel locate the bottom edge of the range to be ob
172. ct All Overlays ViewProcessor Color Look Up Table Views gt Intensity Scaling Experiment Properties 2 138 Page APPLIED OPERATIONS Changing the Image Display Method 2 4 2 2 LUT Graph Editing by Gamma Correction The intensity data of an image can be reallocated Coloring Intensity 3 to make it easier to view 1 Display the Display panel of the image to be subjected to LUT change lt LUT gt button 2 Click the lt LUT gt button in the toolbar at the bottom left of the screen The Color Tool lt Graph display gt button dialog box appears as shown in Fig 2 51 3 Click the lt Graph display gt button on the top right of the Color Tool dialog box The intensity graph of the LUT appears in the Intensity dialog box Ba sassa t 4 Set the range of intensity graph application with the Low and High scales in the Intensity Mapping group box Intensity Mapping TIP S Dragging one end of the graph High 4095 4 a oS makes it possible to change the Chi Ch2 O Ch3 i i V Apply to all views i inclination The set intensity graph isi y grap i OK Cancel immediately reflected in the image in the Displ I l Pe spa Panel costes Fig 2 53 LUT Intensity Graph Display lt TIP Double clicked the Low scale High scale and Gamma text box they are reset to the default values 0 with the Low scale 4095 o
173. ction 2 2 2 Image Acquisition in Other Observation Modes as well as the following procedure SU After Configure the system and confirm the images to be observed XY observation wu errr rr rere rr er reer rr er errr ee rt tt Section 2 2 1 Ce a Sy Set the acquisition parameters Section 2 2 1 4 Observation mode Scanning speed Image brightness snunnng e Multiple section to be observed Area to be observed When noise is noticeable Perform accumulation Section 2 2 1 6 awwinwunanananananaanananananaanananaanananaanananmna ta wamunumunnununnununuuananananananananananananaanananasa a e ee s Go to Save the acquired data in a file or Process the image Rn ammmunuunununununuwunununununuununununununuanunununuanununuanumnununuunununununununuanunuananuanunununuanunun 2 4 Page APPLIED OPERATIONS General Operation Procedure pt 2 1 3 Examples of Operation Procedures Begin using the FLUOVIEW system by acquiring an image or opening an image in a file The procedures for the subsequent operations such as image processing are not in question here For the detailed operation method of each item in the procedure see the section specified in parentheses Example 1 To perform XYZ observation of a cell apply Example 2 To open a previously acquired image in a filter proce
174. ction scanning range 4 While observing the image in the Live panel locate the bottom edge of the range to be observed by moving up the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the upper edge of the range to be observed by moving up the revolving nosepiece using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons 5 When the position is located click the lt Set gt button The Stop Z text box will show the scanning stop position of the range of the multiple sections to be observed Z direction scanning range 2 Setting the numbers of steps and acquired image slices n 1 Set the number of steps using the lt gt or lt Y gt button in the Step Size text box Step Size i lt TIP O 25um Ideal1 1 Saent 1 i Step Size text box ithe system so that the depth scale of the acquired image is identical to the The number of steps shown in the Step Size text box has been calculated by f horizontal scale rs TIP The number of acquired images shown in the Slices text box can also be input Slices from the keyboard a After setting Start Z Z direction scan start position and Stop Z Z direction Slices textbox scanning stop position input the desired number of images in the Slices text box This automa
175. d Confirm Password V User Must Change Password at Next Logon User Cannot Change Password Password Never Expires T Account Disabled m amp Groups Profile Dialin Fig Appendix E 2 New User Dialog Box Page E 1 Appendix E User Registration 5 Enter the user name in the Username text box is not permitted to enter an existing user name or the same user name as e group name The user name can be a character string of up to 20 haracters and can contain any uppercase or lowercase characters except r the following characters f N X X lt gt J i user name composed only of periods or space is not acceptable 6 Enter the full name of the user in the Full Name text box 7 Enter the description on the user in the Description text box 8 Enter a password with up to 14 characters in the Password and Confirm Password text boxes 9 Click the User Must Change Password at Next Login check box to check it he next time you log in with the registered user name you can change the assword g 10 Click the lt Group gt button The Group Memberships dialog box appears Groups lt Group gt button User Cancel Help Member of Not member of Users Administrators Backup Operators Guests Power Users Replicator lt Add fg ee Fig Appendix F 3 Group Memberships Dialog Box 11 Select Administrators in the Net member of
176. dministrator and the user who log in now cannot be deleted 4 Select the user name to be deleted from the list box 5 Click the lt Delete a user gt button NOTE The Administrator cannot be deleted from the list 6 The Delete a user dialog box appears to ask if you really want to delete the user Delete a user Are you sure you wish to delete the user Userl H 4 Page Appendix H USER REGISTRATION OF FV500 Deleting a User 7 Click the lt Yes gt button if you want to delete the user or the lt No gt button if you do not The deleted user name disappears from the list Microscope N Z Stage j Objectives 3 Software Hardware Lasers Coloring Tables Registration Scanning Unit FLUOVIEW Users You are logged in as as Administrator Administrator a Add a user and Reset user to E Factory Defaults Delete a user Save New Setup Quit w o Saving NOTE The user is deleted at the moment the lt OK gt button is clicked 8 Click the lt Save New Setup gt button or lt Quit w o Saving gt button to close the dialog box Page H 5 Appendix Change of Default Folder for File I O Panel Appendix Change of Default Folder for File I O Panel FLUOVIEW FV500 usually opens the default folder for the File I O panel C FLUOVIEW IMAGES to save acquired images or load saved images The default folder for saving an image can be changed or a desired folder can be specified direct
177. dyeing method 2 12 Page APPLIED OPERATIONS Image Acquisition Doo 3 From the panel page tabs shown on the bottom right of the Acquire panel select the Optics sub panel lt Scope Control gt button Displays useful information for the system setup Micri e Control Trans Scope Su Lamp Control Control Fig 2 4 Optics Sub panel 4 Select the lt Scope Control gt button at the bottom of the panel The window as shown below will appear in case of a combination with the IX MMM Md C Fluorescence Homarski Fig 2 5 Microscope Configuration Window Page 2 13 APPLIED OPERATIONS Image Acquisition s BI 5 Select the lt Bl gt button in the Observation group box and select the microscopy lt BI gt button from the option buttons below it The points where system setting is to be changed for microscope observation will blink in red Turret Cube 6 Change the system configuration setup of cube turret etc by following the Lom guidance given by the red blinking light NOTE The Microscope Configuration window is designed to give guidance on system configuration The red blinking light does not stop even after the indicated configuration point has been changed 7 While looking into the microscope move the stage and check the observed image 2 14 Page
178. e Image OR Image Click the lt Image OR Image gt button A new Display panel showing Image OR Kik SS u sa Image in the page tab appears showing the image obtained by the operation Page 2 181 APPLIED OPERATIONS Image Processing pO 2 5 3 8 Image XOR Image Two different images can be XORed The operation method is identical to the AND operation between two different images except for the following point See section 2 5 3 6 Image AND Image Image XOR Image Click the lt Image XOR Image gt button A new Display panel showing Image XOR lt Image XOR Image gt button Image in the page tab appears showing the image obtained by the operation 2 182 Page APPLIED OPERATIONS Image Analysis 2 6 Image Analysis Images can be analyzed using the Analyze panel Display the Analyze panel at the front Displays the icon of the image being displayed image to be subjected to analysis Measurement Results box Shows the measurement data of the specified line or region Intensity Profile box Shows the intensity profile chart of the specified line or region When a line is specified the line profile is displayed and double clicking this field displays the Enhanced Profile Plot window When a region is specified the bird s eye view is displayed and double clicking this field displays the Intensity Map window File WO Y Tile 2rocess analyz Live Y xvecu t
179. e A pop up menu as shown below appears FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Views Experiment Properties 3 Select Views then select Remove a last view in the sub menu Annotate FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Experiment Properties 2 146 Page 4 APPLIED OPERATIONS Changing the Image Display Method A view is removed from the Display panel Acquire File VO Y Tie Y gt roces XYZ 2 TIF XY2CH File Type Any image F Files Name icon x th pitif 800 600 xy2ch tif p e eo pee Tra xyz tif f em eo Scr SB Load Experiment Display Panel Fig 2 60 Display panel after View Removal Page 2 147 APPLIED OPERATIONS Changing the Image Display Method 2 4 6 Switching the Display Method of Multiple Images With images composed of multiple slices such as time lapse images or images acquired by changing the multiple sections the image to be displayed at the front position can be switched or the images can be displayed successively 1 Display the Display panel of the multiple images The buttons as shown below are displayed at the top of the Display panel 2 To switch the image to the image of another multiple sections click the l
180. e For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections Set the dyeing method Section 2 2 1 1 in OPERATION Configure the microscope and scan unit Sections 2 2 1 1 amp 2 2 1 2 in OPERATION Set the objective magnification Section 2 2 1 4 1 in OPERATION Set a lower scan speed Section 2 2 1 4 10 in OPERATION Set the zoom ratio to 1X Section 2 2 1 4 2 in OPERATION If the image If the image don becomes be s clean cle Set the channel to be acquired Section 2 2 1 4 3 in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION Set the XY observation mode Section 2 2 1 4 5 in OPERATION Perform repeated scanning Re adjust the image brightness Section 2 2 1 4 9 in OPERATION Stop repeated scanning Section 2 2 1 4 11 in OPERATION Section 2 2 1 4 6 in OPERATION Set the observation mode Section 2 2 2 2 1 in OPERATION Adjust the Z position to observe the desired cross section Section 2 2 1 4 7 in OPERATION Set the gf vation line Section 2 2 2 2 2 in OPERATION Set the observation range Acquire image Section 2 2 1 4 8 in OPERATION Section 2 2 2 2 3 in OPERATION Adjust the image brightness Save image Section 2 2 1 4 9 in OPERATION Section 2 3 1 in OPERATION Page APPLIED OPERATIONS Image Acqu
181. e Laser is not oscillating Turn the laser unit ON 1 2 1 OPERATION cannot be observed Ensure that the emission key INSTRUCTIONS is set to the ON position An inverted microscope is Setthe light path selector 1 1 1 amp 1 2 4 used and the light path dial to the LSM light path OPERATION selector dial is not set to the INSTRUCTIONS LSM light path When an erecting microscope is used starting scanning sets the LSM light path automatically An excitation tube or Disengage the excitation 1 1 1 amp 1 2 4 analyzer for visual tube and analyzer from the JOPERATION observation is engaged in llight path INSTRUCTIONS the microscope When the AX70A is used starting scanning disengages the excitation cube from the light path automatically The objective is not engaged Stop the objective at a click Instruction manual in the light path position of microscope When the AX70A is used engage the objective correctly in the light path The pinhole diameter is Increase the pinhole size 1 3 2 5 small OPERATION INSTRUCTIONS The barrier filter is not Set the barrier filter 1 2 6 amp 1 3 2 4 optimum OPERATION INSTRUCTIONS The light path is blocked by Stop the light path detection 1 1 1 the light path detection switch at a click position OPERATION switch INSTRUCTIONS The excitation light is weak Increase the transmittance 1 2 7 amp 1 3 2 4 of the ND filter OPERATION INSTRUCTIONS Page 1
182. e Print Save Display Save Experiment Select All Overlays ViewProcessor Views Experiment Properties 2 126 Page APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 6 Saving the Image Information Observation Condition lt Experiment List gt button The image information and observation condition can be saved as ASCII text file in a disk 1 Display the Display panel of the image to be saved at the front position 2 Select the lt Experiment List gt button in the tool bar displayed at the bottom of the File I O panel The Experiments in Memory dialog box as shown below appears Experiments in Memory CAIMAGEW Y 2 3 TIF oc Fig 2 41 Experiments in Memory dialog box Page 2 127 APPLIED OPERATIONS Saving Opening and Shredding Images 3 In the list in the Experiments in Memory dialog box select the file name including the image whose image information and observation condition are to be saved And click the lt Comments gt button The Image Comments dialog box as shown below appears Image Comments C IMAGES WX lt YZ 2 TIF Image Attributes Image Fields Comments Name x Y z Ch Resolution fo Jos Jos Jes Jeo Lens Magnification 40X lt Properties gt button in the Export to File group box Ces r hanqes Saves the image information and observation condition Cancel Changes Import from File Annotations ROls Expo
183. e Save as Type drop down list in the Save Experiment As dialog box Three file types are available as detailed below e Fluoview Multi Tiff tif TIFF format designed for use with FLUOVIEW Used for image analysis processing etc on FLUOVIEW Single TIF s 8 bit tif Single TIF s 24 bit tif TIFF Tagged Image File Format is used for image exchange between applications or computers Two types including the 8 bit and 24 bit types are available Bitmap 8 bit bmp e Bitmap 24 bit bmp The BMP format is the standard raster format of MS Windows Two types including the 8 bit and 24 bit types are available e mVx Files img ani File format for microVoxel Used for exchanging images with microVoxel 2 112 Page e APPLIED OPERATIONS Saving Opening and Shredding Images 5 TIP a In the file types above characters inside indicate the extension when a ifile is saved NOTE when saving a display the file format can be selected from Single TIF s 8 bit tif Single TIF s 24 bit tif Bitmap 8 bit bmp and Bitmap 24 bit bmp NOTE When saving an image obtained by merging more than one channel use a 24 bit file type NOTE it you want to save the image with the comment drawn on it as an image select the file type according to the usage of the image as described below Fluoview Multi Tiff Select when the image will be analyzed or processed after save Openi
184. e View The intensity values on a region in an image can be displayed graphically 1 Display the Single sub panel at the front 2 Display the Display panel of the image to be subjected to the intensity checking at the front 3 When the image was acquired in the multi channel mode select whether the multiple channels are analyzed simultaneously or only one channel is analyzed Jie f To select the target channel s use the lt Display channel switch gt buttons Only the lt Display channel switch gt 4 I button channel s being displayed will be analyzed Example When only the Ch1 image is displayed only the Ch1 image is analyzed For the switching of channels see section 2 4 3 Switching the isplay Channels Click the lt Annotate gt button in the toolbar at the bottom of the Analyze panel A list lt Annotate gt button of buttons appear as shown below l Z A ot DO OIC RYLEY 2 5 From the displayed buttons click the lt Rectangular gt button lt Circle gt button or lt Polyregion gt button Page 2 187 APPLIED OPERATIONS Image Analysis s 6 Specify the region to be checked in the image in the Display panel They can be specified as described below e Tospecify a rectangle On the image drag the mouse pointer along the diagonal line of the desired rectangle from the top left corner to the bottom right corner al lt Rectangu
185. e a a e E 1 58 1 3 Online Help sorro uu ASAS eaae akaa aaa asas 1 60 T31 FUNCHON Helurei E aus ua aa una O u ama e 1 60 1 3 2 Microscope HelDp a a 1 61 1 3 2 1 Configuring the Microscope a 1 64 1 3 2 2 Configuring the Microscope Combination with BX51 BX61 1 66 1 3 2 3 Configuring the Microscope AX70A 1 70 1 3 2 4 Configuring the Filters l u 1 72 1 3 2 5 Setting the C A Diameters u 1 74 2 APPLIED OPERATIONS 2 1 2 1 General Operation Procedure 2 1 2 1 1 Image Acquisition Procedure Section A a 2 3 2 1 2 Image Acquisition Procedure in an Observation Mode Section B 2 4 2 1 3 Examples of Operation Procedures a 2 5 2 2 Image Acquisition I 2 7 2 2 1 Image Acquisition in XY Observation Mode 2 8 2 2 1 1 Configuring the Microscope Y 2 9 2 2 1 2 Setting the Filters a 2 15 2 2
186. e angle 1 Acquire an image in the XY observation mode For the operation procedure see section 2 2 1 Image Acquisition with XY Observation 2 Select the Line XT option button or lt Slant gt under the Depth XZ option button in the Scan Mode group box in the Acquire panel A line indicating the control range appears in the Live panel Scan Mode C Surface XY Horm C Line XT Depth XZ 4 Hormal Slant C Point Size sss by Z z 3 Move the line to the area to be observed To move the line place the mouse pointer on it and drag the mouse 4 Change the line length Place the mouse pointer on the line and click to display square handles on the two extremities of the line Then place the mouse pointer on either handle and drag it to change the line length Handle 5 Change the inclination angle of the line Right click the mouse to display the pop up menu and select Rotate in it Move Size Bait Rotate Properties Page 2 77 APPLIED OPERATIONS Image Acquisition pO 6 The inclination angle of the line can be varied according to the mouse pointer movement Place the moue pointer on an inclining position and click the mouse left button to fix the line inclination angle 7 Acquire the image with the buttons such as the lt XYZ gt lt XYT gt lt XYZT gt buttons 8 After acquiring the image select lt Normal gt under the Surface XY option button in
187. e at specified times and after accumulation is performed the next line is to be scanned Each mode can be selected according to its ability the frame mode is adequate for observing fixed sells and the line mode is adequate for observing living cells 2 2 1 7 Saving the Acquired Image in File 1 Display the File I O panel 2 Click the page tab of the Live panel showing the image to be saved so that the image is displayed at the front 3 Click the lt Experiment gt button in the Save group box in the File I O panel lt Save gt button For details see section 2 3 1 Saving Image 2 30 Page APPLIED OPERATIONS Image Acquisition 2 2 2 Image Acquisition in Other Observation Modes 2 2 2 1 XZ Observation Mode The description in this section will be focused on the image acquisition operations in the PC i e a XZ observation mode that are not used in the XY observation modes which are the operations enclosed in Co in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections Page 2 31 APPLIED OPERATIONS Image Acquisition Set the dyeing method Section 2 2 1 1 in OPERATION Set the range of the multiple sections to be observed the Z direction scanning range Configure the microscope and scan unit Section 2 2 2 1 1 in OPERATION
188. e curve Place the mouse pointer on either handle and drag it to change the line shape The color of the mouse pointer is changed on the handle The handle can be add by clicking the mouse on desired coordinates To delete the handle click the mouse on it m a Right click the mouse on the coordinates but the handles to fix the shape after the change Set the scan speed for image acquisition Set the speed in the Scan speed group box of the Acquire panel Scan Speed Fast i Slow 0 5s Scan 0 5s Image TIP E The speed recommended for this scan mode are as follows 2us The scroll bar indicates Fast position It is useful for th specimen changes rapidly _AUs 8us The scroll bar indicates Slow position It is useful for th i specimen changes slowly I APPLIED OPERATIONS Image Acquisition bs 6 Set the interval time or the number of scans The operating procedure is same as that in the XY observation mode See section 2 2 2 XT Observation Mode for details s TIP S j x Up to 8000 times images can be acquired Set the number of scans ini the N text box in the Time Series sub panel U U UU 7 Click lt XZ gt lt XT gt or lt XZT gt etc to acquire the image x i TIP k The image shown in the Live panel after image acquisition is the image that acquired at the curved line specified on the image and x arranged uprightly
189. e file of the image to extract channels in advance TIP When the image has been saved in more than one image file it is possible to use only some of the slices by setting a slice range See section 2 13 1 E Setting the Range of Multiple Image Slices for the operation procedure NOTE The channel extraction cannot change the image type XYZ XYT etc Therefore it is not possible for example to extract an XY image from an XYZ or XYT image Click the lt Experiment List gt button in the toolbar at the bottom of the File I O panel The Experiments in Memory dialog box appears as shown below lt Experiment List gt button Experiments in Memory CAIMAGES lt Y 1 TIF CAIMAGES Y 2 TIF Fig 2 125 Experiments in Memory Dialog Box Page 2 249 APPLIED OPERATIONS Merger Extraction of Image Channels pO 3 Click the lt ADV gt button in the Experiments in Memory dialog box The Experiment Editor group box appears as shown below lt ADV gt button Experiments in Memory a sn Editor T Extract mei D XYCH CAMAGES lt Y 1 TIF CAIMAGES XY 2 TIF Fig 2 126 Experiment Editor Group Box Displayed by lt ADV gt Button he frame at the top left of the Experiment Editor group box shows th on of the image file displayed at the front of the Display panel 2 250 Page APPLIED OPERATIONS Merger Extraction of Image
190. e laser head for more than 30 minutes press the remote switch connected to the rear of the laser head once The auto alignment starts and completes in some tens of seconds The indicator on the rear of the laser head blinks during auto alignment Do not press the remote switch while the indicator is blinking Do not use the remote switch too frequently The recommend interval is once every few months provided that the UV Ar laser is used frequently When the UV Ar laser has not been used for a long period press it only once at the time of idle running of the laser head aging which should be performed every month Maintenance of Major System Units UV Ar Laser 2 2 2 Checking Refilling and Replacing Water in Chiller 1 Checking Chiller is a water cooled system After long hours of use check the water level and if water is insufficient refill distilled water to the specified level If the water in the tank is polluted change the distilled water The check period is about once every six months Note that the water tends to be polluted when the weather is warm or when the heat exchanger has been left unused for a long period 1 Switch off the main circuit breaker of the heat exchanger and unplug its power cable from the power outlet Top cover Top cover retaining screws x 4 Front panel Cooling hose Air inlet Remote cable Main circuit breaker switch Power cable 2 Remove the 4 top cover
191. e light path Disengaging the FR Frost from the light path may generate interference fringes on an image 6 Universal condenser Condenser for transmitted lighting In addition the rotary turret for the transmitted light DIC prism and the polarizing plate for differential interference observation polarizer are also provided e To perform differential interference observation engage the transmitted light DIC prism matching the objective in use in the light path For both visual observation and laser differential interference observation e To perform visual differential interference observation or laser differential interference observation engage the polarizing plate in the light path 7 Hand switch U HSTR2 U FH is optionally available with BX61 The hand switch corresponding to the BX electric system Connect the filter wheel of BX to connector of the following in NOTE the back of UCB e FWO FW1 e FWR FW2 e FWT FW3 Page 1 3 Getting Started FLUOVIEW Basic Operations Combination with AX70 or AX70WI 1 Light path selector button 3 Analyzer FV5 ANA 4 Transmitted light DIC slider 2 Universal vertical FV5 DICT illuminator mirror cube housing AX URBC 6 Universal condenser U UCDB 5 Filters Filter lever ND Filter levers IF550 LBD 7 Hand switch U HSTR 1 Light path selector button Select the light path between the visual observation and laser micros
192. e rears fice seer rine Mee isa n o E o 200 400 600 800 1000 420 1400 1600 1800 2000 220 2400 2600 280 x e 2 2 Time s I 5 300 The Disabled option button is selected 7 Start the forth and subsequent scan in 10 seconds after the third scan is performed The image of the photobleached specimen is acquired NOTE when performing the forth or subsequent scan be sure to switch the REX mode and Bleach mode between scans NOTE in the TIME COURSE software optional when the REX mask file in the REX or Bleach mode is changed while image scanning is performed obtaining the real time graph the laser is irradiated while the pop up menu is displayed however the real time graph is not plotted Please be aware this before using 2 100 Page APPLIED OPERATIONS Saving Opening and shredding Images 2 3 Saving Opening and Shredding Images Use the File I O panel to save open or shred an image Display the File I O panel at the front Open To open a file select the desired file in the Files list box and drag the file here File Type drop down list Selects the type of the files to be displayed in the Files list box Files list box Lists the image files of the type selected with the File Type drop down list and their acquisition parameters Select the file to be opened from this list The files in the list can be viewed by scrolling
193. e set in the Disable REX mode Page 2 95 APPLIED OPERATIONS Image Acquisition 5 Setting the XYT Observation mode 1 Confirm that XY Norm is displayed in the Scan Mode group box in the Acquire panel 2 Select the XYT option button in the Acquire panel Acaui Ele VOY Tie Y Proce lve YREx tivexo EA XY Repeat I mito m PMT Gain Offset uz 0 e 10x j xY xvt C xz CXZI Mode Surface XY Clip 1 0 23 Normal Clip Sq Cip C Line xT 057s Scan 0575 5 es time eee woj sec 3 300 H KI 1 10 000 n 94 sec Lime serie Z stage Limes Optics 0 min 0 0 sec x 7 20 Di Lasers REX_LivettO 2 96 Page APPLIED OPERATIONS Image Acquisition 3 From the page tabs on the bottom right of the Acquire panel select the Time Series sub panel The panel as shown below appears Interval text box Set the interval with the lt gt and lt Y gt buttons Key entry is also acceptable N text box Nil Set the number of scans with the lt gt and lt Y gt buttons Key entry is also ja acceptable wn Rest Time text box Shows the time after the end of an image acquisition until the start of next acquisition Total Time text box Shows the time required to complete image acquisition Fig 2 27 Time Series sub panel
194. e windows are resized to facilitate the reading and some grayed out characters are printed in readable characters 4 Page SYSTEM OVERVIEW On This Volume This volume describes the outline of the FLUOVIEW FV500 system Please read this volume so that you can understand the system before use CONTENTS pO 1 SYSTEM OVERVIEW 1 1 1 1 Principle visastscisesedeans cece teenscceciacuaaaduaaiass is oeueiai aana anina 1 1 1 2 Features of FLUOVIEW FV500 1 2 1 3 Light Path Diagram 1 1 eats 1 3 1 4 System Configuration 1 4 1 4 1 System Component Units and Their Functions 1 4 1 4 2 Block Diagram sau a Sa ka et Adee in eee 1 6 1 5 Software Functional Configuration 1 9 1 5 1 Function Panel and Display Panel a 1 9 1 5 2 Panel Structure of the Software raras 1 10 1 5 3 Icons Executed by Dragging amp Dropping 1 11 1 5 4 Identification of Images Depending on the Observation Methods 1 12 SYSTEM OVERVIEW Principle 1 SYSTEM OVERVIEW Olympus FLUOVIEW FV500 is a confocal scanning type laser reflected fluorescence microscope whi
195. eck box Enables the Z Motor operation The moving amount assigned to the lt Z stage fine adjustment gt and lt stage coarse adjustment gt buttons can be changed See section 1 3 in MAINTENANCE Setting the System Configuration fo detailed operations Page APPLIED OPERATIONS Image Acquisition bs 1 Check the Locked check box in the Z Stage sub panel Do not turn the fine focus adjustment knob while the Locked check box is checked for this may damage the Z motor 2 While observing the image in the Live panel locate the upper edge of the range to be observed by moving down the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the bottom edge of the range to be observed by moving down the revolving nosepiece using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons 3 When the upper edge position is located click the lt Set gt button The Start Z text box will show the scan start position of the range of the multiple sections to be observed Z direction scanning range 4 While observing the image in the Live panel locate the bottom edge of the range to be observed by moving up the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub pa
196. ection 2 6 1 1 Intensity Values on a Line Line Profile Double click the Region Histogram window The Enhanced Histogram Plot windows appears as shown below lt Properties gt button Displays the Editing dialog box for use in detailed setting of the chart or change of the chart display See section 2 14 Changing the Chart Display Method for details lt Copy gt button Copies the plotted image in the clipboard lt Save gt button Properties Saves the profile data in a file using an Excel cora T compatible format Save lt Close gt button Quit Quits the Enhanced Profile Plot window and returns to the Analyze panel Fig 2 91 Enhanced Histogram Plot Window Line Specification Grey Level ae When a desired area is specified by dragging the left button of the mouse on the graph the specified area can be magnified A TIP i When the right button of the mouse is dragged on the graph the graph ican be scrolled 2 192 Page APPLIED OPERATIONS Image Analysis The magnification or scrolling of the graph can be canceled by dragging the left button of the mouse from the bottom left to the right of 3 TIP When the mouse pointer is placed on a graph line while the Ctrl or Att key is held depressed the coordinates can be displayed TIP x The displayed data can be applied to other applications
197. ections to be observed From the panel page tabs shown on the bottom right of the Acquire panel select the Z Stage sub panel Z Series Current Pos text box Shows the current position of the stage The value can also be entered directly from the keyboard lt Z stage fine adjustment gt buttons lt Z stage coarse adjustment gt Displ the Z st by 0 1 isplace the Z stage by um per buttons step Displace the Z stage by 1 0 um per step Step Size oo P O 40um ideal t 1 Locked Locked check box Enables the Z motor operation Fig 2 9 Z Stage Sub panel Page 2 21 APPLIED OPERATIONS Image Acquisition s TIP The moving amount assigned to the lt Z stage fine adjustment gt and lt stage coarse adjustment gt buttons can be changed See section 1 3 in MAINTENANCE Setting the System Configuration fo detailed operations 1 Check the Locked check box in the Z Stage sub panel Do not turn the fine focus adjustment knob while the Locked check box is checked for this may damage the Z motor 2 While observing the image in the Live panel locate the plane to be observed by displacing the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel 2 22 Page APPLIED OPERATIONS image Acquisition 8 Setting the Area to be Observed When the observation targets are concentra
198. ects the Z Stage sub panel Selects the Time Series sub panel Selects the Dyes sub panel Selects the Config sub panel Selects the Main sub panel Selects the Filter sub panel Selects the Histogram sub panel Selects the Logical sub panel Selects the Single sub panel Selects the Series sub panel Visualize sub panels Target Operation O Selects the Orientations sub panel R Selects the Other options sub panel B 4 Page Appendix C Glossary Appendix C Glossary AOTF AOTF represents Acoustic Optical Tunable Filter AOTF is the sound optical element having optical anisotropy When a sound wave is spread in the element the element behaves as a phase grid and only the light of the wavelength corresponding to the wavelength of a sound wave is diffracted in the specific direction Therefore it is possible to control selection of laser wavelength and adjustment of laser intensity at high speed Active Status of being selected or executable An active window can be distinguished from other windows by the color of the title bar Application Same as software Among software refers to the software used directly by the users Backlash The quantity of play or loosening between gear teeth and part which is produced w
199. ed by clicking the lt gt button 00 Acquire File O Tile Process Focus XY Repeat Once ates IV DAPI M Ftc PMT Gain Offset PMT Gain Offset a Shum 55 60pm 87 Transmitted PMT Gain Offset 186v 43x 0 Uvepth xz Size eoo by 600 z Objective PLaPo 40x z Pan Zoom Scan Speed Fast HH Slow 4 618 Scan 7 61 5 image Acquisition Settings Load Save Focus Mode G x2 C x4 r Hormal C Kalman C Accumulate To Peak z l_ 2 J Display Panel C A and Laser ND LED sliders Set these values independently The laser type used with each channel is displayed below its Laser ND LED slider 1 76 Page Getting Started FLUOVIEW Online Help 4 While observing the images in the Live panel change their pinhole diameter settings Clicking this field allows the value to be changed on a large scale The ND value which are usually used are displayed in green Clicking the lt gt or lt gt button or the field displays the set value The set value can be varied by direct input from the keyboard sy z TIP y TP brash Gain pran Clicking this button allows li l fine adjustment of the 800u 10 0x value C A FITC PMT Gain Offset PMT Gain Offset PMT Gain Offset PMI Gain Offset l 800
200. ed in piscaeooeoe gray click and select the specified region on the Live panel When two or more regions are specified f h d Select all the regions to be masked and let the oe i handles be displayed around each region _ Handle Page 2 89 APPLIED OPERATIONS Image Acquisition Make REX mask 5 Select the lt Make REX mask gt button lt Make REX mask gt button The REX Live panel the REX mask file is made in the Display panel Beau Fie VOY Tie Yero ive Y REXive Y REX Livet0N XY Repeat ca E Once 2 2 lua aot ex Ox Oxz Omer va Make the REX mask file w s which masks the region outside of the specified cem region UPLAPO 20x z E Zoom Pan ae a Scan Speed Fast Slow 0s7s Scan 05 Laser Intensity DEI ar S Ja on Eu TA AOTr REX Function Disabled C REX mode C Bleach mode i i Make REX mask xS REX_LivettO Lases Optics Dyes Tine Sere TIP k When the REX Live panel has already been created on the FV30 software A panel is continuously created after REX_Live0 The saving procedure is completely the same as that of a display For details see section 2 3 1 2 Saving a Display in this manual 2 90 Page APPLIED OPERATIONS Image Acquisition 6 In order to adjust the laser intensity for every region right click the mouse on the wh
201. efault Folder for File I O Panel 2 Select the Environment sub panel System Properties Startup Shutdown Hardware Profiles General Performance System Variables Variable Value ComSpec CAWINNT system32 cmd exe FLUOVIEW C NFLUOVIEW S FLUOVIEWIMAGES C FLUOVIEWSIMAGES NUMBER_OF_PR 1 os Windows NT xj User Variables f Variable JM Value FLUOVIEW CAFLUOVIEW FLUOVIEWBACKUP C FLUOVIEWSystem Backup FLUOVIEWIMAGES C FLUOVIEWSIMAGES ID3 C Program Files 03D User name logging in Windows NT ID3IMAGES C Program Files O3D IMAGES zl Variable Value 3 Select FLUOVIEWIMAGES under Variable in the User Variables for Administrator list box System Properties 21 x Startup Shutdown Hardware Profiles l User Profiles General Performance Environment System Variables Variable Value ComSpec CAWINNT system32 cmd exe FLUOVIEW C AFLUOVIEW FLUOVIEWIMAGES C FLUDVIEWSIMAGES NUMBER_OF_PR 1 os Windows NT x User Variables for Administrator Variable Value FLUOVIEW C AFLUOVIEW FLUOVIEWBACKUP C FLUOVIEW System Backup Ma C AFLUOVIES AGES C Program Files 03D IDSIMAGES C Program Files O3D IMAGES gt l Variable JFLu OVIEWIMAGES Value estu OVIEWSIMAGES Set Delete Wind Page l 3 Appendix Change of Default Folder for File I O Panel 4 In the Value text box enter the path
202. eing method is shown in the pop up display Assign dyes manually Available Dyes Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method lt Prev gt button Sets the dyeing method which was set last time by clicking the lt Apply gt button lt Apply gt button Applies the dyeing method dragged in the Selected Dyes group box to the Ch group in the Acquire panel lt Clear gt button Clear the set dyeing method Fig 2 21 Dyes Sub panel 2 Select the specimen dyeing method by dragging desired dye names in the Available Dyes list box in the Selected Dyes group box to the field immediately above the list box 3 Click the lt Apply gt button to apply the selected dyeing method to the Ch group box on the upper part of the Acquire panel S TIP gt When the dyeing method is selected from the Available Dyes list box and i the lt Apply gt button is clicked a channel for acquiring fluorescence is set automatically according to the changed filter And the dyeing method is Page 2 63 APPLIED OPERATIONS Image Acquisition One Point The Assign dyes manually check box can also be used to set the dyeing method to the desired channel 1 Check the Assign dyes manually check box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes I list bo
203. el display position Sets the angle of label display format Sets the label type Select Auto or None Sets the type color and width of the line along the label Enables or disables the display of coordinate axis scales in the intermediate position between labels Enables or disables the display of scale line in the positions where labels are displayed Sets the length of the scale line from the coordinate axis to the label Sets the number of division between labels Sets the label display APPLIED OPERATIONS Changing the Chart Display Method Position sub panel Used to set the coordinate axis display position Editing Series General Axis Titles Legend Panel Paging Walls 3D Z ShowAxis Scales Title Labels Ticks Position Axis Left Position Z a C Bight x fo Pits Start Z C Bottom End ho C Depth IV Visible Titles sub panel Switches the title display Used to set a chart title position between the header and footer Chart Series Series General Axis Titles Legend Panel Pagi Enables or disables the title wam gt display ver Visible Border Sets the title character font Back Color Pattern IV Adjust Frame Switches the title background display range between the entire width of the chart display area and the character area Enter the title to be displayed here
204. elected as the target of mean value l Handle computation operation while the handles are displayed a d I I k d NOTE If the mouse is clicked in other place than inside the region specified on the image the handles will disappear On the contrary clicking the mouse in a region displays handles around it The operation cannot be executed while the handles are not displayed 2 198 Page APPLIED OPERATIONS Image Analysis 8 Click the mouse A pop up menu as shown below appears Select Properties from the menu Copy Move Size Edit Delete Properties 9 The Properties dialog box as shown below appears Display the Color panel at the front General Properties Color Set Color Standard Colors Color Palette Yellow Edit Custom Color Cancel Apply Fig 2 93 Properties Dialog Box 10 Select the desired color from the Color Palette list box 11 Itis also possible to specify more than one region simultaneously and display their operation results together First specify the regions by repeating steps 7 and 8 above for each Use different colors for the regions After having set the regions click the mouse in the first region With the second regions and after click the mouse while pressing the Shift key depressed 12 Click the lt Annotate gt button so that the list of buttons disappears ZArnolates button 13
205. en the light incident to photomultiplier tube is too bright or PMT voltage is set to a high voltage PMT Over warning may be displayed as shown below iw Error Condition xi Overrange on PMT2 Overrange on PMT1 This warning is displayed to protect photomultiplier tube when the light incident to it exceeds a certain level When it is displayed decrease PMT voltage setting Page 2 25 APPLIED OPERATIONS Image Acquisition c 10 Setting a Lower Scan Speed 1 The scan speed can be decreased using the scale in the Scan Speed group box on the Acquire panel TIP In general setting a lower scan speed allows the acquired image quality t f be improved However a low scan speed also lengthens the time required for imag iacquisition NOTE when the scan speed is decreased during fluorescence observation the saturation of fluorescence may darken the image of certain types of specimens In this case increase the scan speed and increase the PMT Voltage or use accumulation in scanning Scan Speed group box Set the scan speed by clicking a point on the scale line Fast A 3 3 iat 3 2s Scan 3 2s Image Scan Speed 11 Stopping Repeated Scanning 1 After the brightness and gain have been adjusted select the lt STOP SCAN gt button in the Acquire panel to stop scanning temporarily STOP SCAN 2 2 1 5 Acquiring Image 1 Select the lt Once gt button The acquired i
206. ending on the type of the chart The following descriptions take a Line 3D chart and Point 3D chart as examples This is the Format sub panel when the Line 3D chart is selected Select the chart series to be set in the Series panel Enables or disables the shadowed 3D display Series40 JAX Line Series40 Sets whether or not the outer frame of the chart is Format Point General Marks Data Source displayed and sets the I Enables or disables the color width and type of the Line coloring per coordinate axis frame Border Dark 3D s ILK Sets the chart color Gi fal I Color Each stairstep type chart display Lees P Inverted Pattern Sets the chart pattern This is the Format sub panel when the Point 3D chart is selected Editing x Chart SeriesO ba A Point 3D SeriesO Format Paint General Marks Data Source Sets the chart color tee EE C I Color Each Depth 4 i Sets whether or not chart lines are displayed and sets the color width and type Sets the depth of points in the chart Enables or disables the display varying the color at every point in the chart e Page 2 261 APPLIED OPERATIONS Changing the Chart Display Method Points sub panel Used to set the inflection points in the chart Enables or disables the inflection point display Sets the width of inflection Series40 points
207. ent stage position as the scanning start position of the range of the observed cross section Z direction scanning range lt Go gt button Moves to the set scan start position Use this button to check the scan start position Locked check box Enables the Z Motor operation TIP a The moving amount assigned to the lt Z stage fine adjustment gt and lt stage coarse adjustment gt buttons can be changed See section 1 3 in MAINTENANCE Setting the System Configuration fo detailed operations 1 Check the Locked check box in the Z Stage sub panel Do not turn the fine focus adjustment knob while the Locked check box is checked for this may damage the Z motor 2 56 Page APPLIED OPERATIONS Image Acquisition bs 2 While observing the image in the Live panel locate the upper edge of the range to be observed by moving down the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the bottom edge of the range to be observed by moving down the revolving nosepiece using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons 3 When the upper edge position is located click the lt Set gt button The Start Z text box will show the scan start position of the range of the multiple sections to be observed Z dire
208. ents the visual observation light path Fluorescence represents fluorescence observation for both LSM and BI observations and Nomarski represents transmitted light observation 1 65 Getting started FLUOVIEW Online Help BI 3 Select the lt Bl gt button in the Observation group box and select the microscopy lt BI gt button from the option buttons below it The points where system setting is to be changed for microscope observation will blink in red Turret Cube 4 Change the system configuration setup of cube turret etc by following the HUH HIHI guidance given by the red blinking light For the operation of the lever see section 1 1 1 Microscope in this volume NOTE The Microscope Configuration window is designed to give guidance on system configuration The red blinking light does not stop even after the indicated configuration point has been changed 5 While looking into the microscope move the stage and check the observed image 1 3 2 2 Configuring the Microscope Combination with BX51 BX61 When the combination with BX51 or BX61 is in use the microscope and scan unit FV500 can be configured on the FLUOVIEW software Use the following procedure to configure the microscope 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Optics sub panel Light Path BI LSM Tv lt Scope Control gt button Sets t
209. ents and click the moue while holding the comment and after Shift key to select the second After making all of the comments to be deleted active i e handled displayed around them click the right button of the mouse on one of the comments and select Delete from the displayed pop up menu _ 2 11 9 Moving Comment displayed around it Select Move from the menu together with the mouse pointer Click the moues on the comment to be moved to make it active i e handles Click the right button of the mouse A pop up menu as shown in Fig 2 118 appears The mouse pointer turns into a cross Move the mouse to move the comment Click the left button of the mouse to determine the new position Page 2 237 APPLIED OPERATIONS Entering Comment in Image A comment can also be moved by selecting it placing the mouse pointer o it so that the mouse pointer turns into a cross then dragging the mous In this case the final positioning of the comment can be determined b placing the mouse pointer outside the areas enclosed by the handles an clicking the left button of the mouse Handle Z NOTE The mouse pointer may be hardly visible depending on the image being displayed In this case use the method of displaying the pop up menu 2 238 Page APPLIED OPERATIONS Entering Comment in Image pO 2 11 10 Changing the Comment Size 1 Click
210. er transmittance filter Sec 1 2 7 Sec 1 2 2 Set the observation condition Select the light path for 100 binocular tube and focus on the specimen Sec 1 2 3 som Sec 1 2 8 1 Set the zoom ratio to 1X Sec 1 2 8 2 Select the LSM light path e Setthe channels Sec 1 2 8 3 Sec 1 2 4 e Setthe highest scan speed Sec 1 2 8 4 Select the XY observation mode Sec 1 2 8 5 Perform repeated scanning Sec 1 2 8 6 S oooooooo When the combination using the laser combiner is used set the optimum ND filters for the laser Sec 1 2 7 If no image is displayed p eldsip jou Ins s oew y Jl i Image is i displayed in ithe Live panel of the software If an image is Adjust PMT Voltage Sec 2 2 1 4 9 Set the multiple sections to be observed Sec 1 2 8 7 Set the area to be observed Sec 1 2 8 8 Set a lower scan speed Section 1 2 8 9 Stop repeated scanning Sec 1 2 8 10 Acquire an image Sec 1 2 9 Save the image Sec 1 2 10 Exit from the FLUOVIEW software Sec 1 2 11 Turn the system power OFF Sec 1 2 12 Page 1 17 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 1 Turning Power On Set the power switch of each unit to ON As the computer monitor power unit and hard copy unit optional are connected to the switched power o
211. ereo 3D images 4 Watch the two images with two eyes for a while They will look as a single 3D image APPLIED OPERATIONS viewing 3D Image Tie Y2roces Analyze isuatiz live XVZ2TF Y 30Animation a ee es REFERE Visualize Rendered View Extended Focus View C Arbitrary View Method Brightest Stereo pair O Sum Initial view Sequence views Initial Rotation Angle x y ml Display zoom 50 animation Opixel Channel 1 2 Ste Display zoom 50 animation Opixel Channel 1 2 Ste Zz off x Standard Views Left to Right 7 Number of views x 8 e Zio 2 Display Panel Fig 2 106 Panel Showing Stereo 3D Images Page 2 217 APPLIED OPERATIONS viewing 3D Image 2 8 4 Building a 3D Image to be Viewed Through Color Red Green Eyeglasses Images composed of multiple image slices acquired by varying the multiple sections XYZ observation can be built into an image which looks 3D when viewed through a pair of color red green eyeglasses This image can be viewed three dimensionally by watching them with two eyes for a while The operation is similar with animation Perform steps 1 to 10 in section 2 8 2 Animation then proceed to the following steps 1 Check the Stereo Pair check box 2 Click the lt Begin Visualize gt button to start building the images When the building completes the built image is displayed in the 3D Animat
212. es sub panel The Conforcal Aperture value is set appropriately Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 8 2 Setting the Zoom Ratio to 1X Use the Zoom scale in the Acquire panel to set the zoom ratio to X1 Pan Zoom Using the UV Ar laser set the zoom ratio to X2 1 2 8 3 Setting the Channels 1 In the Ch group box make sure that the check boxes showing the applicable dyeing methods are check marked to indicate that the channels are ready for image acquisition If the check box of a channel is not check marked check it to make the channel ready Channel check boxes with which dyeing methods are displayed a Bra tne het FITC V Transmitted PMT r Offset ped Offset a Offset Offset PMT Gain Offset Sa Fa jae ew 800v 10 0x 7 soov 10 0x 7 800v 10 0x A 10 0x z 186v 43x 0 K TIP 1 iTo display the information on all channels simultaneously right click th ene between channel display boxes ITERE AE AEE A EE E AEE E OEE EE E E EEN POE EENEN E E 1 2 8 4 Setting the Highest Scan Speed 1 Set the scan speed to the fastest speed by using the scale in the Scan Speed group box in the Acquire panel Scan Speed group box Set the scan speed by clicking a point on the scale line Scan Speed 3 2s Scan 3 2s Image 1 50 Page e Getting Started
213. es as shown below caulk File vO Tile t Append Next Series Don aalala Transmitted lt Series Done gt button Determines the acquired images Once this button is clicked it is not possible to append an image Click the lt Append Next gt button to append an image An image will be acquired with the same number of steps as the image acquired immediately before and appended to it Click the lt Series Done gt button when it is not required to append an image Page APPLIED OPERATIONS Image Acquisition Doo O 2 2 2 4 XYZ Observation Mode A The description in this section will be focused on the image acquisition operations in the XYZ o observation mode that are not used in the XY observation modes which are the operations enclosed in C in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections e Page 2 45 APPLIED OPERATIONS Image Acquisition Set the dyeing method Section 2 2 1 4 in OPERATION Configure the microscope and scan unit Sections 2 2 1 1 amp BEEZ in OPERATION Set the range of the cross section to be observed the Z direction scanning range Section 2 2 2 4 1 in OPERATION Set the objective magnification Section 2 2 1 4 1 in OPERATION Set the numbers of Z direction steps and acquired image slices Section 2 2 2 4 2
214. es the channel between the selected and deselected status alternately The lines connected to Merger indicate the selected channels APPLIED OPERATIONS Merger Extraction of Image Channels Experiment Editor XY 2 T1F Clicking a channel line in this area switches the channel between the selected and deselected status Up to 6 channels can be selected together To select another channel after having selected 6 channels deselect the unnecessary channels before selecting required channels 7 Click the lt Merge gt button in the Experiment Editor group box A new Display panel showing Merge in the page tab appears and the images including newly merged channel s are displayed in the panel Acquire File uo Y Tie Y roces Live iif XY 4 TIF XY 2 TIF i Merge EP Open Save E XYCH File Type Tift TIF Files 2ndmerge tif Gatesi 512812 b3120sstt 600809 Experiments in Memory Sees Editor F jo oe Extract XYCH A XY CHO tae C NMAGESWY 1 TIF CAIMAGES XY 2 TI eS SSS x slaiZizicl2 Jin ee Fig 2 124 Merge Panel 2 248 Page APPLIED OPERATIONS Merger Extraction of Image Channels 2 13 3 Extracting Channels from Image Desired channels can be extracted from an image Use this facility to extract only the images of required channels from an image acquired from more than one channel 1 Open the imag
215. etup CD ROM in the CD ROM drive Then the dialog box as shown below appears OLYMPUS FLUVIEW Install Program Update from Ver 2 x Start Update Exit Copyright 2004 Olympus Optical Co Ltd Click the option button of the version of FV software installed and click the lt Start Update gt button Update FV Software dialog box is displayed and starts the current settings For save carry out according to a wizard Page 1 1 Software Setup Re setup or Updating of the Software NOTE Current settings are performed after lt Start Update gt button selection Be sure to save the current settings before proceeding to the removal of the software If the software is removed without saving the current settings the settings in the software program obtained by re setup will be reset to the initial settings 4 Continuously deletion of software is started For deletion carry out according to a wizard 5 Continuously installation of the software of a new version is started Refer to 1 2 New Setup of the Software for details 1 2 Page Software Setup New Setup of the Software 1 2 New Setup of the Software The following procedure allows you to set up the FLUOVIEW software in a computer you use 1 Load the FLUOVIEW setup CD ROM in the CD ROM drive 2 Select the Start button to display the Start menu then select commands Settings Control Panel
216. exists the dialog box appears to ask you to overwrite the existing file or not When overwriting is not required click the lt NO gt button and enter another name to save the file 2 3 7 2 Reading the Region File 1 Display the Display panel of the image including the region to be read at the front position 2 Select the lt Experiment List gt button in the tool bar at the bottom of the File 1 0 panel The Experiments in Memory dialog box as shown below appears lt Experiment List gt button F F Experiments in Memory CAIMAGEYS Y Z 2 TIF CAIMAGEWY Z 22 comment pone Fig 2 48 Experiments in Memory dialog box 3 In the list in the Experiments in Memory dialog box select the image file name including the region to be read and click the lt Comments gt button The Image Comments dialog box as shown below appears 2 132 Page APPLIED OPERATIONS Saving Opening and Shredding Images Image Comments C IMAGES X lt YZ 2 TIF Image Attributes Image Fields Comments Name x Y z Ch Size 12 bit 800 600 5 2 lux um um um sasay ama 40K Ej lt Annotations ROIs gt button in the Export to File group box Saves the region into a file Record Changes Cancel Changes lt Annotations ROIs gt button in the Import from File group box Reads the file saving the region Export to File Annotations ROls Properties Fig
217. eyboard objective with keyboard keyboard 3 Click the lt OK gt button after setting is completed TIP E The initial values are automatically set in the Magnification N A n and Recommended Cofocal Pinhole text boxes after the objective is selected 1 3 2 3 Setting the Color 1 Setting the color of the button disengaged from the light path 1 Right click the mouse on the button whose color is to be changed The pop up menu as shown below appears Edit Color 2 Select Color The Color dialog box as shown below appears Color BE Basic colors Sees ET HHHH Hue 40 Red 255 Sat 240 Green 255 CobiSoid pum 120 Blye 0 Add to Custom Colors 3 Select the color which you want to set in the color palette and click the lt OK gt button Page 1 17 Software Setup Setting the System Configuration 2 Setting the color of the button engaged into the light path 1 In BX Control Panel window on the software specify the color of the button of the cube the objective and the condenser engaged into the light path Right click the mouse on the area outside of the buttons in the Mirror Unit Nosepiece Condenser Turret or Filter Turret group box Hole Count Selected Hole Color 2 Select Selected Hole Color The Color dialog box as shown below appears Color Basic colors Jmm ma mr mr LIS I I E Es m F zs zs z zs mi
218. f laser controller to the OFF position 4 Turn the rocker switch of laser controller to 0 OFF Turning the laser power off by force may damage the laser NOTE tube Never omit the step 1 the process for cooling down except for emergency Page 1 59 Getting started FLUOVIEW Online Help 1 3 Online Help The FLUOVIEW application comes with two kinds of online help facility e function help for referencing the function and operation procedure description while controlling the application and e microscope help providing information on the system setup 1 3 1 Function Help This section describes a simple method for displaying and consulting the online help on the functions and operation procedures The figure below shows the initial display table of contents of the FLUOVIEW 2 Online Help window To display this window click the lt Help gt button in the toolbar at the bottom left of the screen lt Help gt button Some words in the displayed information are shown in enhanced display underscored or colored green Clicking one of these words allows you to jump to the meaning of the word or to more information about its meaning amp Fluoview Online Help _ x Fie Edit Bookmark Options Help Gants trier Bek Pa Perso Finger pointer FLUOVIEW Confocal Laser Scanning Microscopes OLYMPUS FLUOVIEW is a family of Confocal Laser Scanning Microscopes The family
219. g Water in Chiller 2 3 Software Setup Re setup or Updating of the Software 1 Software Setup The FLUOVIEW software ha A CD ROM containing the s s been set up before it is delivered to the user oftware program is provided with FLUOVIEW This CD ROM is intended for use when re setup is required due to a fault or when the user wants to set up the software newly When the FLUOVIEW software is updated the user is also requested to update the software in use accordingly For re setup or updating of the software see section 1 1 Re Setup or Updating of the Software below and follow the procedures given i When you want to set up the procedures given in it n it software newly see section 1 2 New Setup of the Software below and follow the P To set up the FLUOVIEW software it is necessary that the computer in use already has Microsoft Windows NT Version 4 0 English version installed lin its hard disk f A vacant space of 100 MB is re 1 1 Re setup or Updating of the Software Save the current settings and remove the current software before starting re setup or updating with the following procedures 1 Update from Ver 3 x option button When updating from FV software after ver3 0 select this option button Update from Ver 2 x option button When updating from FV software of ver2 0 to ver2 9 select this option button Insert the FLUOVIEW s
220. g box For details see the description on the Software panel in section 1 3 1 i MAINTENANCE Overall Setting of FLUOVIEW for detailed operations 2 11 12 Changing the Comment Font 1 Click the mouse on the comment to be changed of font to make the comment active i e handles displayed around it 2 Click the right button of the mouse A pop up menu as shown in Fig 2 118 appears 3 Select Properties from the menu The Properties dialog box as shown below appears Display the Font panel at the front 2 240 Page APPLIED OPERATIONS Entering Comment in Image Properties Ea General ActiveText Color Properties Font Size Arial x j2 gt Effets T Bold Underline T Italic T Strikeout Sample Text Cancel Appi Fig 2 120 Properties Dialog Box 4 Set the font type and size using the drop down lists and select the effect Page 2 241 APPLIED OPERATIONS Image Output at Printer 2 12 Image Output at Printer 1 Display the Display panel of the image to be output at the printer 2 Click the lt Print gt button in the toolbar at the bottom left of the panel A dialog box as shown below appears lt Print gt button Print HES Printer Name Laserqraphics Personal LFR Plus Properties Status Ready Type Lasergraphics Personal LFR Plus Where LPT1 Comment m Print range r Copies All Number of copies 1 ge ee mmm
221. g pictures on image Section 2 11 2 Drawing scales on image Section 2 11 3 Output the image at the printer Section 2 12 Exit from the FLUOVIEW software and turn power OFF Sections 1 2 11 amp 1 2 12 2 2 Page APPLIED OPERATIONS General Operation Procedure 2 1 1 Image Acquisition Procedure Section A This section describes the procedure for acquiring images See sections 2 2 and after for the actual operation methods The detailed operation methods of each item in the procedure are described in the section specified in parentheses Acquire an image in an observation mode FLUOVIEW provides the following observation modes Gi SS CBS SS SS TIME TIME i XY observation XZ observation XT observation XZT observation XYZ observation XYT observation XYZT observation Section 2 2 1 Section 2 2 2 1 Section 2 2 2 2 Section 2 2 2 3 Section 2 2 2 4 Section 2 2 2 5 Section 2 2 2 6 When saving the image in a file With this system the observation z iby XY scanning is called simply as Save the acquired data in a file a obserwator Section 2 3 1 Page 2 3 APPLIED OPERATIONS General Operation Procedure 2 1 2 Image Acquisition Procedure in an Observation Mode Section B As an example of Acquire an image in an observation mode this section describes the procedure in the XY observation mode For the procedures in other observation modes see se
222. ge at the printer lt Exit gt button Exits from FLUOVIEW Page A 1 Appendix A List of Tools List of Tools Appendix A 1 2 Tools at the Top of Display Panel These refer to the horizontal array of buttons at the top of the display panel The functions frequently used with images are gathered here lt Set end position gt button When successive display or frame by frame display is required set the image with which the display should end n 1 assuming that the number of images is n is set when this button is not used This button is also used to set the image for ending a processing operation such as image save lt Set start position gt button When successive display or frame by frame display is required set the image with which the display should start 0 is set when this button is not used This button is also used to set the image for starting a processing operation such as image save lt Display switching gt button Used to display more than one channel simultaneously lt Z T series switch gt button When an image is composed of multiple image slices this button switches between the display according to multiple sections and that according to the progress of time This button is also used to display an extended image lt Magnify reduce gt button Magnifies or reduces the image Magnification or reduction by 3 1 or 1 3 the original image size is possible lt Bound gt lt Loop gt but
223. ge from the end position to the start position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame e Page 2 221 APPLIED OPERATIONS viewing Images Following the Progress of Time s 4 Click the lt Set start position gt button If the start position is not set image No 0 becomes the start image automatically 5 Display the image slice to end the successive display by using the lt Display gt button at the top of the Display panel 6 Click the lt Set end position gt button If the end position is not set image No n 1 assuming that the number of images is n becomes the start image automatically 7 Click the lt Display gt button The images will be displayed successively from the start position to the end position To stop the successive display click the lt Stop gt button again 2 222 Page e APPLIED OPERATIONS transferring Data to Another Application Doo 2 10 Transferring Data to Another Application 2 10 1 Transferring Analysis Data to Another Application Analysis data can be transferred to Excel 1 Display the Analyze panel and executes analysis After it display the Enhanced Profile Plot Intensity Map Enhanced Histogram Plot Average Intensity Trace or Integrated Intensity Trace TIP For t
224. gging and dropping the frame at the top left shows the icon btract from Imag ultiply Image witi of the image file displayed in the Display panel a Log base 10 Multi Bass Deia poe eee pe l a a zmes g gt TIP gt The mouse pointer turns into the image icon during dragging U S btract 2 Image j e Exp riments in h emory J i rN SEY Fry 4 From the Experiments in Memory dialog box select the file name of the second file and drag it to the frame at the top right of the Process panel The icon of the image is displayed in the frame at the top right of the Process panel x ealel Zizi olal fa TIP 5 Click the lt Done gt button in the Experiments in Memory dialog box to close it 2 180 Page APPLIED OPERATIONS Image Processing 6 Click the lt Image AND Image gt button A new Display panel showing Image AND we Image gt Image in the page tab appears showing the image obtained by the operation utton Acquire File vO Y Tile Y2roce lt _ Lie Y xam Y Fiter ImageanDimag ImageANDImage Ee NOT Image Image O Image J S a Histo Fig 2 85 Image AND Image Panel 2 5 3 7 Image OR Image Two different images can be ORed The operation method is identical to the AND operation between two different images except for the following point See section 2 5 3 6 Image AND Imag
225. ght microscope set up Z motor for fluorescence observation Motor for driving the focusing block of the microscope Multi control box Transmitted Controller of the AX70 microscope light unit Unit incorporating a 100 W halogen lamp bulb to provide illumination for transmitted light observation Connected with the microscope Image monitor Video monitor for displaying laser scanning images control panel etc Control unit Unit for driving the through an r optical fiber scanning unit laser cable combiner X motor and transmitted light Laser power detector supply units Computer Used to control the Anti vibration _LSM process image platform files etc Exclusively designed pneumatic Reflected light unit platform Unit incorporating a 100 W mercury burner to provide illumination for reflected light observation Power box Connected with the Light Power Supply Unit Photo 1 1 Combination with AX70 Power supply unit microscope through Power supply unit for the for the AX70 an optical fiber cable mercury burner for reflected microscope light observation Reflected Fluorescence 1 4 Page SYSTEM OVERVIEW System Configuration Microscope Upright microscope exclusively designed for FLUOVIEW FV500 Photo 1 2 Combination with IX Page 1 5 SYSTEM OVERVIEW Ssystem Configuration 1 4 2 Block Diagram MONITOR 6 2m Dsub9pin
226. gia 3 D Line a 3 D Pie a5 D Surface Fig Appendix G 5 Dialog Box of Chart Wizard 2 5 10 Select the desired chart type and click the lt Next gt button The dialog box as shown below appears ChartWizard Step 3 of 5 Select a format for the Line chart Fig Appendix G 6 Dialog Box of Chart Wizard 3 5 Page G 3 Appendix G Converting Analysis Data into a Chart Using EXCEL 11 Select the desired chart format and click the lt Next gt button The dialog box as shown below appears ChartWizard Step 4 of 5 Sample Chart Data Series in C Rows Columns Use First n Column s for Category X Axis Labels Use First fo Row s for Legend Text Fig Appendix G 7 Dialog Box of Chart Wizard 4 5 12 Click the Columns option button under Data Series in 13 Set the data column number that you want to use as the X axis label position in the Use First Column s text box 14 Click the lt Next gt button The dialog box as shown below appears ChartWizard Step 5 of 5 Sample Chart Enhanced Profile Plot Add a Legend C Yes No Chart Title Enhanced Profile Plot Axis Titles Category x Position um Ses Value Y Intensity ite I Second Y Fig Appendix G 8 Dialog Box of Chart Wizard 5 5 15 Enter the chart title in the Chart Title text box 16 Enter the X axis label in the Category X text
227. hannel gt Display Zoom lt display zoom gt Is bounded lt bounds gt Experiment Name lt name gt lt display zoom gt lt z gt lt t gt lt animation gt lt channel gt lt stereo gt lt bounds gt TIP Desired characters can also be entered f 1 Click the character in the drop down list of the dialog box 2 Delete the character by pressing the Delete or Back Space key i TIP When the lt Set as Default gt button is pressed after having selected a label f it is set as the label displayed permanently at the bottom left of the Display f ipanel 4 Click the lt Font gt button The dialog box as shown below appears Font Font style Size rial Regular fi 2 s Cancel Courier T Courier New Fixedsys F Lucida Console FH Lucida Sans Unicode FH Marlett r Effects I Strikeout I Underline Color C Yelow X Script Westem X This is a TrueType font This same font will be used on both your printer and your screen Fig 2 115 Font Dialog Box Page 2 229 APPLIED OPERATIONS Entering Comment in Image pO Select the character font and size using the Font Font Style and Size list boxes 6 Click the lt OK gt button to close the Font dialog box 7 Click the lt Close gt button to close the Active Overlays dialog box 8 Place and click the mouse pointer on the image position you want to enter characters lt
228. he Live panel lt XY Repeat gt button Repeats XY scanning to display images successively in the Live panel The acquired image is displayed cys Offset PMT Gain Offset PMT Goin F og r PMT Gain um J 800v 10 0 Z 800v 100x Z 485v 43x os PMT Gain and Offset LED sliders These can be adjusted independently Ch1 Ch2 Ch3 Ch4 and Ch5 check boxes St low EE Check to select the image acquisition i Ee e s EZ xY CxYT OXYZ C xYzT channel Increasing the PMT voltage improves the sensitivity Increasing the Gain brightens the image Increasing the Offset darkens the image Option buttons C Line xT Check a button to select one of the upas A C Point displayed observation modes gt Em E Select the magnification of the Scan Speed group box objective on the microscope Sets the scan speed Sliding to the left of the scale increases the scan speed and to the right decreases it Scan Z Stage Time Series Dyes and Optics Lasers sub panels lt Load gt button Loads the observation conditions at image acquisition These panels are used to set the information required for image acquisition lt Save gt button Saves the observation conditions at image acquisition xas Enl Display Panel Fig 2 1 Acquire Panel Page 2 9 AP
229. he edges of the area in which the chart is displayed 2 254 Page APPLIED OPERATIONS Changing the Chart Display Method Axis sub panel Used to set the coordinate axes of the chart Displays the chart axes by selecting the optimum scale automatically Enables or disables the display of chart axes r Sets the maximum and Left minimum values of the chart T axes either automatically or manually Selects the chart axes to be set in the Scales Title Labels Ticks and Position panels Right Top Bottom Sets the number of steps of Benth the values displayed in the chart axis labels Enables or disables the chart f Re Logarithmic T Inverted axis display set in the Axis m gt group box TAS Visible Enables or disables the inverted chart display Enables or disables the logarithmic chart display e Title sub panel Used to set the title of the coordinate axis Series General Axis Titles Legend Panel Paging Walls 3D Sets the title of the M ShowAsis Scales Title Labels Ticks Position coordinate axis Sets the angle of displaying Anis the coordinate axis title Title Sets the title character font C Bottom Sets the angle of displaying C Depth the coordinate axis title IV Visible Page 2 255 APPLIED OPERATIONS Changing the Chart Display Method Labels sub panel Used to set the label of the coordinate axis
230. he LUT is the tabulation of this relationship between the input and output m Macro Record of a series of operations When a macro is executed the series of operations defined for it are executed Maximize button The button showing an upward triangle at the right of the title bar Clicking this button displays the window full screen The same operation is also available using the Maximize command in the control menu C 2 Page Mouse A device which was named because it looks like a mouse It is used to give instructions in the window Mouse pointer When the mouse is moved on the desk arrow amp moves in the display along the movement of the mouse This is called the mouse pointer o Offset This function darkens the image by the ratio set at the time of image acquisition Offset should be used before using Gain On line help Manual displayed on the screen that is built into software Option button Small circular button inside a rectangular frame in a window Only one option button item can be selected from the items enclosed in the frame P Panel The large rectangle with a page tab in a window The panel is provided on a per function basis and clicking the page tab displays the panel of the selected function Paste Action of placing the data in the clipboard in an application Piezo Abbreviation of piezoelectric which is the phenomenon of generating electricity when a fo
231. he following characters to set the display method hotspot Display measurement points with markings raw Display data in values between 0 and 4095 calibrated Display data in numerical values um units Display the unit if this has been set Same as the concentration computation in FV TIEMPO Examples lt Input character strings gt lt Displayed strings represent figures gt lt intensity raw value gt r tt rrr tttee eres lt intensity hotspot raw value gt 1 2 When images are overlapped pecs neneenanensaneneaceneneseeneaeaneneesanensaneneaseneserceseaeaseaeeseaeesaneeaseneaseseseseeseaenseaeesaneeaseneaseneseaeaneaeeneaensauentaneneaseasseaeeaeaeeneasesaneesanentaseneasaeeasaeeneasenanennanentaa p TIP E The detailed display procedure is described in section 2 11 2 Viewing th J 4 Page Appendix J List of Functions in the Active Overlays Dialog Box Appendix J 3 Other The following image data can be displayed in addition to the X and Y coordinate positions and intensity value Appendix J 3 1 Channel Number The channel number can be displayed When images are overlapped the displayed channel numbers are connected by the markings Syntax lt Channel gt Appendix J 3 2 Objective Power The objective power used in image capturing can be displayed Syntax lt Objective gt Appendix J 3 3 Date of Image Capturing The
232. he image acquisition 3 In the Channel 3 group box check the check box showing the applicable dyeing method to ready the image acquisition 4 In the Channel 4 group box check the check box showing the applicable dyeing method to ready the image acquisition CH2 of box CH3 an box CH4 check box CH1 check box 4 DAPI v L A Transmitted PMT Gain Offset mmr Offset 2 a Offset Pa Offset PMT Gain Offset LW il H II 800v 10 0x Y 800v 10 0x i 800v 10 0x 800v 10 0 Z 4869 43x 0 TIP lt To display the information on all channels simultaneously right click the boundary between channel display boxes i Click the boundary again to return to the original display U UU UU Page 2 19 APPLIED OPERATIONS Image Acquisition 4 Setting the Highest Scan Speed Set the scan speed to the fastest speed by using the scale in the Scan Speed group box in the Acquire panel Scan Speed Scan Speed group box Fast Slow Set the scan speed by clicking a point on the scale line 3 2s Scan 3 2s Image i TIP he focus mode makes it possible to increase the line skipped scan speed f rom the page tabs on the bottom right of the Acquire panel select the can sub panel elect either option button in the Focus Mode group box Acquisition Settings Load Save j A Focus Mode F Mod b Focus i ode group box x2 C K
233. he operation me iw Enhanced Profile Plot x lt Save gt button Properties Saves the profile data in a file using Copy an Excel compatible format N ae Save lt Close gt button Close Quits the Enhanced Profile Plot _ ii window and returns to the Analyze Position um panel Fig 2 108 Enhanced Profile Plot Window s TIP Microsoft Excel is not included in the FLUOVIEW FV300 system ase purchase i Page 2 223 APPLIED OPERATIONS transferring Data to Another Application 2 Click the lt Save gt button When the Save As dialog box appears as shown below set the file name and click the lt OK gt button to save the analysis data Save As 21x Save in E images v a c ere Filename XYZ 2 xls Save as type Excel file xls Cancel Fig 2 109 Save As Dialog Box 3 Exit from FLUOVIEW or display the Start menu by pressing the Ctrl Esc keys 4 Select Programs and issue the Microsoft Excel command 5 From the File menu of Excel select the Open command and open the file saved in step 2 6 When the dialog box as shown below appears click the Delimited option button in the Original Data Type group box then select Windows ANSI from the File Origin drop down list Text Import Wizard Step 1 of 3 The Text Wizard has determined that your data is Delimited If this is correct choose Next or choose the Dat
234. he operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter Average Click the lt Average gt button lt Average gt button 2 166 Page APPLIED OPERATIONS Image Processing Doo 2 Median filter lt Median gt button 3 Low pass filter lt Lowpass gt button The Median filter reduces noise in image while leaving the edges intact However it may be ineffective in case noise is concentrated in some positions or the image is very noisy The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter Click the lt Median gt button The low pass filter passes the low frequency structures In this way it can eliminate small grains and provide smooth noise reduced image filter format is as shown below 1 2 1 2 4 2 X 1 16 1 2 1 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter e Click the lt Lowpass gt button 2 5 1 3 Image Sharpening 1 Sharpen filter lt Sharpen gt button The sharpen filter turns blurred image into a clear image The filter format is as shown below 0 1 0 1 5 1 0 1 0 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter Click the lt Sharpen gt button
235. he point that you want to start the straight line and drag the mouse from there to the point you want to end it To draw a polygonal line On the image click the points corresponding to the start point peak points and end point of the desired polygonal line then click the right button of the mouse to set the specification To draw a rectangle On the image drag the mouse pointer along the diagonal line of the desired rectangle from the top left corner to the bottom right corner To specify a circle or ellipse On the image assume a rectangle circumscribing the circle to be checked and drag the mouse pointer along the diagonal line between opposite corners of the rectangle To specify a polygonal region On the image click the points corresponding to the corners of the desired polygon After clicking the last corner point click the right button of the mouse to connect the last clicked point to the first clicked point To specify a free region On the image specify a region by dragging Then release the mouse button to complete dragging The point where the dragging was ended will be connected to the point where it was started To specify a free line On the image drag the mouse pointer along the desired line APPLIED OPERATIONS Entering Comment in Image 2 11 5 Drawing a Scale in Image A scale can be drawn between two points in an image lt Scale gt button 1 2 3 4 Display the Display pane
236. he status of the Ch5 check box Ch5 scanned not scanned PMT voltage gain offset adjustments Target Operation Enables and makes variable the LED slider on the left 2 Enables and makes variable the LED slider on the right Decreases the value of an enabled LED slider Fine adjustment Decreases the value of an enabled LED slider Coarse adjustment Increases the value of an enabled LED slider Fine adjustment Increases the value of an enabled LED slider Coarse adjustment An LED slider is enabled and variable when its setting values are displayed in red Page B 1 Appendix B List of Hot Keys Image acquisition Scanning Target Operation Key Acquires another image after completing series image acquisition Append Next Completes image acquisition after completing series image acquisition Series Done Stops scanning Stop Scan Scanning speed and area setting Target Operation Decreases the scan speed Increases the scan speed es Ctrl 4 Decreases the zoom ratio Ctrl T Increases the zoom ratio Z stage setting Target Operation Moves the Z stage up Fine adjustment Moves the Z stage up Coarse adjustment
237. he system r Mroshqo Control Trans cope SU Lamp Control Control Fig 1 6 Optics Sub panel 1 66 Page Getting Started FLUOVIEW Online Help 2 Select the lt Scope Control gt button on the bottom of the Optics sub panel The BX Control window as shown below appears Shutter group box Clicking inside the box switches the EPI shutter to be closed opened DE shutter is opened The shutter is closed LMT group box Selects the light path lt BI gt is for GaS A direct observation lt LSM gt is for LSM observation and lt TV gt is for TV observation Filter Turret group box Clicking the filter for visual observation to be set switches the turret automatically Mirror Unit group box Clicking the cube automatically switches the turret Nosepiece group box Click to change the objective Top Lens group box Clicking inside the box switches the top lens to be engaged into the light path s tp lens is into the H oath The top lens is disengaged from the light path Condenser Turret group box Clicking the universal condenser automatically switches the turret Aperture Iris group box Changes the AS value Filter Turret group box Clicking the filter for transmitted light observation to be set switches the turret automatically BX Options group box Used for optional BX settings lt Link Setting gt button Selects the fu
238. heck box in the Ch1 Ch2 Ch3 Ch4 group box that shows the dyeing method is check marked to indicate that the corresponding channel is ready for image acquisition Ch3 check box Ch4 check box Ch2 check box FITC V Transmitted Ch1 check box r can Offset EMT Offset ma c _ Offset ma Offset PMT Gain Offset 800v 10 0x mi 800v 10 0x i s 10 0 i 800v 10 0x Y 186 43x 0 e Set the image brightness Adjust the brightness of the image of the image acquisition channel by using the PMT Offset and Gain LED sliders in the Acquire panel For details see to section 2 2 1 4 9 Adjusting the Image Brightness in the OPERATION volume 2 62 Page APPLIED OPERATIONS Image Acquisition 2 2 3 2 Dual Fluorochrome Image The wavelength obtained by dual fluorochrome dyeing can be acquired and observed as images of the channels Ch1 or Ch2 The subsequent description deals with the differences in operation between the monochrome dyeing and dual fluorochrome dyeing Set the two or more dyeing methods 1 From the page tabs on the bottom right of the Acquire panel select the Dyes sub panel r Selected Dyes Assign dyes manually check box Checking this enables the manual setting Dragging the dyeing method in the list directly to the Ch group box assigns the dye to the desired channel Place the pointer on the icon displayed in the Selected Dyes and the dy
239. hen the fluorescent cube is selected in the Mirror Unit Link Setting 7 Nosepiece gt is Nosepiece gt x Nosepiece gt TD Lamp N gt FL Mirror Unit gt FL Mirror Unit gt Condenser Turret Z Stage Top Lens OUT DIC Shutter Close AN PO TD Lamp OFF Top Lens OUT Click the desired check box to be checked Select the lt Close gt button to close the dialog box e Selecting the lt Load Setting gt button displays the Open dialog box as shown below Open Look in aseis si c s BX2 ini File name Files of type BX Setting File ini X Cancel When the setting file which you want to read is not displayed in the list box use the Look in drop down list and select the drive or directory where the file is saved Select BX Setting File ini in the Files of type drop down list In the list box select the setting file which you want to read Click the lt OK gt button to close the dialog box Getting Started FLUOVIEW Online Help e Selecting the lt Save Setting gt button displays the Save As dialog box as shown below Save in Setting 7 cl File name Ex2ini Save as type ex Setting File ini z Cancel To change the save destination drive or directory use the Save in drop down list Enter the setting file name into the File name text box Select the lt Save gt button to
240. hen the stage is moved up or down by the Z motor Button Command button Check box A small square which can be either checked with X or cleared The check box indicate an item which can be enabled or disabled The item is enabled when it is checked with X Clear Action of removing check mark X from a check box to disable the item To clear a check box checked with X click the check box Click Action of pressing then releasing the button of the mouse Clipboard The place which relays data when an operation such as Copy Cut and Paste is executed Command button A figure in the shape of button in the window Clicking a command button with the mouse allows the function indicated on the button to be executed Confocal Signifies the possibility of obtaining data on the plane where the irradiated laser beam is focused Contrast Variation change between the brightest and darkest areas in an image Control menu The menu displayed when the control menu box at the left end of the title bar of a window is clicked When the window is minimized the control menu can be displayed by clicking the icon The control menu contains commands for controlling the window Control menu box The square button at the left end of the title bar of a window Clicking this box opens the control menu Copy Action of placing selected data in the clipboard so that the data can be placed in other place later Cursor
241. her The change over time can be viewed at a glance by displaying multiple image slices together For the detailed operation method see section 2 4 7 Displaying Multiple Image Slices Together 2 9 2 Displaying Images Successively The change over time can also be displayed as animation With images composed of multiple slices such as time lapse images the buttons as shown below are displayed at the top of the Display panel 1 Display the Display panel of the image composed of multiple image slices 2 To display XYZT observation images by noticing the progress of time it is required to select the lt XYT series gt button under the lt Z T series switch gt button at the top of the Display panel If the lt XYT series gt button is not displayed click the lt Z T series switch gt button and click the lt XYT series gt button in the displayed list of buttons 3 Display the image slice to start the successive display by using the lt Frame advance gt button at the top of the Display panel 2 220 Page APPLIED OPERATIONS viewing Images Following the Progress of Time lt Loop gt button lt Z T series switch gt button Successive display or frame by frame display is repeated lt Set start position gt button in the same direction i ey When successive display or frame by frame display is required set the image with which the display should start 0 is set when this button is not used
242. hod 4 Click the lt New Page gt button A new Display panel appears showing the same images as the displayed image The number of the displayed images is as set in step 2 above NOTE Use the lt Retile gt button when it is required to re arrange the images in the currently displayed Display panel 5 Click the top left image of the displayed images Buttons appear above the clicked image 6 Switch the image display using the displayed buttons rs a TIP For details on the display switching method see sections 2 4 3 Switching the Displayed Channels 2 4 4 Displaying Images of Multiple Channels Simultaneously and 2 4 6 Switching the Display Method of Multiple Images 7 Perform steps 5 and 6 above for each of the displayed images Eem Tie Y Process XYZ_2 TIF XYZ_2 Tiled o XYZCH Tiling Columns Rows Experiment al elec Tile Over zZ lt Display Type Over and Under Views Reie New Page Delete Page xalan Display Panel SS Fig 2 66 Panel Showing 2 Channel Mode Images in Different Display Methods Fig 2 66 shows a panel wher display methods are varied as shown below he 2 2 2 VE or n zy Z 2 1 4 Page 2 155 APPLIED OPERATIONS Changing the Image Display Method 2 4 7 6 Re arranging Images Using the Same Display Method Al
243. iF mi lt Begin Analysis gt button Starts analysis XY2CH TIF Tntensiy Profile Region Histogram box Shows the histogram of the specified line or region Double clicking this field displays the Enhanced Histogram Plot window Region Histogram xalca Fig 2 86 Analyze Panel 2 183 Page APPLIED OPERATIONS Image Analysis s 2 6 1 Checking the Intensity of a Specific Part 2 6 1 1 Intensity Values on a Line Line Profile The intensity values on a line in an image can be displayed graphically 1 Display the Single sub panel at the front 2 Display the Display panel of the image to be subjected to the intensity checking at the front 3 Click the lt Annotate gt button in the toolbar at the bottom of the Analyze panel A list of buttons appears as shown below lt Annotate gt button yO ey RYLEY 2 4 From the displayed buttons click the lt Line gt button lt Poly Line gt button or lt Free Line gt button 5 Specify the straight line polygonal line or free line on the image in the Display panel They can be specified as described below e To specify a straight line IN On the image place the mouse pointer on the point you want to start the lt Line gt button straight line and drag until the point you want to end it To specify a polygonal line kal lt Poly Line gt button On the image click the points corresponding to the start
244. ialog Box J 1 Appendix J 1 Coordinate Position Data J 1 Appendix J 1 1 X Coordinate eee eet eettttttttteeeeeeeeeeneeeeaea J 1 Appendix J 1 2 Y Coordinate r J 2 Appendix J 1 3 Other U tata J 3 1 Z Positio lfiu u u u uu alaihi sasaqa EE aE e RAE J 3 2T POSiON asin sih aad tear ne Ae ee as J 3 SAE iS a sees R S ete base tan va uuu ui T J 3 Appendix J 2 Intensity Data J 4 CONTENTS a Appendix J 3 Others u ese teastecareitiecetuacteserioc iei misin iei giat hisini dad J 5 Appendix J 3 1 Channel Number a J 5 Appendix J 3 2 Objective Power a J 5 Appendix J 3 3 Date of Image Capturing a J 5 Appendix J 3 4 Time of Image Capturing J 5 Appendix J 3 5 Image File Name a J 5 Getting Started FLUOVIEW Basic Operations 1 Getting Started FLUOVIEW 1 1 Basic Operations 1 1 1 Microscope The following figure shows the major controls of a microscope The actual configuration of the modules including the specimen stage revolving nosepiece and lighting equipment may differ from those shown below
245. if shima tit r pi tif Se DATA Load r Save Experiment Experiment Display Animation Fig 2 98 Panel Showing an Extended Focus Image Page 2 205 APPLIED OPERATIONS Building an Image from a Different Viewpoint s Among the multiple image slices composing the XYZ image the range of image slices to be used in extend image building can be specified 1 Display the image slice to be set as the start image by using the lt Frame advance gt lt Set start position gt or lt Successive display gt buttons at the top of the Display panel sp 2 Click the lt Set start position gt button at the top of the Display panel 3 Display the image slice to be set as the end image by using the lt Frame advance gt or lt Set end position gt lt Successive display gt buttons at the top of the Display panel button 4 Click the lt Set end position gt button at the top of the Display panel NOTE The lt Set start position gt and lt Set end position gt buttons are valid in the pushed in condition To cancel a previously set start or end position click the lt Set start position gt or lt Set end position gt button again NOTE When it is required to analyze the built extended focus image or save it in the Fluoview Multi Tiff format create the extended focus image as a separate image from the original image See section 2 7 1 2 Turning Built Image into Single I
246. ified or reduced The buttons as shown below are displayed on the top of the Display panel Usually the lt Auto gt button is displayed and clicking it displays the list of buttons shown below Us these buttons to magnify or reduce the image lt 3 1 gt button Magnifies the original image by 3 times lt 2 1 gt button Magnifies the original image by 2 times lt 1 1 gt button Displays the image in the original size lt 1 2 gt button Reduces the original image by 1 2 lt 1 3 gt button Reduces the original image by 1 3 lt AUTO gt button Magnifies or reduces the image according to the current Display panel size Fite uo Y Tie Y ocessYanayz File Type XYZ TIF XYZCH Titt TIF K Files Name Icon a moma tif fitc pitit ida011 tit ptk2 tit shima tif tr pitit u40fpz2c tit 800 800 800 800 600 600 600 600 800 800 800 600 600 600 le DA TA ie Load Experiment Save Expenment Diy Animation ajaz Drag Drop images on input basins Fig 2 70 Panel Showing a 2 1 Magnified Image Page 2 161 APPLIED OPERATIONS Image Processing 2 5 Image Processing Images can be processed using the Process panel Display the Process panel at the front Use the Filters sub panel in the Process panel to apply filtering to images 1 Display the Filte
247. image file mode Use these icons to identify the observation XY observation XY observation 2 channel mode XYt observation sede gy Suman cote anit E x TIP In all observation modes the icons methods used in image acquisition i i apane for 3 or more channels are identical mode XYZ observation l XYZ observation 2 channel mode XYZt observation XYZt observation 2 channel mode Animation image Stereo 3D image Image to be viewed with color eyeglasses 3 or more channels 1 12 Page OPERATION INSTRUCTIONS On This Volume This volume describes the operating procedures of the FLUOVIEW FV500 system Getting Started FLUOVIEW contains information on the basic operation flow until acquisition of XY images APPLIED OPERATIONS provides detailed operating procedures of the system Please read this volume so that you can understand the system before use CONTENTS 1 Getting Started FLUOVIEW 1 1 1 1 Basic Operations u ncn ened 1 1 Test MICrOSCoODe zu a asua itaypaq qapaq yatisasa eee 1 1 1 1 2 General Mouse Operation Procedures 1 13 1 1 3 Names of Major Panel and Window Controls and Their Functions 1 14 1 2 Outline of LSM Observation Procedures 1 16 1 2 1 Turning Power On a nsssssssssssssssa 1 18 1 2 2 Starting the Software
248. image was acquired in the XY observation mode the Single Slice Integration group box shows the option buttons for selection of the X or Y direction When the selected image was acquired in the XT observation mode the option buttons for selection of the X or T direction are displayed Example When the direction of interest is selected in the Single Slice Integration group box line by line computation operation starts on the perpendicular lines to the selected direction For example an image acquired in XY observation is checked as shown below When the X direction is selected When the X direction is selected Y Y T T gt X gt X 2 196 Page APPLIED OPERATIONS Image Analysis Ss p I TIP a When the image is composed of multiple image slices the range lt Set start position gt of image slices to be subjected to the operation can be set using Buen the lt Set start position gt and lt set end position gt buttons above the images First display the image slice to start the operation using E lt Set end position gt the lt Display gt button and click the lt Set start position gt button butt i 2 ee i ahi i Then set the image slice to end the operation in the same way as M j above lt Display gt button Y TIP With an image acquired in XYZT observation the slice images Py cross section Z time lapse T to be subjected to the operation
249. in OPERATION Set the zoom ratio to 1X Section 2 2 1 4 2 in OPERATION Set the channel to be acquired Section 2 2 1 4 3 in OPERATION Set a lower scan speed Section 2 2 1 4 10 in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION If the image e oooi ebia become clean Set the XY observation mode Section 2 2 1 4 5 in OPERATION Re adjust the image brightness Perform repeated scanning Section 2 2 1 4 9 in OPERATION Section 2 2 1 4 6 in OPERATION Adjust the Z position to observe the desired cross section Stop repeated scanning Section 2 2 1 4 7 in OPERATION Section 2 2 1 4 11 in OPERATION Set the observation range Set the observation mode Section 2 2 1 4 8 in OPERATION Section 2 22 gy OPERATION Adjust the image brightness Acquire image Section 2 2 1 4 9 in OPERATION Section 2 2 2 4 4 in OPERATION Save image Section 2 3 1 in OPERATION 2 46 Page APPLIED OPERATIONS Image Acquisition 1 Setting the Z direction scanning range While acquiring image move the Z stage according to the range of the multiple sections to be observed Z direction scanning range From the panel page tabs shown on the bottom right of the Acquire panel select the Z lt Go gt button Stage sub panel Stop Z text box Shows the scan stop position in the range of the observed cross section Z direction scanning range lt Z
250. independently Increasing the PMT Voltage improves the sensitivity Increasing the Gain brightens roim the image Increasing the Offset darkens the image Fess e u 800v 10 0x C xvz C xYzT lt v gt button PLAPO 10X 7 Click to display the pinhole diameter and the laser ND filter setting Zoom gt a Scan Speed MA a B x1 Fast I Slow 4 7s Scan 4 7s image p Acquisition Settings Load C Accumulate To Peak xa e 2 Display Panel Page 1 75 Getting started FLUOVIEW Online Help 3 Click the lt Y gt button of the channel you want to change the pinhole diameter The group boxes for setting the pinhole diameters and laser ND filter values of the channels are displayed below the Ch group box They are not displayed for channels set for transmitted light observation ch1 Ch2 Ch3 Ch4 and Ch5 group boxes Select the channels from which you want to acquire images PMT Gain and Offset LED sliders Set these values independently Increasing the PMT Voltage improves the sensitivity Increasing the Gain brightens the image Increasing the Offset darkens the image lt gt button Click to close each group box showing the pinhole diameter and laser ND filter value The pinhole diameter and laser ND filter value are valid even before the group box is clos
251. ing this button allows fine adjustment of the value IV cys V Transmitted PMT aa Offset PMT Gain Offset el III III 800v 10 0x Ej 800v 10 ox Z 186v 43x 0 Clicking this field allows the value to be changed on a large scale The ND value which are usually used are displayed in green j we 800v 10 0x Z Clicking the lt gt or lt gt button or the field displays the set value The set value can be varied by direct input from the keyboard 1 While observing the image in the Live panel change the setting values of the PMT voltage PMT Offset Offset and Gain Gain in the Acquire panel The functions of these parameters are as described below PMT Voltage Increasing this value improves the sensitivity However too high a sensitivity makes image noise noticeable If sufficient brightness cannot be obtained by setting the PMT voltage to 800 V leave it as it is and increase Gain This will usually provide a better result than using a PMT voltage over 800 V Offset Darkens the image at the ratio set during image acquisition This value can be set independently from the Gain Gain Brightens the image at the ratio set during image acquisition This value can be set independently from the Offset For example When the acquired image is dark or the observation targets are hardly visible increase PMT voltage to improve the sensitivity If the image resulting f
252. ion panel s TIP o During the building the lt Begin Visualize gt button turns into the lt Cance f Visualize gt button Click the lt Cancel Visualize gt button if you want to cancel the building 3 The following buttons are displayed at the top of the Display panel 3D image for color red green eyeglasses 4 Watch the image through a pair of color red green eyeglasses B TIP k The color red green eyeglasses can also be used to view the animatio 2 218 Page APPLIED OPERATIONS Viewing 3D Image Live Y XYZ 21F__ Y 30Animation e DERESE __Tite Y2reces Analyze Y isualiz Emi Make Mutti Plane View Begin Visualize Rendered View E Extended Focus View C Arbitrary View Method Brightest Stereo pair O Sum Initial view O Sequence views Initial Rotation Angle 2 af 304 i oi g Standard Views Left to Right Z Number of views x l el l Display Panel isplay zoom 100 animation 0pi8 l Channel 1 Stereo Pair Fig 2 107 Panel Showing 3D Image to be Viewed Through Color Eyeglasses Page 2 219 APPLIED OPERATIONS viewing Images Following the Progress of Time pO 2 9 Viewing Images Following the Progress of Time Images composed of multiple image slices XYT XYZT or XZT observation can be displayed following the time lapse to show the change over time 2 9 1 Displaying Images Toget
253. ion gt or lt Free region gt button And drag on the image to specify the region of laser excitation Sle lt Rectangular gt button gt lt Circle gt button lt Polyregion gt button a lt Free Region gt button TIP region with holding down the SHIFT key after a region is specified Bequiry Ele WY Tie Yprce tive Y Fecuvee Y REX Lvem Y Focus xw Repeat Once Foou El a P M Mito PMT Cain Offeet Specify the region TEST 690 10x v Gx OxT Oxz O xYZT i Surface XY Clip 4 0 a s Normal Cip Sa Cio line xT depth xz Point size p50 ny 256 Cup Box Z objective ooo JUPLAPO 20 zi Pan aa Cine Ser Z Stage Opies Dyes 2 88 Page APPLIED OPERATIONS Image Acquisition Doo 4 From the page tabs on the bottom right of the Acquire panel select the Lasers sub panel Laser Intensity EP naa lt gt 4 EJ a gt Pay F eetan 50 Rdy Anot E gt 2 23 l a AOTF a REX Function Disabled al C REX mode 4 lt Make REX mask gt button Bleach mode Makes the REX mask file of the specified region ake REX mask Fig 2 26 Lasers sub panel lt TIP When the lt Make REX mask gt button is display
254. ion is suspended STOP SCAN A Cg File O Tile Proce lt Resume gt button A Resume Series Done gt lt Series Done gt button Restarts image acquisition at Determines the acquired the frame next to the frame fe Vv Viiv Transmitted images Once this button is where the acquisition is clicked it is not possible to suspended append an image Page 2 53 APPLIED OPERATIONS Image Acquisition IU j 2 2 2 6 XYZT Observation Mode ra The description in this section will be focused on the image acquisition operations in the XYZT observation mode that are not used in the XY observation modes which are the operations enclosed in CO in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections 2 54 Page Set the dyeing method Section 2 2 1 1 in OPERATION Configure the microscope and scan unit Sections 2 2 1 1 amp 2 2 1 2 in OPERATION Set the objective magnification Section 2 2 1 4 1 in OPERATION Set the zoom ratio to 1X Section 2 2 1 4 2 in OPERATION Set the channel to be acquired Section 2 2 1 4 3 in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION ut Set the XY observation mode Section 2 2 1 4 5 in OPERATION Perform repeated scanning Section 2 2 1 4 6 in OPERATION Adjust the
255. ious screen returns 5 To enter all the required cubes repeat the above procedure 6 The currently displayed values can be stored as one set 4 cubes Stored data can then be loaded stored and confirmed for future 1 To display the screen shown in Fig D 3 press CUBE MEM 6 Fig D 3 2 Press STORE 7 Using the JOG dial select the MEMORY number in which to store 3 When IEXECUTE 8 is pressed the confirmation screen will appear If correct press ENTER If something is wrong press CANCEL 4 When ENDJ is pressed storage is performed and the previous screen will return D 2 Page Appendix D U MCB Setting Objective Data Input OB Appendix D 2 Objective Data Input OB 1 To display the OB LENS DATA SET screen Fig D 7 press OB 1 on the INIT input screen Fig D 6 TFC TAFO TION S gt DATA 2 When the objective number 2 to be entered is pressed the screen shown in Flg D 8 will appear The revolving nosepiece will Bt Plan Apo 12 PH Bz UR In tee Z rotate and the objective at the specified nosepiece position will be 083 Plan Apo 42 PH B4 Ulan Ano 120K Di1PH engaged 3 Rotating the JOG dial select the designation 3 of the objective engaged into the light path 4 When END 4 is pressed the first objective designation is
256. is required to exit from FLUOVIEW exit from Windows and restart OS 2 3 Start the image transfer destination application Paint etc 4 Inthe application open the file saved in step 1 e Page 2 227 APPLIED OPERATIONS Entering Comment in Image TT 2 11 Entering Comment in Image Comment can be entered in an image for use in presentation or slide creation Use the lt Annotate gt button in the toolbar at the bottom left of the screen Click the lt Annotate gt button A list of buttons appears as shown below lt Annotate gt button IN A oS OO Ol na EE 2 2 11 1 Writing Characters in Image This facility is used to enter the title acquisition parameters and or notes in an image Some labels are provided in advance Characters can be written either by using these labels or entering desired characters at will lt Text gt button 1 Display the Display panel of the image in which you want to write characters Click the lt Text gt button in the displayed list of buttons The dialog as shown below appears Active Overlays x Set as Default Enter an annotation or pick one from the list click on the image to place it Close Fig 2 114 Active Overlays Dialog Box 2 228 Page APPLIED OPERATIONS Entering Comment in Image 3 From the drop down list in the dialog box select one of the following labels Z Position lt z gt Time Instant lt t gt Channel lt c
257. isition bs 1 Setting the observation mode 1 Inthe Scan Mode group box in the Acquire panel select the Line option button 2 In the Acquire panel select the XT observation mode option button 2 Setting the observation line 1 Aline is displayed on the image in the Live panel Place the mouse pointer arrow on the line and drag it to the position you want to observe 3 Acquiring image 1 Click the lt XT gt button in the Acquire panel The acquired image will be displayed in the Live panel The XT observation acquires the images of the same position line for 2000 times successively at the set scanning speed For reference the time required for the image acquisitions is shown in the frame on the bottom left of the Scan Speed group box Scan Speed Fast i Slow 10 2s Scan 40 2s Image The number of image acquisitions 2000 times can be changed by changing the value in the N text box in the Time Series sub panel While acquiring an image in the XT observation mode clicking the lt STOP SCAN gt button changes the buttons at the upper part of the Acquire panel as shown below The lt Resume gt button restarts image acquisition at the frame next to the frame where the acquisition is suspended STOP SCAN Acan File vO Tile Proce lt Resume gt button Resume Series Done lt Series Done gt button Restarts image acquisition at Determines the acquired
258. ite region on the Display panel where the REX mask file is displayed The pop up menu as shown below appears Copy Move Size Edit Set intensity Delete Properties 7 Select Set intensity in the menu The Laser Intensity dialog box as shown below appears Laser Intensity lt Cancel gt button Cancels the value set in the Laser Intensity dialog box and closes the dialog box lt Accept gt button Accepts the value set in the Laser Intensity text box and closes the dialog box 8 Set the value for the laser intensity on percentage and click the lt Accept gt button The brightness of the region is changed according to the value set in the Laser Intensity dialog box TIP The laser intensity of the REX mask file is setup on percentage to 100 TIP The region becomes brighter as the value is close 101007 U NOTE when the laser intensity has been set to other than 100 in the Laser Intensity group box in the Lasers sub panel the value can be decreased in the Laser Intensity dialog box However the laser intensity value does not equal to the value obtained by multiplying the value in the Laser Intensity group box by the percent in the Laser Intensity dialog box Page 2 91 APPLIED OPERATIONS Image Acquisition 2 2 11 2 Example of FRAP experiment The example of procedure of FRAP experiment using AOTF is described in this section In order to perform ph
259. ition is 217 MB Create partition of size 217 MB Cancel Help 5 Set the value of the Create partition of size edit box in the Create Primary Partition dialog box to Maximum size and click the lt OK gt button 6 From the Partition menu of the Disk Administrator window select the Commit Change know command 7 When the confirmation message as shown is displayed click the lt Yes gt button Changes have been made to your disk configuration l Do you want to save the changes 8 When the following message is displayed click the lt OK gt button Disk Administrator xi Q Disks were updated successfully It is recommended that you update the emergency repair configuration information and create a new Emergency Repair Disk You can do this with the system utility RDISK EXE 9 In the Disk Administrator window select the MO disk drive Click F 2 Page Appendix F Formatting of Magnetic Optical Disk 10 From the Tool menu of the Disk Administrator window select the Format command The dialog box as shown below appears Format E N xi Capacity Removable Media Unknown Size x File System FAT hs Allocation Unit Size Default allocation size ha Volume Label Format Options T Quick Format Jo Eneble Compression Fig Appendix F 2 Format Dialog Box 11 Select Removable Media Unknown Size from the Capacity drop down list i
260. ix D 4 Setting PC Communications Set the RS232C communication DIP switch as follows SW1 OFF SW2 ON SW3 ON SW4 OFF SW5 OFF SWE OFF SW7 ON Swe ON With the above settings the communication rate is set to 9600 bps data length to 7 bits parity check to None stop bit to 2 bits and delimiter code to CR LF Refer to the instruction manual of your AX microscope for details e Page D 7 Appendix E User Registration Appendix E User Registration FLUOVIEW manages the system setting data on a per user basis Use the following procedure to register yourself as a user TIP Register yourself as a new user 1 From the Start menu select Programs Administrative Tools Common User Manager 2 The User Manager window appears i User Manager Mm E User Policies Options Help Username Guest Built in account for quest access to the computer domain LSM Groups Description Administrators Members can fully administer the computer domain Backup Operators Members can bypass file security to back up files Guests Users granted guest access to the computer domain Power Users Members can share directories and printers Replicator Supports file replication in a domain Users Ordinary users Fig Appendix E 1 User Manager Window 3 Select New User from the User menu 4 The New User dialog box appears Username Full Name Cancel Description Help Passwor
261. k box of the Ch OPERATION with which Transmitted is INSTRUCTIONS displayed The transmitted light filter is Disengage the filter from the 1 3 2 4 engaged in the light path of light path OPERATION the microscope INSTRUCTIONS 3 Image is disturbed The installation location is Consult Olympus subject to large vibrations External light from a Darken the room before fluorescent lamp etc is acquiring image detected rage Ensure that the top cover of the scan unit is closed tightly 4 Image is striped The laser reflection light is The barrier filter selection is 1 2 6 amp 1 3 2 4 OPERATION INSTRUCTIONS 5 Image looks poor The analyzer is engaged in Disengage the analyzer from 1 2 4 OPERATION the light path the light path INSTRUCTIONS The scan speed is high Decrease the scan speed to 1 2 8 9 amp 2 2 1 6 an optimum speed or OPERATION integrate the image INSTRUCTIONS penetrating 1 2 Page TROUBLESHOOTING GUIDE Phenomenon Manual Ref Pages 5 Image looks poor The fluorescent dye does not Select the barrier filter 1 2 7 OPERATION match the barrier filter matching the fluorescent INSTRUCTIONS dye Change the ND filters by reading 1 2 7 Selecting the ND Filter in OPERATION INSTRUCTIONS The excitation light is weak Increase the transmittance 1 2 7 OPERATION of the ND filter INSTRUCTIONS The installation location is Consult Olympus s
262. k the lt Finish gt button Text Import Wizard Step 3 of 3 This screen lets you select each column Column Data Format and set the Data Format General General converts numeric values to Text numbers date values to dates and all Date MDY remaining values to text C Do Not Import Column Skip Data Preview ile Ls Jee ji spe ven I Dan Fig 2 112 Dialog Box When the File is Opened by Excel 3 3 Page 2 225 APPLIED OPERATIONS transferring Data to Another Application x lt TIP a or detailed operation procedures of Excel refer to the Excel manuals see At Line Intensity Profile A B CEST EE al ae E H 1 Line Intengity Profile 2 112 000000 to 2176000000 3 Position um 0 000000 to 181 048776 4 j No Position 5 0 0 1315 6 1 0 707107 1358 7 2 1414214 1353 8 3 2 12132 1334 9 4 2 62132 1365 10 5 3 328427 1404 11 6 4 035534 1276 2 10 2 Transferring the Plot Image of Analysis Data to Another Application The plot image of analysis data can be transferred to an application handling images such as Paint The following description takes Paint as example 1 Display the Analyze panel and executes analysis After it display the Enhanced Profile Plot Intensity Map Enhanced Histogram Plot Average Intensity Trace or Integrated Intensity Trace ia Enhanced Profile Plot x
263. kness The value displayed here in advance has been calculated by the system so that the scale in the planar or XY direction of the image is identical to the scale in the depth or Z direction Usually this value does not need to be chanqed Threshold range 0 High ossa el Low Orientation epth Weight D ll Stereo Factor _ m Factor EE Fig 2 104 Other options Sub panel of Visualize Panel 2 211 Page APPLIED OPERATIONS viewing 3D Image 2 8 1 Successive Display of Images Images composed of multiple image slices acquired by varying the multiple sections XYZ observation XYZT observation can be displayed successively using the buttons at the top of the Display panel as shown below 1 Display the Display panel of the image composed of multiple image slices 2 The buttons as shown below are displayed at the top of the Display panel If the lt XYZ series gt button is not displayed under the lt Z T series switch gt button while the images were acquired in XYZT observation mode click the lt Z T series switch gt button and click the lt XYZ series gt button in the displayed list of buttons lt Loop gt button lt Z T series switch gt button Successive display or frame by frame display is repeated in the same direction lt Set start position gt button When successive display or frame by frame display is required set the image with which the display should
264. l l The mouse pointer transforms to an image icon during dragging Place the mouse pointer on the desired image file name in the Files list box and double click it 2 3 3 Shredding Images Shredding an image refers to removing it from the objects of processing including display Shredding does not actually deletes the image saved in the disk 1 Click the lt Experiment List gt button in the toolbar at the bottom of the File 1 0 panel The Experiments in Memory dialog box appears as shown below lt Experiment List gt button Experiments in Memory C IMAGE SY 2 3 TIF commens pone Fig 2 35 Experiments in Memory Dialog Box e Page 2 119 APPLIED OPERATIONS Saving Opening and Shredding Images o 2 In the Experiments in Memory dialog box select the file name of the image to be prg shredded and click the lt Shredder gt button lt Shredder gt button The file can also be shredded by placing the mouse pointer on it and dragging it to the lt Shredder gt button lt TIP 3 Click the lt Done gt button in the Experiments in Memory dialog box to close it PEAS Multiple images displayed can be shredded simultaneously i 1 Display the Experiments in Memory dialog box while showin x multiple images in the Display panel 2 Pressing down the Shift key select multiple image files to b i x shredded x 3 Click the lt Shredder gt button Pro i
265. l of the image in which you want to draw a scale Click the lt Scale gt button in the displayed list of buttons Click the image position you want to draw a scale Change the scale size See 2 11 8 Changing the Comment Size for the operation procedure NOTE To display a correct scale it is required that the image has been TIP acquired while the objective magnification setting in the software matches the magnification of the actually used objective ae A vertical scale can be drawn as well as a horizontal scale i 4 Click the mouse on the scale to turn the scale active i e handles x displayed around the scale x 2 Click the right mouse button i 3 Select Edit from the displayed menu A sub menu as shown below appears Copy Move Size Delete Properties 4 _ Select Vertical Scale to display a vertical scale a Page 2 233 APPLIED OPERATIONS Entering Comment in Image 5 TIP The size of characters in the scale can also be changed A The size of characters in the scale is determined by the size of character lt Text gt button written using the lt Text gt button Perform the following operation before startin to draw the scale Click the lt Text gt button in the list of buttons 1 When the lt Annotation gt dialog box is displayed click its lt Font gt butto f The Font dialog box will appear 2 Change the character font and size using the Font Fon
266. l of the images displayed together in a Display panel can be rearranged simultaneously based on the same display method channels magnification scroller position 1 Click one of the images displayed together Two sets of buttons appear above and below the clicked image 2 Change the image display method using the displayed buttons 3 Click the lt Retile gt or lt New Page gt button All of the images in the panel are re displayed using the same display method as that set in step 2 above 2 4 7 7 Displaying Different Images Together 2 156 Page Two completely different images can be displayed together The two images to be displayed together should be acquired by observation or loaded by opening a file If two images are not available prepare them by image acquisition or file opening 1 Display the Display panel of either image to be displayed with another image The icon of the image is displayed in the frame at the top left of the Tile panel and the acquisition parameters used in image acquisition are displayed in the Experiment panel lt Experiment List gt button Acquire Fite zo Y Tile Yebroces XYZ 2 TIF Columns 1 Experiment F Animate all views Tite Over Experiments in Mem sry ey CAIMAGE XY2 TIF rE CAIMAGENTR LTIE ASSE 222 xalas Drag Drop images on input basins 2 3 APPLIED OPERATIONS Changing the Image
267. lar gt button e To specify a circle or ellipse On the image assume a rectangle circumscribing the circle to be checked is and drag the mouse pointer along the diagonal line between opposite corners lt Circle gt button of the rectangle To specify a polygonal region Ol On the image click the points corresponding to the corners of the polygon to lt Polyregion gt button be checked After clicking the last corner point click the right button of the mouse to connect the last clicked point to the first clicked point To specify a free region Q On the image specify a region by dragging Then release the mouse button skree Region button to complete dragging The point where the dragging was ended will be connected to the point where it was started A region is displayed on the image together with handles on p g 4 the perimeter The region is selected as the target of the i i bird s eye view while the handles are displayed I i k__ d Handle NOTE If the mouse is clicked in other place than inside the region specified on the image the handles will disappear The bird s eye view cannot be displayed while the handles are not displayed 2 188 Page APPLIED OPERATIONS Image Analysis Doo TIP The checked region can be moved deleted or changed of size or Color i This is possible with the same method as entering comment in the image x For details see sections 2 11 6 2
268. lated to the keyword specified in lt gt and displays the data values on the image The following setups are required to enable the use of Active Overlays Appendix J 1 Coordinate Position Data Appendix J 1 1 X Coordinate The X coordinate position with respect to the top left corner of the screen is displayed Syntax lt x hotspot raw calibrated value units gt Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied Setup Procedure 1 Enter x as the first character inside lt gt 2 Add the following characters to set the display method hotspot Display measurement points with markings raw Display data in pixel values calibrated Display data in numerical values um units Display the unit pixels um Examples lt Input character strings gt lt Displayed strings represent figures gt lt x raw value gt cccttt ttre eee lt x hotspot raw value gt ssstt ttt tte terres lt x hotspot calibrated value units gt um TIP The detailed display procedure is described in section 2 11 3 Viewing the X or Y Coordinate Position of Image in Volume OPERATION Page J 1 Appendix J List of Functions in the Active Overlays Dialog Box Coordinate Position Data Appendix J 1 2 Y Coordinate The Y coordinate position with respect to the top left corne
269. le at present RedMgneric List of dyes that can be used during FLUOVIEW operation Dio x Please drag Dyes from here and drop them here to identity those Dyes you actually use r For TIEMPO Double click to edit Generate Probe j Include previous FLUOVIEW DataBase dye names in Probe DataBase Save New Setup Quit w o Saving The setting procedure is similar to the objective setting procedure Objectives Calcium Green 5N YFP Calcium Orange panel 7 Display the Coloring Tables panel in the front position In this panel select the characters to be displayed in each of the 8 buttons displayed in the Color Tool dialog box of the FLUOVIEW application and the LUT file to be read out when the button is pressed Software f Hardware j Lasers Users Microscope Z Stage Objectives Fluorescence Look Up Tables Click the button to change the name and Look Up Table that will be used in FLUDVIEW for that button Standard Color LUTs Gray Red Green Blue Hilo Fall Spect Spec2 Fall Specl Spec2 Spec3 Save New Setup Quit w o Saving Page 1 9 Software Setup Setting the System Configuration lt To change the button settings gt Press the button to indicate that you want to change the setting When the dialog box as shown below appears specify the button name and LUT file name and select the lt OK gt button The LUT files
270. lected dyeing method to the Ch group box on the upper part of the Acquire panel hen the dyeing method is selected from the Available Dyes list box and TP e lt Apply gt button is clicked a channel for acquiring fluorescence is seti utomatically according to the changed filter And the dyeing method is 2 60 Page APPLIED OPERATIONS Image Acquisition One Point The Assign dyes manually check box can also be used to set the dyeing method to the desired channel 1 Check the Assign dyes manually check box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the i Acquire field of the Ch check box ere e cramer Eia Assign dyes manually check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set ene i I The dyeing method is set r con Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Page 2 61 APPLIED OPERATIONS Image Acquisition pO e With types of dyeing of the specimen to observe the barrier filter will be set automatically to the optical path To change the barrier filter types see section 1 3 2 4 Configuring the Filters and change by Optical System Configuration window Make the channel ready for image acquisition In the Acquire panel make sure that the c
271. lected in the Series Title box Changes the type of the chart selected in the Series Title box Page 2 253 APPLIED OPERATIONS Changing the Chart Display Method TeeChart Gallery sub panel Displayed when the lt Add gt or lt Change gt button is clicked Used to set the chart type The chart types that cannot be set cannot be selected fi TeeChart Gallery olx Standard Functions a Extras Line Horiz Bar Area Point Fast Line Shape Bubble Arrow LZ ko he OK Cancel Enables or disables the 3D display General sub panel Used to set general items related to the chart display Previews the printed chart or sets the orientation and Chart Series margins of the print paper Enables or disables zooming Series General Axis Titles Legend Panel Paging alls 3D Zoom Saves the chart in the format Print Preview V Allow Zoom of an image file or in the clipboard Export When a chart is displayed by IV Clip Points zooming or scrolling selects J whether the data of only the Margins Z area contained in the chart axes is to be displayed or not When zooming is enabled enables or disables gradual zooming according to the step count set in the Steps dialog box vV Animated Zoom Steps F Sets the chart scrolling C None direction C Horizontal C vertical U Both Sets the margins from t
272. led Image lt Include Overlay Fig 2 30 Save Display Dialog Box 4 When it is required to save the comment drawn on the image together with the image check the Include Overlay check box 5 Click the lt Display gt button in the Save group box The Save Experiment As dialog box as shown below appears Save Experiment As Save in a Images z El E 86 3d1 tif 96xt1 tif SBxyt ti B6xyz2 2 ti E 86 3d2 tif E 36xt2 tif E 86xyt2 tif E 86xyz3 tif fa 86 3d3 tif E 86xt3 tif E 86xyt3 tif E 86xyz3 2 ti E 86 3Dst1 tif E 86 y1 tif E 86xyz1 tif E 86xyzt1 tif E 86 3Dst2 tif E 36xy2 tif E 86syz1 2 ti OBxyzt2 tif E 86 3dst3 tif E 86xy3 tif E 86xyz2 tif E 86xyzt3 tif gt Filename lt 2CH TIFF Save as type FLUDVIEW MultiTit tif x Cancel Fig 2 31 Save Experiment As Dialog Box 2 106 Page APPLIED OPERATIONS Saving Opening and Shredding Images When it is required to change the save destination drive or directory use the Save in drop down list When it is required to change the saved file type use the Save as Type drop down list See section 2 3 1 4 File Types Available for Save for details Enter the file name in the File Name text box Click the lt Save gt button NOTE If a file with the same name as the entered file name already exists a dialog box is displayed to ask if you want to overwrite the existing file If you do not want to
273. lices Together With images composed of multiple slices such as time lapse images or images acquired by changing the multiple sections the image slices can be displayed together for simultaneous viewing However note that the size per image reduces when the number of displayed image slices increases Use the Tile panel for displaying images together Display the Tile panel TE Acquire Y File YOY Tile Y roces _ Icon of the displayed images image XYZ 2 TIF slices shown together No i Tiling group box Shows how the images are displayed in the Display panel Live XYZ_2 TIF lt gt Ulla a Rows text box Sets the number of rows or the number of image slices displayed per vertical column Columns text box Sets the number of columns or the number of image slices displayed in a horizontal row Columns Experiment Experiment Shows the acquisition parameters used in acquisition of the displayed images Display Type drop down list Select the display method T Animate all views Single View Side by Side Views abies or Over and Under Views can be Tile Over drop down list selected Select the acquisition parameter to be Disa nas based on when arranging the images See Seem El Self Z or T can be selected New Page button Displays images by arranging them in a new Display panel Retile New Page lt Retile gt button
274. lick the lt Color Bar gt button in the displayed list of buttons lt Color Bar gt button 3 Draw color bars in the image by dragging the mouse pointer along the desired position in the image 4 To change the color bar size see section 2 11 8 Changing the Comment Size E TIP x The labels of the color bars can be switched to display or hide i 4 Click the mouse on inside the color bars to select The handle appear arround the color bars 2 Right click the mouse 3 Select Edit in the menu to display the sub menu as shown below Copy Move Size Edit 4 Hide Labels Delete Show Labels Properties 4 In the sub menu select Show Labels to display the labels or Hid Labels to hide the labels 4095 3071 2047 The labels are shown The labels are hidden 2 236 Page APPLIED OPERATIONS Entering Comment in Image 2 11 8 Deleting Comment 1 handles displayed around the comment Handle A 2 Copy Move Size Edit gt Delete Properties Fig 2 118 Pop up Menu 3 Select Delete from the menu TIP 1 Click the mouse on the comment to be deleted to make the comment active i e Click the right button of the mouse A pop up menu as shown below appears ee To delete more than one comment simultaneously Select multiple comments and select Delete from the pop up menu To select click the mouse on or in the middle of one of the comm
275. list box 12 Click the lt Add gt button 13 Click the lt OK gt button 14 Click the lt OK gt button To register other users repeat steps 5 to 13 for each E 2 Page Appendix F Formatting of Magnetic Optical Disk Appendix F Formatting of Magnetic Optical Disk Use the Disk Administrator tool of Windows for formatting a magnetic optical MO disk Note that this tool can be used only by a person who have the authority of Administrator 1 Click the lt start gt button at the bottom of the Windows screen 2 When the start menu is displayed select commands Programs Administrative Tools Common Disk Administrator The window as shown below appears g Disk Administrator Partition Tools View Options Help SE ail Disk 0 Cc D BACKUP NTFS NTFS 2067 MB 1819 MB 248 MB Disk 1 Free Space 217 MB 207 MB CD ROM 0 F isi Primary partition Partition 217 MB Unknown E Fig Appendix F 1 Disk Administrator Window 3 Select the MO disk drive in the Disk Administrator window NOTE The drive to which the MD disk is assigned is variable depending on computers Page F 1 Appendix F Formatting of Magnetic Optical Disk 4 From the Partition menu of the Disk Administrator window select the Create command The dialog box as shown below appears Create Primary Partition xi Minimum size for the partition is 1 MB Maximum size for the part
276. liter bottles e Page 2 5 TROUBLESHOOTING On This Volume This volume describes the treatment against possible troubles In case of trouble please read volume before calling for service If the normal operation cannot still be restored please contact your local Olympus representative CONTENTS O 1 TROUBLESHOOTING GUIDE 1 1 1 Fluorescence image cannot be observed 1 1 2 Transmitted image cannot be observed 1 2 3 Image is disturbed U l ee ete 1 2 4 Images striped uuu uuu usus ussinisussssssssmsssyassasasqssuswsiswasasqasaq 1 2 Ds limage l0okS POON isisisi innar aa aaa a aai 1 2 6 Image is irregularly blurred or the brightness is uneven 1 3 7 a t Ce ERE EE AE A ote es a 1 3 8 Circular flare is observed at the center of image 1 3 9 Image is blurred or out Of focus 1 3 10 Image is dark and noisy 1 3 11 The position reproduction of the Z motor is poor 1 3 12 The Acquire panel cannot be displayed 1 4 13 The scale of the Z Stage sub panel in the Acquire panel cannot DO MOVE Gis ie R N S haasi ha hua ass Qua 1 4 14 The fine movement knob of microscope cannot be turned or is WOE SIMO Uy Z u
277. liz Live __x 2 2 11F Y 3DAnimation 3 lt ORAHE T ia x AB j y asaf os Standard Views Default Se Number of views al E xlelel7iee ol 2 DisplayPanel Fig 2 105 Panel Showing the Built Image CG 12 Click and hold the lt Display gt button at the top of the 3D Animation panel The lt Display gt button images will rotate so that they can be viewed three dimensionally lt TIP Click the lt Stop gt button to stop the image rotation UU e Page 2 215 APPLIED OPERATIONS viewing 3D Image s 2 8 3 Building Stereo 3D Images Images composed of multiple image slices acquired by varying the multiple sections XYZ observation can be built into a pair of stereo 3D images These images can be viewed three dimensionally by watching them with two eyes for a while The operation is similar with animation Perform steps 1 to 10 in section 2 8 2 Animation then proceed to the following steps 1 Check the Stereo Pair check box 2 Click the lt Begin Visualize gt button to start building the images When the building completes the built images are displayed in the 8D Animation panel Cancel Visualize _TIP k During the building the lt Begin Visualize gt button turns into the lt Canc lt Cancel Visualize gt button 2 216 Page Visualize gt button Click the lt Cancel Visualize gt button if you want to cancel the building St
278. lso provided here For DIC observation engage the transmitted light DIC slider optional matching the objective in use in the light path This applies to both visual and laser DIC observations For visual DIC observation or laser DIC observation engage the polarizer in the light path 6 Filter Used in adjustment of the transmitted light illumination 1 12 Page Getting Started FLUOVIEW Basic Operations 1 1 2 General Mouse Operation Procedures Use the mouse to select a command character string or button Use the left button of the mouse unless otherwise specified To select or execute something Clicking To click the mouse place the mouse pointer on the desired function and press the mouse button once Pressing the right button of the mouse is referred to as right clicking To select something and execute its function Double clicking To double click place the mouse pointer on the desired function and press the mouse button successively twice To move something Dragging To drag place the mouse pointer on the desired function and while pressing and holding the mouse button move the mouse to the desired destination At the desired destination release the mouse button Dragging by pressing the right button of the mouse is referred to as right dragging One Point When the mouse is moved the picture of arrow on the screen moves accordingly The picture which moves o
279. lt OFF gt button Click lt gt button to brighten the filter by one step is selected Click lt gt button to make the filter brighter 3 After completing the setup click the lt Close gt button x TIP To control the microscope with the U MCB while keeping the software active click the lt Use MCB gt button in the AX Control window before x proceeding to the setup x Selecting the lt Use MCB gt again enables you to operate the microscope ini i this software Getting started FLUOVIEW Online Help TIP E f During image acquisition the lt XY Repeat gt and lt Once gt buttons are set asi x follows provided that the setting is not altered by the operator e Observation mode group box The lt DIC gt button is selected Cube group box Set to OUT f e Shutter group box Set to Close x e AS group box The value of AS is set to 100 This is variable depending on the objective type x e FS group box The value of FS is set to 120 This is variable depending on the objective type x e Lamp group box The transmitted light lamp is set to OFF x e FILTER group box The value of ND is set to 100 x e FILTER group box Both color correction filters are set to 1 3 2 4 Configuring the Filters The barrier filters excitation filter and beam splitter are set automatically to the light path according to the dyeing method selected for the specimen
280. lt XYZ series gt I f i button ican be selected using the lt XYZ series gt and lt XYT series gt G N series gt 4 When the image was acquired in the multi channel mode select whether the utton Q multiple channels are operated simultaneously or only one channel is operated 4 2 3 4 5 To select the target channel s use the lt Display channel switch gt buttons Only lt Display channel switch gt the channel s being displayed will be analyzed buttons Example When only the Ch1 image is displayed only the Ch1 image is analyzed x TIP k For the switching of channels see section 2 4 3 Switching the Page 2 197 APPLIED OPERATIONS Image Analysis s 5 Click the lt Annotate gt button in the toolbar at the bottom of the Analyze panel A list of buttons appear as shown below lt Annotate gt button IS iOu lt Rectangular gt button lt Circle gt button lt Polyregion gt button ki gt C3 lt Free Region gt button RY gE Pa 6 From the displayed buttons click the lt Rectangular gt button lt Circle gt button lt Polyregion gt button or lt Free Region gt button 7 Specify the region to be checked in the image in the Display panel For the specification method see section 2 6 1 2 Intensity Values on a Planar Region Bird s Eye View A region is displayed on the image together with handles on the lt perimeter The region is s
281. ly when loading a saved image i TIP Change the default folder only when required i x There is no need of change if folder C FLUOVIEW IMAGES is all right TIP Folder names are delimited with V For example C NFLUOVIEW NIMAGES means folder IMAGE in folder When the default folder is C N FLUOVIEW IMAGES gt Ea a p a a a T Acquire File uo Y Zie Y Proces _ Live L XY Ch 1ot2 File Type Tif TIF Files Name icon X N 4 samplet TIF 800 600 sampie2 TIF fe 160 420 test tif m 1040 682 4 Default folder C EG Sy FLuoview FLUOVIEW IMAGES is being open Load Save Experiment Experiment Display Animation xian File Selection A different default folder can be set for each of the users logging in Windows NT Page l 1 Appendix Change of Default Folder for File I O Panel 1 Click the Start button to open the Start menu Then select commands Settings Control Panel System The System Properties dialog box appears as shown below System Properties 1 21 x Hardware Profiles User Profiles Performance Environment System Microsoft Windows NT 4 00 1381 a E Registered to z Olympus STT 50036 415 0285411 50781 Computer 86 Family 6 Model 5 Stepping 2 AT AT COMPATIBLE 523 688 KB RAM l 2 Page Appendix Change of D
282. mage 2 206 Page APPLIED OPERATIONS Building an Image from a Different Viewpoint 2 7 1 2 Turning Built Image into Single Image From XYZ multiple sections image an extended focus image can be built as a separate image from the original image Use the Visualize panel to build the image First display the Visualize panel Display panel Shows the image The file name of the image is shown in the page tab of the panel _Tile Y Proces Analyze isuatiz Make Multi Plane View Begin Visualize button eae Vani Starts building the extended focus image Rendered View Extended Focus View C Arbitrary View Rendered View group box slat Brightest e Stereo pair Extended Focus View option button Builds the extended focus image Orientation Arbitrary View option button Builds the image for stereo display C IMAGES DEMO ZYUSU TIF Fig 2 99 Visualize Panel 1 Display the Display panel of the XYZ multiple sections image 2 Click the Extended Focus View option button in the Rendered View group box 3 Click the lt Begin Visualize gt button to start the image building When it completes the built image is displayed in the Extended panel e Page 2 207 APPLIED OPERATIONS Building an Image from a Different Viewpoint Tile Proces analyze Y jeualiz Y Live ZYUSU TIF Y Extended J Rendered View Ex
283. mage will be displayed in the Live panel lt Once gt button 2 26 Page APPLIED OPERATIONS Image Acquisition 2 2 1 6 Acquiring Image in Accumulation Mode When the image is dark or noisy use an accumulation mode in image acquisition to improve the image quality Kalman Accumulation and Peak Accumulation The Kalman accumulation acquires images for the specified number of times while averaging the images This operation is effective for reduction of noise The Peak accumulation acquires images for the specified number of times while adding the images and stops image acquisition when any intensity value on the image reaches the peak 4095 This operation is effective for acquiring an image with the dark lower part in the XYZ observation and observation of an extremely dark image Kalman Accumulation Algorithm Every time an image is acquired the pixel values are rewritten based on the following formulae where it is assumed that n number of image acquisitions I n Result of n times of Kalman accumulations Intensity values of pixels I new New intensity value obtained after every image acquisition The result of the first Kalman accumulation is identical to the result of ordinary image acquisition I 1 I new The result of the n th n gt 1 Kalman accumulation is I n I n 1 n 1 I new n Page 2 27 APPLIED OPERATIONS Image Acquisition 1 Acquiring Image in Accum
284. mage with Divide Imaqe by Multi Image Operations Aad 2 Imanes _ Subtract 2 Images Multiply 2 Images Divide 2 Images Display zoom 100 z 0um Channel 1 Display zoom 100 z 0um Channel 2 xee Fig 2 128 Display Panel Showing Pop up Menu Page 2 265 APPLIED OPERATIONS Pop up Menus Pop up menu of comment When the right button of mouse is clicked on an comment in image a pop up menu appears to allow editing or deleting the specified comment File 0 Y Tie Y rocess anayz i Y szam Measurement Results Line Length 20 488 um x 42 um Y 15 8 um Statistics Total 44577 25353 Average 564 27 320 92 Std Dev 473 07 379 06 Intensity Profile Channel 2 Region Histogram A L Copy Move Size Edit Delete Properties um Channel 1 2 Fig 2 129 Image Comment with Pop up Menu Copy Copies the comment Move Moves the comment Size Resizes the comment Edit h Edits the selected arrow or scale Delete Deletes the comment Properties Edits the comment color and font 2 266 Page APPLIED OPERATIONS Pop up Menus Pop up menu of image When the right button of mouse is clicked on the image a pop up menu appears to allow selection of image operations full screen display printer output image save LUT setting number of image divisions comment editing Tie Y Process af Analyze Live Y wvz 2 nr I XYZ 2
285. many other analysis operations hen opening and analyzing an image file creased with anothe pplication on FLUOVIEW enter the lengths per image pixel and th umber of steps in the Z direction if these values are known then click th lt Set Attributes gt button NOTE During modification of the lengths per image pixel the number of steps in the Z direction or the objective setting if it is required to restore the previous setting click the lt Restore Attributes gt button However once the lt Set Attributes gt button is pressed the original setting cannot be restored NOTE The objective setting can be changed even after having acquired the image If the objective is not set with the Acquire panel before image acquisition the analysis and measurement results of the image may be erroneous In such a case set the objective again NOTE When the objective setting is changed the lengths per image pixel will be re calculated automatically and changed 4 Display the Image Fields panel at the front The panel as shown below appears where the information on the selected image is shown 2 124 Page Range of intensity values mapped for each channel APPLIED OPERATIONS Saving Opening and Shredding Images Image Comments C IMAGESWXYZ2 TIF Map Ch0 Range 00000 to 04095 Map Ch1 Range 00000 to 04095 Image Fields LUT status Acquisition parameters Version information Comment Change
286. me successive display or frame by frame display in a direction ends at the start or end position the display is restarted in the opposite direction lt Display gt button Press to start display of n slices of image from the start position to the end position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame lt Rabbit gt button Press for successive display of image slices without pause lt Clock gt button Press for successive display of image slices at the time interval set in the Time Series sub panel lt Turtle gt button Press for successive display of image slices at an interval of 0 8 sec lt Display gt button Press to start display of n slices of image from the end position to the start position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by 2 148 Page advancing frame by frame APPLIED OPERATIONS Changing the Image Display Method TIP E Click and hold the lt Display gt button for successive display To stop it click the lt Display gt button again i _For frame by frame display simply click the lt Display gt button U 2 4 7 Displaying Multiple Image S
287. n dialog Spee box is not displayed but the system is automatically started for the Appendix H 3 Deleting a User 1 Double click the FLUOVIEW icon on the desktop The dialog box for entering the user name appears as shown below iw FLUOVIEW User Login FLUOVIEW Setup icon O LY RE US User None O 2 Enter Administrator in the User Name text box and click the lt OK gt button The FLUOVIEW Setup dialog box appears as shown below FLUOVIEW Setup Se ee a eae Microscope O r Configuration c s COAX FV Review Station C es g s C Fv Upgrade Csxm Fv300 o C P500 IX C 2cH C 3CH Extra Magnification 1 00 4CH TD Auto installed D lt Yes No Save New Setup Quit w o Saving Page H 3 Appendix H USER REGISTRATION OF FV500 beleting a User 3 Display the Users panel at the front Microscope Z Stage A Objectives Software Hardware Lasers Coloring Tables Registration Scanning Unit FLUOVIEW Users You are logged in as as Administrator Administrator a userl lt Add a user and Log in gt button Used to add a new user Add a user and Reset user to Log in Fa Save New Setup lt Reset user to Factory Defaults gt button Quit w o Saving lt Delete a user gt button Resets the system setups of the users selected in Deletes the user selected in the list box the list box to the factory defaults The A
288. n image acquired by observation or opened from a file can be changed as described below 2 4 1 Displaying an Image in Simulated Colors 1 Display the Display panel of the image to be colored at the front px 2 Click the lt LUT gt button in the toolbar at the bottom left of the screen The Color lt LUT gt button Tool dialog box appears Color Tool Live Standard Color LUTs Fy Gray Speci Spec2 Gray Gray Intensity Mapping ces o af gt Gamma High 4095 4 gt l ES Chi C Ch2 C Ch3 C Ch4 C Ch5 V Apply to all views OK Cancel Fig 2 51 Color Tool Dialog Box 3 When the image was acquired from more than one channel select the channels to be colored using the Ch1 Ch2 Ch3 Ch4 and or Ch5 option buttons The Ch1 Ch2 and Ch3 option buttons are displayed only when an image acquired from more than one channel mode is displayed selected 4 From the Standard Color LUTs group box select the desired color button The selected LUT will be applied immediately to the image in the Display panel 5 Click the lt OK gt button 1 TIP The Apply to all view check box can be selected while the Display panel showing multiple images created in the Title panel is displayed l When this check box is checked all changes are applied to all of the e Page 2 135 APPLIED OPERATIONS Changing the Image Display Method One Point The Color Tool dialog box can al
289. n the Format dialog box Then select FAT from the File System drop down list and select FAT and select Default Allocation Unit Size from the Allocation Unit Size drop down list 12 Click the lt Start gt button 13 When the confirmation message as shown below is displayed click the lt OK gt button Format E 4 xi WARNING Formatting will erase ALL data on this disk fe Select OK to format the disk CANCEL to abort Cancel 14 When the following message is displayed after completion of formatting click the lt OK gt button Formatting E xi i Format Complete 15 Click the lt Close gt button in the Format dialog box then select Partition Exit in the Disk administrator window to exit from the operation Page F 3 Appendix G Converting Analysis Data into a Chart Using EXCEL Appendix G Converting Analysis Data into a Chart Using EXCEL 1 Start up Excel 2 From the File menu of Excel select the Open command to open the analysis data file saved after analysis using FLUOVIEW 3 When the dialog box as shown below appears click the Delimited option button in the Original Data Type group box then select Windows ANSI from the File Origin drop down list Text Import Wizard Step 1 of 3 The Text Wizard has determined that your data is Delimited If this is correct choose Next or choose the Data Type that best describes your data Original
290. n the Scan Mode group box to set the scanning range to the original setting Page 2 71 APPLIED OPERATIONS Image Acquisition s 2 2 6 Acquiring Finer Image Sequential Scan An image without containing superimposition of fluorescence can be obtained by acquiring the image slice of each laser excitation or channel sequentially With this image capturing method the image of a multiple dyed specimen can be obtained by sequentially acquiring image slice of each type of fluorescence 1 Select the Surface XY Note option button in the Scan Mode group box in the Acquire panel then select lt Sequen gt in the list shown below Scan Mode tf Surface X Norm Normal Sequen C Line XT C Lem XT C Pemt Size 800 by 600 z Selecting lt Sq Clp gt lt Sq Rct gt or lt Line Sq gt enables the image acquisition of the desired rectangle area or that of the rectangle area at desired angle or a line sequential scan Line Sequential Scan Ordinary sequential scan is performed for every one frame for every group whereas line sequential scan is performed for every one line for every group Therefore line sequential scan is of advantage in minimizing the time lag between wavelength and acquiring a fewer cross talk image NOTE Image acquisition in the line mode can be performed when you use the FV300 system with AOTF FV5 COMBA 2 Select the observation mode with the option button
291. n the screen as the mouse is moved is referred to as the mouse pointer Page 1 13 Getting Started FLUOVIEW Basic Operations 1 1 3 Names of Major Panel and Window Controls and Their Functions The window as shown below is displayed when FLUOVIEW starts up FLUOVIEW uses panel type windows This section describes the names of the major controls displayed in panels and windows by taking the Acquire panel and Microscope Configuration window as examples Page tab Page tab scroll marking Click to switch the panel for executing the When there are a large number of indicated processing function panels it is not possible to display all Right clicking a page tab displays the pop up of them In this case clicking this menu of all items under it so the desired one can marking scrolls the panels one by one be selected mauk Piero Y tie afl uw wd XY Repeat fee Panels A separate panel is provided for each function Ta row PEE J PF I F transmitea i Drop down list Click W to display tAhe list of available m items for selection x 320 mo Oh To select an item in the list click the item xy C xYT1 C xyz C xYzT Buttons B 123 Normal ret oo Click each button to Coline xr Scale execute the Pam The scale is used to set a value which is continuously processing indicated See ete ed variable in a certain range on it preen z Clicking a point in the scale area allows the value to Zoom
292. name of the default folder to be changed by delimiting the drive and folder names using backslashes The figure shows an example in which folder IMAGE in drive C is entered Startup Shutdown Hardware Profiles l User Profiles l General Performance Environment System Variables Variable Value ComSpec CAWINNT system32 cmd exe FLUOVIEW C NFLUDVIEWS FLUOVIEWIMAGES C FLUOVIEWSIMAGES NUMBER_OF_PR 1 os Windows NT z User Variables for Administrator Variable Value FLUOVIEW CAFLUOVIEW FLUOVIEWBACKUP C FLUOVIEW System Backup FLUOVIEWIMAGES C FLUOVIEWSIMAGES ID3 C Program Files 03D IDSIMAGES C Program Files O3D IMAGES Variable JFLu OVIEWIMAGES Value SiC IMAGES Value text box Delete Enter the path name of the default OK Cancel Appi folder 5 Click the lt Set gt button System Properties x Startup Shutdown Hardware Profiles User Profiles l General Performance Environment System Variables Variable Value ComSpec CAWINNT system32 cmd exe FLUOVIEW C AFLUOVIEW S FLUOVIEWIMAGES C FLUOVIEWSIMAGES NUMBER_OF_PR 1 os Windows NT gt Change is reflected FLUOVIEW CAFLUOVIEW FLUOVIEWBACKURQC FLUOVIEWSystem Backup FLUOVIEWIMAGES C IMAGES ID3 C Program Files 03D IDSIMAGES C Program Files O3D IMAGES H Variable Value JJ ese Delete Cancel lt OK gt button Saves
293. nction to change settings corresponding to the change of BX settings lt Load Setting gt button Loads the BX settings registered to reflect it lt Save Setting gt button Saves the current BX settings already EPI lamp Indicates the EPI lamp Filter Turret group box Clicking the filter for reflected light observation to be set switches the turret automatically Te OLYMPUS BX Options Link Setting Load Setting Save Setting Lamp group box Clicking inside the box switches the TD lamp ON OFF Lamp e The TD lamp is set to OFF l The TD lamp is set to ON Page 1 67 1 68 Page Getting started FLUOVIEW Online Help e Clicking the lt Link Setting gt button displays the Link Setting dialog box as shown below Checking here links the objective in the Nosepiece group box with the condenser turret Checking here escapes the stage when the objective is selected and changed in the Nosepiece group box Checking here disengages the top lens from the light path when the objective of x4 or lower magnification is selected in the Nosepiece group box Checking here closes the FL shutter and engages Analyzer and Polarizer into the light path when the TD lamp is set to ON in the Lamp group box Checking here sets the TD lamp to OFF when the fluorescent cube is selected in the Mirror Unit Checking here disengages the top lens from the light path w
294. nd setting laser type excitation wavelength and emission wavelength Add the dyeing method using the Fluorescence panel in the Fluoview Setup dialog box 1 Double click the FLUOVIEW Setup icon on the desktop The FLUOVIEW User Login dialog box as shown below appears iw FLUOVIEW User Login OLYMPUS User Name FLUOVIEW Setup icon A A o te 2 Enter user name into the User Name text box and click the lt OK gt button to log into FLUOVIEW FV500 w TIP X When using the system for one user the FLUOVIEW User Login dialo 1 20 Page Double click here to add the dyeing method Name of the Dye text box Enter the name of the dyeing method Laser Type drop down list Select the laser to use Software Setup Adding the dyeing method 3 Display the Fluorescence panel at the front position in the FLUOVIEW Setup FLUOVIEW Setup dialog box Software Hardware Microscope Z Stage Fluorescent Dyes Colors All Known Dyes Calcium Crimson Calcium Green 1 Calcium Green 2 Calcium Green 5N YFP Red Generic Green Generic Calcium Orange Calcium Orange 5N Please drag Dyes from here For TIEMPO Generate Probe Vv Include previous FLUOVIEW DataBase dye names in Probe DataBase Scanning Unit i Lasers Coloring Tables 3 Registration Objectives Users Dyes on your Samples ble click here to make a new Dye gt Calcium Crimson
295. nel 2 150 Page APPLIED OPERATIONS Changing the Image Display Method Acquire Zero Y Te Proces XVZ_2 TIF XVZ 2 Tiled XYZ 2 TIF jo Tiling Columns Rows ria fe Experiment a 4 CH I Animate all view Tile Over Z Display Type Side by Side Views X Retile New Page Delete Page xaea Drag Drop images on input basins Fig 2 62 Panel Displaying Images Per Channel e Page 2 151 APPLIED OPERATIONS Changing the Image Display Method 2 4 7 2 Displaying Images of Two Channels Together Images acquired in a multi channel mode can be displayed together for simultaneous view The operation method is identical to the method for displaying images per channel except for the following point See section 2 4 7 1 Displaying Multiple Images Per Channel With images acquired in a multi channel mode select the display method from the Display Type drop down list Eer Tile Process _ XYZ_2 TIF XYZ_2 Tiled mee lt Til gt U cz ei o Columns Rows zi E I Experiment zZ 4 CH 2 T Animate all view Tile Over Z Display Type Retie New Page Delete Page xalan Drag Drop images on input basins gt Fig 2 63 Panel Displaying Images by Overlaying Different Channels 2 152 Page APPLIED OPERAT
296. nel When using the FLUOVIEW system with an inverted microscope locate the upper edge of the range to be observed by moving up the revolving nosepiece using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons 5 When the position is located click the lt Set gt button The Stop Z text box will show the scanning stop position of the range of the multiple sections to be observed Z direction scanning range 2 Setting the observation mode 1 In the Scan Mode group box in the Acquire panel select the Depth option button 2 In the Acquire panel select the XZT observation mode option button e Page 2 41 APPLIED OPERATIONS Image Acquisition pO 3 Setting the observation line 1 Aline is displayed on the image in the Live panel Place the mouse pointer arrow on the line and drag it to the position you want to observe 4 Setting the numbers of steps and acquired image slices 1 From the page tabs on the bottom right of the Acquire panel select the Z Stage sub panel Step Size 2 A number of steps is displayed in the Step Size text box This number can be changed using the lt gt or lt Y gt button in the Step Size text box O25um Ideal 1 1 Step Size text box TIP e The number of steps shown in the Step Size text box has been calculate by the system so that the depth scale of the acquired image is identical to ithe horizon
297. nel s use the lt Display channel switch gt buttons Only the channel s being displayed will be used in the animation building Example When only the Ch1 image is displayed only the Ch1 image is used lt _TIP E For the switching of channels see section 2 4 3 Switching the Displa Click the Arbitrary View option button in the Rendered View group box Click the Brightest or Sum option button in the Method group box Select the image rotation direction from the Standard Views drop down list Click the Initial view option button DNO Or Be G Set the angle at which the rotation should start using the Initial Rotation Angle scale 8 Click the Sequence views option button 9 Set the angle per rotation step using the Rotation Angle Increment scale Page APPLIED OPERATIONS viewing 3D Image 10 Set the number of images to be rotating using the Number of views scale 11 Click the lt Begin Visualize gt button to start building the animation When the building completes the built image is displayed in the 3D Animation panel TIP The status bar shows the progress of building processing U Cancel Visualize TIP ts During the building the lt Begin Visualize gt button turns into the lt Cancel lt Cancel Visualize gt pera Visualize gt button Click the lt Cancel Visualize gt button if you want to button i 3 a A A e NREN EA RE i Title Y2roces Y Analyze Y jisua
298. ng the image also opens the comment which can be changed or measured as required 24 bit 8bit file type Select when the image will be handed to another application The following table shows the list of image save status by taking the below mentioned acquisition parameters as an example Observation mode XYZ observation Number of channels 3 channels Number of acquired images 4 slices Save file name abcdefgh Page 2 113 abed VLL z snjejs p pu x uou eu 6Buo y u panes s eGew u 1 J y uo s Beuui pebiey pue soebew apis Aq apls uonipuoo 0 4O Z4D LUO si yByopoge pexvids popun QWEU lIJ BAS jauueyo e Bunes 99S aip panes ale uO pue Jo Jaquinu Buipuods uuo5 0 0 S0N u 249 LUD ZUD LUD Jo sabeu ul 0 0 son uo f ay ule uo5 skemje 4r yBsopoqe SMOIA sy YByepoge JOU s op djl e JEU JON OWEU ld SAPS saqe y u pasn sjauueUd WEU Oil SACS panes uonipuoo jauueyo p l5 l s panes ale gud y JO s o is oew au panes aq uep y ul panes si abewi y pue zu LUD Jo sebew ay suiejuoo aBew paves eu s gy9 juo Jo aBew y HLL giny AS IAOn d cae efef LH ser suo ng yoyms jauueyo Aeldsiq suo ng yoyms j uueyo Aeldsiq suoynq apis Aq apis PEWS sebewi uolinq uols s u s 1 7 padeidsip ole y9 pue j gy9 Ajuo u y mM 9lduiexg uo UMEJp ZUD LUD uayM 9lduuexg sebeul u
299. ng up the FLUOVIEW application User default intensities option button Select to use the default intensity set in the Default Laser Intensity scale above Use most recent intensities Option button Set to use the intensity that was used in the last observation 11 Display the Scanning Unit panel in the front of position Set the filters to be used with the Scanning unit Beam Splitter FLUOVIEW Setup Sets the beam splitter of each Microscope Z Stage j Objectives Fluorescence j Coloring Tab e Registration channel aaa BNI Software Hardware Lasers Users Scanning Amit From the left this shows the acral DMs amp Filters in SU beam splinter of Ch1 Ch2 and Sets the excitation filter of each p TE waman Ch3 channel i And corresponds to the order of the scanning unit from the top to bottom And corresponds to the order of the scanning unit from the top to bottom BA505 525 BA6TOIF Empty Empty BA560 600 Empty Empty Empty Empty Em Empty Empty I w Empy Empty Empty Barrier Filter ay ao ks Sets the barrier filter of each channel And corresponds to the order gt of the scanning unit from the Shows each channel Save New Setup Quit w o Saving top to bottom 1 12 Page Software Setup Setting the System Configuration 12 The Registration panel have been set at the factory and do not need to be changed here 13 After com
300. ngaging the FR Frost from the light path may generate interference fringes on an image 1 Cube turret 2 Analyzer U MDICT3 3 Transmitted light DIC slider U DICTS WI DICTHRA when using BX61WI optional 5 Universal condenser 4 Filters LBD ND6 ND25 6 Hand switch When using U HSTR2 U FH BX61WI LBD FFR This figure shows the case of BX 1 32 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 4 2 Combination with Inverted Microscope BX50WI 1 From the page tabs on the bottom right of the Acquire panel select the Optics sub panel Microscope Control Trans Scope SU Lamp Control Control Fig 1 7 Optics Sub panel 2 Select the lt LSM gt button in the Light Path group box The lt LSM gt button looks pushed in to indicate that it is selected When scanning is started while the lt BI gt button is selected the LSM light path is selected automatically It is switched back to the visual observation automatically when scanning completes 3 Rotate the cube turret 2 of the universal vertical illuminator to CO 4 When the FV5 ANU analyzer 3 is in use disengage it from the light path by setting the switch to the pulled out position 5 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider 4 by setting the switch to the pulled out position When transmitted light differenti
301. nge the excitation filter and beam splitter of each channel Confocal Aperture The optimum confocal aperture is set automatically to each channel according to the dye and objective in use and the set aperture number is displayed Clicking this area causes the Microscope Configuration window to appear Barrier Filter drop down list Changes the barrier filter of each channel TD Unit group box Shows the transmitted light detection Dyes Shows the dyeing method set for each channel Z Resolution Shows the Z resolution set for each channel al rrier Filter ea A Getting Started FLUOVIEW Online Help 2 Click the lt SU Control gt button at the bottom of the panel The window as shown below will appear Optical System Configuration Laser Unit F N or FaN NH DMs amp Filters in SU eel DM Beam Splitter r spusso Ad spusso N N BS2 8 Confocal Aperture C A 1 2 3 4 5 x Dye DAPI FITC o3 Cys Transmitted Z Resolution 3 60um 4 02um 4 33um 511um Close Fig 1 22 Optical System Configuration Window 3 To change the displayed filter types use the Excitation DM Beam splitter and or Barrier Filter drop down lists 4 After completing the system setup click the lt Close gt button to close the window 1 73 Page Getting started FLUOVIEW Online Help 1 3 2 5 Setting the C
302. nit 1 0 Delay for shutter changes Sec 3 0 Delay for bulb changes Sec Automatic Black level adjustment Timing for bidirectional scanning i i Maxi duration fe Sa epee en arenas or Sens ecto scanning Sec Select Enabled Disabled to C Disabled enable disable the automatic black level adjustment Save New Setup Quit w o Saving Timing for bidirectional scanning group box Enter the maximum duration for continuous bi directional scanning in seconds when setting fast scan mode Page 1 11 Software Setup Setting the System Configuration 10 Display the Lasers panel in the front of position Set the lasers to be used with the FLUOVIEW application and the initial ND filter values here Laser Install check box Select and check the lasers to be used with the FLUOVIEW application FLUOVIEW Setup Microscope ZStage Pobjectives fluorescence Coloring Tables Software Hardware Lasers Users Scanning Unit Default Laser Intensity scale Set the initial ND filter value at the start of the FLUOVIEW application ers Installed Default Laser Intensity igon fio af C Krypton HeNeG 50 Kii fi gt Vv o sf a I uv Argon ir T Heca Laser Intensities at Startup Use default intensities Use most recent intensities Save New Setup Quit w o Saving Laser Intensities at Startup group box Set the laser intensities when starti
303. nt cannot be restored 5 Click the lt Save Comments To Image gt button NOTE At the moment the lt Save Comments To Image gt button is clicked the comment in the Comments panel is saved simply but it is not yet saved in the image file To save the comment in the image file use the lt Save gt button in the File I O panel Click the lt OK gt button Click the lt Done gt button in the Experiments in Memory dialog box to close it Display the File I O panel at the front ON o Click the lt Experiment gt button in the Save group box One Point The Image Comments dialog box can also be displayed by a mouse operation Display the image to be saved at the front of the Display panel and right click a point in the image A pop up menu as shown below is displayed Select Experiment Properties from the menu FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Views Experiment Properties 2 122 Page APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 5 Checking the Image Information Acquisition Parameters 1 Click the lt Experiment List gt button in the toolbar at the bottom of the File I O panel The Experiments in Memory dialog box appears as shown below Experiments in Memory lt Experiment List gt button CAMAGEWSY 2 3 TIF E conmens pone Fig 2 38 Experiments in Memory Dialog Box
304. ntrol Fig 1 6 Optics Sub panel 2 Select the lt LSM gt button in the Light Path group box The lt LSM gt button looks pushed in to indicate that it is selected When scanning is started while the lt BI gt button is selected the LSM light path is selected automatically It is switched back to the visual observation automatically when scanning completes 3 Push the hand switch button to 8 set LO to be displayed in the cube display window 1 on the reflected light fluorescence vertical illuminator 4 When the U MDICT3 analyzer 2 is in use disengage it from the light path by setting the switch to the pulled out position e Page 1 31 Getting started FLUOVIEW Outline of LSM Observation Procedures 5 When only fluorescence observation is required disengage the U DICTHR WI DICTHRA transmitted DIC 3 by setting the switch to the pulled out position When transmitted light differential interference observation or simultaneous fluorescence transmitted light differential interference observation is required engage the U DICTHR WI DICTHRA and the optimum transmitted light DIC for the objective in the light path by operating the universal condenser 5 6 For transmitted light observation disengage any filter 4 from the light path When you perform transmitted observation using laser with BX61WI use the filter knob to disengage the LBD from the light path and engage the FR Frost into the light path Dise
305. nuously Simulated colors Colors used to display the image data acquired by observation on a display Original simulated colors can be created by editing the LUT Status bar The line showing information at the bottom of a window It shows the information on the operation or the description of the function selected with the mouse pointer Text A file expressed with the ASCII codes such as characters and numerals and with some control codes such as the line feed code This format is referred to as the text format The text is usually input from the keyboard Page C 3 Appendix C Glossary Text box A box in the window that accepts the input of character strings Clicking a text box displays blinking This indicates the position where the input character is inserted Title bar The horizontal bar at the top of the window that shows the title of the window or dialog box Toolbar The toolbar provides frequently used functions in the form of buttons It can be used any time during execution of any function w Window A large rectangle with a title A window can be opened or closed C 4 Page Appendix D U MCB Setting Appendix D U MCB Setting Please set U MCB before using this system Switch the display for setting of the multi control box to MAIN to operate it through the FLUOVIEW software after the setting has been completed 1 Press the INIT 1 mode selector button to display
306. ocedures 1 2 4 5 Combination with Inverted Microscope IX 1 Turn the light path selector 1 to SP 2 Set the intermediate magnification knob 7 to 1X The 1 5X position cannot be used Rotate the cube turret of the reflected light fluorescence unit to LO 4 When the FV5 ANI analyzer 3 is in use disengage it from the light path by setting the switch to the pulled out position 5 When only fluorescence observation is required disengage the FV5 DICT transmitted DIC slider 4 by setting the switch to the pulled out position When transmitted light differential interference observation or simultaneous fluorescence transmitted light differential interference observation is required engage the FV5 DICT and the optimum transmitted light DIC slider for the objective in the light path by operating the universal condenser 5 With simultaneous fluorescence transmitted light differential interference observation leaving the FV5 DICT engaged in the light path will degrade the fluorescence image resolution somewhat 6 For transmitted light observation disengage filters 6 other than the FR frost filter from the light path Be sure to engage the FR frost filter in the light path Page 1 39 Getting started FLUOVIEW Outline of LSM Observation Procedures 6 Filters 4 Transmitted DIC slider FV5 DICT ZZ Left side view 1 Light path selector 2 Cube turret 3 Analyzer FV5 ANI
307. ollowing operations are available in this condition x e Moving the chart x Place the mouse pointer on the chart and drag it x e Resizing the chart x Place the mouse pointer on one of the handles around the chart and drag it To return to the original condition click a place outside the chart To enter reference text use the reference text style Page Appendix G Converting Analysis Data into a Chart Using EXCEL q TIP When the mouse is double clicked on the chart a frame composed of oblique lines appear aroun ithe chart The frame may become a window when the chart is large The format settings are available in this condition x 1 Click one of the following positions to display black handles Data series format setting Click a place on the plot data eo Plot Axis label chart title format setting Click the characters of the axis label or chart title Enhance SOON SOOO D w iri ian aAa s Chart area format setting Click a margin area inside the chart frame Axis format setting Click a place on a chart axis Position um Plot area format setting Click a place in the plot area 2 Double click The dialog box for setting the selected format appears Set the desired format an click the lt OK gt button Refer to the Excel manuals for details G 6 Page Appendix H USER REGISTRATION OF FV500 Appendix H USER REGISTRATION OF FV500
308. omatically when scanning completes 3 Seta cube in the Universal vertical illuminator mirror cube housing 1 by pressing the desired cube conversion button 6 on the U SHTR hand switch 4 When the FV5 ANA analyzer 2 is in use disengage it from the light path by setting the switch to the pulled out position 5 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider 3 by setting the switch to the pulled out position When transmitted light differential interference observation or simultaneous fluorescence transmitted light differential interference observation is required engage the FV5 DICT and the optimum transmitted light DIC slider for the objective in the light path by operating the universal condenser 5 6 For transmitted light observation disengage any filter 4 from the light path Page 1 35 Getting started FLUOVIEW Outline of LSM Observation Procedures 2 Analyzer FV5 ANA 3 Transmitted light DIC slider 1 Universal vertical FV5 DICT illuminator mirror cube housing AX URBC 5 Universal condenser 4 Filters Filter lever ND Filter levers IF550 LBD Hand switch U HSTR 1 36 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 4 4 Combination with Upright Microscope AX70A 1 From the page tabs on the bottom right of the Acquire panel select the Optics sub panel Microscope Control Tr
309. on ml D Section 2 2 1 4 4 in OPERATION Set the XY observation mode Stop repeated scanning Section 2 2 1 4 5 in OPERATION Section 2 2 1 4 11 in OPERATION Set the observation mode Perform repeated scanning p 9 Section 2 2 2 5 1 in OPERATION Section 2 2 1 4 6 in OPERATION Adjust the Z position to observe the Set the interval time desired cross section Section 2 2 2 5 2 in DPERATION Section 2 2 1 4 7 in OPERATION Set the number of scans Set the observation range Section 2 2 2 5 3 in DPERATION Section 2 2 1 4 8 in OPERATION Acquire image Adjust the image brightness Section 2 2 2 5 4 in DPERATION Section 2 2 1 4 9 in DPERATION Save image Section 2 3 1 in OPERATION Page 2 51 APPLIED OPERATIONS Image Acquisition 1 Setting the observation mode 1 In the Scan Mode group box in the Acquire panel select the Surface option button 2 In the Acquire panel select the XYT observation mode option button 2 Setting the interval time 1 From the page tabs on the bottom right of the Acquire panel select the Time Series sub panel A panel as shown below will be displayed Interval text box Set the interval time using the lt gt or lt w gt button or by input from the keyboard Z Stage N text box Set the number of scans using the lt a gt or lt w gt button or by input from the keyboard wn v Rest Time tex
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311. ons Displaces the Z stage on a fine scale Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Step Size text box Set the number of steps using the lt a gt or lt Y gt button This number can also be input directly from the keyboard Recommended step size Shows the number of steps calculated by the system so that the scale of depth in the Z direction of the acquired image is identical to the scale of the plane in the X and Y directions Slices text box Shows the number of images acquired This number can also be input directly from the keyboard 2 40 lt Go gt button Moves to the set scanning stop position Use this button to check the scanning stop position lt Set gt button Sets the current stage position as the scanning stop position of the range of the observed cross section Z direction scanning range Current Pos text box Shows the current position of the stage lt Set Zero gt button Sets the current stage position as the home position Pressing this button also clears the Stop Z and Start Z values lt Set gt button Sets the current stage position as the scanning start position of the range of the observed cross section Z direction scanning range lt Go gt button Moves to the set scan start position Use this button to check the scan start position Locked ch
312. otobleaching to a specimen the value of laser intensity is raised to 100 to acquire an image since strong laser intensity is temporarily required When 100 or more of laser intensity value is required zoom magnification is gathered to acquire an image It is because the square power of zoom magnification per time can be secured Moreover using the objective of larger NA concentrates the laser irradiation spot in Z direction and stronger laser can be irradiated Therefore a preliminary experiment is required to set up the laser intensity the objective the zoom ration of the REX mask file according to a specimen 1 Preliminary Experiment 1 Set the laser intensity the objective and the zoom ration in the Acquire panel so that they are suitable for a specimen 2 Acquiring Image in XY mode 1 Confirm that XY Norm is displayed in the Scan Mode group box in the Acquire panel 2 Select the XY option button in the Acquire panel 2 92 Page APPLIED OPERATIONS Image Acquisition Doo 3 Select the lt Once gt button in the Acquire panel and acquire an image If necessary set the range for image acquisition with clip scan for example after 3 Making REX Mask File See section 2 2 11 1 Making REX Mask File for the procedure to make the REX mask file If the setup of the region of laser excitation remains after making the REX mask file the intensity change of the region can be observed in
313. oup box to the lt Prev gt button Ch group in the Acquire panel Sets the dyeing method which was set last time by clicking T the lt Apply gt button Prey Clear Apply 3 Click the lt Apply gt button to apply the selected dyeing method to the Ch group box on the upper part of the Acquire panel e Page 1 43 Getting started FLUOVIEW Outline of LSM Observation Procedures ae TIP When the dyeing method is selected from the Available Dyes list box an aig the lt Apply gt button is clicked a channel for acquiring fluorescence is s automatically according to the switched filter And the dyeing method i shown in the Ch group box The Confocal Aperture value is also set automatically according to th 7 TIP lf you have changed the objective click the lt Apply gt button in the Dye ro sub panel The Conforcal Aperture value is set appropriately s A A E RR AEREE ENA EE GE EE ENAS E E AE AEE N E AS OE AENA EAE E RAT A E 1 44 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures One Point The Assign dyes manually check box can also be used to set the dyeing method to the desired channel 1 Check the Assign dyes manually check box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box Game XY Repeat Once C chamei CO PMT Gain Offset
314. pears confirm the setup destination drive name and directory and select the lt Next gt button Choose Destination Location xi Setup will install FLUOVIEW in the following directory To install to this directory click Next To install to a different directory click Browse and select another directory You can choose not to install FLUOVIEW by clicking Cancel to exit Setup Destination Directory cwtuowew Browse 1 4 Page Software Setup New Setup of the Software 7 The Start Copying Files dialog box appears Confirm the installation destination directory and select the lt Next gt button This will start copying of files Start Copying Files x Setup has enough information to start copying the program files If you want to review or change any settings click Back IF you are satisfied with the settings click Next to begin copying files Current Settings Setup Type Complete Target Folder c Mluoview User Information Name Development Group 4 Company OLYMPUS 8 When the setup has completed the Setup Complete dialog box appears Select the Yes want to restart my computer now option button and press the lt Finish gt button This will restart the computer Setup Complete Setup has finished copying files to your computer Before you can use the program you must restart Windows or your computer C No will restart my computer later
315. peated scanning Section 2 2 1 4 11 in OPERATION Set the XY observation mode z Section 2 2 1 4 5 in OPERATION Set the aggrvation mode Section 2 2 2 3 2 in OPERATION Perform repeated scanning B Set the observation line Section 2 2 2 3 3 in OPERATION Section 2 2 1 4 6 in DPERATION et the numbers of Z direction steps and acquired image slices Adjust the Z position to observe the desired cross section Section 2 2 1 4 7 in OPERATION Section 2 2 2 3 4 in OPERATION Set the observation range Section 2 2 1 4 8 in OPERATION Set the interval time Section 2 2 2 3 5 in OPERATION Adjust the image brightness Section 2 2 1 4 9 in OPERATION Set the number of scans Section 2 2 2 3 6 in OPERATION Acquire image Section 2 2 2 3 7 in OPERATION Save image Section 2 3 1 in OPERATION Page 2 39 APPLIED OPERATIONS Image Acquisition 1 Setting the Z direction scanning range While acquiring image move the Z stage according to the range of the multiple sections to be observed Z direction scanning range From the panel page tabs shown on the bottom right of the Acquire panel select the Z Stage sub panel Stop Z text box Shows the scan stop position in the range of the observed cross section Z direction scanning range lt Z stage coarse adjustment gt buttons Displaces the Z stage on a large scale lt Z stage fine adjustment gt butt
316. pend Next Series Done images Once this button is acquired immediately before clicked it is not possible to z append an image FIPTUPTUFIFRERS 9 Click the lt Append Next gt button to append an image An image will be acquired with the same number of steps as the image acquired immediately before and appended to it Click the lt Series Done gt button when it is not required to append an image 2 50 Page APPLIED OPERATIONS Image Acquisition Doo 2 2 2 5 XYT Observation Mode Fs The description in this section will be focused on the image acquisition operations in the XYT observation mode that are not used in the XY observation modes which are the operations enclosed in CO in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections Set the dyeing method Section 2 2 1 1 in OPERATION Configure the microscope and scan unit Sections 2 2 1 1 amp 2 2 1 2 in OPERATION Set the objective magnification Set a lower scan speed Section 2 2 1 4 1 in OPERATION Section 2 2 1 4 10 in OPERATION Set the zoom ratio to 1X If the image Section 2 2 1 4 2 in OPERATION ao does not cleaft become clean Set the channel to be acquired Section 2 2 1 4 3 in OPERATION Re adjust the image brightness Section 2 2 1 4 9 in OPERATION Set the highest scan speed Secti
317. played Syntax lt z raw calibrated value units gt Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied 2 T Position With time related images such as XYZ images the T position with respect to the first image can be displayed Syntax lt t raw calibrated value units gt Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied 3 Animation With animation images created in the Visualize panel the position with respect to the first image can be displayed Syntax lt Animation raw calibrated value units gt Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied e Page J 3 Appendix J List of Functions in the Active Overlays Dialog Box intensity Data Appendix J 2 Intensity Data The intensity value can be displayed When images are overlapped the intensity value of each image is accompanied with the channel number placed after it Syntax lt intensity hotspot raw calibrated value units gt Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied Setup Procedure 1 Enter intensity as the first character inside lt gt 2 Add t
318. playing the image obtained by the addition operation Acquire File vo Y Tile roc Live Y Xv JdTF Fiter Image image Multi Image Operations Image Image DAE Scalar Operations Scalar Value Add to Image Subtract from Image Multiply Image with Divide Image by Log base 10 Add 2 Images Subtrach images Hmp Fig 2 80 Image Image Panel Page 2 175 APPLIED OPERATIONS Image Processing pO 2 5 3 2 Image Subtraction Subtraction of an image from an image constant from an image is possible as described below The operation method is identical to Image Image Image Constant except for the following point See section 2 5 3 1 Image Addtion To subtract an image from an image Subtract 2 Images Click the lt Subtract 2 Images gt button in the Multi Image Operations group box A lt Subtract 2 Images gt button new Display panel showing Image Image in the page tab appears displaying the image obtained by the subtraction operation To subtract a constant from an image Subtract from Image Click the lt Subtract from Image gt button in the Scalar Operations group box A new lt Subtract from Image gt button Display panel showing Image Const in the page tab appears displaying the image obtained by the subtraction operation 2 5 3 3 Image Multiplication Multiplication of an image by an image image by a constant is possible as described
319. pleting the setup select the lt Save New Setup gt button on the bottom left of the panel Selecting lt Quit w o Saving gt exits the panel without saving the system setup Page 1 13 Software Setup Setting the System Configuration 1 3 2 Setting the BX Control Panel The lt Microscope Setting gt button as shown below appears when selecting BX 51 52 or BX51 52WI and checking the Automatic check box or selecting BX61 62 or BX61 62W1 in the Microscope group box in the Microscope panel in the FLUOVIEW Setup dialog bor a fa ff s r Configuration C PV Review Station CR C FV Upgrade C F 300 G Fv500 2cH C 3CH lt Microscope Setup gt button ACH Displays the BX Control Panel window TD Auto installed x Yes No Save New Setup Quit w o Saving Clicking the lt Microscope Setup gt button displays the Microscope Setup window as shown below Filter Turret group box Sets up the name and color of the filter turret for reflected light observation Filter Turret group box Sets up the name and color of the filter turret for visual observation Mirror Unit group box Sets up the name and color of the cube turret Nosepiece group box Sets up the name color of the objective and sets the magnification N A refractive index n number of the confocal pinhole and the condenser worked with to each objective
320. point peak points and end point of the desired polygonal line then click the right button of the mouse to set the specification 2 184 Page APPLIED OPERATIONS image Analysis Doo e To specify a free line On the image drag the mouse pointer along the line lt Free Line gt button to be checked The line is displayed on the image together with the handles on it The intensity profile can be displayed Fal Handle while the handles are displayed NOTE When the moues is clicked in other place than on the specified line the handles on the line disappear The intensity profile cannot be displayed when the handles are not displayed The checked line can be moved deleted or changed of size or color x This is possible with the same method as entering comment in the image For details see sections 2 11 6 2 11 7 2 11 8 and 2 11 9 in section 6 Click the lt Annotate gt button so that the list of buttons disappears XYZ 2 TIF 7 Click the lt Begin Analysis gt button The intensity profile of the specified line will lt Annotate gt button s in the Intensity Profile box of the Analyze panel i Total 44577 25353 laverage 564 27 320 92 Std Dev 473 07 379 06 intensity Profite 0 Fig 2 87 Panel After Analysis Line Specification e Page 2 185 APPLIED OPERATIONS Image Analysis 8 Double click the Intensity Profile button The Enhanced P
321. pressed the screen shown in Fig D 11 will appear The turret will rotate and the specified turret aperture will be placed at the position of the turret s z When setting and specifying the turret aperture the optical element is placed at the position of the turret s optical element is placed in the optical axis 3 Using the JOG dial select the designation of the optical element procedure 6 Select either the type Fig D 10 HIGH the previous screen will return 5 or LOW 3 positioned at the turret s optical element mounting hole 4 When END 4 is pressed the first optical element is entered and 5 To enter the data of other optical elements repeat the above 6 magnification top lens NOTE For details on using either high or low magnification top lens refer to the instruction manual for the condenser Page D 5 Appendix D U MCB Setting Transmitted light DIC prism Setting UCD The following table shows which DIC prism is suitable for a objective lens Objective Transmitted light DIC prism PLAPO 60X0 ODPAO60 UPLAPO 10X DP10 UPLAPO 20X DPA20 UPLAPO 20X0 DPA20 UPLAPO 40X DPA40 UPLAPO 40XOl DPAO40 UPLAPO 100XOI DP100 UPLFL 10X DP10 UPLFL 20X DP20 UPLFL 40X DP40 UPLFL 100XO DP100 UPLFL 100XOI DP100 D 6 Page Appendix D U MCB Setting Setting PC Communications Append
322. ptics sub panel Light Path 7 qc BI LSM TV J Microscope Control Trans Scope SU Lamp Control Control Fig 1 5 Optics Sub panel 2 Select the lt BI gt button in the Light Path group box The lt BI gt button looks pushed in to indicate that it is selected 3 Engage a cube in the light path by pressing the desired cube conversion button 2 on the hand switch for transmitted reflected light 1 4 Focus on the specimen by looking into the eyepiece Be sure to adjust the diopter of the eyepiece in advance Refer to the instruction manual of the AX microscope When the Z motor is in use clear the check mark in the Locked check box in the Z Stage sub panel in the Acquire panel see section 2 2 1 4 7 of this volume then focus on the specimen by operating the fine focus adjustment knob of the microscope Be sure not to operate the fine focus adjustment knob while the Locked check box is checked for this may damage the Z Motor Getting Started FLUOVIEW Outline of LSM Observation Procedures The specimen ma float durin oil immersed NOTE 7 observation In this case prepare an optional clip for immersion objective and attach it as shown on the left NOTE To observe a TV image when the system is combined Tv with a BX or AX microscope click the lt TV gt button in the lt TV gt button Light Path group box in the Optics sub panel of the Acq
323. r 255 with igh scale an 5 The gamma value can be changed by dragging on the graph The set intensity graph is immediately reflected in the image in the Display panel TIP k The gamma value can also be changed by entering a value in the Gamma text box in the Intensity Mapping group box ama e Page 2 139 APPLIED OPERATIONS Changing the Image Display Method 6 If it is required to save the LUT in a file click the lt Save LUT gt button in the Color LUT Tool group box TIP 2 4 3 Switching the Displayed Channels Ch1 Ch5 The buttons on the bottom left of the screen can be used to select where the image of a single channel or images of multiple channels are to be displayed For the simultaneous display of multi channel images see section 2 4 4 Displaying Images of Multiple Channels Simultaneously 2 140 Page APPLIED OPERATIONS Changing the Image Display Method 1 Display the Display panel of for the image obtained from multiple channels at the front N t 2 s o s Click the image to display the lt Display channel switch gt buttons on the bottom left lt Display channel switch gt of the image buttons 3 Select the channels which should be displayed by pushing lt Display channel 1 D22231 switch gt buttons The color of a selected channel s button becomes darker lt Channel 1 gt button 4 Press the previously pressed lt Display channel switch gt
324. r Differential interference slider 8 Excitation filter spectral filter and barrier filter Inside the scan unit The definitions of the term maintenance by IEC EN60825 are quoted below for reference EN60825 Maintenance The performance of those adjustments or procedures specified in user information provided by the manufacturer with the laser product which are to be performed by the user for the purpose of assuring the intended performance of the product It does not include operation or service e Page 2 1 Maintenance of Major System Units UV Ar Laser 2 2 UV Ar Laser The definitions of the term maintenance by CDRH 21CFR are quoted below for reference CDRH Maintenance means performance of those adjustments or procedures specified in user information provided by the manufacturer with the laser product which are to be performed by the user for the purpose of assuring the intended performance of the product It does not include operation or service as defined in paragraph b 27 and 38 of this section 2 2 1 Auto Alignment Operation Remote Switch 2 2 Remote Switch Page The UV Ar laser is provided with an auto alignment facility which automatically optimizes the laser oscillation by adjusting the unstability in output due to mirror alignment error resulting from long period of use It is recommended to perform auto alignment about once a month After warming up th
325. r the frame 2 94 Page APPLIED OPERATIONS Image Acquisition Doo 4 Select the Bleach mode option button in the AOTF group box of the Laser sub panel AOTF REX Function C Disabled C REX mode Bleach mode Make REX mast 5 Aframe to specify the REX mask file appears on the right side of the Bleach mode option button Right click the mouse inside the frame to display the pop up menu as shown below Select the image to be masked in the menu The REX mask file displayed RE Live in the Display panel is pop RE Livet0 up displayed E TIP In order to use the already saved file as a REX mask file open the image beforehand i The opening method of the REX mask file is completely the same as that of fan image For details see section 2 3 2 Opening Previously Saved Images in this manual 6 The icon of the selected REX mask file is displayed inside the frame AOTF REX Function REX_Live 0 Disabled The icon of the selected REX mode REX mask file is displayed inside the frame And the 9 Bleach mode file name and the observation mode are also MEKERES masr 2 displayed above and under the frame 7 Set each laser ON OFF and their intensity If necessary use the Laser Intensity dialog box in the Lasers sub panel to set the laser ON OFF And the value set in the Laser Intensity dialog box can be obtained apart from the valu
326. r example i When 1 0 mm is set as the maximum range for Z series the minimum value for Z starting becomes 500 um and the maximum value for z Page 1 7 Software Setup Setting the System Configuration 1 8 Page 5 Display the Objectives panel in the front position In this panel select the items to be included in the list of objectives used by the FLUOVIEW application Each user should set the objectives that the user wants to use with FLUOVIEW Software Hardware Users Scanning Unit Microscope ZStage Objectives Fluorescence Coloring Tables Registration Objective Lenses All Known Objective Objectives on your nses Microscope PLAPO 40x F Double click here to make a new obja PLAPO 60x0 PLAPO 40x PLAPO 100x0 PLAPO 60x0 PLAPO 40XWLSM PLAPO 100X0 PLAPO 60XWLSM PLAPO 40XWLSM PLAPO 60XOLSM PLAPO 6OXWLSM UPLAPO 10X PLAPO 60XOLSM UPLAPO 10XD UPLAPO 10X UPLAPO 20x UPLAPO 20X UPLAPO 20x0 UPLAPO 20X0 UPLAPO 40x UPLAPO 40X UPLAPO 40x0 UPLAPO 40X0 UPLAPO 60X El UPLAPO 60 El Please drag Dbiectives from here and drop them here to identify those Objectives you actually use Double click to edit Save New Setup Quit w o Saving lt To delete an unnecessary objective from the list gt In the list on the right double click the objective to be deleted from the list When the dialog box as shown below appears select the lt Delete gt button Edit Objective
327. r i Or asuacsacsasoraee enue apus opis sss ma TIP Use the lt FOCUS gt button to acquire image at an even higher speed ee the specimen is already being scanned stop scanning with th lt Focus gt button _ lt STOP SCAN gt button before selecting the lt XY Repeat gt button NOTE The Focus function reduces the scanning time by line skipped scan As a result the acquired images become coarse NOTE Do not move FLOUVIEW FV500 Menu while acquiring an image 1 2 8 7 Setting the Cross section to be Observed While acquiring image move the Z stage to select the cross section to be observed 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Z Stage sub panel r Z Series BX Current Pos text box Shows the current position of the stage The value can also be entered directly from the keyboard lt Z stage fine adjustment gt buttons Displace the Z stage by 0 1 um per step lt Z stage coarse adjustment gt buttons Displace the Z stage by 1 0 um per step Step Size Slices a s O40um ideait Locked Locked check box Enables the Z motor operation Fig 1 12 Z Stage Sub panel 1 52 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures 2 Check the Locked check box in the Z Stage sub panel Do not turn the fine focus adjustment knob while the Locked check box is checked for
328. r of the screen is displayed Syntax lt y hotspot raw calibrated value units gt Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied Setup Procedure 1 Enter y as the first character inside lt gt 2 Add the following characters to set the display method hotspot Display measurement points with markings raw Display data in pixel values calibrated Display data in numerical values um units Display the unit pixels um Examples lt Input character strings gt lt Displayed strings represent figures gt lt y raw value gt irr treet este eters eeeeees lt y hotspot raw value gt rrrtereereeeee lt y hotspot calibrated value units gt um lt x hotspot raw value gt lt y raw value gt he detailed display procedure is described in section 2 11 3 Viewing the or Y Coordinate Position of Image in Volume OPERATION J 2 Page Appendix J List of Functions in the Active Overlays Dialog Box Coordinate Position Data Appendix J 1 3 Other Positions can also be displayed by entering the following keywords in the syntax in place of the X and Y coordinate positions described in sections J 1 1 and J 1 2 1 ZPosition With cross section related images such as XYZ images the Z position with respect to the first image can be dis
329. ragged in the Selected Dyes group box to the Ch group in the Acquire panel 2 Select the specimen dyeing method by dragging desired dye names in the Available Dyes list box in the Selected Dyes group box to the field immediately above the list box 3 Click the lt Apply gt button to apply the selected dyeing method to the Ch group box on the upper part of the Acquire panel TIP I When the dyeing method is selected from the Available Dyes list box an the lt Apply gt button is clicked a channel for acquiring fluorescence is se _ automatically according to the changed filter And the dyeing method i APPLIED OPERATIONS Image Acquisition One Point The Assign dyes manually check box can also be used to set the dyeing method to the desired channel 1 2 Check the Assign dyes manually check box in the Dyes sub panel Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box Foal ae mes x Ox r Sean Mode Surface X Se Normal Fast Assign dyes manually check box After dragging the icon appears on the right of the Ch check box and the dyeing method is set M FITC k i The dyeing method is set can Ofs con i pem EN 800v 10 0x Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Page 2 67 APPLIED OPERATIONS Image Acquisition e
330. ranges from the original FLUOVIEW through the new FV300 and F Y500 systems The original FLUOVIEW can be added to an OLYMPUS BX or IX fluorescence microscope The F300 can be used with a very wide range of OLYMPUS microscopes and FV500 can also be used with the fully automatic AX70 auto In all cases FLUOVIEVY provides the advantages of confocal laser scanning microscopy without limiting any of the versatility of the base microscope FLUOVIEW turns your fluorescence microscope into a five dimensional digital image capture system with integrated image processing and analysis functions The FLUOVIEW system consists of the following components optical hardware computer hardware software documentation To learn more about the different aspects of the FLUOVIEW system please click on any one of the topics listed below Overview Enhanced display cquisition on F 300 lisition on FN and Loading Files Tile and Print Fig 1 16 Initial Window 1 60 Page Getting Started FLUOVIEW Online Help hen the mouse pointer is placed on a word in enhanced display the ouse pointer turns into a finger pointer One Point Click the lt Contents gt button to the initial display Click the lt Back gt button to return to the previous information page 1 3 2 Microscope Help The microscope scan unit and laser types can be set up from the FLUOVIEW software by selecting the dyeing me
331. ration of selecting an icon image file or observation method dragging it to the desired position and dropping it there Selected Dyes lt XYZ 2 TIF TR 4 TIF Scalar Ops Scalar Value 2 Add to Image J Subtract from Image Rhodamine Phalloipii Multiply Image with Texas Red Divide Image by Prev Clear Apply Multi Image Ops XYZ 2 TIF Add 2 Images gt Save Subtract 2 Images XY 2 GB 1of 2 Multiply 2 Images Experiments in Me mory C IMAGE XY2Z 2TIF CAIMAGE TR 1 TIF Name live tif th pitif tr 1 tif xy 1 tif xy2ch tif 2 nen ae Page 1 11 SYSTEM OVERVIEW Software Functional Configuration 1 5 4 Identification of Images Depending on the Observation Methods On many occasions FLUOVIEW displays Image Icon image icons to allow identification of the XZ observation i observation method used when each image is XZ observation 2 channel mode acquired See table on the left When the File I O Tile Process Analyze Xt observation or Visualize panel is selected the icon of the Xt observation 2 channel mode image selected in the Display panel is displayed in a frame at the top of the function XZT observation XZT observation 2 channel panel The image icons are also displayed in the Icon field in the Files list box in the File I O panel or during dragging of an
332. rce is applied This phenomenon is utilized in electric spark generators for use with gas appliances etc Pixel The minimum graphic unit of screen display Also referred to as the dot PMT Voltage Increasing this setting increases the sensitivity When a bright image cannot be obtained even by setting PMT Voltage to 800 V do not vary PMT Voltage any more but increase Gain This usually provides a better effect than increasing PMT Voltage above 800 V Appendix C Glossary R Resolution Number of dots composing the image on a screen or printer When the number of dots is increased i e when the resolution is increased the gradation can be displayed in more details Reversed display Display method which indicates that an item or character string is selected and has become the target of the user s next operation The reversed displayed characters are shown in a different color from other characters s Scroll Action of moving text or a picture up or down in order to view the other part of information than the information which can be displayed at once in the window Scroll bar The bar displayed at the bottom or right of a window containing more information than can be displayed at once The scroll bar has a knob and two arrow buttons Dragging the knob scrolls the information directly and clicking one of the arrow buttons scrolls the information line by line Holding an arrow button scrolls the information conti
333. re m m Custom colors Bees Po ms EER s s za z zs Define susto alors gt Cancel Red 255 Green 255 Blye p Hue fao Sat 240 ColorlS olid Lum 120 Add to Custom Colors 3 Select the color which you want to set in the color palette and click the lt OK gt button 1 18 Page Software Setup Setting the System Configuration 1 3 2 4 Finishing the setting Finishing the setting and closing the Microscope Setup window Microscope Setup lt Quit w o Saving gt button lt Save New Setup gt button Gaye New Setup Gu WS Samm 1 Click the lt Save New Setup gt button to save the setup and close the window Or click the lt Quit w o Saving gt button to cancel the setup and close the window Then the dialog box as shown below appears If you want to save click the lt Yes gt button if you do not want to save click the lt No gt button a The setting has changed Do you want to save the changes Ee NOTE After saving is selected in the Microscope Setting window the original setting is not returned even if the lt Quit w o Saving gt is clicked in the FLUOVIEW Setup dialog box Page 1 19 Software Setup Adding the dyeing method 1 4 Adding the dyeing method Newly add a dyeing method and set laser type excitation wavelength and emission wavelength This section describes a simple example of adding CFP as a dyeing method a
334. re than one channel it is possible to select whether images from more than one image are saved simultaneously or only one of the images is saved Use the lt Display channel switch gt buttons to select the images to be saved The selected images will be saved under the conditions set for each channel Example When only the image of Channel 1 is displayed only the image of Channel 1 will be saved Click lt Experiment gt button in the Save group box The Save Experiment As dialog box will open Save Experiment As Fix Save in imas i E cl E E 86 3d1 tif 86xt1 tif BBxyt tif E S6xy22 2 ti E 86 3d2 tif F 86xt2 tif Fa 86xyt2 tif S 86xyz3 tif E 86 3d3 tif F 86xt3 tif 86xyt3 tif 86xyz3 2 ti E 86 3Dstl tif 86xy1 tif E 86xyz1 tif E S6xyztl tif fa 86 3Dst2 tif E 86xy2 tif E 86xyz1 2 tif 86xy2t2 tif E 86 3dst3 tif 86xy3 tif E 86xyz2 tif E S6xyzt3 tif gt Filename lt v2CH TIF Save as type FLUOVIEW Multi if tif Cancel Fig 1 15 Save Experiment As Dialog Box Enter the file name in the File name text box Select FLUOVIEW MultiTif from Save as type Click the lt Save gt button Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 11 Exiting from the Software 1 Click the lt Exit gt button in the toolbar at the bottom of the screen This will allows you to exit the software The Shut Down FLUOVIEW dialog box as shown below
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336. retaining screws and remove the top cover e Page 2 3 Maintenance of Major System Units UV Ar Laser 3 Remove the water tank cover and check the level and pollution of water in it Water tank Distilled water Specified level 2 4 Page Maintenance of Major System Units UV Ar Laser 2 Refill and Replacement If the water level in the water tank is below the specified level refill distilled water until the specified level If the water is polluted by algae or moth replace the water to ensure cooling Replacement procedure 1 Set COOL DOWN CYCLE to O OFF COOL DOWN CYCLE Set to O OFF 2 Pump water out 3 To eliminate water remaining in the cooling hose switch POWER of the heat exchanger I ON run it idle and pump out water which comes inside the tank POWER Set to I ON 4 Refill new distilled water until the specified level Run the heat exchanger manually and check the water level again If it is below the specified level refill additional distilled water 5 Close the water tank with the lid place the top cover and tighten the 4 retaining screws tightly using a screwdriver 6 Plug the power cord into the power outlet and set the main circuit breaker to ON 7 Set up for normal operation For details see section 1 2 1 Turning Power ON in Volume OPERATION INSTRUCTIONS Consumable supply Distilled water about 10 liters commercially available in 20
337. rightness of the image of each channel by using the PMT Offset and Gain LED sliders in the Acquire panel For details see section 2 2 1 4 9 Adjusting the Image Brightness Page 2 65 APPLIED OPERATIONS Image Acquisition 2 2 3 3 Transmitted Image Images obtained by transmitted light observation can also be acquired or observed 2 66 Page simultaneously with images obtained by fluorescence observation When observing fluorescence images simultaneously set the dyeing method 1 From the page tabs on the bottom right of the Acquire panel select the Dyes sub panel Assign dyes manually check box Checking this enables the manual setting Dragging the dyeing method in the list directly to the Ch group box assigns the dye to the desired channel Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method lt Prev gt button Sets the dyeing method which was set last time by clicking the lt Apply gt button r Selected Dyes Assign dyes manually Available Dyes Mito Tracker Pl Rhodamine Phalloidin Texas Red Fig 2 22 Dyes Sub panel Place the pointer on the icon displayed in the Selected Dyes and the dyeing method is shown in the pop up display lt Clear gt button Clear the set dyeing method lt Apply gt button Applies the dyeing method d
338. ro gt button Sets the current stage position as the home position Pressing this button also clears the Stop Z and Start Z values lt Set gt button Sets the current stage position as the scanning start position of the range of the observed cross section Z direction scanning range lt Go gt button Moves to the set scan start position Use this button to check the scan start position Time Serie Step Size Slices v Locked check box Enables the Z Motor operation Fig 2 13 Z Stage Sub panel The moving amount assigned to the lt Z stage fine adjustment gt and lt stage coarse adjustment gt buttons can be changed l See section 1 3 in MAINTENANCE Setting the System Configuration for 1 Check the Locked check box in the Z Stage sub panel Do not turn the fine focus adjustment knob while the Locked check box is checked for this may damage the Z motor 2 33 Page APPLIED OPERATIONS Image Acquisition fe 2 While observing the image in the Live panel locate the upper edge of the range to be observed by moving down the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the bottom edge of the range to be observed by moving down the revolving nosepiece using the lt Z stage coarse adjus
339. rofile window appears as shown below lt Properties gt button Displays the Editing dialog box for use in detailed setting of the chart or change of the chart display See section 2 14 Changing the Chart Display Method for details lt Copy gt button Copies the plotted image in the clipboard lt Save gt button Saves the profile data in a file using an Excel compatible format lt Close gt button Quits the Enhanced Profile Plot window and returns to the Analyze panel i TIP x TIP TIP 2 TIP k l TP When the mouse pointer is placed on a graph line while the Ctrl ori w Enhanced Profile Plot x Properties Copy Save Position umj Fig 2 88 Enhanced Profile Plot Window When a desired area is specified by dragging the left button of the mouse on the graph the specified area can be magnified i When the right button of the mouse is dragged on the graph the graph The magnification or scrolling of the graph can be canceled by dragging the left button of the mouse from the bottom left to the right of Ait key is held depressed the coordinates can be displayed U 1 The displayed data can be applied to other applications See section 2 10 Transferring Data to Another Application for 2 186 Page APPLIED OPERATIONS Image Analysis Doo O 2 6 1 2 Intensity Values on a Planar Region Bird s Ey
340. rom this becomes too bright first try decreasing PMT voltage slightly If the image background is still too bright increase Offset to darken the background As this also reduces the brightness of the observation target increase Gain as required so that the observation target is clearly visible Gamma Correction provides you more brightened image even if it was acquired with darkness See 2 4 2 2 LUT Graph Editing by Gamma Correction for details in this operation 2 24 Page APPLIED OPERATIONS image Acquisition fs i TIP Each click of the lt gt or lt gt button of the PMT LED slider increases or I decreases the PMT voltage by 5 V Each click of the slider section of the PMT LED slider varies the PMT voltage by 25 V in the transmitted channel case each click of the lt gt or lt gt button of the PMT LED slider increases or decreases the PMT voltage by 1V x In the transmitted channel case each click of the slider section of the PMT x u LED slider varies the PMT voltage by 10V Each click of the lt gt or lt gt button of the Gain LED slider increases or decreases the Gain by X0 5 x Each click of the slider section of the Gain LED slider varies the Gain by X0 5 x Each click of the lt gt or lt gt button of the Offset LED slider increases or decreases Offset by 1 x Each click of the slider section of the Offset LED slider varies the Offset NOTE wh
341. rs sub panel in the Process panel at the front Live Fieuo Y Tile 2rocess Anaiz Filter Icon of the image being displayed 0 ei Image to be processed by filtering XY CH 1082 Y xvecu tie Smoothing Filters Smoothing Filters group box Bee Provides a command button for each Eere noise filter Median Sharpening Filters edishpsss 22 Sharpening Filters group box ea Provides a command button for each of the Eemien contour enhancement and image Sobel sharpening filters E Prewitt Sharpen DIC Image Filter DIC leveling DIC Image Filter group box OEE Used for correction of DIC level irregularities BEER eJel l l2 Drag Drop Dyefs to select setup Drop Dye s to select setup Fig 2 71 Filters Sub panel 2 162 Page APPLIED OPERATIONS Image Processing Es 2 5 1 1 Contour Enhancement When an image is blurred by the boundaries between image grains becoming unclear it can be sharpened by applying contour enhancement Five types of filters are provided for use in the contour enhancement 1 Laplacian filter This filter enhances the contours of the image grains If the image contains noise the noise is also enhanced By adding the original image to the image processed with Laplacian filtering it is possible to obtain an image with stronger contour enhancement The filter format is as shown below 1 1 1 1 8 1
342. rt to File AnnotationsiROIs Properties Fig 2 42 Image Comments dialog box 4 Select the lt Properties gt button in the Export to File group box The Save to txt dialog box as shown below appears Saves the image size and resolution irents lt Save gt button Intensity Mapping Which of the Experiment s Saves the checked item Saves the range of intensity mapped to every channel Acquisition Parameters Properties would you like to into the text file u nim save to an ASCII text file Saves acquisition parameters for every channel Description Saves version information Has oe lt Cancel gt button Saves observation conditions LUT Ch2 ear Cancels the checked LUT Ch3 item and closes the Saves the condition of LUT for Channel1 LUT Ch4 dialog box sae E LUT Ch5 Cancel Saves the condition of LUT for Channel2 m e Fig 2 43 Save to txt dialog box lt TIP The LUT Ch1 to LUT Ch6 check boxes are displayed according to the channel used for image acquisition 5 Check the check box of the item to be saved 2 128 Page APPLIED OPERATIONS Saving Opening and Shredding Images 6 Select the lt Save gt button The Save As ASCII Text dialog box as shown below appears Save in ja images ce O tif B CFP tit OON tit CFP2 tif D1V tif El CFP3 tif 1 txt z DA gt Z ls 2P femtowaveGS000 tif fast avi AdT est log
343. s panel x TIP o Before the dragging and dropping the frame at the top left show the icon of the image file displayed in the Display panel 4 From the Experiments in Memory dialog box select the file name of the second file and drag it to the frame at the top right of the Process panel The icon of the image is displayed in the frame at the top right of the Process panel No second file dragging is needed when the image processing is by xsena Drag Drop Dyefs to select setup value TIP The mouse pointer turns into the image icon during dragging 5 Click the lt Done gt button in the Experiments in Memory dialog box to close it 2 174 Page Add 2 Images lt Add 2 Images gt button Add to Image lt Add to Image gt button APPLIED OPERATIONS Image Processing Enter the constant for use in operation in the Scalar Value text box in the Scalar Operations group box This step is required only for operation between an image and a constant To add an image to an image Click the lt Add 2 Images gt button in the Multi Image Operations group box A new Display panel showing Image Image in the page tab appears displaying the image obtained by the addition operation To add a constant to an image Click the lt Add to Image gt button in the Scalar Operations dialog box A new Display panel showing Image Const in the page tab appears dis
344. s contents of Image Field NB These are non editable text fields Annotations ROls Annotations ROls Properties Fig 2 40 Image Fields Panel Import from File Export to File 5 Select the information items to be checked from the list box on the right The kind of information displayed when each item is selected is shown below hts C IMAGESWXYZ 2 TIF RGB 0 000 000 000 RGB 1 000 001 RGB 2 000 002 RGB 3 001 003 FEA RGB 4 001 004 d nerpa RGB 5 001 005 T Cho RGB 6 001 pog Channel 1 Fluorescence Acquisition Paramete a Channel 1Dye FITC RGB 7 002 007 y LUT Chi RGB 8 002 pog Channel 2 Fluorescence Version Info RGB 9 002 009 Channel 2Dye Rhodamine Description DED 1n_nn gt nin PMT Voltage Ch2 800 Offset Ch2 0 RRR 1n_nin gt are non editable text fields File Version 2 FLUOVIEW Version 1 09 OLYMPUS i After checking the information click the lt OK gt button Properties 6 7 Click the lt Done gt button in the Experiments in Memory dialog box to close it Page 2 125 APPLIED OPERATIONS Saving Opening and Shredding Images One Point The Image Comments dialog box can also be displayed by a mouse operation Display the image to be saved at the front of the Display panel and right click a point in the image A pop up menu as shown below is displayed Select Experiment Properties from the menu FullScreen Display Past
345. s in the Acquire panel 2 72 Page APPLIED OPERATIONS Image Acquisition bs 3 Group which means each laser and the lt gt or lt Y gt buttons appear on the lower part of each CH group box v DAPI iv FITC Transmitted PMT Offset PMT Offset Aagi Offset ie Offset PMT Gain Offset Click to increase the acquired group No era s r 800v TE a 10 0x 0 800v rr 0 Fr Ox 0 186v 43x Group 74 Group 4a _w Group v 22 Grup wiz w Grup v 42 o Shows the group to Click to decrease the be acquired acquired group No 4 Set the group number according to the reagent in use with the lt gt or lt Y gt buttons This setting is not required if the desired number is already displayed TP To prevent unwanted color fading it is recommended to acquire the image from the reagents with longer wavelengths 5 Click the lt Seq Once gt button to acquire the image 6 When transmitted observation is also attempted the FLUOVIEW dialog box and the Lasers sub panel appear to specify the laser type which has not been specified yet w FLUOVIEW x Please select the lasers for Seq Scan Group 1 Then press any key to begin the scan Cancel Fig 2 24 FLUOVIEW Dialog Box Page 2 73 APPLIED OPERATIONS Image Acquisition 2 74 Page 10 lt Off gt button Select the laser type
346. selected cube is displayed 4 Focus on the specimen by looking into the eyepiece Be sure to adjust the diopter of the eyepiece in advance Refer to the instruction manual of the BX microscope 1 24 Page e Getting Started FLUOVIEW Outline of LSM Observation Procedures When the Z motor is in use clear the check mark in the Locked check box in the Z Stage sub panel in the Acquire panel see section 2 2 1 4 7 of this volume then focus on the specimen by operating the fine focus adjustment knob of the microscope Be sure not to operate the fine focus adjustment knob while the Locked check box is checked for this may damage the Z Motor NOTE The specimen may float during oil immersed observation In this case prepare an optional clip for immersion objective and attach it as shown on the left Clip for immersion objective If you want to use a differential interference unit in transmitted light observation refer to the instruction manual of your microscope NOTE With transmitted light differential interference observation using an immersion objective set the microscope s field diaphragm so that it circumscribes the field of view Otherwise the contrast may become degrade e Page 1 25 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 3 2 Combination with AX 1 26 Page 1 From the page tabs on the bottom right of the Acquire panel select the O
347. served by moving up the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the upper edge of the range to be observed by moving up the revolving nosepiece using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons 5 When the position is located click the lt Set gt button The Stop Z text box will show the scanning stop position of the range of the multiple sections to be observed Z direction scanning range 2 48 Page APPLIED OPERATIONS Image Acquisition fs 2 Setting the numbers of steps and acquired image slices Step Size 1 Set the number of steps using the lt gt or lt Y gt button in the Step Size text box b 0 25um ea 7 TIP iThe number of steps shown in the Step Size text box has been calculated Step Size text box S a ae i i by the system so that the depth scale of the acquired image is identical to the horizontal scale TIP The number of acquired images shown in the Slices text box can also be Slices ee w input from the keyboard i Slices text box After setting Start Z Z direction scan start position and Stop Z Z direction scanning stop position input the desired number of images in the Slices text box This automatically sets Step Size number of steps
348. smitted light DIC slider FV5 DICT optional This is a prism for use in differential interference observation e Engage the FV5 DICT in the light path for laser differential interference observation or visual transmitted light differential interference observation Leaving the FV5 DICT engaged during laser fluorescence observation will degrade image quality somewhat We recommend disengaging the FV5 DICT from the light path when simple laser fluorescence observation is required 1 8 Page Getting Started FLUOVIEW Basic Operations NOTE With transmitted light differential interference observation using an immersion objective set the microscope s field diaphragm so that it inscribes the field of view Otherwise the contrast may degrade This applies to both visual observation and laser differential interference observation 5 Condenser polarizing plate Condenser for transmitted lighting In addition the rotary turret for the transmitted light DIC slider and the polarizing plate for differential interference observation analyzer are also provided e To perform differential interference observation engage the transmitted light DIC slider optional matching the objective in use in the light path For both visual observation and laser differential interference observation e To perform visual differential interference observation or laser differential interference observation engage the polarizing plate in the light
349. so be displayed by a mouse operation Display the image to be colored at the front of the Display panel and right click a point in the image A pop up menu as shown below is displayed Select View Processor from the menu then select Intensity Scaling from the displayed sub menu Annotate FullScreen Display Paste Print Save Display Save Experiment Select All Overlays ViewProcessor Color Look Up Table Views gt Intensity Scaling Experiment Properties 2 136 Page APPLIED OPERATIONS Changing the Image Display Method 2 4 2 Editing the LUT Look Up Table An original LUT can be created by editing an existing LUT 2 4 2 1 LUT Graph Editing According to Colors 1 Display the Display panel of the image that you want to edit the LUT 2 Click the lt LUT gt button in the toolbar at the bottom of the screen The Color Tool lt LUT gt button dialog box appears as shown in Fig 2 51 3 Click the lt Graph display gt button on the top right of the Color Tool dialog box The dialog box shows the LUT intensity graph lt Graph display gt button 4 Select the LUT color to be edited by checking the check boxes below the Coloring Intensity 1 Fy graph in the Color LUT Tool group box 5 Set the range of intensity graph application using the Low and High scales in the Intensity Mapping group box TIP Double clicked the Low scale High scale and Gamma text fh Red
350. software FLUOVIEW software Sections 1 2 1 amp 1 2 2 Sections 1 2 1 amp 1 2 2 Open an image in a file Open an image in a file Section 2 3 2 Section 2 3 2 Acquire an image Convert the contrast Section 2 2 Section 2 5 2 Display the opened and acquired images side by side and compare them Section 2 4 6 Draw comment text on the image Section 2 11 Save the image Section 2 3 1 Exit from the FLUOVIEW software and turn power OFF Sections 1 2 11 amp 1 2 12 Output the image at the printer Section 2 12 Exit from the FLUOVIEW software and turn power OFF Sections 1 2 11 amp 1 2 12 2 6 Page APPLIED OPERATIONS Image Acquisition x 2 2 Image Acquisition Confirm the image to be acquired using the microscope and acquire its image using FLUOVIEW The image can be saved in a file as required NOTE Do not move FLOUVIEW FV500 Menu while acquiring an image Page 2 7 APPLIED OPERATIONS Image Acquisition pO 2 2 1 Image Acquisition in XY Observation Mode This section describes the basic operation procedure from the system configuration to the image acquisition in the XY observation mode and image saving in a file as shown in the following chart The details of each operation will be described in the subsequent sections Set the dyeing method Section 2 2 1 1 in OPERATION Configure the microscope and scan unit Sections 2 2 1 1 amp 2 2 1 2 in
351. splay switching gt button will change to the icons of the lt Side by side views gt button or lt Over and under lt Over and Under views gt button views gt buttons Acquire File vo Y Tile Y gt roces _ Live Y xvz21iF 3 XYZ 2 TIF s Is xaea Display Panel Fig 2 55 Panel Displaying Images of Two Channels Side By Side 2 142 Page APPLIED OPERATIONS Changing the Image Display Method 2 4 4 2 Displaying Merged Image of Multiple Channels Single View E 1 Display the Display panel of the merged image of multiple channels at the front lt Display switching gt button 2 Click the lt Display switching gt button at the top of the Display panel The list of buttons as shown below appears E 3 From the displayed list of buttons press the lt Single View gt button The Display lt Single View gt button panel will show the images of multiple channels side by side At the same time the icon shown in the lt Display switching gt button will change to the icon of the lt Single 1 2 3 4 5 lt Display channel switch gt View gt button buttons 4 Click the image to display the lt Display channel switch gt buttons at the bottom left of z the image lt Channel 1 gt button 5 Select the channels which should be displayed by pushing lt Display channel switch gt buttons The color of a selected channel s button becomes darker 2 File 0
352. splayed in the frame at the Xt observation top of the function panel such as the File I O panel Xt observation 2 channel mode In the File I O panel image icons are also Fi IP eae XZT observation displayed in the Icon field in the Files list box XZT observation 2 channel mode XY observation TIP all observation modes the icons for XY observation 2 channel mode 3 or more channels are identical x USE SS SS s ibana XYt observation 2 channel mode XYZ observation 2 channel mode XYZt observation XYZt observation 2 channel mode Point Scan Stereo 3D image Image to be viewed with color eyeglasses 3 or more channels 2 102 Page APPLIED OPERATIONS Saving Opening and Shredding Images lt Files List Box gt The enter view of this list box cannot be displayed Scroll the entries when viewing this list box Name Image file names Icon Icons showing the image observation modes X Image sizes in the X direction Y Image sizes in the Y direction Z Number of images acquired in the Z direction T Number of images acquired over time Name tA St Size bytes Date xy2ch tif E 800 600 1 1 2 k 1 1838942 Mon Feb 05 19 43 40 1996 xyttit fr 800 600 1 3 2 1 1 5789492 Tue Mar 19 17 28 06 1996 xyz 2 tif fil 800 600 4 1 2 1 1 7714964 Tue Mar 19 13 47 14 1996 OLYMPUS FLUOVIEVY xyz tit mil 800 600 3 1 1 1 1 2901710 Ved Nov 22 10
353. ssing to the image display file measure the intensity distribution and animation to identify the cell shape then edit data with Excel calculate the area and save the obtained image data Turn power ON and start the Turn power ON and start the FLUOVIEW software Sections 1 2 1 amp 1 2 2 FLUOVIEW software Sections 1 2 1 amp 1 2 2 Acquire an image Open an image in a file Section 2 2 Section 2 3 2 Apply filter processing cUe eer eeeereeaenenaeeenenenennenenenes Section 2 5 1 Specify the area to be measured When wuuunununnununununununununuununuununuunnuununununununuunun Display the animation thereis Measure the intensity distribution Section 2 8 2 another Section 2 6 2 area to be measured Measure area Save the measurement data in the Section 2 6 3 2 Excel format Section 2 10 1 Read the saved measurement data with Excel and edit it Save the image Section 2 3 1 Exit from the FLUOVIEW software and turn power OFF Sections 1 2 11 amp 1 2 12 Exit from the FLUOVIEW software and turn power OFF Sections 1 2 11 amp 1 2 12 Page 2 5 APPLIED OPERATIONS General Operation Procedure pO Example 3 To acquire an image and compared it with Example 4 To open an image in a file improve its a previously acquired image contrast and create a presentation image by entering comment etc Turn power ON and start the Turn power ON and start the FLUOVIEW
354. t button Saves the profile data in a file using an Excel compatible format lt Close gt button Quits the Average Intensity Trace window and returns to the Analyze panel TIP p APPLIED OPERATIONS Image Analysis Average Intensity Trace Fig 2 95 Average Intensity Trace Window When a desired area is specified by dragging the left button of the When the right button of the mouse is dragged on the graph the graph x ITP iThe magnification or scrolling of the graph can be canceled by dragging the left button of the mouse from the bottom left to the right of When the mouse pointer is placed on a graph line while the Ctrl ori Alt key is held depressed the coordinates can be displayed 1 The displayed data can be applied to other applications i See section 2 10 Transferring Data to Another Application for Page 2 201 APPLIED OPERATIONS Image Analysis 2 6 3 4 Measuring the Change in Integrated Intensity The total value of the intensity in a region specified in an image can be measured and displayed graphically The operation results are displayed graphically in the Integrated Intensity box in the Series sub panel The operation method is completely identical to that for obtaining the mean value of intensity See section 2 6 3 3 Measuring the Change in Mean Value of Intensity Double click the In
355. t Finish gt button Text Import Wizard Step 3 of 3 This screen lets you select each column Column Data Format and set the Data Format General General converts numeric values to Text numbers date values to dates and all C Date MDY remaining values to text C Do Not Import Column Skip Data Preview Fig Appendix G 3 Dialog Box Displayed When File is Opened with Excel 3 3 6 Drag the data to be converted into a chart yon aa a eS B5 0 Aga bn Cae E Line Intensity Profile 73 000000 to 1658 000000 Position um No Position jej 2 a gt gt sy N aim mo n a a a a a a a IN SISSIDE G 2 Page Appendix G Converting Analysis Data into a Chart Using EXCEL 7 Click the lt ChartWizard gt button lt ChartWizard gt button 8 On the sheet drag the area where the chart is to be inserted The dialog box as shown below appears ChartWizard Step 1 of 5 If the selected cells do not contain the data you wish to chart select a new range now Include the cells containing row and column labels if you want those labels to appear on the chart Range ERANI 76 Fig Appendix G 4 Dialog Box of Chart Wizard 1 5 9 Click the lt Next gt button The dialog box as shown below appears ChartWizard Step 2 of 5 Select a chart type A G E 4 K D Bar 3 D Column
356. t Style an x Size list boxes 3 Click the lt OK gt button to close the Font dialog box 4 _ Click the lt OK gt button to close the Annotation dialog box U U U 2 234 Page APPLIED OPERATIONS Entering Comment in Image 2 11 6 Drawing an Arrow in Image lt Arrow gt button This facility is used to draw an arrow for indicating a point in interest in image or adding explanation in it 1 Display the Display panel of the image you want to draw an arrow 2 Click the lt Arrow gt button in the displayed list of buttons 3 Drag the mouse pointer from the start point to the end point of the desired arrow 4 To change the arrow size see section 2 11 8 Changing the Comment Size 3 TIP 5 The direction indicated by a previously drawn arrow can be changed i 1 Click the mouse on the arrow to make the arrow active i e handles f displayed on the arrow U 2 Click the right button of the mouse 3 Select Edit from the displayed menu A sub menu as shown below appears Copy Move Size up Delete Upper Right Properties Right Bottom Right Down Bottom Left Left Upper Left 4 Select the desired arrow direction fromthe sub menu II Page 2 235 APPLIED OPERATIONS Entering Comment in Image fe 2 11 7 Drawing Color Bars in Image This facility is used to draw color bars in an image 1 Display the Display panel of the image you want to draw color bars Eul 2 C
357. t Z T series switch gt button then from the displayed list of buttons click the lt XYZ series gt button To switch the image to the image of another moment in the elapsed time click the lt Z T series switch gt button then from the displayed list of buttons click the lt XYT series gt button The icon in the lt Z T series switch gt button will change to the icon of the selected button 3 Display the image to be displayed at the front by using the lt Display gt buttons at the top of the Display panel lt Loop gt button lt Z T series switch gt button Successive display or frame by frame display is repeated in the same direction lt Loop Bound switch gt button lt Set start position gt button When successive display or frame by frame display is 5 i required set the image with which the display should lt Display speed switch gt button start 0 is set when this button is not used Set the speed of successive display lt Set end position gt button When successive display or frame by frame display is required set the image with which the display should end Assuming that the number of images is n n 1 is set when this button is not used lt XYZ series gt button Displays one of multiple images by selecting it according to the multiple sections Z lt XYT series gt button Displays one of multiple images by selecting it according to the time T lt Bound gt button Every ti
358. t box Shows the time after end of acquisition of an image until the start of next acquisition Dyes Total Time text box Shows the total time required for image acquisitions f Opties Fig 2 17 Time Series Sub panel 2 Set the interval time using the lt gt or lt Y gt button in the Interval text box TIP I n image with no interval can be acquired by entering 0 in the Interval xt box this case the Interval text box shows Free Run message 2 52 Page APPLIED OPERATIONS Image Acquisition 3 Setting the number of scans 1 Set the number of scans using the lt gt or lt Y gt button in the N text box in the Time Series sub panel 4 Acquiring image Click the lt XYT gt button in the Acquire panel The acquired image will be displayed in the Live panel When the built in power supply for transmitted light illumination is used for a long period in the XYT mode the metallic parts may be expanded by heat causing the focusing to be deviated To acquire precise image data it is recommended to set the power switch of the transmitted light illumination to O OFF While acquiring an image in the XYT observation mode clicking the lt STOP SCAN gt button changes the buttons at the upper part of the Acquire panel as shown below The lt Resume gt button restarts image acquisition at the frame next to the frame where the acquisit
359. t the FLUOVIEW software Sections 1 2 1 amp 1 2 2 Acquire an image According to the selected Open an image in a file observation mode Section 2 3 2 For detailed operation procedures for image acquisition see section 2 1 1 Image Acquisition Procedure Section A Process the image Filtering Section 2 5 1 Contrast conversion Section 2 5 2 Inter image operation Section 2 5 3 Observe the image Observation of image shape Observation of image change Image display in simulated colors over time Section 2 4 1 Side by side image display LUT change Section 2 4 2 Section 2 9 1 Simultaneous display of multi channel Continuous display Frame by frame Images Section 2 4 4 display Section 2 9 2 Side by side image display Section 2 4 7 Magnified reduced image display Section 2 4 9 Stereo 3D image display Section 2 8 3 Color eyeglass 3D image display Section 2 8 4 Animation display Section 2 8 2 Continuous display Frame by frame display Section 2 8 1 Measure the image Measurement of image shape Measurement of image intensity Length measurement Section 2 6 3 1 value and distribution Area measurement Section 2 6 3 2 Intensity on a line Line profile Section 2 6 1 1 Intensity on a plane Bird s eye view Section 2 6 1 2 Intensity distribution Section 2 6 2 Save the image Section 2 3 1 Compile the presentation data Drawing characters on image Section 2 11 1 Drawin
360. tal scale Slices w The number of steps calculated by the system may be erroneous Slices text box unless the XZT observation mode has been set previously TIP The number of acquired images shown in the Slices text box can also b x input from the keyboard After setting Start Z Z direction scan start position and Stop Z Z direction scanning stop position input the desired number of images in th Slices text box This automatically sets Step Size number of steps 2 42 Page 5 Setting the interval time APPLIED OPERATIONS Image Acquisition 1 From the page tabs on the bottom right of the Acquire panel select the Time Series sub panel 2 Set the interval time using the lt gt or lt Y gt button in the Interval text box Interval text box Set the interval time using the lt A gt or lt v gt button or by input from the keyboard N text box Set the number of scans using the lt gt or lt gt button or by input from the keyboard Rest Time text box Shows the time after end of acquisition of an image until the start of next acquisition Total Time text box Shows the total time required for image I I TIP f text box gt GES 1 2000 68sec End 12 0 sec Fig 2 15 Time Series Sub panel p An image with no interval can be acquired by entering 0 in the Interval x In this case the Interval text box sho
361. ted in a narrow area or when observation of a specific area detail is required the image of a limited area can be selected The 4 buttons represent directions and clicking a button moves the acquired image area in the direction indicated by the button Clicking the square button on the center returns the acquired image area to the center e Click a point inside the circle to change the s gt position of the acquired image area directly Pan Zoom me Fig 2 10 Pan Zoom Group Box Clicking a point in the scale area to change the value on a large scale Clicking the top or bottom arrow button allows fine adjustment of the value Dragging the square knob allows the value to be changed directly For instance let us assume that the observation target is deviated at the top left of the acquired image With this example the area containing the observation target can be observed using the following procedure 1 Increase the zoom ratio using the Zoom scale in the Acquire panel 2 The light blue circle to the left of the Zoom scale represents the field visible through the microscope and the blue frame indicates the acquired image area Move the blue frame inside the circle using the lt Pan gt buttons so that the desired observation targets are displayed in the Live panel Page 2 23 APPLIED OPERATIONS Image Acquisition pO 9 Adjusting the image brightness Click
362. tegrated Intensity box The Integrity Intensity Trace window appears as shown below Integrated Intensity Trace lt Properties gt button Displays the Editing dialog box for use in detailed setting of the chart or change of the chart display See section 2 14 Changing the Chart Display Method for details lt Copy gt button Copies the plotted image in the Properties clipboard Copy lt Save gt button Save Saves the profile data in a file using an Excel compatible format put lt Close gt button Quits the Integrated Intensity Trace window and returns to the Analyze panel Fig 2 96 Integrated Intensity Trace Window HE C z I TIP lt When a desired area is specified by dragging the left button of the mouse on the graph the specified area can be magnified TIP When the right button of the mouse is dragged on the graph the graph ican be scrolled 2 202 Page When the mouse pointer is placed on a graph line while the Ctrl or APPLIED OPERATIONS Image Analysis The magnification or scrolling of the graph can be canceled by dragging the left button of the mouse from the bottom left to the right of lt k is held dep The displayed data can be applied to other applications See section 2 10 Transferring Data to Another Application for Page 2 203 APPLIED O
363. tended Focus View C Arbitrary View Method Brightest FE Stereo pair Sun Standard Views Default Number of views 2 gt Extended Fig 2 100 Panel Showing Extended Focus Image 2 208 Page APPLIED OPERATIONS Building an Image from a Different Viewpoint 2 7 1 3 Turning Built Image into time series image From XYZT image an extended focus image can be built as a separate time series image from the original image Use the Visualize panel to build the image First display the Visualize panel 1 Display the Display panel of the XYZT image 2 Click the Extended Focus View option button in the Rendered View group box 3 Click the lt Begin Visualize gt button to start the image building When it completes the built image is displayed in the Extended panel Pie Proces Analyze isustiz Live __ZvusutiF Y Extended i Rendered View Extended Focus View C Arbitrary View Method e Orientation Initial Rotation ne x asaf gt j n asaf gt j Kl E Standard Views Default Number of views xisi Extended Fig 2 101 Panel Showing Extended Focus Image 2 7 2 Building line images to be viewed in Z direction The images acquired by cutting the XYZ image off vertically and horizontally can be displayed with the information on each line Each line images is created as a separate image from the original one
364. tensity Analog 0 10V VIS1 and VIS2 are for invisible laser UV is for UV laser IR is for IR laser and Blanking is for switching laser ON OFF UV laser and IR laser are optional 1 8 Page Either the Kr laser or HeNe G laser can be used 1 Control cable 2 Interlock cable 3 Optical fiber cable 4 Analog signal cable 5 I O cable 6 Galvano control cable 7 Galvano X Y cable 8 Power control cable 9 Power cable 10 Laser power cable 11 RS 232C cable For connection to AOTF FV5 COMBA Coaxial connector Input and output terminals on FV5 LCU back Service connector For connection to FV5 PSU TD Dsub 25 nin male terminal For connection to FV5 TD Dsub 25 pin male terminal IS1VIS2 UV IR Blankin OOOO MQA QAQABE For switching signals output from the left connectors VIS1 and VIS2 are for invisible laser UV is for UV laser IR is for IR laser and Blanking is for switching laser ON OFF UV laser and IR laser are optional Event output signal 1 TLL Event output signal 0 TLL Trigger input signal 1 TTL Trigger input signal 0 TTL Horizontal sync signal TTL Vertical sync signal TTL SYSTEM OVERVIEW Software Functional Configuration 1 5 Software Functional Configuration This software uses panel type windows Usually it is required to select a menu then select the command to be executed in order to execute a function
365. ter is placed on a graph line while the Ctrl Alt key is held depressed the coordinates can be displayed 1 The displayed data can be applied to other applications See section 2 10 Transferring Data to Another Application fo mouse on the graph the specified area can be magnified APPLIED OPERATIONS Image Analysis a 2 6 3 Measuring a Part 2 6 3 1 Length Measurement The length between two points in an image or the perimeter of a region in an image can be measured The Measurement Results box in the Single sub panel shows the measurement results such as the length between 2 points Length or perimeter of the region Perimeter and the horizontal and vertical lengths X Y Z or T of the region and the statistic result of each channel such as the total Total average Average and standard deviation Std Dev The operation method for measuring the length between 2 points is identical to that for displaying the intensity profile of a line See section 2 6 1 1 Intensity Values on a Line Line Profile The operation method for measuring the perimeter of a region is identical to that for displaying the bird s eye view of a region See section 2 6 1 2 Intensity Values on a Planar Region Bird s Eye View A TIP The measurement results can be written in comment by copying and pasting them in the Image Comments dialog bo
366. tes until the oscillation stabilizes 4 Open the shutter lever of the laser tube 2 5 For the method of turning an IR 750 nm laser on refer to the instruction manual of the IR laser by MELLESGRIOT 2 6 HeCd laser 1 Turn the rocker switch 1 of laser controller to I ON 2 Turn the key switch 2 of laser controller to the ON position 3 Turn the key switch 3 of remote interface module to the ON position 4 Turn the lt ON gt button of the remote interface module ON After the key is set to ON it takes tens of seconds before the laser oscillation starts To stabilize the laser beam output we NOTE recommend leaving the system for warm up Remote interface module y after turning the laser power ON The warm up period should exceed 10 minutes when the laser system in use uses an Ar laser or exceed 30 minutes when it uses a Kr laser Green HeNe laser or HeCd laser 3 Turning the reflected light power supply unit 1 Set the power switch to ON Once the reflected light power supply unit is turned OFF do not turn it ON again for at least 10 minutes Otherwise the service life of the mercury burner will be shortened 1 20 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures Turning the computer PC ON When the power outlet unit is used turning the computer power ON also turns ON the power supplies to the microscope monitor power supply unit
367. the environment data input screen Fig D 1 NOTE During setting of the environment data the controls of the hand _ switch U HS U HSTR and the AX70 microscope frame e g lamp ON OF button will be inoperative NOTE Always remember to press the STORE button 2 after setting all the INIT data Fig D 1 If the STORE button is not pressed all the already set data will be erased when another screen MAIN PHOTO etc is selected with the mode selector buttons However the STORE operation is not required after setting the time only Page D 1 Appendix D U MCB Setting Mirror Cube Data Input CUBE Appendix D 1 Mirror Cube Data Input CUBE Please register the fluorescence cubes for fluorescence observations NOTE Please do not do full registration Leave at least one position NONE for use in LSM observation 1 When the CUBE button on the INIT input screen is pressed the CUBE DATA SET screen will appear as shown in Fig D 2 The data field 1 shows the currently entered values 2 When the cube button 2 corresponding to the cube to be engaged is pressed the screen shown in Fig D 4 will appear and the specified cube will be engaged into the light path 3 Rotating the JOG dial select the name of the cube engaged into the light path 4 When END 5 is pressed the first cube name is specified and the prev
368. the lt Divide Image by gt button in the Scalar Operations group box A new Be by gt Display panel showing Image Const in the page tab appears displaying the image obtained by the division operation 2 5 3 5 NOT Image The NOT operation of an image allows the bright and dark areas of the image to be reversed 1 Display the Logical sub panel of the Process panel at the front Sets the image to be subjected to Acquire File Vo Y Te Yroce iw Y wam 3 operation The icon of the image is eRe S disolaved here xY lt NOT Image gt button NOT Image Executes a NOT operation Image AND Image J Image OF Image Image XOR Image J xala Fig 2 81 Logical Sub panel 2 Display the Display panel of the image to be subjected to NOT operation at the front The icon of the image is displayed in the frame at the top left of the Process panel e Page 2 177 APPLIED OPERATIONS image Processing pO 3 Click the lt NOT Image gt button A new Display panel showing ImageNot in the eee page tab appears showing the image obtained by the operation u Live xvi Y imagetiot I Acquire File 0 Y Tile Yhroces ImageNot Drag Drop Dyefs to select setup Fig 2 82 ImageNot Panel 2 178 Page APPLIED OPERATIONS Image Processing 2 5 3 6 Image AND Image Two different images can be ANDed 1 Display the Logical sub panel of the Process
369. the lt Save gt button Page 2 109 APPLIED OPERATIONS Saving Opening and Shredding Images 12 A dialog box as shown below appears Size of the specified area Saving a subset of Experiment s Data P 8 3 gt b You are saving a rectangular sub region of sizd Sure you want to overwrite the existing file Fig 2 33 Saving a subset of Experiment s Data Dialog Box TIP he Saving a subset of Experiment s Data dialog box does not appear if ino area is specified in the image on the Display panel I 13 Click the lt Yes gt button 2 110 Page APPLIED OPERATIONS Saving Opening and Shredding Images 2 3 1 4 Saving Animation Images Transform a created animation image into the AVI file format and save it in the file type which can display the animation image without this software 1 Create an animation image Refer to 2 8 2 Animation for details 2 Display the Display panel of the created image at the front TIP The AVI file is created to save the animation at the display speed mode selected here For varying the animation display speed see section 2 8 1 3 Select whether saving images from multiple channels simultaneously or only an image from a channel if the images to be saved are acquired from multiple channels Use the lt Display Channel Switch gt button for this selection The image is saved according to the condition of the selected channel
370. the background thickness Dark 3D check box Enables or disables the shadowed 3D display This check box can be selected when the background thickness is specified P Transparent Size fo Dark 3D Page 2 259 2 260 Page Switches between the 3D and 2D chart display Sets the 3D chart display effect by varying the angle Enables or disables the 3D display in the depth wise direction Enables or disables the zooming display of character strings in the chart such as label characters together with the chart display 3D sub panel eries Used to set the 3D display APPLIED OPERATIONS Changing the Chart Display Method ries General Anis Titles Legend Panel Paging Wal Ds gt f azpo A Orthogonal Zoom Text Zoom Rotation Elevation Horiz Offset lt Vert Offset Perspective Change the zoom ratio Rotates the chart in the horizontal direction with respect to the bottom side Rotates the chart in the vertical direction with respect to the bottom side Moves the chart display position in the horizontal direction Moves the chart display position in the vertical direction Distorts the chart to change the display effect APPLIED OPERATIONS Changing the Chart Display Method 2 14 2 Series Panel Format sub panel Used to set the display method of a series of images The set items are variable dep
371. the change and closes the dialog box lt Apply gt button Saves the change without lt gt cancel button closing the dialog box Closes the dialog box without saving the change l 4 Page Appendix Change of Default Folder for File 1 0 Panel 6 Click the lt OK gt button to close the System Properties dialog box Now the default folder for the File I O panel will be changed from the next time you start FLUOVIEW e Wh en the default folder is changed to C IMAGES Acquire Y File VO Tile Y Proces XYZ 2 TIF Live XY Ch 1062 File Type Tift TIF 3D2 tif 120 3D4bf Ei 320 240 3D5 tif 330 323 3D6 bif Ei 400 300 aa Ltt Hi New default folder C IMAGES is being Em open Open Save Save Display File Selection TIP gt To reset the default folder to the factory setup enter C FLUOVIEW ag ae T me NOTE Do not change the setup after FLUOVIEWIMAGES in the User Variables for lt username gt list box If erroneous setting is made here the system will be unable to be started up Page l 5 Appendix J List of Functions in the Active Overlays Dialog Box Coordinate Position Data Appendix J List of Functions in the Active Overlays Dialog Box Active Overlays is a kind of overlay function displayed on an image Active Overlays does not simply show the entered characters but searches the image data re
372. the mouse on the comment to be resized to make it active i e handles displayed around it 2 Click the right button of the mouse A pop up menu as shown in Fig 2 118 appears 3 Select Size from the menu 4 The menu pointer turns into x When the mouse is moved the comment is magnified or reduced according to the mouse pointer 5 Click the left button of the mouse to determine the size Handle gt TIP s A comment can also be magnified or reduced by selecting it placing the mouse pointer on one of the handles so that the mouse pointer turns into g then dragging the mouse mmm e Page 2 239 APPLIED OPERATIONS Entering Comment in Image pO 2 11 11 Changing the Comment Color n O_ 0 11 anat Changing the Comment Geter _______________ the Comment Color Click the mouse on the comment to be changed of color to make the comment active i e handles displayed around it 2 Click the right button of the mouse A pop up menu as shown in Fig 2 118 appears 3 Select Properties from the menu The Properties dialog box as shown below appears Display the Color panel at the front Properties Color Set NI Color Standard Colors Color Palette General Edit Custom Color Cancel Apply Fig 2 119 Properties Dialog Box 4 Selectthe desired color from the Color Palette list box TIP To change the comment color automatically set in the FLUOVIEW Setu dialo
373. this may damage the Z motor 3 While observing the image in the Live panel locate the plane to be observed by displacing the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel 1 2 8 8 Setting the Area to be Observed When the observation targets are concentrated in a narrow area or when observation of a specific area detail is required the image of a limited area can be selected The 4 buttons represent directions and Pan Zoom Clicking a point in the scale area clicking a button moves the acquired image a to change the value on a large area in the direction indicated by the button scale Clicking the square button on the center Y Clicking the top or bottom arrow returns the acquired image area to the center button allows fine adjustment of the value Y Dragging the square knob allows Click a point inside the circle to change the x1 the value to be changed directly position of the acquired image area directly Fig 1 13 Pan Zoom Group Box e Page 1 53 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 8 9 Setting a Lower Scan Speed The scan speed can be decreased using the scale in the Scan Speed group box on the Acquire panel TIP in general setting a lower scan speed allows the acquired image quality t x be improved However a low scan speed also lengthens the time required for im
374. thod and following the displayed guidance information e Selecting the Dyeing Method 1 From the page tabs on the bottom right of the Acquire panel select the Dyes sub panel F Selected Dyes Assign dyes manually check box Checking this enables the manual setting Dragging the dyeing method 7 directly to the Ch group box assigns gt Assign dyes manually the dyes to the desired channel Available Dyes Place the pointer on the icon displayed in the Selected Dyes and the dyeing method is shown in the pop up display lt Clear gt button Clear the set dyeing Available Dyes list box method Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method lt Apply gt button Applies the dyeing method dragged in the Selected Dyes group box to the Ch group in the Acquire panel lt Prev gt button Sets the dyeing method which was set last time by clicking the lt Apply gt button Fig 1 17 Dyes Sub panel 2 Select the specimen dyeing method by dragging desired dye names in the Available Dyes list box in the Selected Dyes group box to the field immediately above the list box 3 Click the lt Apply gt button to apply the selected dyeing method to the Ch group box on the upper part of the Acquire panel e Page 1 61 Getting started FLUOVIEW Online Help K TIP When the dyeing me
375. thod is selected from the Available Dyes list box an the lt Apply gt button is clicked a channel for acquiring fluorescence is s automatically according to the changed filter And the dyeing method i shown in the Ch group box The Confocal Aperture value is also set automatically according to th w velength and the 1 62 Page Getting Started FLUOVIEW Online Help One Point The Assign dyes manually check box can also be used to set the dyeing method to the desired channel 1 2 Select the dyeing method in the Available Dyes field of the Ch check box XY Repeat Once TR Chanel T T T PMT Gain Offset Assign dyes manually check box 3 M FITC f Gain offa E ex 800v 10 0 Y Check the Assign dyes manually check box in the Dyes sub panel ist box and drag it directly to the Acquire After dragging the icon appears on the right of the Ch check box and the dyeing method is set The dyeing method is set Icon Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Page 1 63 Getting started FLUOVIEW Online Help 1 3 2 1 Configuring the Microscope 1 Display the Acquire panel Focus XY Repeat Once Hin DII M Transmitted PMT Gain Offset f i 320v x10 0 xY CxYT C xYz CxYzT Sean Mode
376. tically sets Step Size number of steps III Page 2 57 APPLIED OPERATIONS Image Acquisition 3 Setting the observation mode 1 In the Scan Mode group box in the Acquire panel select the Surface option button 2 In the Acquire panel select the XYZT observation mode option button 4 Setting the interval time 1 From the page tabs on the bottom right of the Acquire panel select the Time Series sub panel A panel as shown below will be displayed Interval text box Set the interval time using the lt x gt or lt Y gt button or by input from the keyboard N text box Set the number of scans using the lt gt or lt v gt button or by input from the keyboard 1 2000 Rest Time text box Shows the time after end of 0 8 sec acquisition of an image until the f start of next acquisition Total Time 12 0 sec Total Time text box Shows the total time required for image acquisitions Fig 2 19 Time Series Sub panel 2 Set the interval time using the lt gt or lt Y gt button in the Interval text box s4 TIP n image with no interval can be acquired by entering 0 in the Interval xt box i this case the Interval text box shows Free Run message sing lt v gt button also sets the Interval text box to Free Run 2 58 Page APPLIED OPERATIONS Image Acquisition I 5 Setting the number of scans
377. tion dichroic mirror for spectroscopy confocal aperture and emission filters are set automatically 12 When using the system with AOTF FV5 COMBA the following functions are available Line Kalman scanning Line sequential scanning Image acquisition in the REX Bleach mode FRAP experiment 1 2 Page SYSTEM OVERVIEW Light Path Diagram 1 3 Light Path Diagram PMT 1 Pinholes1to4 _ Emission DM turrets 1 to 3 Confocal ps lenses 1 to 4 PMT3 za f vA a sat Bb PMT4 lt oy ag ra ee Sy s v cai ae lt gt t b aN Emission filter turret Ti ss s Ys OS COS is a r gt To IR laser unit i IR gt lt Ou UV laser unit f x Fa UY XK i b Ces laser VIS combiner DA P a ak ae J i XIY galvano Excitation NAS lt 22 k 3 Mirrors a to 2 DM turret gt lt Mirrors Page 1 3 SYSTEM OVERVIEW Ssystem Configuration 1 4 System Configuration 1 4 1 System Component Units and Their Functions Laser combiner Scan unit Unit for merging laser beams from the lasers Heart of a laser microscope composed Transmitted light detector such as an Ar laser Kr laser and HeNe laser of the light scanner and light detector unit into a single beam to be transmitted through Unit for acquiring transmitted the optical fiber cable images Connected with the microscope through an optical fiber cable Microscope AX70 upri
378. tiple sections Z lt XYT series gt button Displays one of multiple images by selecting it according to the time T lt Bound gt button Every time successive display or frame by frame display in a direction ends at the start or end position the display is restarted in the opposite direction lt Display gt button Press to start display of n slices of image from the start position to the end position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame lt Rabbit gt button Press for successive display of image slices without pause lt Clock gt button Press for successive display of image slices at the time interval set in the Time Series sub panel lt Turtle gt button Press for successive display of image slices at an interval of 0 8 sec lt Display gt button Press to start display of n slices of image from the end position to the start position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame 2 244 Page I I APPLIED OPERATIONS Merger Extraction of Image Channels Doo 3 Display the image slice to start the range at the front using the lt
379. tment gt and lt Z stage fine adjustment gt buttons 3 When the upper edge position is located click the lt Set gt button The Start Z text box will show the scan start position of the range of the multiple sections to be observed Z direction scanning range 4 While observing the image in the Live panel locate the bottom edge of the range to be observed by moving up the stage using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the upper edge of the range to be observed by moving up the revolving nosepiece using the lt Z stage coarse adjustment gt and lt Z stage fine adjustment gt buttons 5 When the position is located click the lt Set gt button The Stop Z text box will show the scanning stop position of the range of the multiple sections to be observed Z direction scanning range 2 Setting the observation mode 1 In the Scan Mode group box in the Acquire panel select the Depth option button 2 In the Acquire panel select the XZ observation mode option button 3 Setting the observation line 1 Aline is displayed on the image in the Live panel Place the mouse pointer arrow on the line and drag it to the position you want to observe 2 34 Page APPLIED OPERATIONS Image Acquisition Doo 4 Setting the numbers of steps and acquired image slices
380. tons Set the repetition direction of the successive display or frame by frame display lt Display gt button Press to start display of n slices of image from the start position to the end position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame lt Display speed switch gt button Set the speed of successive display lt Display gt button Press to start display of n slices of image from the end position to the start position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame A 2 Page Appendix B List of Hot Keys Frequently used FLUOVIEW functions scanning panel switching can be controlled from the keyboard without using the mouse Image acquisition related keys Image acquisition channel selection Target Operation Cttrl Switches the status of the Ch1 check box Ch1 scanned not scanned Cttrl Switches the status of the Ch2 check box Ch2 scanned not scanned Cttrl Switches the status of the Ch3 check box Ch3 scanned not scanned Cttrl Switches the status of the Ch4 check box Ch4 scanned not scanned Cttrl Switches t
381. tted light observation be sure to disengage the filter 6 from the light path Page 1 41 Getting started FLUOVIEW Outline of LSM Observation Procedures 4 Transmitted light DIC slider FV5 DICT optional 1 Light path selector knob 2 Cube turret 3 Analyzer FV5 ANI 1 42 Page e Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 5 Selecting the Dyeing Method 1 From the page tabs on the bottom right of the Acquire panel select the Dyes sub panel Selected Dyes oo Assign dyes manually Available Dyes Rhodamine Phalloidin Texas Red Fig 1 10 Dyes Sub panel 2 Select the specimen dyeing method by dragging FITC and TRITC in the Available Dyes list box in the Selected Dyes group box to the field immediately above the list box Assign dyes manually check box Selected Dyes Checking this enables the manual setting Dragging the dyeing method in the list directly to the Ch group box assigns the dye to the desired channel Place the pointer on the icon displayed in the Selected Dyes and the dyeing method is shown in the pop up display Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method lt Clear gt button Clear the set dyeing method lt Apply gt button Applies the dyeing method dragged in the Selected Dyes gr
382. u susu A T Seats apes aus ies ceshcenec neces 1 4 15 The image cannot be saved in the disk 1 4 16 The image cannot be output at the printer 1 4 17 The FLUOVIEW software cannot be started 1 4 18 The mouse pointer in the screen cannot be moved by moving the IOS Soe cece ac su N Su CUA ua E 1 5 19 The power supply of Power Unit FV5 PSU is not turned on 1 5 20 The current limit indicator on the UV Ar laser power supply is DIINKINO U Noe aster ete Ra SGS a fe anne ree Paar T aes err 1 5 21 The error indicator on the UV Ar laser power supply will not turn OTE aa ees ess bec esas os es labakaghnayhikukesiqhtaphiuusica TE 1 5 22 Bios some check error message appears at the moment of Starting up the computer J 1 5 TROUBLESHOOTING GUIDE 1 TROUBLESHOOTING GUIDE Under certain conditions performance of this system may be adversely affected by factors other than defects If a problem occurs please review the following list and take remedial action as needed If you cannot solve the problem after checking the entire list please contact your local Olympus representative for assistance Before contacting Olympus please fill the Inquiry Table at the end of this volume and inform Olympus of its contents Phenomenon Cause Treatment Manual Ref Pages 1 Fluorescence imag
383. ubject to large vibrations 6 Image is irregularly The specimen is tilted Set the specimen Instruction manual blurred or the horizontally of microscope brightness is uneven 7 Image is flared Non fluorescent glass is not Use non fluorescent glass The cover glass thickness is Use a cover glass with a not optimum thickness of 0 17 mm The specimen is over Stain it properly again or 2 2 1 4 9 stained increase the OFFSET value OPERATION INSTRUCTIONS 8 Circular flare is The excitation wavelength Select suitable barrier filter 1 2 6 amp 1 3 2 4 observed at the center does not match the barrier for the excitation length OPERATION INSTRUCTIONS of image filter type or the barrier filter is not used 9 Image is blurred or out The focusing is not correct Adjust focusing with visual Instruction manual of focus observation of microscope 10 Image is dark and The fluorescent dye is pale Apply optimum fluorescent noisy dye The pinhole diameter is too Adjust to an optimum size 1 3 2 5 OPERATION INSTRUCTIONS The HV of photomultiplier Set HV at no more than 800 2 2 1 4 9 tube exceeds 800 If the image is still dark OPERATION adjust GAIN INSTRUCTIONS The scan speed is too high Decrease the scan speed or 1 2 8 9 amp 2 2 1 6 integrate the image OPERATION INSTRUCTIONS The excitation light is weak Increase the transmittance 1 2 7 1 3 2 4 amp 2 of the ND filter 2 1 4 10 O
384. uire panel e Page 1 27 Getting started FLUOVIEW Outline of LSM Observation Procedures 1 2 3 3 Combination with IX 1 Turn the light path selector 1 on the right side of the microscope to 2 Engage the optimum cube for specimen dye by operating the cube turret 2 3 Focus on the specimen by looking into the eyepiece Be sure to adjust the diopter of the eyepiece in advance Refer to the instruction manual of the IX50 70 microscope When the Z motor is in use clear the check mark in the Locked check box in the Z Stage sub panel in the Acquire panel see section 2 2 1 4 7 of this volume then focus on the specimen by operating the fine focus adjustment knob of the microscope Be sure not to operate the fine focus adjustment knob while the Locked check box is checked for this may damage the Z motor Oe f s The specimen may float during oil immersed observation In this case prepare a stage clip U SCL and attach it to the microscope as shown on the left 1 28 Page e Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 2 3 4 Combination of IXBP Bottom Port 4 Turn the light path selector 1 on the right side of microscope to 2 From the index tabs on the bottom right of the Acquire panel select the Optics sub panel Light Path BI LSM Microscope Control Trans Scope SU Lamp Control Control Fig 1 4
385. uisition SCAM werner Sets the zooming ratio and observation mode for image acquisition Sets the Z direction scanning range for image acquisition Sets the interval period for image acquisition Sets the fluorescence dye method for image acquisition and reagent for each channel Sets the microscope light path for image acquisition Z Stage Time Series Dyes Sets the ND of laser for image acquisition File Q uuu aaa aaa aaa Saves loads and deletes images Miles usan Sousa alae Changes the image display method Proces Sadoni Processes the acquired images Math 1 Performs mathematical operations between images al ke Filters images Histogram Changes the image contrast ROGICA eniinn Performs logical operations between images Analyze a Analyzes image data Obtains the intensity values intensity distribution length area and average intensity in images Obtains the change in the sum of intensity values in images Constructs an image from a different viewpoint or displays a 3D image L Orientation 9 Sets the image rotation angle direction and number Other Options Sets the various 3D Rendering 1 10 Page SYSTEM OVERVIEW Software Functional Configuration 1 5 3 Icons Executed by Dragging amp Dropping This software selects image files and observation methods dye name by means of dragging amp dropping This allows simple selection based on an intuitive ope
386. ulation Mode Frame mode 1 From the page tabs on the bottom right of the Acquire panel select the Scan sub panel Acquisition Settings Load Save 5 wl Focus Mode X2 C x4 Filter group box Select the accumulation mode Filter Two accumulation modes Kalman and Normal F Accumulate To Peak are available C Kalman 4 C Accumulate To Peak Fig 2 11 Scan Sub panel 2 Inthe Filter group box select the Kalman or Accumulate To Peak option button 3 When Kalman is selected enter the accumulation count in the text box And select the Frame Kalman option button displayed r Acquisition Settings Load Save m Focus Mode x2 Ox4 Filter O Hormal Kalman Frame Kalman option Mode button Frame Kalman Line Kalman Enter the accumulation count S TIP The accumulation count can be set up a maximum of 63 times ee hen 0 is set as the number of times of accumulation an ordinary image i acquisition is to be performed 2 28 Page APPLIED OPERATIONS Image Acquisition bs 4 When Accumulate To Peak is selected enter the addition count in the text box 5 Click the lt Once gt button in the Acquire panel The acquired image will be displayed in the Live panel 2 Acquiring Image in Accumulation Mode Line mode NOTE Image acquisition in the line mode can be performed when you use the FV3
387. uonnq pis q pis sobew apis Aq apis Bunes ON uO Ji u6j poqe 0 ON uO si QUByapoge SOWEU IJ BACS CUD WO Ajuo pares ase sobeul jefe suo j ng Yyoyms jauueyo Aeldsiq paxe dsip s uo luo u y mM 9lduiexs uonipuoo3 p e dsip J pun uugu2 e unes abeg Hq 7c QLbL Z panes aq ueg sebew Buipp ius pue Buiuedo Bunes SNOILYN dO a3llddY LLL obed b ON 49 HY L LAuBJ poqe C ON Lu9 J 6Au6J poqe Z ON uO J eA YyByopoge Z ON LUD sl QAYByepoge L ON uO J GAYUBjopoge snyejs L ON LuO pepue x9e uou eulBUO Jy eaybyepoqe y Ul pores s Hew GUL on uO ZuO Luo 0 ON uO A y y uo sabeu Ue ee n p s pue SEBEW on suns zuo iuo JF OAUByepoge pis q pis uonipuoo Jy QUBjepoqe S WLU lJ SACS p eldsip Jepun lt SOWEU Oj SACS uo jouueyo e Bunes s g ON eyo IguIBlio pue ZYD LUO 40 yore SIOWGEUS duuiq u6j poqe i u uey Jood ae sobew woud panes ase s Bgui y ooN eu jo 19quinu Buipuods Jio5 ay uie uo5 seme elo one pee ie sQ SA IA BAOGE Y duuq ou6j poqe JOU s op aI e yey ON panes ase s BEuul Ul pasn sj uueyo y JO SOWEU Oil SACS panes uonipuoo jauueyo p 5 s eyo pue zy yo BulBiew sass abe y sujezuoo SUD wo q ueg y u panes s oBew oul Aq p uiezqo sobew ew pa
388. utlet unit their power is turned ON or OFF simultaneously according to the power ON OFF of the computer When it is required only to read data it is not required to turn the laser power supply and reflected light power supply units ON Power unit supply 1 Set the power switch of the computer PC ON The power to the following units is turned ON in interlocking with the power of the computer wass e Power supply unit e Monitor Refer to the instruction manual of your monitor a ul XVN Since these units are permanently connected to the computer leave their power switches set to the ON position NOTE It takes about 2 minutes after power supply unit turns ON until it is initialized Before proceeding to any other operation wait for about 2 minutes after turning power ON Ar laser supply 2 Turning the laser power ON 2 2 1 Ar laser 1 Set the power switch to ON 2 Turn the key to the ON position The laser fan will start 3 Turn the key further in the clockwise direction The key will return to the ON position The laser starts oscillation in some tens of seconds after the key is set to ON 1 18 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures 2 2 Kr laser 1 Set the power switch to ON 2 Turn the key to the ON position The laser fan will start 3 Turn the key further in the clockwise direction The key will return to the ON position
389. v 40 0x 186 43x 0 C A Ar Each click of the lt gt or lt gt button of the C A LED slider increases or decreases the pinhole diameter by 5 nm Each click of the slider section of the C A LED slider varies the pinhole diameter by 25 nm Each click of the lt gt or lt gt button of the Laser ND LED slider increases or decreases the laser ND value by 1 x Each click of the slider section of the Laser ND LED slider varies the laser ND value by 5 To set to the Confocal Aperture value to its primal value click the lt Apply gt button in the Dyes sub panel of the Acquire panel Page 1 77 APPLIED OPERATIONS General Operation Procedure 2 APPLIED OPERATIONS 2 1 General Operation Procedure This section describes the general image acquisition procedure with the aim to get accustomed with the operation Begin using the FLUOVIEW system by acquiring an image or opening an image from a file The procedures for the subsequent operations such as image processing are not in question here For the detailed operation method of each item in the procedure see the section specified in parentheses NOTE The following is the general operation procedure of FLUOVIEW Many other functions that are not shown in the following are also available Please also study their description Page 2 1 APPLIED OPERATIONS General Operation Procedure Turn power ON and star
390. vation The lt DIC gt button performs transmitted light DIC observation The lt BF gt button performs transmitted light brightfield observation The lt PO gt button performs polarized light observation lt Use MCB gt button Select to set the items other than those shown in the AX Control window by using the U MCB After the setup using the U MCB click this button again to apply the set up contents in the AX Control window Enable MCB during BI TV Observation check box The operation with MCB is possible when this check box is checked and the current observation mode is BI TV observation Shutter group box Opens or closes the shutter of the transmitted light lamp The shutter opens when lt OPEN gt anak button is selected The shutter closes when lt CLOSE gt button is selected Objective group box Selects the objective from OLymPus the drop down list AX70 Oo AS group box Changes the value of AS FS group box Changes the value of FS T Engble MCB during BILTV Observation Filter group box Lamp group box lt LBD gt and lt IF550 gt buttons Switches the transmitted lighting lamp Selects whether color correction filter is engaged or not ON or OFF Click lt gt button to make the filter darker ibe Amp is ON when lt ON gt button is 7 selected Click lt q gt button to darken the filter by one step The lamp is OFF when
391. ws Free Run message _Using lt gt button also sets the Interval text box to Free Run 6 Setting the number of scans 1 Set the number of scans using the lt gt or lt Y gt button in the N text box in the Time Series sub panel Page 2 43 APPLIED OPERATIONS Image Acquisition 7 Acquiring image 1 Click the lt XZT gt button in the Acquire panel The acquired image will be displayed in the Live panel While acquiring an image in the XZT observation mode clicking the lt STOP SCAN gt button changes the buttons at the upper part of the Acquire panel as shown below The lt Resume gt button restarts image acquisition at the frame next to the frame where the acquisition is suspended STOP SCAN lt Resume gt button Restarts image acquisition at the frame next to the frame where the suspended 8 Appending image lt Append Next gt button Acquires another image and appends i to the image acquired immediately before 2 44 mode acquisition is Ag File YO Tile Proce m Sq T rensmimea lt Series Done gt button Determines the acquired images Once this button is clicked it is not possible to append an image An additional image can be appended to the image acquired in the XZT observation Immediately after acquisition of an image in the XZT observation mode the buttons at the upper part of the Acquire panel chang
392. x and drag it directly to the field of the Ch check box e EE To assign dyes Drag and drop channel either flo or from the dye i Assign dyes manually kene check box After dragging the icon appears on the right of the Ch check box and the dyeing method is set M FITC The dyeing method is set Gain Offs i pa Se 800v 10 0x 7 Icon Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method 2 64 Page e APPLIED OPERATIONS Image Acquisition bs e With types of dyeing of the specimen to observe the Barrier Filters will be set automatically to the optical path To change the Barrier Filter types see to section 1 3 2 4 Configuring the Filters and change by Optical System Configuration window Make the channels ready for image acquisition In the Acquire panel make sure that the check box in the Ch1 Ch2 Ch3 Ch4 group boxes that show the dyeing methods are check marked to indicate that the corresponding channels are ready for image acquisition Ch2 check box y Ch3 wassap ama box Ch4 check box FITC an Transmitted Ch1 check box Sar cn Offset ea Offset ma c Er offset pi Offset PMT Gain Offset 800v 10 0x a 800v 10 0x Z ma 10 0x i 800v 10 0x Y 186v 43x 0 When observing a fluorescence image s simultaneously also set the brightness of the fluorescence image s Adjust the b
393. x in the Experiments in Memory dialog box 2 6 3 2 Area Measurement The area of a region in the image can be measured The Measurement Results box in the Single sub panel shows the measurement results such as the area of the region Area perimeter of the region Perimeter and the horizontal and vertical lengths X Y Z or T of the region and the statistic result of each channel such as the total Total average Average and standard deviation Std Dev The operation method is identical to that for displaying the bird s eye view of a region See section 2 6 1 2 Intensity Values on a Planar Region Bird s Eye View Page 2 195 APPLIED OPERATIONS Image Analysis pO 2 6 3 3 Measuring the Change in Mean Value of Intensity The mean value of the intensity in a region specified in an image can be measured and displayed graphically 1 Display the Series sub panel at the front 2 Display the Display panel of the image that you want to check the intensity at the front 3 When the image is composed of multiple image slices operations will be applied to each slice of the image When the image is composed of a single image slice or when one of the multiple image slices is selected the Single Slice Integration group box appears as shown below Select the direction of interest X or Y using the option buttons Intensity vs X l Single Slice Integration e x Oy NOTE When the selected
394. y to the OFF position and wait for the fan to stop automatically when the laser unit has cooled down Then set the power switch to OFF Refer to the instruction manual of your laser unit e HeNe Green Red laser Turn the key to the OFF position UV laser power supply UV Arlaser 1 41 Turn the key switch on the UV laser power supply to the O OFF position 1 2 Set the MAIN POWER lever on the UV laser power supply to the O OFF TIP Chiller is switched to auto cooling operation by interlocking When it is left in auto cooling it will automatically turn off i LASER POWER While the heat exchanger is running do not shut off the power supply to the power outlet where the chiller is MAIN POWER Key switch connected If the power supply is turning on the UV Ar laser tube may be destroyed by high temperature After the heat exchanger has turned off leave it for more than an hour until the laser tube has cooled down before turning it on again 1 58 Page Getting Started FLUOVIEW Outline of LSM Observation Procedures IR laser Refer to the instruction manual of the IR laser manufactured by MELLES GRIOT HeCd laser 1 Turn the lt OFF gt button of remote interface module OFF Wait till LASER OFF message appears on digital display For about 5 minutes to cool it down 2 Turn the key switch of remote interface module to the OFF position Turn the key switch o

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