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DeltaVision® Core and personalDV

1. wt Reso Nead ee File view Options Calibration Meip7 The Resolve3D Menu includes N a a comma nds for acquinng Acquire Experiment Seyings images controlling marked Excitation CFP oy Sos 10 points specifying image and ee m display settings and calibration Emission CFP 470 SQ see Page 183 T BLOCK1 x EX Shutter Ex o Q Q The Resolve3D Toolbar allows aoe N Y you to acquire images open Exposure 1 000 TRG Calibrate the Design Run Experiment Image size 512x512 dia log to set up and run r experiments and open the Lens 10 v wo Settings dialog to control Bin ixi Fg dete Wham display image and file options see Page 186 Pixel size 0 6680 um o zaa 24 dx 10 00 Image Control FRelds allow you a to choose filters exposure time a lenstype image size and binning settings see Page 187 dy 10 00 Stage Position Contol Fields and 2 Buttons can be used to move S the sage and mark points of E interest see Page 189 Xo Blue box and barcan be dragged to move the sage in X Y and Z see Page 193 Pr eee OE MSE EEEE We O00 6 00 0 00 0 Max 0 Mean ie Image Intensity and Scale Values display image properties in numencal and graphical formats see Page 193 eee Scale min Scale max 65535 A arre The Message Pane reports the status
2. ppliedPrecision Chapter 7 Facility Requirements and Components 101 Desktop Components Desktop Components include the flat panel display monitor the keyboard the mouse the keypad and a joystick DeltaVision Core includes a vibration isolation table personalDV includes a bench top microscopy isolation platform Hat Panel Display Monitor All DeltaVision systems are equipped with Flat Panel LCD monitors These monitors offer a very high level of performance in several areas pertaining to the quality of displayed images the most critical of which is contrast ratio LL Note For instructions that show how to adjust the monitor see the Flat Panel Display manual CAUTION DeltaVision is configured to work with the monitor that is included with the system Other monitors are not necessarily supported The Keypad and J oystick Many of the functions accessible through Resolve3D are also available on the keypad joystick see Keypad Joystick Operation on Page 211 04 720104 Rev D 1008 102 DeltaVision Core and personalDV User s Manual Vibration Isolation Table Within the vibration isolation table is a breadboard surface that is supported by mechanical vibration isolators These components provide optimal performance without an external air source The isolators are sized for the system weight as delivered If significant additional weight is added to the breadboard higher capacity isolation may be requ
3. 5 Press the power button on the IC MIC once to shutdown 6 Turn off the monitor 7 Turn off the power strip bar switch LU Note For personalDV there isno power rip bar For personalDV skip this step 8 Clean the objective see Turning DeltaVision Off on Page 30 9 Lower the objective Starting the System Use the following instructions to start the system after a total shut down To start the DeltaVision System 04 720104 Rev D 1008 134 DeltaVision Core and personalDV User s Manual 1 Turn on the power strip bar Note For personalDV there isno power strip bar Begin the power up process with Step 2 2 Turn on the IC MIC 3 Turn on the workstation 4 Turn on the monitor 5 Follow the instructions for turning on DeltaVision on Page 19 Replacing the Xenon Bulb WARNING Ensure the xenon lamp isoff and hashad plenty of time to cool before starting this procedure Referto the Xenon Lamp Safety section in Chapter 2 of this manual for details on safety issuesand proper disposal of the lamp Follow these steps to replace the xenon bulb on DeltaVision 1 If the system is on exit Resolve3D and ensure that the IC MIC is off prior to proceeding The fan on the lamp housing must be off before you begin this procedure 2 Loosen the three hex screws in the flange of the xenon lamp housing 3 Gently slide the lamp housing away from the flange to remove it from the DeltaVision excitatio
4. Lt Note Aligning for Critical and Kohler illumination is an iterative process The alignment will improve with each iteration 5 When the image is centered tighten the clamping screws on the Fiber Alignment Disk 04 720104 Rev D 1008 150 6 f Delta Vison Core and personalDV Users Manual gt F r 2 gt FOC Alignment Clamping Screws E X Axis FOC Alignment Aq 3 Screw Clamping Screws n Ra Fully close the Field Aperture and slowly rotate the microscope Z focus knob in and out of focus The light should evenly expand around the aperture If the light tends in one direction call the API Customer Service Hotline 1 800 862 5166 Move the Fiber Optic Module between the Kohler and Critical position several times and make sure that the positions are repeatable within about 1 If the Kohler and Critical positions are not repeatable call the API Customer Service Hotline 1 800 862 5166 Replacing IC MIC Fuses Follow these instructions to replace a fuse in the Microscope Interface Chassis To replace fuses for other components follow the instructions in the manuals that are provided for those components To replace a fuse 1 2 CAUTION Installation of improperly rated fusescan cause damage to the system Shut down the system Unplug the power cord on the back of the IC MIC Remove the fuse holder Test the fuses with a continuity meter Replace any bad fuse
5. 6 Specify the area around the cell the region of interest or ROI that you want DeltaVision to use for image recognition For more about this option see ROI Percent on Page 72 7 If you are performing an experiment on a single cell use the stage controls to center the cell laterally in the field of view If you are performing a point 04 720104 Rev D 1008 70 DeltaVision Core and personalDV User s Manual visiting experiment make sure that the points that you are monitoring are centered before you start your experiment 8 Click the Run Experiment tab and then click Start Scan to run the experiment Guidelines for specifying Cell Tracking Options You can specify several parameters that DeltaVision uses to track cells a Tracking Method Reference Channel Move Threshold a ROI Percent Trac king Method The tracking method is the method that DeltaVision uses to determine the center of the feature This center is recalculated after each image is acquired You can choose from two tracking methods a Center of Intensity calculates the center of the feature based on intensity values a Center of Geometry calculates the center of the feature based on its geometry Center of Intensity Center of Geometry Reference Channel DeltaVision uses the Reference Channel for pattern recognition If you are acquiring more than one channel choose the channel that has the most distinctive features LL Note The tem Re
6. micropositioning technology Before long Dr Sedat contacted Applied Precision cofounder Ron Seubert for detailed information about Nanomover performance The relationship between UCSF and Applied Precision grew steadily Later in 1993 Applied Precision licensed the image restoration technology from UCSF and began development of DeltaVision Collaboration between Applied Precision and UCSF still continues for the benefit of both parties In October of 1993 Applied Precision shipped the first Applied Precision UCSF hybrid to Michael Paddy at the University of Florida In 1994 Applied Precision designed built and delivered a DeltaVision prototype to Paul Goodwin at the Fred Hutchinson Cancer Research Center Seattle In August 1994 the first commercial ppliedPrecision Chapter 1 Introduction DeltaVision microscope was shipped to Bethe Scalettar at Lewis amp Clark College Portland Oregon All three of these systems are still active The DeltaVision software has grown continuously since 1983 with contributions from scientific programmers faculty and graduate students at UCSF Applied Precision s contribution to the software started in earnest in 1994 The advances in computer and camera technology in the early 1990s resulted in the emergence of optical sectioning technology For example in 1993 Applied Precision s benchmark deconvolution 512 x 512 x 64 required 3 hours of processing time on a 35 000 workstation The
7. Bright Light Exposure While the transmitted light source installed in the microscope does not present possible UV exposure it could cause discomfort under certain conditions You must be aware of the eyepiece filter wheel when viewing a specimen through the microscope oculars Make sure that the proper filters are in place so that your eyes are not suddenly exposed to a bright flash of light when the transmitted light shutter is opened Do not look through the eyepiece while switching filters Your eyes can be exposed to unfiltered light during the filter transition The xenon arc lamp reaches a very high temperature when lit Never touch the housing during operation Never remove the housing during operation or before allowing it to cool completely Carefully follow the directions found in Chapter 9 Maintenance tor changing the lamp Shock Hazardous voltages are present even when the system is disconnected from the AC main power outlet To replace the transmitted light source LED follow the instructions in the manuals that are included with your microscope To replace the xenon arc lamp follow the instructions found in Chapter 9 Maintenance Refer also to the following section Xenon Lamp Safety for additional related safety issues No other system components contain user serviceable parts and do not warrant disassembly If the High Res Camera coolant fluid is leaking shut down the system and contact Applied Precisio
8. Cy5 645 30 705 72 Emission filter only Cameras DeltaVision Core comes standard with the CoolSNAP HQ CCD charge coupled device camera referred to as the High speed Camera and also supports an upgrade option of the Cascade II EM CCD camera For more about the Cascade II camera see EM CCD Camera on Page 111 04 720104 Rev D 1008 98 DeltaVision Core and personalDV User s Manual personalDV comes standard with the CoolSNAP ES Camera High Speed Camera The High Speed CCD camera CoolISNAP HQ collects digital image data from the microscope This camera is designed to collect image data at a high frame rate At the fastest speed the camera can collect 30 frames per second of a 64 x 64 pixel image Increased acquisition speed is useful when collecting images of live cells that deteriorate over time The High speed Camera The High speed Camera is air cooled The cooling apparatus is incorporated into the camera assembly therefore when the High speed Camera is turned on the cooler is also operating When using cooled cameras in humid conditions it is possible for condensation to form on the CCD camera window If this happens a mottled pattern is superimposed on the images You will need to lower the ambient humidity level to avoid condensation Notes 1 The High speed Camera controller is built into the camera head The High Speed Camera hasno userserviceable parts inside 2 If no imagesare acquired o
9. Design PK Experiment Run Experiment Experiment name Resolves Estimated File Size 96 03 Mb Enable Fast Acquisition fast Acuuisition Uintons Sectioning l Channels l Time lapse l Points Panels Actions F Collect Panels Overlap pixels f Separation um L067 Start Coordinates o oo 0 00 Get Start End Coordinates 0 00 0 00 Get End 04 720104 Rev D 1008 202 Delta Vison Core and personalDV Users Manual Collect Panels Specifies to use a panel collection macro Overlap pixels Specifies the amount of overlap in pixels between adjacent panels Start Coordinates Specifies the XYZ coordinates at which to start collecting panels Use the Get Start button to obtain the current XYZ stage coordinates End Coordinates Specifies the XYZ coordinates at which to finish collecting panels Use the Get End button to obtain the current XYZ stage coordinates The Design Run Expenment Dialog Box The Design Run Experiment dialog box provides tools to select design edit and execute experiment macros Experiment macros are scripts of commands that guide the DeltaVision system to collect images To open the Design Run Expenment dialog box gt From the Resolve3D window click Experiment and click the Run Experiment tab Figure E 18 The Design Run Expenment dialog box E Design Run Experiment Resolve3D exp aa Note The Design PK Experiment tab is available only for systems that
10. Green 525nm Orange 605nm Infrared 705nm Blue Green 470nm Yellow Green 535nm Appropnate Probes DAPI Hoechst Coumanin Fluorescein GFP CY3 Rhodamine Texas Red Phycoerythnn CY 5 CFP YFP The optional Live Cell filter wheel module includes four of the most common sets of filters used for live cell imaging Alter Na me CFP YFP mC heny EG FP Excitation Deep Blue 430nm Blue Green 500nm Yellow 572nm Blue 470nm Emission Blue 470nm Yellow Green 535nm Red 632nm Green 525nm 2 Data for filters provided by Chroma Technology Corp Appropnate Probes Cyan GFP CFP Yellow GFP DsRed Express DsRed2 EGFP sgG FP ppliedPrecision Appendix D Reference Information 173 Reference List Selected references are provided on the following pages Contact Applied Precision for the most recent list If you notice omissions from the list please inform Applied Precision Microscopy Agard D A Sedat J W 1983 Three dimensional architecture of a polytene nucleus Nature 302 676 681 Agard D A 1984 Optical Sectioning Microscopy Cellular Architecture in Three Dimensions Ann Rev Biophys Bioeng 13 191 219 Agard D A Hiraoka Y Sedat J W 1988 Three dimensional light microscopy of diploid Drosophila chromosomes Cell Motility amp Cytoskeleton 10 18 27 Agard D A Hiraoka Y Shaw P J Sedat J W 1989 Fluorescence microscopy in three dim
11. OMS LI A docs uses uaa E and angie acaba tam a cae ana 151 Movin TS SY SUSI soars estat deine E eect tea i E eases ae aes 151 Appendix A The Immersion Oil Kit sscssssscsnssecnssseensssensssensssens LOS THE Oil C Alea Ors foc asec serene thse ood es ce tee ein tev erste eee ees eco 153 Appendix B Toubleshootng ccsssscsnssecnnsseenssecnsseennessenssseenssses LOD Diag nosing System Problems ssscnins cides E N ohne 155 Troubleshooting the Controleert n tani AE Ae 155 Troubleshooung the Work stalin sssini a ii iia EE eet 156 Analyzing Reasons for Poor Image Quality eee eeceeeeeseeeecseesseeseeseeeseeeseeeeeees 156 Delta Vision Problemi Report PORN eeen n a densi tsoassstsaninevedss 159 Appendix C Acquinng a PSF sssscssssccnssccnssenssecnssennssecnssennssennsss LOL Ol OTC O O Ea a E E T ETE 161 Acquario a PoEsia a E wade day eee oa aaa 161 Ea E EEA E E DT EENE A AE E AA E E A E AE PA EENEN 162 Podine DOGS ates cose teased an E ae eaketaael nisatenan tne enataes atte uaadatahesee 164 Selecting the Correct Mimers Oil assis secede itenstenssttehstdo vst seemeisioentoptesaiiabioanetent 165 COMVe reine PORTO OTE nora a E yaaa ata gnaranuace saunas eau aunaaiaes 166 Appendix D Reference InfOnnatiONn ccsssscssssscsssssenssseensssesessees LIL Standard Filename EX tensions voviscesiinwieewa O ean aad aa news 171 Standard Fl grescenc Fier renaires nea ieccsieseatianeslavomes Seals E enema 172 Eve cel Piller Sets aa
12. Specifies the list of points described by number to visit during the experiment All sectioning and wavelength procedures are repeated at each of the listed points A point list can be entered as a series of numbers separated by commas or dashes Separating two numbers with a dash as shown in the following example indicates that all point numbers in between should also be visited For example Lg 2p Op LHL Note When you use Point Visiting with Zsectioning the microscope uses the Zvalue of the current focus of each point asthe initial reference forthat point Values specified for Zsectioning are incremented relative to that point Autofocus before imaging Automatically focus the camera at each visited point before acquiring an image Panel Collection Setup Use the Design Experiment Panels tab to create panel collection macros These macros are useful when you want to scan a large area with a relatively high magnification lens You can use the panels as a means of reviewing a large area of a slide or as data that you want to stitch together to form a single large image Panel collection macros are sensitive to microscope settings such as image size and magnification Because the number of panels required depends upon many factors it is usually not a good idea to reuse panel collection macros Figure E 17 Expenment Designer Panel Collection Setup Options Ww Design Run Experiment File Help Design Experiment
13. Pergamon Oxford 1970 Hecht E Optics Addison Wesley Reading MA 1987 Hopkins H H 1955 The frequency response of a defocused optical system Proc Royal Society A 231 91 103 Jenkins F A White H E Fundamentals of Optics McGraw Hill New York NY 1976 Streibl N 1985 Three dimensional imaging by a microscope Journal of the Optical Society of America A 2 121 127 ppliedPrecision Appendix E Resolve3D and Keypad Options This chapter describes the following Resolve3D windows and dialog boxes The Resolve3D Window is the main window for data acquisition The Design Run Experiment Dialog provides tools to select design edit and execute experiment macros The Experiment Designer is used to generate experiment command macros The Settings Dialog used to control how images are displayed select camera settings and specify file output Keypad Joystick Operation is a reference for the buttons on the keypad Many of the Resolve3D functions are also available on the keypad and joystick 182 DeltaVision Core and personalDV Users Manual The ResolvesD Window The Resolve3D window is the main data acquisition window In addition to providing many of the acquisition options and controls it provides access to the other dialogs and windows that are used for data acquisition To open Resolve3D gt From the softWoRx menu choose File Acquire Resolve3D Figure E 1 Resolve3D Window
14. Set up a 3D Sectioning experiment see Sectioning Specimens for 3D Images on Page 35 2 In the Design Run Experiment window click the Channels tab and select the channels for your experiment see Selecting Filters on Page 42 3 Select the Reference Image option 3 In the Z position list choose whether to acquire the reference image at the top middle or bottom of the sample ppliedPrecision Chapter 5 Acquinng Data From Live Specimens 77 4 Under the Reference Image select which filters to use 5 In the EX Shutter field select the type of light to use for the image EX is the Excitation or the arc lamp and TRANS is the transmitted light d Design Run Experiment Resolve3D exp modified File cial g Oe Design Experiment Design PK Experiment j Run Experiment Experiment name Resolveatl Estimated file size 1 50 Mb 22 28 Gb Available Pest acquisition ipio Lamps Off when finished Sectioning Channels Time lapse Points Panels Actions Conventional d Multiplexed Exp EX Filter EM Filter T Ex Shutter a a ee W Reference Image Z Position Middle of Sample Bi oo evs evs v ffevs ioo TRans Refresh exposure conditions This expenment specifies using the DAPI and FITC filtersto acquire imagesof each section and using the transmitted light to acquire a reference image in the middle of the sample 6 Save and Run the Experiment After DeltaVision
15. Since DeltaVision is an inverted microscope the sample must be placed with the cover slip facing down toward the objective If you are using a standard microscope slide 1 x 3 or 25 mm x 75 mm you can use the Repeatable Slide Holder to hold the slide To do this pull the return spring to the left and place the Slide Cover Slip down onto the Slide Holder so that the upper right corner is pressed laterally against the brass locators shown as A and B in Figure 5 and the upper left edge is pressed laterally against the brass locator C Then gently release the return spring to secure the slide Push down on the slide to make sure that it is fully seated in the holder Figure 5 The Repeatable Slide Holder C A Retum Spring s s 3 Choose the desired eyepiece EP filter 4 Select the desired excitation EX neutral density T and emission EM filters These parameters should automatically be in place after selecting the eyepiece filter unless non standard arrangements are needed 5 Rotate the Beam Selector at the base of the microscope to direct the light collected by the objective to the eyepiece 6 To use the transmitted light to locate the specimen open the transmitted light shutter by pressing TRANS SHUTTER on the keypad This allows white light to transilluminate the sample The intensity for the transmitted light is controlled using the T field on the Resolve3D menu 7 Set the stage speed using the k
16. The Design Expenment Tab The Design Experiment tab is used to generate experiment command macros To open the Design Expenment tab gt From the Resolve3D window choose Experiment The Design Run Experiment window opens with the Design Experiment tab selected ppliedPrecision Appendix E Resolve3D and Keypad Options 195 Figure E 12 Design Expenment J Design Run Experiment is iy File Design Experiment Design PK Experiment Run Experiment Experiment name ResolveaD Estimated File Size 32 01 Mb Enable Fast Acquisition fast Acquisition Cities Sectioning Channels Time lapse l Points l Panels Actions F Z Sectioning Z Scan Options Focus point when scan starts Middle of Sample ha Optical section spacing 0 20 Number of optical sections B4 Sample thickness i20 Get thickness Enable OAI Scan Expenment name Specifies the name of the experiment macro Enable Fast Ac quisition Enables fast acquisition experiments see Page 196 Sectioning Specifies sectioning for 3D images see Page 196 Channels Specifies channels filters and exposure time see Page 198 Time lapse Specifies criteria for time lapse experiments see Page 199 Point Visiting Specifies a list of marked points see Page 200 Panels Allows you to set up panel collections that you can use to review a large area of a slide or to stitch together to form a single large image see Pag
17. Use the following instructions for shutting the system down during a power outage or other occasions that require total shutdowns 132 DeltaVision Core and personalDV User s Manual DeltaVision Power Switches The main DeltaVision power switches are shown below The Master Switch on the isolation transformer that plugs into the wall is not shown TOLEK Piik Workstation Switch Main Components Switch TIF iA QLM Switch optional ppliedPrecision Chapter 9 Maintenance 133 Guidelines for Using Switches Main Components Use this switch to turn power on and off for the DeltaVision Workstation Instrument Controller and Microscope Interface Chassis IC MIC the Fast Camera Power Supply and the DeltaVision Microscope Workstation IC MIC and Monitor Leave these switches on except on rare occasions such as power outages when you need to shut down the entire system Shutting Down the System In some situations such as power outages you will need to shut down the entire DeltaVision system To shut down the DeltaVision system 1 Save all data on the workstation 2 Turn off the xenon light source using the bulb E icon on the Resolve3D main menu Clicking the icon will switch it to the off ra state 3 On the softWoRx menu bar choose File Exit Then exit all other workstation applications 4 From the main menu button choose Logout and then Shut Down Wait until the monitor displays Power Down
18. activate the Lamps Off when finished checkbox Note Use discretion when activating the Lamps Off when finished checkbox feature Cycling the DeltaVision lamp on oroff more than is necessary can reduce the xenon bulb life 4 Enter the desired time interval in the Time lapse field Note The minimum time interval is limited by acquisition time If you specify a time that is less than the acquisition time the expenment proceedsat the quickest possible rate 5 Set up the remaining parameters for your experiment in one of the following ways e Enter the desired number of time points in the Time Points field The Total Time field displays the total time that will elapse during the experiment e Enter the total time in the Total Time field The Time Points field displays the number of time points for the experiment ppliedPrecision Chapter 4 Setting Up and Running Expenments 47 Point Visiting You can use point visiting to monitor areas of the slide that are in different fields of view Instead of recording one cell or field in a single experiment multiple sites can be imaged in a single experiment increasing data collection efficiency In practice the number of sites is limited only by the minimum acceptable time interval between each time point at a single site This makes time lapse imaging much more efficient and allows you to collect enough data to generate statistically significant results In addition variab
19. shutter from the EX Shutter field EX is the standard fluorescence light source TRANS is the transmitted light source EX2 is an optional second fluorescence light source and LASER is an optional laser source using the QLM module 7 If you are repeating an experiment and you have entered new exposure times or T in the Resolve3D window click Refresh exposure conditions 8 Repeat Steps 2 6 for each set of filters that you want to use 9 Save and run the experiment Setting up Time lapse Expenments Time lapse images are very useful for analyzing live specimens Time lapse images showing cell mitosis 04 720104 Rev D 1008 Delta Vison Core and personalDV Users Manual To design a time lapse expenment 1 Select the Time lapse tab under the Design Experiment tab on the Design Run Experiment window Design Experiment Design PK Experiment Run Experiment Experiment name Resolves Estimated file size 64 02 Mb 22 29 Gb Available Use Fast Acquisition ast Acuuisiion ipio Lamps Off when finished Sectioning Channels Time lapse a Points Panels Actions W Time lapse Hours Minutes Seconds Milliseconds Time lapse e e fo fo Total Time fo lo fo fo Time Points f W Enable Cell Tracking Cell Tracking Options W Autofocus before imaging Autofocus Options 2 Activate the Time lapse checkbox 3 If you want the DeltaVision lamp to turn off when the experiment is completed
20. with X Y and Z coordinates that you want the system to remember These points can be interactively visited at any time and they can be used in experiments Clear Stage Trails Clears the Stage Trails history The system maintains a history of the paths of motion that you take while exploring your sample These paths are displayed as Stage Trails on the Stage View Clear Stage Thumbnails Clears all of the thumbnail images currently displayed on the stage view Blank Screen Turns the computer screen blank This is useful when you are imaging under very light sensitive conditions Clicking anywhere on the screen restores it ppliedPrecision Appendix E Resolve3D and Keypad Options 185 Command Line Interface Opens the Command Line dialog box that provides advanced users with the ability to issue individual Resolve3D commands to the system CAUTION The Command Line Interface should be used carefully because it can put the system in an unstable state The Resolve3D Options Menu Use the Options menu to open the Settings dialog box where you can set display and image options and to save configuration settings and state information Figure E 4 The Resolve3D Options Menu or toe Resalvesd 5 x File View Options Calibration Help settings save Settings Settings Opens the Settings dialog box which allows you to control display imaging and file output options Save
21. 274 1732 1736 ppliedPrecision Appendix D Reference Information 177 Ma X Ehrhardt D W Margolin W 1996 Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein PNAS USA 93 12998 13003 Marshall W F Dernburg A F Harmon B Agard D A Sedat J W 1996 Specific interactions of chromatin with the nuclear envelope Positional determination within the nucleus in Drosophila melanogaster Molecular Biology of the Cell 7 825 842 Marshall W F Fung J C Sedat J W 1997 Deconstructing the nucleus Global architecture from local interactions Current Opinion in Genetics amp Development 7 259 263 Marshall W F Straight A Marko J F Swedlow J R Dernburg A Belmont A Murray A W Agard D A Sedat J W 1997 Interphase chromosomes undergo constrained diffusional motion in living cells Curr Biol 7 930 939 Mathog D Sedat J W 1989 The three dimensional organization of polytene nuclei in male Drosophila melanogaster with compound XY or ring X chromosomes Genetics 121 293 311 Minden J S Agard D A Sedat J W Alberts B M 1989 Direct cell lineage analysis in Drosophila melanogaster by time lapse three dimensional optical microscopy of living embryos Journal of Cell Biology 109 505 516 cover article Moritz M Braunfeld M B Sedat J W Alberts B Agard D A 1995 Microtubule nucleation by gamma tub
22. 38 editing 96 98 Maintenance adjust fiber optic module tilt 156 57 centering field stop aperture 155 56 changing MIC fuses 161 checking fiber optic cable alignment 153 55 cleaning DeltaVision 162 critical illumination position 158 filter wheel calibration 135 39 illumination path alignment 150 61 Kohler spring adjustment 157 58 moving DeltaVision 162 system shutdown 143 testing illumination path alignment 159 Mark Point button 229 Mark Point setting 203 Marked Points List button 205 Marking points 50 52 52 Replacing points in a list 52 saving a point list 53 Visiting marked points 52 Master switch 142 Material Safety Data Sheets MSDS 162 Maximum intensity 206 Maximum Z test range setting 223 Mean intensity 206 Medium button 227 Message pane 207 Microscope interface chassis 143 replacing fuses for 161 Microscope Interface Chassis 113 Middle of sample 41 Middle of the sample option 40 Minimum intensity 206 Monitor switch 142 Monitoring data acquisition 91 96 Monitoring point visiting 54 Move threshold 75 Moving DeltaVision 162 Multiplexed wavelength experiment design 35 66 filter set activation 58 62 option 35 66 122 overview 58 ND arrows 227 Network connection 103 Neutral density filters 18 No button 227 Number of optical sections field 41 OAI See Optical axis integration OAI setting 210 Objective ppliedPrecision Appendix E Resolve3D and Keypad Options cle
23. Cell 62 89 106 Paddy M R Chelsky D 1991 Spoke a 120 kD protein associated with a novel filamentous structure on or near kinetochore microtubules in the mitotic spindle Journal of Cell Biology 113 161 171 Paddy M R Saumweber H Agard D A Sedat J W 1996 Time resolved in vivo studies of mitotic spindle formation and nuclear lamina breakdown in Drosophila early embryos Journal of Cell Science 109 591 607 Periasamy A Day R N 1997 PIT 1 protein localization at different optical sections in a single living cell using FRET microscopy and green fluorescent proteins Microscopy amp Microanalysis submitted for publication Periasamy A 1998 Digital Deconvolution FRET Microscopy 3D Visualization of Protein Protein Interactions in a Single Living Cell Invited Paper SPIE 3260 1 9 Rossi F Charlton C A and Blau H M 1997 Monitoring protein protein interactions in intact eukaryotic cells by beta gt galactosidase complementation PNAS USA 94 8405 8510 Rykowski M C Parmelee S J Agard D A Sedat J W 1988 Precise determination of the molecular limits of a polytene chromosome band regulatory sequences for the Notch gene are in the interband Cell 54 461 472 Scalettar B A Swedlow J R Sedat J W Agard D A 1996 Dispersion aberration and deconvolution in multi wavelength fluorescence images Journal of Microscopy 182 50 60 Schwartz K Richards K Botstein D 1997 BIM1 encodes a a microtubu
24. DANK Move to start of sectioning from initial point Remove backlash DEACTIVATELASERS FOCUS 14 30 DISBANK FOCUS 7 80 DISPLAY FOCUS 0 10 DISHIN Focus 0 10 f x A use The Experiment Macro Editor Menu Bar The menu bar File menu contains commands to open and save experiment macro files The Save amp Quit option saves the macro file with the current name The Edit menu has commands for manipulating text The Search menu provides search and replace capabilities and the Help menu displays additional information Search amp Replace These text fields are used to specify a search and replace pattern You will need to enter strings in these fields before you use the Search Replace Text menu command Status Area The text area below the Search and Replace fields is used by the Macro Editor to supply you with status and hint information The Macro Text Area The Macro Text Area below the Status Area is the main working area of the Macro Editor Commands List This is the list of available commands When you highlight a command in the list by clicking it you are provided with information about the command in the Status Area If you double click or select the Use Item button below this list the command is inserted in the Macro Text Area at the current cursor location 04 720104 Rev D 1008 90 DeltaVision Core and personalDV User s Manual To edita reference macro 1 On the Resolve3D Experiment Macro Editor wi
25. Image file name field enter the file name Notes 1 Do not use file names that include spacesorspecial characters e g l a amp f F 2 The image file name hasan _R3D dv extension This isthe unprocessed i e raw Resolve3D image A file with a log extension is also created The log file isa text file containing information about the image file including the macro and objective that were used to collect the image 7 In the toolbar at the top of the screen click the green arrow button to start the scan The macro collects the image and saves it in your data folder see Setting up a Personal Data Folder on Page 29 To process the image use the deconvolution module in the softWoRx program as described in the softWoRx Imaging Workstation User s Manual Sectioning Specimens for 3D Images You can acquire images of multiple sections This data can be used to create 3D projections view cross sections and create volumetric and line models 04 720104 Rev D 1008 DeltaVision Core and personalDV Users Manual Layer 1 Layer 2 Layer 3 Layer 4 Layer 5 When you set up a Z scan on DeltaVision you normally begin by defining a focus range to use This is accomplished by marking the top and bottom of the sample You then focus on a plane of interest The focus range is taken above and below the plane of interest If you are new to 3D microscopy you may have never had to deal with
26. New high resolution 3 D microscope avoids damage to live samples Biophotonics International 3 40 44 cover article Welnhofer E A Zhao L Cohan C S 1997 Actin Dynamics and Organization During Growth Cone Morphogenesis in Helisoma Neuron Cell Motility and the Cytoskeleton 37 54 71 cover article Wilson S M Datar K V Paddy M R Swedlow J R Swanson M S 1994 Characterization of nuclear polyadenylated RNA binding proteins in Saccharomyces cerevisiae Journal of Cell Biology 127 1173 1184 04 720104 Rev D 1008 180 Delta Vision Core and personalDV Users Manual Zimowska G Aris J P Paddy M R 1997 A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes Journal of Cell Science 110 927 944 Linux Operating System Siever Ellen Linux in a Nutshell Third Edition O Reilly and Associates 2001 Image Processing Bracewell R N The Fourier Transform and Its Applications 2nd Edition McGraw Hill New York NY 1986 Goodman J W Introduction to Fourier Optics McGraw Hill New York NY 1968 Castleman K R Digital Image Processing Prentice Hall Englewood Cliffs NJ 1979 Gonzales R C Wintz P Digital Image Processing 2nd Ed Addison Wesley Reading MA 1987 Pratt W K Digital Image Processing Wiley New York NY 1991 Russ J C The Image Processing Handbook CRC Press Boca Raton FL 1992 Optics Born M Wolf E Principles of Optics
27. On the desktop double click the DV icon to open softWoRx 8 On the softWoRx menu choose File Acquire Resolve3D and follow the prompts allowing the system to initialize 04 720104 000 Rev D 1008 20 DeltaVision Core and personalDV User s Manual 9 Release the Focus Lock by turning it clockwise when facing the lock until it is loose 10 Lower the objective by turning the Coarse Z Focus knob away from you clockwise when facing the knob CAUTION Always lowerthe objective before you initialize the system to prevent damage to the objective lens 11 A dialog box is displayed prompting you to lower the objective before continuing After you lower the objective select Initialize to initialize the system ppliedPrecision Chapter 3 Getting Started 21 Resolve3D Acquisition Parameters Resolve3D Stage Contols The Resolve3D window the Data Collection Window and the Filter Monitor dialog box open on the desktop MP Beach ID mad Acquire Experiment seringa Data Folder Image Windows 1 21 J mear E 5 Alter Monitor Exckation CFP 0 10 feb Space MT 30 22 Gb tree Data Space M 30 22 Gb tree A Emission CFP ama sT 100 j EX Shumer EX 9 l 7 Exposure fh nog Find J Calibrate image size 512512 f i Lens an mw 2 Bin 1x1 ov Awe i eee Fim sce 0 1638 um AO Tals 7 d pija Data Collection Window Jer Indl Initialt F
28. Reseat motorcableson excitation module Power up system Stalled sequence of Computer Exit the software and restart IC MIC Images misa lloc ated memory Troubleshooting the Workstation Other system troubles are indicated by messages or readings in the software The Resolve 3D message window displays Resolve 3D activity Observe the messages in this window when troubleshooting This table shows possible problems and corrective actions Table B 2 Workstation Troubleshooting Chart Indication Cause Conection Delete unwanted files Save image filesto CD or DVD or LAN See softWoRx Imaging Workstation User s Guide for more information There isno more storage space for image data File system full message when trying to save images Camera not found message Power up sequence was incorect Shut down the system and then restart it using the steps described in Chapter 9 Maintenance Lack of Communication between the Resolve 3D settings are not updating changesmade Shut down the system and then restart it using the stepsdescnbed in Chapter 9 Maintenance using the keypad or Instrument joystick for Controller and example the filter Workstation selection orZ position User hasnot been added At login username not recognized Add user See the softWoRx Imaging Workstation User s Guide Analyzing Reasons for Poor Image Quality The following table documents the most common acquisiti
29. a width that is 50 of the width of the field of view In the first image the width of the ROI is defined as 50 of the width of the field of view In the second image the stage has moved to keep the cell of interest within the ROI Perimeter cellshave also moved almost entirely out of the field ppliedPrecision Chapter 5 Acquinng Data From Live Specimens 73 Q Tip If DeltaVision loses the cell because it moves out of the field of view you can update the point coordinates by manually finding the cell and centering it in the field of view The point list is automatically updated Acquinng 3D Z Projections with OAI Optical Axis Integration or OAI also referred to as Continuous Z Sweep is useful for acquiring 3D Z projections of live specimens Instead of collecting an individual image at each focal plane OAI collects and integrates one continuous image through an extended Z movement WY ERR cD 9 3 e9 YW p _ F D e Se ax eS S i o 8 O gt Optical Sectioning Real Time ZSweep Instead of acquinng multiple images Real Time ZSweep acquires one image dunng a continuous tage movement and instantly createsa 3D ZProjection Real Time Z Sweep has significant advantages for applications such as Leading Edge Motion Analysis Fast Organelle Dynamics Microtubule Dynamics and Fluorescence in situ Hybridization FISH It is especially useful for studies of objects that are moving in 3D space e g kineticho
30. acquires the data it creates two image files One file contains the reference image data and the other file contains all of the other data Q Tip After you acquire the images you can use the Image Fusion tool in softWoRx to combine the reference images with the rest of the images 04 720104 Rev D 1008 Chapter 6 Data Collection Techniques Data Collection Techniques This chapter provides guidance and suggestions for m Finding a Specimen and Recording Its Position m Finding Exposure Time a Using Kohler and Critical Illumination a Monitoring Data Acquisition a Editing Experiment Macros Finding a Specimen and Recording its Position Recording the position of your slide is useful when you are conducting a point visiting experiment and you need to remove the slide before you are finished Use the following instructions to find a sample and record its position on the slide with the Repeatable Slide Holder When you resume your experiment you can place the slide in the position that you recorded 04 720104 Rev D 1008 79 80 Delta Vison Core and personalDV Users Manual Note Ifyou are using an oil immersion lens you must apply immersion oil to the sample See Appendix A The Oil Immersion Kit for more information about the oil calculator To find and center the specimen 1 Choose the objective by rotating the objective turret Be sure to select the same objective in the Resolve3D window 2 Secure the slide
31. and view the primary light path 4 Use the EX2 formerly CAMERA SHUTTER button on the keypad to open the EX2 shutter and view the secondary light path Select the EX filter currently in the primary light path Ra Note With the Multiplexed Wavelength option you can view both selected wavelengths simulta neously by opening both EX shutters at the same time Designing a Multiplexed Wavelength Expenment Use the following procedure to begin the design process for Multiplexed Wavelength experiments To design a Multiplexed Wavelength expenment 1 From the Resolve3D main menu click the Experiment button to open the Design Run Experiment window ppliedPrecision Chapter 4 Setting Up and Running Expenments Na Resolves File Options Calibration Help Acquire Experiment Settings View Excitation F TSA Experiment Emission CFP 470 30 Button oT BLOCK Exposure 1 000 Find _I Calibrate Image size 512x512 x Lens fiox e Info Bin fxr Aux Mag Pixel size 0 6680 um O zala X l a al ax hooo aj bso al di 10 00 ayj ea 7 N all 2 Ifthe Multiplexed Wavelength option is enabled on your DeltaVision system you ll see the Multiplexed tab in the Design Run Experiment window Click on the Multiplexed tab to view the options for Multiplexed Wavelength experiments m Design Run Experiment Resolve3D_Startup exp a Agu Oe D
32. as shown below ppliedPrecision Chapter 9 Maintenance 149 r7 Az 2 Adjust the center alignment in the Kohler position as follows a Slide the Focus Control to the Kohler position set the eyepiece to CT and use the Centering Telescope Focus to focus on the objective back aperture You should see the area between the back aperture of the objective lens the darker area and the fiber tip bright green a b Use a 1 5 mm hex key to adjust the X and Y adjustment screws on the top and left sides of the Fiber Optic Module until the fiber tip image is at a point that is midway between its original center and the actual center the point where the centers of the two circles are aligned 3 Adjust the tilt of the Fiber Optic Module as follows m a Make sure that the viewing knob on the microscope base is set to direct the light to the eyepiece and verify that the 60X objective is in detent and in its lowest position a b Remove the oil from the objective a c Place a piece of paper on the stage with a piece of glass on top to keep it flat m d Press EX SHUTTER on the keypad to open the EX shutter and set the neutral density filter to 100 light transmittance in Resolve3D a e Usea3 mm hex key to adjust the X and Y tilt screws so that the bright spot on the paper is circular and is centered in the dim field 4 Repeat steps 1 3 in this procedure until the image is centered in both the Critical and Kohler positions
33. become active The Autofocus parameters in this dialog box are described in Settings Dialog Box Autofocus Options on Page 208 Tracking Cells Cell Tracking moves the stage laterally to follow cells as they move during a time lapse experiment With the Enable Cell Tracking option selected DeltaVision automatically keeps cells in the field of view as they move during a time lapse experiment After you specify an ROI around an object of interest the software determines the center of the cell and establishes a recognizable pattern within the ROI In subsequent images the software recognizes this pattern and recalculates the center of the cell on the fly The position of the new center is compared with the position of the previous center and if the cell has moved beyond a specified threshold the system automatically moves the stage and re centers the cell in the field of view 04 720104 Rev D 1008 DeltaVision Core and personalDV User s Manual x y 100 0 100 0 x y 100 0 100 0 Time Point 3 Time Point 4 x y 100 0 100 0 x y 40 0 100 0 At Time Points 1 and 2 the cell is moving through the ROI the inner square but the center Is till within the Move Threshold the circle At Time Point 3 once the center of the cell touches the threshold boundary the stage movesto re center the cell within the ROI Note the new stage position displayed in the lower nght comer of Time Point 4 To use Cell Tracking 1 Set up a t
34. benchmark deconvolution is 16 times larger than the original deconvolutions performed in 1983 Although only a few laboratories were able to afford 35 000 for a computer the lower cost warranted commercialization of a deconvolution microscope In 1996 the same deconvolution required about 1 4 of an hour with a 14 000 workstation and 1 2 of an hour with an 8000 machine This thirty fold increase in performance price allowed a more widespread acceptance of deconvolution microscopy Advances in computing power and data storage have continued to benefit DeltaVision users In 2003 a 2500 workstation could perform the standard benchmark in less than four minutes representing another twenty fold increase in performance price since 1996 The current workstations can now perform this benchmark in less than 30 seconds under 20 seconds for DeltaVision Core and under 30 seconds for personalDV Vast amounts of data generated by these experiments can be stored on local hard drives by burning DVD s or by transferring the data to other locations using high speed network connections What Can You Use DeltaVision for DeltaVision Core and personalDV use research grade microscopes to collect optical images in one of the following imaging modes Fluorescence Brightfield Phase contrast Differential Interference Contrast DIC Phase contrast requires additional equipment that is available from Olympus distributors 04 720104 000 Re
35. click Acquire Leave Data Collection window 21 open In the Image window choose Tools Measure Distance Then set the Units to Pixels in the Measure Distance dialog box Draw a line across the image from a point on the top left square to a point in the same relative position on the top right square If the vertical delta is more than four pixels re align the slide Repeat this process at the middle and bottom Then count and record the number of grid elements and record the distance in pixels Repeat Steps 7 and 8 in the vertical direction Calculate the pixel size for each of the six measurements top middle bottom left center and right as follows Pixel Size um 9 995 um box Sum of Measured pixels Number of grids per measurement Your calculation should be accurate to four decimal places Average the six pixel sizes to obtain the correct pixel size To acquire a PSF 1 Obtain the objective lens ID number see the softWoRx online Help for information about lens identification numbers From the softWoRx main menu choose Utilities Revise Microscope Configuration Then enter the root password to open the RESOLVE3D SYS file In the RESOLVE3D SYS file under the Microscope Specifications section a Increase the number of lenses next toMS Number Lenses by 1 b Add the name of the objective toMS Lens Names e g 100Xoil 60Xwater c Add the lens ID toMS Lens ID Numbers d Enter the pixel
36. click the Refresh button Xe Lamp bulb age hours Displays a count for the current number of hours on the xenon bulb Use photosensor Enables use of the Photo sensor Settings Dialog Box Files Options Use the Settings dialog box File options to set the image output directory set the directory where experiment macros are stored and select auto incrementing for file names Figure E 21 The Settings Dialog Box Files Options licens Scns OOOO AG Display imaging Files Autofocus Mise Gi ht Data folder ata Experiment macros folder momewo Data folder is temporary F Auto increment file names Convert to 2 byte signed integer Done save Settings Data Folder Specifies the destination directory for Resolve3D output images If you supply a directory that you do not have permissions to use you will be warned If you provide a directory name that does not exist you will be presented with the option of creating the directory You can also change the destination directory by changing the global softWoRx Image Data Directory in the User Parameters tool Expenment macros folder Specifies the directory where your experiment macros are stored Data folder is temporary Specifies that any directory entered in the Data Folder field only applies to the current Resolve3D session In subsequent Resolve3D sessions the Data folder 04 720104 Rev D 1008 208 Delta Vison Core and personalDV Users Manual
37. distance from the microscope is correct for Kohler Illumination and if required for Critical Illumination To check alignment 1 Move the Beam Selector to direct the light to the eyepiece and make sure that the 60X objective is lowered and in position as shown previously in Before You Check or Adjust Illumination Path Alignment 2 Use the Eyepiece Filter Wheel to select the FITC eyepiece filter 3 Inthe Resolve3D window select the following filters In this Field Select Excitation TRITC Emission FITC AT 0 1 4 Apply immersion oil to the objective and mount the mirror slide 5 On the keypad press EX SHUTTER to open the Excitation shutter 6 Use the Eyepiece Focus on the oculars to focus on scratches on the mirror slide 7 Fully close the Field Stop Aperture and slowly rotate the fine Z Focus knob in and out of focus in both directions The light should evenly expand around the aperture If it flares in one direction go to Step One Center the Field Stop Aperture on Page 144 8 Check for alignment in K hler Illumination as follows a Open the Field Stop Aperture Then loosen the Locking knob and slide the focus end of the Fiber Optic Module into the K hler Illumination position see Page 84 Then tighten the Locking Knob b Switch the Eyepiece Selection Wheel to the Centering Telescope position CT Use the Centering Telescope Focus control to focus on the objective back aperture the outside edge of the middle cir
38. ed Wiley Press http www wiley com legacy cp cpcb Cells A Laboratory Manual Spector Goldman Leinwand ed Cold Spring Harbor Press 1998 Video Microscopy The Fundamentals 2nd Edition Inoue and Spring Plenum Press 1997 Digital Microscopy 3rd Edition Methods in Cell Biology Vol 81 Sluder and Wolf Academic Press 2007 Papers on Sample Preparation Rines DR He X Sorger PK Quantitative microscopy of green fluorescent protein labeled yeast Methods Enzymol 2002 351 16 34 Hutchins JR Moore WJ Hood FE Wilson JS Andrews PD Swedlow JR Clarke PR Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis Curr Biol 2004 Jun 22 14 12 1099 104 Courses in Microscopy and Cellular Imaging In Situ Hybridization Immunocytochemistry and Live cell Imaging Dernberg Hu and Murray Course Directors Cold Spring Harbor October annual ppliedPrecision Chapter 3 Getting Started 15 Analytical and Quantitative Light Microscopy Sluder and Wolf Course Directors Marine Biological Laboratory Woods Hole MA May annual Getting Familiar with DeltaVision Before you acquire an image become familiar with the key DeltaVision controls for e Controlling the Light Path e Focusing e Choosing Filters e Using the Keypad and Joystick Most of the manual controls for controlling the light path focusing and choosing filters are similar to those that you will find on any microsco
39. expenment Launches an Image window when the experiment is finished Note that if more than two files are created in a point visiting experiment the Image windows will not launch even if this option is selected Images ac quired requested Displays the current number of images acquired compared to the total number requested During fast acquisition the reported number of acquired images may not be updated regularly Disk space required Displays the estimated size of the experiment i e the size of the image file Resolve3D checks the disk space to make sure that enough space is available before it runs the experiment Elapsed time Displays the elapsed time of the experiment in seconds 04 720104 Rev D 1008 204 Delta Vison Core and personalDV Users Manual Estimated Finish Displays the estimated clock time in which a running time lapse experiment will finish Current command Reports each macro command as it is executed Start Scan Starts the selected macro to run an experiment Cancel Scan Terminates a scan while the experiment is running The images that are collected before the experiment is cancelled are automatically saved Help Opens the Help for the Design Run Experiment dialog box The Settings Dialog Box Use the Resolve3D Settings dialog box to control how images are displayed select camera settings and specify file output To open the Settings dialog box gt From the Resolve3D Window
40. for the list using eight characters or less Since there is really no method of displaying a directory of these names write the name of the list down to keep a record of it 3 Press Enter or click OK To visita point using Resolve3D Use one of the following methods to visit a point e In the Stage view double click on the point e Inthe Point List dialog box click the point that you want to visit and click Visit Point Loading a Point List and Specifying Points to Visit If you are using a previously saved point list you will need to load the point list in Resolve3D before you specify which points to visit If you are using a point list that is already open in the Point List dialog box you can specify which points to visit ppliedPrecision Chapter 4 Setting Up and Running Expenments 51 Q Tip You can markand then vist a point list without saving it when you are setting up an expenment To load a point list into Resolve3D 1 On the Resolve3D dialog box click EJ or choose View Points List to open the Point List dialog box 2 Click Open List 3 Inthe Prompt dialog box enter the name of the desired list and click OK Note You may need to load a list when the list of points has been cleared from the Point List dialog box and you want to reload the saved list To specify which points to visit 1 Make sure that the point list is loaded in the Point List dialog box as shown above 2 Open the Design Run E
41. have the QLM module ppliedPrecision Appendix E Resolve3D and Keypad Options 203 Image file name Specifies the file name to use for the image in this text field If you do not provide a file name you will be asked to provide one when the experiment starts running If you have the Auto increment file names setting in the Settings dialog box turned on you will only need to provide a name once for each session The names will have incrementing numbers appended to them automatically Image ttle Specifies text to save in the header of the image file created by the experiment The title can be viewed later using the Header Labels button of the Image Window s Image Information dialog box Add note to log Inserts a note in the experiment s log file at any time before or while an experiment is running You will need to click Do It to insert the note Change time lapse Changes the time lapse value while an experiment is executing You will need to type the desired time in seconds in the text field and click the Do It button to change the value This value only affects experiment macros that contain a TLAPSE command It does not permanently change the macro Show PK Progress graph See the QLM Getting Started Guide Show images dunng acquisition Displays images in a Data Collection window as they are collected In cases where acquisition speed is important deselecting this option may increase performance Launch viewer after
42. instructions for collecting image data You will use experiments to acquire almost all of your data This chapter shows how to set up and run experiments that use several DeltaVision data collection options It includes Creating and Running an Experiment Macro a Sectioning Specimens for 3D Images a Selecting Filters a Setting Up Time lapse Experiments Point Visiting a Collecting Panel Images Over Large Areas a Using the Multiplexed Wavelength Option Creating and Running an Expenment Macro DeltaVision Experiment macros are files that include settings and commands for acquiring data with DeltaVision Macros allow you to automate data collection After you use the DeltaVision interface to create a macro you can run the macro to 34 Delta Vison Core and personalDV Users Manual automatically collect data Macros can be very simple or they can be very complex and include numerous options After you create a macro you can use it as a template to modify and create other macros To Create and run an expenment macro 1 At the top of the Resolve3D window click the Experiment button to open the Design Run Experiment window 2 Click the Design Experiment tab v Design Run Experiment Design Experiment Design PK Experiment Run Experiment Experiment name ResolvesDi Estimated file size 32 01 Mb 22 30 Gb Available W Use Fast Acquisition Fast Acquisition Options Lamps Off when finished Section
43. of this dialog box As a general rule of thumb the Z step size is approximately 1 3 of the Z resolution for each objective 6 Specify either the thickness or the number of sections as follows ppliedPrecision Chapter 4 Setting Up and Running Expenments 39 e To get the thickness of the specimen as defined in Step 2 click Get thickness softWoRx adjusts the number of sections to span the specimen thickness e To specify the number of sections enter the number of sections in the Number of optical sections field 7 Save and run the experiment Guidelines for Designing and Running 3D Expenments Use the following guidelines to determine values for the fields on the Sectioning tab under the Design Run Experiment window Design Experiment tab Foc us point when scan starts This list allows you to specify the focal plane of the microscope at the start of an experiment Choose one of the following options Middle of Sample The focal plane is midway between the top and bottom of the sample This is the default and is usually the plane of best focus for the sample This is also the best point to set for Point Visiting Bottom of Sample This represents a plane where the Z stage is farthest away from the objective Top of Sample This represents a plane where the Z stage is closest to the objective Figure 1 ZTop and Z Bottom _ Slide Top Focal Plane Curent Sage postion Bottom Focal Plane Coversli
44. other camera from the spare camera tray and place the camera that you just removed on the camera tray AN CAUTION Always place the camera in the camera tray when it is not secured to the system Dropping the camera will cause severe damage Install the new camera by sliding it into place and then pushing in and pressing down on the end of the camera Replace the camera cover To selectthe camera 1 In the Resolve3D window click Settings to open the Resolve3D Settings dialog box ppliedPrecision Chapter 8 Changing Camerasand Filters 117 2 On the Imaging tab click the Camera list and select the camera that is currently installed Display imaging Files Autofocus Mise Camera COOLSNAP_ESZ ICXz65 Frames to average po Gain ico Transfer speed 20000 Target temperature 0 00 Current temperature 0 00 Refresh se Lamp bulb age hours 58 7 Refresh Reset F Use photosensor Settings change based on Excitation Filter Done save Settings Help BA Notes 1 The EM CCD camera is lised twice in the Camera list CASCADE2 512 Conv setsthe camera in Conventional mode Cascade 512 EMCC D setsthe camera in electron multiplication mode 2 When the camera ischanged the binning numberis reset to 1 3 Ifthe camera cables loosen orare accidentally disconnected you may need to restart the IC software To reseat the cables consult the camera documentation
45. receives instructions from the workstation and issues commands to the motors through the Microscope Interface Chassis The Microscope Interface Chassis MIC side of the computer provides power and control for the filter motors stage drive and shutters It also contains the Photo sensor which is connected to the microscope through a fiber optic cable Notes 1 The Instrument Controller Microscope Interface Chassishas no userserviceable parts inside 2 Occasionally you may need to replace fuses on the back panel of the chassis Consult Chapter 9 fora description of this procedure Unless there isan obvious 04 720104 Rev D 1008 104 DeltaVision Core and personalDV User s Manual reason why the fuse blew you should contact Applied Precision when you need to replace fuses Workstation The workstation hosts the softWoRx application that is the primary interface used to control the system The workstation can also provide a server for data storage when used with the optional Data Management Software DMS Note The workstation hasno userserviceable parts inside DVD R Recording The DVD R CD Drive supports DVD R format recording DVD R and CD ROM are not supported Other Standard Components Other standard components include the Repeatable Slide Holder the Slide Holder Adapter the Fiber Optic Module the keypad and the joystick The Repeatable Slide Holder The Repeatable Slide Holder holds the slide on the st
46. size for the new lens 04 720104 Rev D 1008 164 10 11 DeltaVision Coreand personalDV User s Manual For example If the desired lens is 40X 1 35 with ID 10403 the third lens in the list then the pixel size is 0 1656 MS Lens ID Numbers 10105 10205 10403 10602 10002 Mount a bead slide on the microscope and focus on the beads to obtain the maximum intensity Find a bead that is located by itself Refer to Finding Beads immediately following this procedure Use the Center Object tool to center a single bead in the X and Y directions It is helpful to collect large images such as 1024x1024 Adjust the CCD exposure time so that the maximum intensity at the plane of best focus is at least 2000 counts Make sure that the camera does not saturate at the plane of best focus Now use a 256x256 image Ensure there is only one bead in the field of view and that as you go out of focus no rings from other nearby beads enter the image Verify that your microscope and software are accurately configured for lens and auxiliary magnification Execute the Standard PSF Measurement Macro described in the online Help to measure the standard point spread function or run a Z series through the bead consisting of 128 sections acquired in 0 1 um Z increments Run the softWoRx PSF to OTF program that converts the optical sections into an OTF Refer to Converting PSF to OTF later in this chapter Finding Beads It is ea
47. the specimen s three dimensional size except in relation to the rest of the specimen Manually operated microscopes provide very little information about real distances in Z or even X and Y Once you start viewing your specimens in three dimensions however you will become accustomed to the relationship between the microscope stage sample and optics and will find that designing a Z scan is usually straightforward Use the following procedure to create and run a 3D experiment To determine the Focus point when scan starts Optical section spacing Number of optical sections and Sample thickness refer to Guidelines for Designing and Running 3D Experiments on Page 39 LU Note The softWoRx softwar reports all distances in microns To design and run a 3D expenment 1 Manually acquire images of your specimen and adjust acquisition parameters and settings until you are satisfied with the images This includes focusing on the specimen and determining what channels to use 2 In the Resolve3D window use the Z Slider on the right side of the Resolve3D window to find and center the object of interest Drag the slider up to find the top of your sample When you release the mouse button an image is acquired ppliedPrecision Chapter 4 Setting Up and Running Expenments 37 and displayed in the Data Collection window Drag the slider and acquire images until you are satisfied with the position Then press the button to mark th
48. the fluorescent probes that you want to use The spectra for the standard DeltaVision filter set and the optional Live Cell set are provided in Appendix D Notes l1the lists of probes in Figure 3 and Figure 4 above do not include all of the probes that can be used with these filter sets 2 Additional filter sets may be purchased from Chroma Technologies or Semrock 3 The CY 5 filter set does not include an eyepiece filter due to the low sensitivity of 04 720104 Rev D 1008 44 Delta Vison Core and personalDV Users Manual the human eye at this wavelength 4 The standard configuration forthe six postion filter wheels includes DAPI FITC TRITC CY 5 a polanzer and a block position For instructions on how to change filter wheel modules and calibrate filter wheels see Changing Filter Wheel Modules and Calibrating the Filter Wheels in Chapter 8 To specify which filters to use 1 On the Design Run Experiment window click the Design Experiment tab Then click the Channels tab Select a check box on the far left next to an Exp field The EX Filter field controls which excitation filter will be placed into the excitation pathway between the T filter and the fiber optic module The T Filter attenuates the intensity of the excitation light The EM Filter field controls which filter will be placed in the emission pathway between the microscope and the camera The EX Shutter field specities which light source to
49. the way out to close it The bright area on the paper should be circular and centered in the dim field Use a 3 mm hex key to loosen the Tilt screws and adjust the screws so that the bright spot on the paper is circular and is centered in the dim field as shown in the previous step Tighten the Tilt screws ppliedPrecision Chapter 9 Maintenance 147 Step Three Set the Kohler Spring Position 1 Move the Eyepiece Selection Wheel from the open O to the Centering Telescope CT lens 2 Use the Centering Telescope Focus on the ocular to focus on the objective back aperture the outside edge of the middle circle Middle Circle Inside Focuson the outside edge of Circle the middle circle 3 Loosen the Locking Knob and use a 2 5 mm hex key to loosen the two Kohler Spring screws Then move the Focus Control back and forth until the edge of the inner circle is in focus After you focus the edge of the inner circle the edges of both the inner and middle circles should be in focus ICE circle These edges should both be in focus 4 Holding the Focus Control still tighten the Locking Knob 04 720104 Rev D 1008 148 5 DeltaVision Core and personalDV User s Manual Slide the Kohler Illumination spring so that it is in the groove on the Focus Control and tighten the Kohler spring locking screws to set the Kohler position Step Four Set the Cntical Illumination Position 1 Move the Eyepiece Sele
50. to the IC MIC DeltaVision displays a message that indicates the cable is removed Do not close this message Position SensorCable gt 2 Holding the oculars in one hand use a 3 mm hex key Olympus provides one with the microscope to loosen the set screw that holds the oculars to the Eyepiece filter wheel and set the oculars on the table Loosening the set screw and removing the oculars 3 Loosen the set screw that holds the Eyepiece filter wheel to the beveled mount on the stand and remove the Eyepiece filter wheel 04 720104 Rev D 1008 120 DeltaVision Core and personalDV User s Manual Removing the Eyepiece filter wheel 4 Place the new Eyepiece filter wheel on the beveled mount on the stand and tighten the set screw that holds it in place 5 Connect the Position Sensor cable to the new Eyepiece filter wheel The IC MIC will automatically start to re initialize the EM and EX filter wheels even though the current EX and EM filter wheels have not yet been replaced You can ignore this re initialization 6 Place the oculars on the microscope and tighten the set screw that holds them in place 7 Ifthe IC MIC is ON click OK on the message box that indicates the cable is removed If the IC MIC is OFF you will need to manually update the filter set in the Resolve3D Settings Misc tab window WARNING Before you unplug any motor cables make sure that the xenon lamp and the IC MIC are tumed OFF To
51. value reverts back to the global softWoRx Data Directory as specified in User Parameters Use this option in environments where several users are running the microscope and using a single system login Data folders can be created for each user under a common parent folder As users start Resolve3D they can choose to use their own folders In most cases you should not use this option if each user has their own system login in your environment With the option deselected the Data folder assignment modifies the global softWoRx Data Directory definition and the system remembers the directory used in the last Resolve3D session Auto increment file names Creates a new file name by appending a serialized number to the base file name each time you run an experiment Convertto 2 byte signed integer If toggled On Resolve3D saves all images as signed 16 bit the pre 3 5 0 default This setting is designed to increase compatibility with older software Note that this setting cannot be On for EMCCD cameras Settings Dialog Box Autofoc us Options Use the options on the Resolve3D Autofocus tab to set up Autofocus parameters Display imaging Files Autofocus Mise QLA Automatically determine parameters Test Cannel f ALGRA LURE k Done nave settings Automatically determine parameters Autofocus parameters are determined from the depth of field of the objective lens which is calculated from the lens numerical aperture You m
52. with a novel chromatin organization Cell 76 901 912 Dawe R K Cande W Z 1996 Induction of centromeric activity in maize by suppressor of meiotic drive 1 PNAS USA 93 8512 8517 Dernburg A F Daily D R Yook K J Corbin J A Sedat J W Sullivan W 1996 Selective loss of sperm bearing a compound chromosome in the Drosophila female Genetics 143 1629 1642 Dernburg A F Sedat J W Hawley R S 1996 Direct evidence of a role for heterochromatin in meiotic chromosome segregation Cell 86 135 146 Dernburg A F Broman K W Func J C Marshall W F Agard D A Sedat J W 1996 Perturbation of nuclear architecture by long distance chromosome interactions Cell 85 745 759 Gertler F B Niebuhr K Reinhard M Wehland J Soriano P 1996 Mena a Relative of VASP and Drosophila Enabled Is Implicated in the Control of Microfilament Dynamics Cell 87 227 239 Goodwin P C 1996 Wide Field Deconvolution vs Confocal Microscopy of Living Cells Scanning 18 3 144 145 ppliedPrecision Appendix D Reference Information 175 Hartley W J How to Use a Microscope Doubleday 1964 Herman B Jacobson K ed Optical Microscopy for Biology Wiley Liss New York NY 1980 Hiraoka Y Agard D A Sedat J W 1991 Temporal and spatial coordination of chromosome movement spindle formation and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos Journal of Cell Biology 111 2815 2828 cover arti
53. 0 selecting cameras 125 27 Settings dialog box 217 25 shortcuts 207 Snapshot 196 Time lapse tab 212 13 toolbar 199 200 window 194 Retrace arrows 228 Reviewing large areas 55 57 ROI percent 76 Run Experiment tab 36 38 Running experiments 35 57 Sadat Dr John W 2 Safety 8 12 Bright Light Exposure 9 Danger of Shock 9 using proper filters 9 UV Exposure 9 Warning Labels 12 Sample thickness 43 44 Sample thickness setting 210 Saturation 87 89 88 170 Save Image button 228 Saving images 30 32 Scale settings 206 Scale values 206 Scaletter Beth 3 Secondary light path 58 Sectioning specimens 38 44 Sectioning tab 209 10 Sedat polychroic filter 152 Settings dialog box 217 25 action buttons 225 Autofocus tab 222 23 Display tab 218 19 Files tab 221 22 Imaging tab 219 20 Misc tab 223 25 Seubert Ron 2 Shutting down DeltaVision 32 33 143 Slide holder 114 16 adapter 115 Slow button 227 Snapshot 196 softWoRx 1 softWoRx Explorer 123 softWoRx software 117 softWoRx Suite 118 advanced option 123 softWoRx Workstation 114 Troubleshooting 167 Space requirement of system 101 Spherical aberration minimizing 177 Stage controls 202 6 Stage speed 86 Standard filename extensions 183 Standard filter sets 107 Standard microscope slide 85 Start Coordinates setting 215 Statistics 96 Step Decrease and Increase buttons 228 Stopping DeltaVision 32 33 Sub image 180 Sym
54. 13 Increase exposure time but consider the amount of exposure the specimen can tolerate Intensity values increase in direct proportion to exposure time e g doubling the exposure time doubles the intensity values 14 Repeat for all desired wavelengths 04 720104 Rev D 1008 84 DeltaVision Core and personalDV User s Manual Using Kohler and Critcal Illumination DeltaVision Core allows you to easily switch between two types of illumination Kohler Illumination is the most commonly used form of illumination It provides very even specimen illumination across the field of view The light uniformly drops off as the distance from the focal plane increases Critical Illumination directs the entire light source to the size of the detected area and not the entire sample With Critical Illumination more light is directed to the focal plane and the out of focus light drops off more rapidly than in Kohler Illumination based on the size of the field of view Critical illumination also provides better axial Z and lateral X Y contrast You will typically use Kohler Illumination for most of your data collection However Kohler Illumination can be insufficient for faint signals For low abundant probes that require more light and better contrast use Critical Illumination To switch to Cntical Illumination gt Loosen the Locking Knob and pull the Focus Control back away from the stand until it snaps into place in the Critical Il
55. 512 x Lens jiox Into Bin ia A Aux Mag Pixel size 0 6680 um at A j l Ar al dx 10 00 A A os ay ial fies dZ foso x The Resolve3D Settings window is displayed 2 From the Resolve3D Settings window open the Misc tab ppliedPrecision Chapter 4 Setting Up and Running Expenments e hesovesD Settings Be Display l imaging Files Autofocus Nise QLA Stage Motion W Allow Lost Motion Compensation LMC Misc tab Stage View Options W Show stage trails W Show stage thumbnails Wo Show paint numbers Filter Wheel Sets Excitation filter wheel LiveCell gt Emission filter wheel LiveCell Eyepiece filter wheel Livecell bd ACHES Piter heta Done save Settings 3 In the Resolve3D Settings window select the Excitation Emission and Eyepiece filter sets you want to use When these fields are changed the lt lt lt Pending Activation message is displayed in the window as shown ba ResolvesD Settings BE Display Imaging Files Autofocus Misc QL m stage Motion Allow Lost Motian Compensation Liig stage View Options Show stage trails Wo Show stage thumbnails Wo Show point numbers Select the EX EM and EP filter sets Filter Wheel Sets Excitation filter wheel Standard bd Emission filter wheel Standard Eyepiece filter wheel Standard ea Message displayed when filter wheel setti
56. 6 Swedlow J R Hirano T 1996 Chromosome dynamics Fuzzy sequences specific attachments Current Biology 6 544 547 Swedlow J R Sedat J W Agard D A 1993 Multiple chromosomal populations of topoisomerase II detected in vivo by time lapse three dimensional wide field microscopy Cell 73 97 108 Swedlow J R Sedat J W Agard D A 1997 Deconvolution in optical microscopy Deconvolution of images and spectra 2nd Edition PA Jansson ed Academic Press NY pp 284 309 Takizawa P A Sil A Swedlow J R Herskowitz I Vale R D 1997 Actin dependent localization of an RNA encoding a cell fate determinant in yeast Nature 389 90 93 Tempel B Wang H Goodwin P C 1996 BioTechniques 20 cover image Winner of 1996 BioTechniques Cover Photo Contest Urata Y Parmalee S J Agard D A Sedat J W 1995 A three dimensional structural dissection of Drosophila polytene chromosomes Journal of Cell Biology 131 279 95 Van Wye J Ghori N Webster P Mitschler R R Elmendorf H G Haldar K 1996 Identification and localization of rab6 separation of rab6 from ERD2 and implications for an unstacked Golgi in Plasmodium falciparum Molecular and Biochemical Parasitology 83 107 120 Vodicka M A Koepp D M Silver P A Emerman M 1998 HIV 1 Vpr interacts with the nuclear transport pathway to promote macrophage infection Genes amp Development 12 175 185 cover article Waters D Brown C 1996
57. AH Used in some older Delta Vision systems Bulbs Component API Part Number 250W xenon Arc Lamp Bulb 34 100390 000 LED Transmitted Light must replace entire assembly 52 851243 000 04 720104 Rev D 1008 114 DeltaVision Core and personalDV User s Manual British Power Cord Component Fuse for Component API Part Number Power cord with Bussman 10A 19 210046 000 TDC 180 10A built into plug Must meet Bntish Standard BS1362 ppliedPrecision Changing Cameras and Filters This chapter provides the following instructions for cameras and filters Changing Cameras Using Live Cell or Custom Filter Wheel Modules Changing Filter Wheel Modules Calibrating Filter Wheels Changing Cameras DeltaVision Core includes a High speed Camera as a standard component If you have the optional EM CCD camera you can change cameras to better meet your imaging needs These instructions show how to change the camera and how to select the new camera in Resolve3D CAUTION Contamination from fingerprints or dust on the camera window orinside the Emission filter wheel will degrade image quality 116 DeltaVision Core and personalDV User s Manual To change the camera 1 Remove the camera cover by lifting it up and sliding it away from the microscope F CE es 2 2 Remove the camera by pushing in and then pulling up on the camera end You do not need to remove cables from the camera Remove the
58. C stage View Options Show stage trails Show stage thumbnails Show point numbers Filter Wheel Sets Excitation filter wheel LiveCell bd Emission filter wheel Livecell Eyepiece filter wheel Livecell Reinitialize Filter vvheels Done save Settings 04 720104 Rev D 1008 210 Delta Vison Core and personalDV Users Manual Allow Lost Motion Compensation LMC Enable Lost Motion Compensation to remove the effect of hysteresis in the stage This option should be selected for most applications When it is turned off the Z series range may shift Clear this option if you want to improve speed at the expense of position repeatability Show stage trails Display the path of stage movement on the Stage View Show stage thumbnails Display a thumbnail image of each image on the Stage View as the image is acquired Show point numbers Displays the number of each point in a point list on the Stage View Exc itation filter wheel Allows you to select the name of the Excitation filter wheel that you are using This option list appears only if you have configured your system for alternate filter wheels Emission filter wheel Allows you to select the name of the Emission filter wheel that you are using This option list appears only if you have configured your system for alternate filter wheels Eyepiece filter wheel Allows you to select the name of the Eyepiece filter wheel to use This option list appears o
59. Chapter 6 Data Collection Techniques describes how to determine the proper exposure time and provides guidelines for finding the areas of interest on a sample The remaining chapters provide information on how to maintain and configure the system a Chapter 7 Facility Requirements and Components lists requirements and describes the DeltaVision system components a Chapter 8 Changing Cameras and Filters describes how to replace cameras and how to install or replace filters a Chapter 9 Maintenance shows how to change and align the xenon lamp align the light path clean the system and change fuses The appendices include reference information and procedures for configuring the system Document Conventions To make the information provided in this manual as easy as possible for you to locate and use the following conventions are observed Lists e Round bullets indicate options in procedures 1 Numbered items are sequential steps for completing a procedure Square bullets indicate items in a list gt Arrows indicate single step procedures ppliedPrecision Notes Wamings and Cautions Note Indicates information about the previous paragraph orstep ina procedure i Important Indicates important or critical information about the previous paragraph orstep ina procedure Q Tip indicates helpful advice AN WARNING Indicates important infomation regarding potential injury WARNIN
60. Coordinates setting 215 End of scan 204 Enviromental chamber 119 Environmental requirements 101 4 102 EX arrows 228 EX Filter field 211 EX Shutter button 19 229 Ex Shutter field 212 Exact method for Z scan settings 44 Excitation filters 18 26 46 specifications 184 Excitation shutter 19 safety 27 Exp field 211 Experiment Macro Editor 96 98 Experiment macros 36 38 Experiments 2D image in 3 wavelengths 17 19 3D 38 44 and autofocus macros 69 71 and focal planes 41 and macros 36 38 and marking points 50 52 and optical section spacing 41 44 and panel collection 55 57 and sample thickness 43 44 Design tab 207 8 editing macros for 96 98 guidelines for 3D 41 macros folders 221 photokinetic 120 point visiting 49 55 running 35 57 setting up 35 57 time lapse 44 49 71 77 Explorer 123 Exposure time 87 89 ppliedPrecision Appendix E Resolve3D and Keypad Options Eyepiece filter wheel 26 129 30 filter wheel and safety 9 filters 18 focus 17 Fast button 227 Fiber Optic Cable 11 Fiber optic module 116 151 adjust tilt of 156 57 checking alignment r 153 Field stop aperture 155 56 File system full error 167 Filename extensions 183 Filter sets 17 19 Filters activating multiplexed sets 58 62 and changing wheels 128 35 and safety 9 and wheel modules 128 35 changing emission wheel 130 choosing 17 19 17 19 cleaning 162 emission 18 excitation 18 excitation and emissio
61. Delta Vision Core and personalDV Restoration Microscopy System User s Manual Revision D built with precisionware fppliedPrecision DeltaVision Core and personalDV User s Manual Legal Notices Revision D of the DeltaVision Core and personalDV Restoration Microscopy System User s Manual Part number 04 720104 000 Rev D 1999 2008 Applied Precision Inc All rights reserved No part of this manual may be reproduced transmitted stored in a retrieval system or translated into any language in any form by any means without the written permission of Applied Precision Inc Information in this document is subject to change without notice DeltaVision softWoRx and Applied Precision are registered trademarks of Applied Precision Inc CoolSNAP is a trademark and Cascade is a registered trademark of Roper Scientific All other registered names and trademarks referred to in this manual are the property of their respective companies Applied Precision Inc 1040 12 Ave NW Issaquah WA 98027 425 557 1000 FAX 425 557 1055 Other Reference Documents User instructions for DeltaVision Core include the following publications Document Purpose Available for Online Help Provides reference information All softWoRx workstations for softWoRx and procedures that show how to use softWoRx tools Product Notes Provide examples and tips for All softWorRx users Online at using softWoRx www appliedprecision com Soft
62. Determine binning parameters Get pixel size Figure E 8 The Resolve3D Image Contol Fields File View Options Calibration Acquire Experiment Settings Excitation CFP w 430 10 Emission crP sy 470 30 arlo gt EX Shutter EX Lampis Off Exposure ooo Find Calibrate Image size 12512 v Lens jiox Info Bin ia 1 Aux Mag Pixel size 0 6680 um 04 720104 Rev D 1008 188 Delta Vision Core and personalDV Users Manual Excitation Specifies an excitation filter When this filter is selected a corresponding emission filter is automatically selected and the following parameters are set to the last values that were used for that excitation filter Exposure time m Neutral Density filter a Active illumination shutters Number of frames to average a Target intensity Camera gain You can choose Save Settings from the Options menu to store the configuration The wavelength bandwidth of the selected filter is indicated to the right of the filter choice box Emission Specifies an emission filter The wavelength bandwidth of the selected filter is indicated to the right of the filter choice box oT Specifies a Neutral Density filter The relative illumination intensity is indicated in the menu This value indicates the amount of light passing through the filter A value of 100 indicates the light is unfiltered Exposure Specifies camera exposure time in seconds Th
63. E TE OON 172 Rereren ce LSC ec ccicskicn antic einen ead ee ee 173 WITCEOSCODY aeiio erin r EE tint ieiain ete O 173 ppliedPrecision Contents vil Loux Operdnno Oys cya g Netrernen ener tes E Ror EnT A nen a een ren eer reer 180 Mriace TP FOCCS SUNS pc och satanedatspnicsauincendasloutiiaalateds EE E 180 NCS oo E act acetates E E T E T 180 Appendix E Resolve3D and Keypad Options sscsss LBL The Resolves D Wind ON sroin ana ra E A T A aes 182 THERESE DOMENU naisi dees E E E R 183 The Resolves D Toolbar ssavccssigccestcrestiodnaioeianteamienteseieuausnstareaceo ERE OTO NRA 186 mage Contool eile els er REE re ee raster errr errr ay 187 Stage Position Control Fields and Buttons s nacniier inn cote nnn 189 image Intensity and Scale V ales axsisids scart Gesiie dk orale eiiler cep SE 193 Ihe Message GING oyran E 193 Resolve o D NOTT CLS iy E E neuen tact da eae ee assess 194 The Desien Experiment Ia Dorerin r ENTE 194 Experiment name and Enable Fast Acquisition eeeeeeseeseseeserrerrererrerrersesresrerrersese 196 DOCH OMI DEU P psoe an rA EEEE TEE EE A E E 196 Channels SC Peio a E A NO ere 198 Peda pse SCTU Penn s E E T E ete 199 TO VISAE DOLE D a A 200 Pinerc ole con Se Upeo mann E O 201 The Design R n Experiment Dialog BOX sissies crniwe shania a 202 ENE Sere s Dialog BOK nisni na a E T E ieee 204 ACHOn DUON Gspan a a a a S 210 Keypad Joysuck Opera ON suisse soi etait a E oat tseaees 211 Lao C senesssnne
64. Explorer application Consumable Parts lists the fuses and other components that you will need to replace to maintain the system Delta Vision Core and personalDV Users Manual Electnical and Environmental Requirements An important aspect of collecting high quality images is meeting the proper electrical and environmental requirements for the system Electical Requirements Line Requirements Operating Frequency 50 60 Hz Operating Power 100 127 VAC requires 8A 200 240 VAC requires 4A Transients Transient over voltages in accordance with Installation Category II in IEC 664 Maximum Power 1200 VA Power Cord Set Requirements The power cord set received with DeltaVision meets the requirements for use in the country where you purchased the equipment General Requirements The requirements listed below are applicable to all countries a The length of the power cord set can be a maximum of 9 75 feet 3 0 m a All power cord sets must be approved by an acceptable accredited agency responsible for evaluation in the country where the power cord set will be used a The power cord set must have a minimum current capacity of 10A for 230 VAC systems or 15A for 100 120 VAC systems as required by each country s power system a The appliance coupler must meet the mechanical configuration of an EN60320 IEC 320 Standard Sheet C13 connector for mating with the appliance inlet on the Isolation Transformer Assembly ppliedPrecisi
65. G Indicates risk of explosion AN WARNING Indicates risk of shock AN CAUTION Indicates important information regarding potential damage to equipment or software User Interface Desc nption Conventions Boldface indicates the names of buttons menus dialog box options and fields Initial Capitals indicate the names of windows dialog boxes and tabs ALL CAPITALS SAN SERIF indicates the name of a key on your keyboard or keypad such as ENTER DELETE or STEP INC REASE Uniform width font indicates text to enter on a command line or in the GUI 04 720104 000 Rev D 1008 Xl Preface xii Delta Vison Core and personalDV Users Manual Contacting Applied Precision Inc If you have questions about DeltaVision first refer to this manual or consult the online Help system If you don t find the information you need contact us at one of the following addresses Customer Service Hotline Phone 800 862 5166 email servicehotline api com Hours 8 00 AM 5 00 PM Pacific Time Monday Friday Corporate Office Applied Precision Inc 1040 12 Avenue NW Issaquah WA 98027 USA Phone 425 557 1000 Fax 425 557 1055 Intemet Address www appliedprecision com Technical Publications email address techpubs api com ppliedPrecision Intoduction This chapter provides an introduction to DeltaVision a What is DeltaVision introduces the DeltaVision system and provides a history of its dev
66. Green 555nm Orange 605nm Red Cy3 CY 5 CY 5 Red 645nm Infrared 705nm Appropnate Probes Filter Name Exc ita tion Emission CFP CFP Deep Blue 430 Blue 470nm optional nm YFP YFP Blue Green 500 Yellow Green optional nm 535nm 04 720104 000 Rev D 1008 26 DeltaVision Core and personalDV User s Manual mC heny mC heny Yellow 572nm Red 632nm optional EG FP EG FP Blue 470nm Green 525nm optional See Optical Filters on Page 97 for more information Finding the Sample To set the focal plane for imaging you will need to find the sample in the eyepieces and then focus on the sample To find the sample 1 With the sample on the stage and the filters selected shown in the previous procedure set the Beam Selector to Eye lt O Turn off the lights in the lab If you cannot turn off the lights place a box over the sample to reduce the amount of ambient light Open the Excitation shutter by pressing the EX SHUTTER button in the lower left corner of the keypad You should see light through the objective The light on the stage should be the same color as the Excitation filter that you selected For example if you selected DAPI the light should be very deep violet It may be hard to see Be sure the eyepiece filter position matches the desired excitation filter position LL Note The Excitation shutter is designed to protect your eyes Each time the filter wheel position ischanged the sh
67. Panels Actions Conventional j Muitipiexed F Do Multiplexed Channel Imaging Exp EX Filter EM Filter T Ex Shutter 1 hh 000 GFP GFP EX 2 f 000 mCherry mCherry EX2 Place mCherry filter in the secondary Filter Slot Be sure that Beam Combiner is at position 4 500 LP Be sure that the Polychroic is at position 2 GFP mCherry Dual MPx J Reference Image 4 88 S25 PeH fej H eA AALS See See ir Bet Ae Nf ne Refresh exposure conditions At this point you have completed activating the Multiplex Wavelength filter set You should now continue with the steps in the next procedure for viewing a sample with the Multiplexed Wavelength operation To view a sample using the Multiplexed Wavelength opton 1 Rotate the eyepiece filter wheel to the POL or BLANK position 2 From the Resolve3D main menu select the Excitation filter currently in the primary light path CFP or GFP 04 720104 Rev D 1008 60 DeltaVision Core and personalDV Users Manual File View Options Calibration Help Acquire Experiment settings Excitation CFP a 430 10 Emission cFP 470 30 aro Ex Shutter eX o arene Exposure ooo Find Calibrate Image size 512x512 Lens o Info Bin jia 1 Aux Mag Pixel size 0 6680 um Os WA arl al dx 0 00 A Al dz dY i 0 00 el Y E ia Y E all 3 Use the EX button on the keypad to open the primary EX shutter
68. SHUTTER button Trans LED Source Toggles the transmitted light LED between off and on Subsequent to changing the transmitted light source from halogen to LED for DeltaVision Core and personalDV an actual shutter is no longer necessary Slow Medium and Fast Control the speed that the stage is moved by the joystick or keypad arrows It s usually best to start with medium The J oystick Controls stage movement Use the joystick to move the stage in the direction that you point with the joystick for example moving the joystick up moves the stage away from you moving joystick left moves the stage to the left and so on ppliedPrecision Chapter 3 Getting Started 19 Tuming DeltaVision On Use the following instructions to turn the system on for day to day use For instructions that show how to turn on DeltaVision after a system shutdown see Shutting Down and Starting the System on Page 131 To tum on DeltaVision 1 Turn on the power strip bar Note For personalDV there isno power strip bar Begin the power up process with Step 2 2 Turn on the IC MIC aa IC MIC Power Switch Workstation Power Switch e Power Strip Bar The Main Power Switches in the DeltaVision Cabinet acrylic cover not shown 3 Ifthe monitor is off turn it on 4 Ifthe Workstation is off turn it on and wait for it to boot up 5 Log on to the Workstation 6 Remove any slides from the stage igp 7
69. Saves the last acquired image to the currently open file STEP DEC REASE INC REASE Changes the step size of the movement controlled by the arrow keys located beneath the joystick Pressing either button many times will change the step size to the minimum maximum There are 8 possible step sizes 50nm 100nm 200nm 400nm 800nm 2250nm 4500nm and 9000nm X AND Y ARROW KEYS Move the stage in the direction shown Z ARROW KEYS Move the stage in the direction shown ZL MARK Z2 MARK Define the boundaries of the Z series 04 720104 Rev D 1008 214 DeltaVision Core and personalDV Users Manual EX SHUTTER Toggles the excitation shutter between open and closed TRANS SHUTTER Toggles the shutter for the transmitted light source between open and closed POINT MARK Adds the stage position to the marked points list ppliedPrecision Index T Filter field 212 3D images 4 38 44 3D Z projections 78 80 Acquire Image button 19 228 Acquire Image setting 204 Acquire images 22 27 28 30 Acquire Mode arrows 227 Active Wavelength buttons 211 Agard Dr David A 2 Altitude requirements 103 Ambient illumination 104 Applied Precision LLC contacting xii 7 Aquiring a PSF 173 78 Auto histogram range 219 Auto intensity scale 219 Autofocus 5 before imaging 213 214 options 222 23 setting in Resolve3D 204 within experiment macros 69 71 Autofocus button 52 Autofocus Z test step option 223 Automated Opti
70. Settings Saves the configuration settings and information current filters image size etc to be used the next time Resolve3D is opened The Resolve3D Calibraton Menu Use the Calibration menu to make calibration tables read calibration tables and designate which calibration tools are active Figure E 5 The Resolve3D Calibration Menu ResolvesD Make Opens the Calibration tool used to create flat field calibration tables and optional tables of bad pixels These tables may be applied to images either when they are acquired or at a later time To apply the tables to images after they are acquired use the Calibrate utility available from the Process item on the main softWoRx menu 04 720104 Rev D 1008 186 Delta Vison Core and personalDV Users Manual Read Opens the Read Calibration Files dialog box that you can use to read calibration tables Manage Opens the Manage tool that you can use to designate which calibration tables are active and to remove tables from the system s session memory The Resolve3D Help Menu Use this menu to turn ToolTips on or off get help on the Resolve3D window or find out which version of softWoRx you are using Turn ToolTips Oniot On Window Version Tum Tooltips On Off Turns tool tips on or off Tool tips display pop up information about buttons on the interface They open when the mouse pointer is held over a button for a few seconds On Window Opens Help for
71. Stick oori e o E A E RR 18 04 720104 000 Rev D 1008 DeltaVision Core and personalDV User s Manual Tomine Derav omr Oesen e bsSiostnsnuaiol vi actin saidet ses susaetesei ea vigtuee esses en oreaa wat Viniutses 19 PCG UNITING ATA SAVIN Dalies toate avo Sata abu dsannispsoansaunslaniy E 21 Setting Up the Sample for Image ACQUISITION cece cece eee eeeeeeeeeetseeesseeeeeeeeees 21 dshGq re T ie gl 8 cae nepnrnN marae Sue N Sis a AAC EST eee e A Smee 26 Acquiring Ah NAC e ata tie entrant ena ae oe eae 27 meena e baa te y amp c co DJ cep Nera ee meme rete ener neon eo eres mre Pree reer eee One rer errr ee tre er ren cere 28 Setting up a Personal Data Foldet xix iacecteinsceseciaanesaesictavandenbevacvednansaunmeeeensemenes 29 Savine a ole le Multi Channel Image wsikesccsceia carte tees Giricanteaicicauiedassd de tiseebiemastidts 29 Turine DCA V 1510 Ue oe sacts sus tieteatscis ened Gs mealiniaesnsaassh cuit NA 30 Setting Up and Running EXpenme nts c ssscssssssssssseessssseeesss 3D Creating and Running an Experiment Macro cee cssceesecesecesecesecsseceseseseseseseseeeeeesees 33 DECLIONING SPECIMENS FOF QD MAGES ein aa a a nay seis asecae nas ea dae adeno setae 35 Guidelines for Designing and Running 3D Experiment cece eeceeseeseeeeeees 39 CI BUICa SECTIONS PACU 2a asiahisa ce Uiea ie uatnastittad con uss ities eaten dasa saagteneaas arse reaseraensosmastiouss 39 DVS LEC LSP ULES oan na BO Ge sae outa tou E arash polos sl
72. To Calibrate the Neutral Density Excitation and Emission filter wheels 1 On the Resolve3D window click Settings to open the Resolve3D Settings dialog box Then click the Misc tab 04 720104 Rev D 1008 126 DeltaVision Core and personalDV User s Manual Display Imaging files Mise QIM Stage Motion A E Allow Lost Motion Compensation LMC Stage View Options E Show stage trails E Show stage thumbnails E Show point numbers Filter Wheel Sets Excitation filter wheel lLivecell Emission filter wheel lLivecell Eyepiece filter wheel lLivecell Reinitialize Filter Wheels Done save settings 2 Verify that the Excitation Emission and Eyepiece filter wheel fields all display the same filter wheel module name if any of the names are different you may need to select or replace one or more of the filter wheels 3 Press Save Settings and close Resolve3D 4 Click on the Instrument Controller icon on the workstation desktop as shown Neighborhood e 8 es a a o Lc D a D an D on 14 28 2006 0 08 5 Log on to the Instrument Controller with the user name worx and password 4delta if prompted ppliedPrecision Chapter 8 Changing Camerasand Filters 127 6 Remove the filter wheel to be calibrated Excitation Emission or Neutral Density from the system so the opening is visible 7 Press Alt H The filter wheels will rotate as they are being init
73. Using Live Cell or Custom Filter Wheel Modules A filter wheel module includes an Excitation filter wheel an Emission filter wheel and an Eyepiece filter wheel 04 720104 Rev D 1008 118 Delta Vison Core and personalDV Users Manual DeltaVision Filter Wheel Module Excitation 10 position filter wheel Emission filter wheel Eyepiece filter wheel A filter wheel module includes the Excitation Emission and Eyepiece filter wheels and can support up to six filter pairs If your system has an alternate filter wheel module you can swap modules to meet your imaging needs You can purchase a module of Live Cell filter wheels that includes factory installed filters You can also purchase a module of empty filter wheels and customize it with your own filters Changing Filter Wheel Modules Changing the filter wheel modules for your system includes changing the Eyepiece and Emission filter wheels and depending on how many filter pairs are supported may also include changing the Excitation filter wheel If you purchased a filter wheel module as a separate component after you purchased your system you must configure the instrument controller for the new module the first time that you use it see the instructions included with your filter wheel module ppliedPrecision Chapter 8 Changing Camerasand Filters 119 To change the Eyepiece filter wheel 1 Disconnect the Position Sensor cable that connects the filter wheel
74. Wo Rx Shows how to process visualize All softWoRx workstations Imaging and analyze data Workstation User s Manual Getting Started Shows how to acquire Acquisition workstations that have with QLM photokinetic data with the QLM the optional QLM module module In addition to the above Applied Precision documentation you will be provided with manufacturers manuals for the microscope components You may also be provided with manufacturers manuals for cameras and other devices depending on your system configuration Some manufacturers do not provide manuals for these devices ppliedPrecision Contents lil Contents PECE sarno a a a a a baaa aaia DC About Ims Marial ssrin A E A E OTE ix Document CONV SITIONS asea na A EA E E E x NS TS US E E O T E acca gaa E den PET E cata E E E x Notes WV arises andG aU HONS ceriosan a AAR xi User Interface Description Conventions cee esseceseceseceseseseseseeessecssecsssessesseeeeeeoegs x1 Contacting Applied Precision 11 Cosi sis scosasotateacns a xii Customer oeie HOUE usean i E E oteniearanteachawneb eto xii Corporate OI CE haan he aad headache hf dca T a xii MEMEO CIC THOM ioe Sic cdccae adie a accie diez acecwnaasuntadeieaiawauduesesdavckeweediiceaiatesaawcall Wat as De HAN 1S 1 Otis ste cotter ig heed cee hates t het aes E osnetanteauteandeaaea asada 1 FUSTO eyen A T E T AEE 2 What Gaty You Use Della Vision TOF nirani e E AA 3 Staridarc Data Colector OPON risien uae vont mi
75. ade epifluorescence inverted microscope Each objective is qualified by Applied Precision to guarantee the highest possible quality The point spread function PSF is measured in order to uphold the image quality The microscope supports a Differential Interference Contrast DIC module this is an optional component A transmitted light shutter is included with your system to enable automated DIC and Brightfield image acquisition Optical Alters The system uses a four band polychroic beam splitter and filter wheels rather than a simple dichroic filter cube The excitation neutral density and emission filters are selected in one of three ways a Selecting options in the Resolve3D acquisition software a Selecting a mode from the keypad Rotating the eyepiece filter wheel Note The eyepiece filter wheel isoperated manually but reports its position to the instrument controller which in tum adjusts the excitation and emission filters Depending upon the environment filters last between one and three years To ensure optimal performance filters should be periodically inspected and replaced if necessary Consult Chapter 9 Maintenance for a detailed explanation of this procedure The following standard DeltaVision Filter set is included in all DeltaVision Core systems Table 2 DeltaVision Standard Filter Set Filter Excitation Emission Eyepiece Name CWL BP CW BP DAPI 350 50 455 50 FITC 490 20 525 36 TRITC 555 25 605 52
76. age It also allows you to move the slide across the stage and to mark the slide position when you remove the slide The ability to move the slide across the stage allows you to view the full slide on the 1 x 1 stage area With the slide held against the three brass locators by the Return Spring you can use the Slide Adjustment Knob to move the slide laterally Marking the position of your slide is useful when you are conducting a point visiting experiment and you need to remove the slide before you are finished You can use the Position Indicator the letter scale at the bottom of the slide holder to record the position of your slide When you resume your experiment you can place the slide in the position that you recorded ppliedPrecision Chapter 7 Facility Requirements and Components 105 Figure 6 The Repeatable Slide Holder Brass Locators Retum Spring Slide Adjustment Knob Position Indicator Note If you are using a Petri dish or any other fomat that is not smilarto a 1 x3 cide you will need to remove the Repeatable Slide Holder or use the Slide Holder Adapter For more about using the Repeatable Slide Holder see Finding a Specimen and Recording its Position on Page 79 Slide Holder Adapter The Slide Holder Adapter holds a chambered coverglass It is mounted on the Repeatable Slide Holder NUNC Lab Tek chambered coverglasses with one two four or eight wells are available The Slide Ho
77. age to the position marked as the bottom of the sample Visit Middle Visits the point in the middle of the defined scan region AF Autofoc us Automatically focuses Acquire Image Collects and displays an image from the microscope The current settings are used to collect the image This image is only displayed in the Data Collection window It is not saved in a file for later use a gt XY Stage Contols Moves the stage in the X and Y axis The left and right arrows move the stage in the X axis in the increment set in the dX field and the up and down arrows move it in the Y direction in the increment set in the dY field Zstage Motion Contols Moves the stage in the Z axis in the increment set in the dZ field ET pan Moves the stage view Note that this tool is sticky To disable it click the Pan tool again RE xl Clear Stage Trails Clears all of the stage trail lines from the stage view xe Clear Thumbnails Clears all of the thumbnail images currently displayed on the stage view 04 720104 Rev D 1008 192 Delta Vison Core and personalDV Users Manual i Marked Points list Opens the Point List dialog box to manage the list of marked points Zoom Tool Controls zoom Drag the thumbwheel down to zoom in up to zoom out The button under the thumbwheel returns the zoom to 1 1 ZSlider Moves the stage up or down dX Specifies the X step size in microns dY Specifies the Y step size
78. al Belmont A S and K Bruce 1994 Visualization of G1 chromosomes a folded twisted supercoiled chromonema model of interphase chromatid structure Journal of Cell Biology 127 287 302 Charlton C A Mohler W A Radice G L Hynes R O Blau H M 1997 Fusion Competence of Myoblasts Rendered Genetically Null for N Cadherin in Culture Journal of Cell Biology 138 331 336 Chen H Sedat J W Agard D A 1989 Manipulation display and analysis of three dimensional biological images in Handbook of Biological Confocal Microscopy Pawley J ed pp 127 135 IMP Press Madison WI Chen H Hughes D D Chan T A Sedat J W Agard D A 1996 IVE Image Visulization Environment A Software Platform for All Three Dimensional Microscopy Applications Journal of Structural Biology 116 56 60 Chikashige Y Ding D Q Funabuki H Haraguchi T Mashiko S Yanagida M Hiraoka Y 1994 Telomere led premeiotic chromosome movement in fission yeast Science 3 270 273 Cooke C A Schaar B Yen T J Earnshaw W C 1997 Localizationof CENP E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase Chromosoma Berl 106 446 455 cover article Csink A K Henikoff S 1996 Genetic modification of heterochromatic association and nuclear organization of Drosophila Nature 381 529 531 Dawe R K Sedat J W Agard D A Cande W Z 1994 Meiotic chromosome pairing in maize is associated
79. aning 32 damage prevention 11 Oil calculator 164 65 Oil immersion See Immersion oil Optical axis integration 68 78 80 Optical components 105 10 Optical filters 106 damage prevention 11 specifications 184 Optical section spacing 210 Optical sections 41 44 Optical transfer function 173 81 conversion from PSF 178 81 file 180 sample image 179 Optional components 118 23 Optional software 123 OTF See Optical transfer function Overlap setting 215 Overscan method for Z scan settings 44 Pan tool 204 Panel 1 and 2 Mark buttons 228 Panel collection 5 55 57 214 15 Personal data folder 30 32 Phase contrast 3 Photo sensor 110 Photo sensor port 151 Photo bleaching 87 Pixel size 174 75 setting 202 Point arrows 227 Point list 49 55 197 dialog box 52 editing 53 Loading 53 Saving 53 Point Mark key 51 Point marking 50 52 Point spread function 173 81 acquiring 173 78 conversion to OTF 178 81 file 180 finding beads 176 tools needed 174 wavelength 180 Point visiting 4 5 41 49 55 monitoring 54 Points tab 213 14 Polychroic mirror 162 Post autofocus Z offset setting 223 04 720104 Rev D 1008 219 Power switches 142 Prerequisites 15 Previous experience method for Z scan settings 44 Primary light path 58 Probes 17 19 PSF See Point spread function OLM switch 142 Quantifiable laser module 26 Quantitative Processing 4 Quit button 227 Radial frequency 179 Recommended Z
80. anogaster interphase nuclei I Tissue specific aspects of polytene nuclear architecture Journal of Cell Biology 104 1455 70 Hochstrasser M Sedat J W 1987 Three dimensional organization of Drosophila melanogaster interphase nuclei II Chromosome spatial organization and gene regulation Journal of Cell Biology 104 1471 83 Ikeya T Shinohara A Sato S Tabata S Ogawa T 1996 Localization of mouse Rad51 and Lim15 proteins on meiotic chromosomes at late stages of prophase 1 Genes To Cells 1 379 389 Inoue S Video Microscopy Prenum Press New York NY 1986 04 720104 Rev D 1008 176 DeltaVision Core and personalDV User s Manual Jongens T A Ackerman L D Swedlow J R Jan L Y Jan Y N 1994 Germ cell less functions early in the germ cell specification pathway of Drosophila Genes Devel 8 2123 2136 Kam Z Agard D A Sedat J W 1997 Three dimensional microscopy in thick biological samples a fresh approach for adjusting focus and correcting spherical aberration Bioimaging 5 40 49 Kam Z Chen H Sedat J W Agard D A 1991 Analysis of three dimensional image data display and feature tracking in Electron Tomography Frank J ed Plenum Press New York Kam Z Jones M O Chen H Agard D A Sedat J W 1993 Design and construction of an optimal illumination system for quantitative wide field multidimensional microscopy Bioimaging 1 71 81 Kam Z Minden J S Agard D A Sedat J W Lepti
81. at location Next use the slider to find the bottom of the sample When you are satisfied press the x button to mark that location Note To reduce the risk of running the objective lens into the sample the Zslider is limited to 5um ata time File View Options Calibration Help File View Options Calibration Help File View Options Calibration Help oe eee we ee a te ae dx 10 00 10 00 dx 10 00 IEN ajte dz 2 00 g Eo ajte dz 2 00 z Ta ajte dz 2 00 Ks 8 xil io kin Use the Zsliderto find the top and bottom of a sample The sample thickness is indicated by the wide line on the Zslider shown on the nght image Note The Zstage view displays the relative position of the objective lens to the sample foran inverted microscope Dragging the Zslider down focuses the microscope closerto the cover glass negative direction Dragging the Zsliderup moves the focus from the cover glass toward the microscope slide positive direction 3 Click Experiment to open the Design Run Experiment window and click the Design Experiment tab Then click the Sectioning tab 04 720104 Rev D 1008 38 Delta Vison Core and personalDV Users Manual hg Design Run Experiment sa gA Design Experiment Design PK Experiment Run Experiment Experiment name Resolves Estimated file size 32 01 Mb 22 30 Gb Available W Use Fast Acquisition Fast Acquisition Options Lamps Off when fini
82. at the xenon lamp s internal pressure be relieved prior to disposal This is accomplished by squeezing off the tip see illustration below with needle nosed pliers until the pressurized gas escapes 04 720104 000 Rev D 1008 10 DeltaVision Core and personalDV User s Manual Pinch off the tip with needle nosed pliers to relieve pressurized gas before disposal Relieving xenon lamp s intemal pressure before disposal Pressurized xenon lamps should not be incinerated but disposed of in a landfill Damage Prevention The following actions could damage the system Moving the stage to the home position with the objective up could break or scratch the objective The stage could be driven into the objective and may potentially scratch the top lens or compress the lens housing causing a leak crack or lens misalignment Disconnecting cables before the system is completely shut down will damage one or more of the electronic circuit boards Disconnecting cables to the camera when the power is on can damage the camera a Touching optical filters or the polychroic beam splitter contaminates them with oil and can lead to premature failure or poor image quality For cleaning information see Chapter 9 Maintenance Bending the fiber optic cable into a coil with a diameter less than 24 will damage the cable m Using improperly rated replacement fuses can create a fire hazard and may result in damage to components Use on
83. au E 42 CHOOSING WINICO TULETS TOUSE aiei a T E T 43 Seting ap Umea pse Experi MCW S irrin cs e A N E E OO 45 POMEN ISTO set ENET T A E A ceyaeeLs 47 Markina TOn S cia Men Mere yer ean ore ee ce ee moe renner ye rere n Tr me eer tr ene ren 48 Editing a Point EiStesnresiirii ra Tegan T O ean asec u eens canes eee 50 Loading a Point List and Specifying Points to Visit eeeeeeeeeeeeeeeeereereerrsrrrrrreereesses 50 Monitonng POmt Visiting EX PSII sssnisenassannik 51 Collecting Panel Images over Large ArcdiSuresen ana a a a a 53 Determine Border RONOM Voxel isecsrsrs sainn E NR 53 Collecting Panel Imag esunianan ie n A 54 Using the Multiplexed Wavelength OPtOn siissiissiicsecsicisirosdierirsisiinersiisensiisinsescisiiearisis 55 Setting Up the Multiplexed Wavelength Option seesessessesserseriereeresrerresresresresressersees 55 Designing a Multiplexed Wavelength Experiment eeeessesresrerrrrrerrerrrrrrrrrrerrereesees 60 Acquinng Data from Live SpeciMenS ssccsssseenssseensseeesseensss OD Using A tofoc s in Experiment NI ACTOS ap veiiscesetnsctishinastssiten aguas avedanaessegucaswastaeavercuntessecwaes 66 Tracking Oe Ds peepee mre rer oreo cere orn er ere ore re a eee eer reer eee errr rere arene 67 Guidelines for specifying Cell Tracking Options cece ees eeeeeseeeseeeeeeeeeeseeees 70 AMequirine DAZ PLOJECHONS WIT OA sassincethiseesicosanozauaadunsstunsuincdssdaiteraicusentatauanlecctsstowad pes diates 73 Continuous Z Sweep versu
84. ble Lamps Off when finished Time lapse Points Panels Actions Z Scan Options Focus point when scan starts Middle of Sample v Sample thickness 2 80 Get thickness W Enable OAI Scan Click Experiment to open the Design Run Experiment window Then click the 4 Select the Z Sectioning and Enable OAI Scan options 5 Click the Run Experiment tab and click the green arrow button in the toolbar at the top of the screen to start the experiment Note When deconvolving OAI images select the More Options button on the Deconvolve window and then select the Deconvolve Projections option in the More Deconvolution Options dialog box 04 720104 Rev D 1008 go Eer it Osis Hal Al a ale apo 5 Z 75 Zslider 76 Delta Vision Core and personalDV Users Manual Acquiring a Reference Image You can use an alternate filter or the transmitted light to acquire a reference image that can be combined with other images This option is useful for Differential Interference Contrast DIC analysis It can also be useful for other types of reference images A reference image of yeast cells You can specify to acquire the reference image at the top middle or bottom of the sample Top of Sample Middle of Sample Bottom of Sample Note It isimportant to optimize the reference image settings priorto defining them in the Expenment Designer To Create a reference image 1
85. cal Sectioning 4 Axial frequency 179 Bead slides 177 Beads finding for PSF measurement 176 measuring in immersion oil 177 78 Bibliography 15 Bin setting 202 Blank Screen button 227 Border rolloff 180 determining 56 Bottom of sample 41 Bottom of Sample setting 204 Brightfield 3 Bulb icon 32 143 Bulbs 124 Cabinet components 112 14 Calculating OTF See Optical transfer function Calculating pixel size 174 75 Camera changing 125 27 damage prevention 11 EM CCD 87 121 22 high speed 87 107 8 power supply 108 saturation 87 170 setting in Resolve3D 220 supported for DeltaVision 107 8 Camera not found message 167 CCD camera 107 8 damage prevention 11 Cell mitosis 47 Cell tracking 71 77 Center Object setting 203 Center of geometry 74 Center of intensity 74 Channel for Autofocus setting 223 Channels tab 211 12 Choosing filters 17 19 Cleaning DeltaVision 162 Coarse focus 17 23 26 Collect Panels setting 57 215 Collecting data 84 98 Collecting panel images 55 57 Command line interface 197 Components 99 124 cabinet 112 14 camera 107 8 consumable 123 24 desktop 110 12 EM CCD camera 121 22 enviromental chamber 119 fiber optic module 116 fluorescense microscope 106 instument controller 113 optical 105 10 optional 118 23 optional software 123 photo sensor 110 repeatable slide holder 114 16 software 117 softWoRx Workstation 114 TIRF module 121 tool kit 116 Xenon
86. cans Be lees AN a A 97 C E E E E asta seed body eu sexaseeasncea 97 LON OOU ES aa tau saan tsetse ei eeasanateon seu sciagtineasan at bees eeutesoreaaadaarageein 99 DCS UO ComPoneilS yrrir o T E EE TEE E 101 Flat Panel Display M ntoiieosssnnniieinen o E AE Sedeieies 101 IIIS IRC VAG AIG Joys u Keran a A NET 101 Vibration Isolation Vales sii feign ste o n a eae EE auebacaeasucdetaes 102 Cabinet Compone NiS rrna n a T E E 102 Instrument Controller Microscope Interface Chassis IC MIC 0 eee 103 WN OTK SEA UO Besse sh acs a S 104 DVD R IRE COM 1G rantonnei a a a R a N 104 Other Stand ard Componer a a idcdah essa do tieaahaeaat since 104 Whe Repeatabie Sid Folder onccestsscasavesmuuesseassstaiscousvayseseusautensndeiensteeisenseeSeacdein deans 104 oide Holder Adapte essri ops ueaabodsausduapeuartacepascananeeseoueoreee 105 CUDAN O E EEEE E ONTA TOENE 105 Ihe Fiber Opie Modal sser a a A 106 A AE T saa ets EE E ae tobe SoA N eye E E anaes 106 SOWA r E E E N O OEE 107 Ophonal COMPOMeries iisa E E A A E O 108 The Environinental Chambe ourensana cate E E A TN A 109 Addinonal Filter Modules xis asco cased stn cods Gla sccumtabaaneuss T E 109 Quantifiable Laser Module Components cece escesseessecseesseceseseseseseeeseseseeesees 110 Total Internal Reflection Fluorescence TIRF Module eccseccceeenteeeeeeneeees 110 EM CDC AIM OL I cos bodexeaysanaiieon E E E dons besuaemnsereneoase dees 111 Analysis Work sla OMS uia eaters a eNote
87. ce the shutter open mode the camera readout mode and the starting Zlocation forthe scan See the online Help for more information Sectioning Setup Once your microscope is focused near the middle of the vertical zone of interest of your sample you can use the parameters in the Design Experiment Sectioning tab to control the Optical Sectioning procedure Note The standard scan direction moves the objective lens towards the specimen ppliedPrecision Appendix E Resolve3D and Keypad Options 197 Figure E 13 Design Expenment Sectioning Setup Options QJ design Run Experiment File Design Experiment Design PK Experiment Run Experiment Experiment name Resolvea0 Estimated File Size 32 01 Mb Enable Fast Acquisition fast Acquisition Uintians Sectioning Channels l Time lapse l Points l Panels Actions F Z Sectioning Z Scan Options Focus point when scan starts Middle of Sample ha Optical section spacing fo 20 Number of optical sections B4 Sample thickness i280 Get thickness Enable OAI Scan Foc us point when scan starts Specifies the Z location of the sample when the experiment starts The recommended option is Middle of Sample Optical section spacing Specifies the spacing in microns between each optical section The focal point will be changed by this value after each image is collected Number of optical sections Specifies the number of sections to c
88. change the Emission filter wheel 1 Turn off the IC MIC 2 Remove the camera cover Then remove the camera by pushing in and pulling up on the camera end as shown below ppliedPrecision Chapter 8 Changing Camerasand Filters 121 3 On the Emission filter wheel motor disconnect the cable that connects the motor to the IC MIC Use a 3 mm hex key to loosen the set screw that holds the Emission filter wheel to the microscope Cable to IC MIC Set Screw IC MIC Cable Connector Ss 5 Install the alternate Emission filter wheel Tighten the Emission filter wheel set screw and connect the cable from the IC MIC to the new Emission filter wheel 6 Reinstall the camera by sliding it into place and then pushing in and pressing down on the camera Note If you still need to change the EP or EX filter wheels do so now before continuing with this procedure 04 720104 Rev D 1008 122 DeltaVision Core and personalDV User s Manual 7 When you are finished changing filter wheels restart the IC MIC 8 If Resolve3D does not automatically display the correct filter module there are two ways to expressly let the system know you have a new filter set e From the Resolve3D main menu select Settings change to the Misc tab and select the current filter set e Unplug and then re plug in the Eyepiece filter wheel position sensor cable see below The system will automatically recognize which filter set the Ey
89. cle Hiraoka Y Dernburg A F Parmalee S J Rykowski M C Agard D A Sedat J W 1991 The onset of homologous chromosome pairing during Drosophilia melanogaster embryogenesis Journal of Cell Biology 120 591 600 cover article Hiraoka Y Haraguchi T 1996 Fluorescence imaging of mammalian living cells Chromosome Research 4 3 173 176 Hiraoka Y Minden J S Swedlow J R Sedat J W Agard D A 1989 Focal points for chromosome condensation and decondensation from three dimensional in vivo time lapse microscopy Nature 342 293 296 Hiraoka Y Rykowski M R Lefstin J A Agard D A Sedat J W 1990 Three dimensional organization of chromosomes studied by in situ hybridization and optical sectioning microscopy Proc Soc Photo electro Intrument Engin 1205 11 19 Hiraoka Y Sedat J W Agard D A 1987 The use of a charge coupled device for quantitative optical microscopy of biological structures Science 238 36 41 Hiraoka Y Sedat J W Agard D A 1990 Determination of three dimensional imaging properties of a light microscope system partial confocal behavior in epifluorescence microscopy Biophysical Journal 57 325 333 Hiraoka Y Swedlow J R Paddy M R Agard D A Sedat J W 1991 Three dimensional multiple wavelength fluorescence microscopy for the structural analysis of biological phenomena Seminars in Cell Biology 2 153 165 Hochstrasser M Sedat J W 1987 Three dimensional organization of Drosophila mel
90. cle as shown below 04 720104 Rev D 1008 144 10 DeltaVision Core and personalDV User s Manual Middle Circle Focus on the outside edge of the middle circle After you focus on the outside edge of the middle circle the outside edge of the inner circle should also be in focus and the centers of the inside and middle circles should be aligned c Ifthe image is not if focus go to Step Three Set the Kohler Spring Position on Page 147 If you are using Critical Illumination check for alignment by moving the eyepiece to the Open position O loosening the Locking knob and sliding the Focus Control on the Fiber Optic Module out to the Critical Illumination position Then tighten the Locking Knob Both the scratches and the edge of the circle should be roughly centered and in focus as shown below If not go to Step Four Set the Critical Illumination Position on Page 148 If you observe any alignment problems in Steps 7 8 or 9 use the instructions in the next section to align the illumination path Path Alignment This procedure shows how to align the Fiber Optic Module and the fiber optic cable with the light path It also shows how to set the positions of the two springs that allow you to switch between Kohler and Critical Illumination Step One Center the Field Stop Aperture 1 Apply immersion oil to both the objective and the mirror slide and mount the mirror slide ppliedPrecision Chapter 9 Main
91. click the Settings button Figure E 19 The Settings Dialog Box Display Options dill E Display imaging Files l Autofocus Mise QLA a Reso lve3D Settings Image display mode None Window ler Wave i Calculate statistics _ Calculate histogram _ Aute histogram range Display images J Deconvolve preview images F Auto intensity scale Acquire after paint visit Done save Settings LL Note The QLM tab is available only for systems that have the QIM module ppliedPrecision Appendix E Resolve3D and Keypad Options 205 Settings Dialog Box Display Options The Settings dialog box Display tab options allow you to control how the images are displayed Image Display Mode Specifies which window and wave are used for image display Five modes are currently available Model Desc nption None Displays images in the current window and wave Scratch Displays all imagesin the specified window Wave 1 Auto Grayscale Displays imagesin a separate window for each Emission EM filter Auto Color Displays imagesin a different wave foreach Emission EM filter This option should be used only when you are running an expenment Point Track Opensa separate window foreach visited point in a point visiting expenment Window Specifies the number of the active display window Select a new window to change where the next image will be displayed Temporary windows are numbered 21 or higher Wave Speci
92. ction Fluorescence module available for DeltaVision Core only includes the Olympus TIRF module and special adapters that allow you to install it on DeltaVision a The EM CCD Camera available for DeltaVision Core only is an optional camera that provides high signal to noise ratios for Low light Fluorescence TIRF or single molecule fluorescence a Analysis Workstations include the softWoRx Linux workstation and the softWoRx Suite Windows workstation Software includes two optional modules for either the DeltaVision Core or personalDV the softWoRx Explorer and the softWoRx Suite advanced option softWoRx Suite for Windows is also available as an option for personalDV ppliedPrecision Chapter 7 Facility Requirements and Components 109 m A selection of Differential Interference Contrast DIC components are supported by DeltaVision Core and personalDV a The optional Multiplexed Wavelength module for DeltaVision includes a second shuttered xenon light source and provides nearly simultaneous two channel imaging a The following optional objectives are tested by Applied Precision to ensure that they meet our rigid quality standards Optional Objectives U APO 40X Oil 0 65 1 35NA 0 10mm WD U PLAN SAPO 100X Oil 1 4NA 0 12 WD U PLAN APO 60X W PSF Water 1 20 NA 0 25mm WD The following subsections describe each of the optional DeltaVision components The Environmental Chamber Environmental control is an essential fa
93. ction Wheel to Open O and fully open the field aperture Apply immersion oil to both the objective and the mirror slide and mount the mirror slide Loosen the Locking Knob Then move the Focus Control on the Fiber Optic Module out approximately 5 mm and then slide it back and forth until the edge of the circle snaps into a sharp focus You should see both the scratches and the edge of the circle in focus as shown below Ra Note Critical Illumination is typically 5mm out from K hler Illumination Tighten the Locking Knob Check the focus If the edges on the circle are not crisp loosen the locking knob re adjust the Focus Control and tighten the Locking Knob again Slide the Critical Illumination spring into the groove on the Focus Control and tighten the Critical Illumination spring screws with a 2 5 mm hex key Step Five Adjust and Test the Alignment 1 Fully close the Field Aperture and slowly rotate the microscope Z focus knob in and out of focus The light should evenly expand around the aperture a If the light does not expand evenly around the aperture make sure that e The shutter is open e The Eyepiece Selection Wheel is set to 0 e The mirror slide is mounted in immersion oil and in focus e The Fiber Optic Module is in Critical position see Step 3 b Use a 1 5 mm hex key to adjust the X and Y adjustment screws on the top and left sides of the Fiber Optic Module until the image is centered in the reticle
94. ctions Sample thickness Optical section spacing x Number of optical sections A number of methods have been used to determine the necessary Z scan settings The best method for your work depends largely on preference and experience and there is not really a single best way to approach the problem Listed below are some of the methods that have been used Exact Method Use the exact method when you want to make a perfect Z scan Use the position buttons in the middle of the Resolve3D window to find and center the object of interest as follows Use the _4 button to move the Z focus to the top of the sample and mark the top location with the 4 button Then use the _ button to move to the bottom of the object and mark the bottom location with the l button On the Sectioning tab use the Get thickness button to calculate the thickness of the sample The thickness is entered into the Sample Thickness field Note that the experiment created by Design Experiment uses relative motion for Z and 04 720104 Rev D 1008 42 Delta Vison Core and personalDV Users Manual not absolute coordinates All motion in Z is relative to the starting point and does not refer to an absolute Z position Ra Note The top and bottom can be viewed by pressing the J button or the button respectively The middle section can be viewed by pressing the button m Overscan Method As the name implies the overscan method involves using a Z range
95. ctor in live cell microscopy or any other type of experiments requiring system stability Focus instability can be difficult to resolve for critical experiments Small changes in ambient temperature can lead to thermal expansion or contraction in the microscope stand stage and objective changing the plane of focus The Environmental Chamber is designed to precisely control temperature and CO levels making it ideal for long term time lapse applications and high resolution imaging The Environmental Chamber includes a temperature controller a CO2 humidifier and a CO2 chamber For instructions that show how to install remove and operate the environmental chamber see the Environmental Chamber User s Manual included with your system Additional Alter Modules You can purchase additional DeltaVision Filter modules and use them to quickly change the filter sets to meet different imaging requirements Each filter module holds up to six filters You can purchase a Live Cell Filter Module that is preloaded with four filter sets that are commonly used for live cell imaging You can also purchase empty filter modules and insert customized filter sets into those modules 04 720104 Rev D 1008 110 DeltaVision Core and personalDV User s Manual The Live Cell Filter Module The Live Cell Filter Module includes the following filters CFP 430 24 470 24 YFP 510 20 535 30 EGFP 470 40 525 50 Quantfiable Laser Module Components The Quant
96. d Running an Experiment Macro on Page 33 2 In the Resolve3D window use the Z Slider on the right side of the Resolve3D window to find and center the object of interest Drag the slider up to find the top of your sample When you release the mouse button an image is acquired and displayed in the Data Collection window Drag the slider and acquire images until you are satisfied Then press the button to mark that location Next use the slider to find the bottom of the sample When you are satisfied press the l button to mark that location ppliedPrecision Chapter 5 Acquinng Data From Live Specimens Q Tall X l arj al dx 10 00 A A dZ l i dY i 0 00 al pE SE y Z mise File View Options Calibration Help O Tal A l arj l dX 10 00 Al z l o a E gt dz 2 00 x Z jo ki YZ 405 80 118 21 3 33 File View Options Calibration Help dx f 0 00 dY f 0 00 Use the Zsliderto find the top and bottom of a sample The sample thickness is indicated by the wide line on the Zslider shown on the nght image Sectioning tab a Design Run Experiment Resolve3D exp modified du g OO Design Experiment Design PK Experiment l Bun Experiment Experiment name Resolves Estimated file size 1 00 Mb he een syrgir iia rhiz fo tysbiesinc FMRI Sav hake DIRINA ESE AREQZE E D Sectioning Channels W zZ Sectioning 22 28 Gb Availa
97. d approximate and save the point list Then in Resolve3D specify a more exact location for each point The points marked using the keypad are automatically communicated to Resolve3D and displayed in the Stage view You can view the list of points by opening the Point List dialog box To mark points using the keypad and Resolve3D 1 Use the arrows on the keypad or the joystick to move to the desired point 2 Press the POINT MARK key on the keypad The number of the marked point is displayed in the Stage view O Faia Hilal At al dx o oo a A dZ dY 10 00 Aly y Z ouno ORRA ATZ bb3 30 Min 130 Max 150 Mean 138 2 lo Marked point in the Stage View 3 Repeat steps 1 and 2 to mark the desired points ppliedPrecision 49 Chapter 4 Setting Up and Running Expenments As you mark points the stage trails and points along with their point numbers are displayed in the Resolve3D Stage view Q rials H 7a wl oe aje eo E dy 10 00 Point Numbers Min 131 Max 150 Mean 138 6 lo 4 To view all of the points use the Zoom Wheel to zoom out on the stage view To pan the stage view use the I button O Tlajt x al af ol OC Tala HH al ael a dx 10 00 a a 10 ro ee a e t 2 Z d 10 00 0 enman XYZ 665 38 753 69 0 23 Min 131 Max 150 Mean 138 6 lo _ T The stage view after zooming in left and panning ng
98. des a functional infrastructure for the storage of biological images and their associated metadata in a centralized database Like softWoRx Suite softWoRx DMS includes the Browser and the Explorer programs but unlike softWoRx Suite does not have the ability to perform any type of image processing The DMS product actually has two distinctive parts One part is the DMS Server and the other part is represented by the DMS clients The DMS Server represents different configurations of what the centralized database looks like The DMS clients are the different programs and methodologies of accessing the image data within the centralized database ie softWoRx softWoRx Suite and softWoRx DMS Optional Components You can purchase several optional DeltaVision components Some options are available for both DeltaVision Core and personalDV but many are available only for DeltaVision Core as noted below a The Environmental Chamber provides a controlled temperature environment for live cell imaging The chamber also supports CO injection which allows you to control humidity and pH by maintaining a CO flow over the sample Additional Filter Modules can be loaded with custom filters or with the Live Cell filter set to provide greater flexibility for your lab Quantifiable Laser Module components available for DeltaVision Core only allow you to use lasers to perform FRAP and other photokinetic experiments The TIRF Total Internal Refle
99. dow bd Design Run Experiment modified File 2 t Z Design Experiment Design PK Experiment l Run Experiment Experiment name ResolvesDi Estimated file size 64 02 Mb 22 29 Gb Available _ Use Fast Acquisition Fest tcquisition piora Lamps Off when finished Sectioning Channels Time lapse Points Panels l Actions W Time lapse Hours Minutes Seconds Milliseconds Time lapse e e o o Total Time o jo o 0 Time Points f W Enable Cell Tracking Cell Tracking Options W Autofocus before imaging Autofocus Options ppliedPrecision Chapter 5 Acquinng Data From Live Specimens 67 2 Click the Design Experiment tab and then click the Time lapse tab 3 Select the Autofocus before imaging option When this option is selected the software automatically determines the parameters for the Autofocus process 4 To change the various Autofocus parameters click the Autofocus Options button The Resolve3D Settings dialog box is displayed with the Autofocus tab selected Display imaging l Files Autofocus Mise QLA _ Automatically determine parameters Test Channel for Autofocus EFP Contrast calculation method Autofocus Z test step umj ae 0 Maximum Z test range umj B000 Fost autofocus lt offset um aoo Done save Settings 5 Deselect the Automatically determine parameters option The remaining parameters on the dialog box
100. e e g 60X to find the sample The approach for finding a sample with a fluorescence microscope is different than what you would use fora transmitted light microscope With transmitted light a typical approach isto find the sample with low powered objectivesand then increase powerafter you find the sample With a fluorescent light however the sample is often not visible at low powers The best approach for finding a sample with fluorescent light isto start with a high power objective 3 In the Resolve3D Lens list select the same objective ppliedPrecision Chapter 3 Getting Started 23 File View Options Calibration Help Acquire Experiment Settings Excitation CFP v 430 10 Emission CFP v 470 30 LT BLOCK EX Shutter EX WS La Exposure nooo Find F Calibrate mage sie 512512 y l Lens 50x ba Info Bn 1x1 J Aux Mag Pixel size 0 1103 um AN CAUTION Make sure that the objective is selected in the Resolve3D Lens list 4 Rotate the dichroic filter wheel to select the appropriate dichroic mirror For standard filters use position 1 5 In the Resolve3D Window click the Info button to open the Lens Information window 04 720104 000 Rev D 1008 24 DeltaVision Core and personalDV User s Manual File Lens Lens ID Name Olympus 10 0 40 D Plan Apo UY phase fluor free Ih Manutacturer Olympus Manufacturer P N 1 LP331 APLLE P N Unknown Magnification fio
101. e 201 Actions Lets you designate laser events Autofocus pause wait time lapse ratio imaging and other specific actions to occur during the experiment 04 720104 Rev D 1008 196 Delta Vison Core and personalDV Users Manual Expenment name and Enable Fast Ac quisition Expenment name Specifies the name of the file that is generated by the Experiment Designer A file extension of exp will be added to the name Enable Fast Ac quisition Enables fast image acquisition of 2D images This is only available when you are using an interline camera This data acquisition mode should be used carefully Fast Acquisition uses a single command to set up a data stream to the Instrument Controller Because no checking for user input occurs once the command is executed you cannot stop an acquisition sequence once it is started There is an upper memory limit for data collection that is based on the size of RAM memory for the Instrument Controller typically about 350 400 MB If you run into this limitation turning Fast Acquisition off allows you to collect larger data sets If the system is unstable after collecting a large data set with Fast Acquisition restart the workstation or stop using Fast Acquisition unless it is strictly necessary Sometimes Fast Acquisition isn t much faster than standard acquisition and more is risked than gained by using it Note You can set Fast Image Acquisition options to control the scan sequen
102. e and personalDV User s Manual a ree fe es ug TTED N T yh Transmitted Illumination Source Connection back of IC MIC 5 Re insert the new transmitted light assembly Part 52 851243 000 into the housing and tighten the two hex screws 6 Re connect the new transmitted light assembly cable to the Transmitted Illumination Source connection on the back of the IC MIC shown above Aligning the Illumination Path To get the best images with DeltaVision make sure that the light path is aligned in both Kohler and Critical positions Use the instructions in this section to test and adjust alignment This section contains the following topics a The Fiber Optic Module describes the settings and adjustments on this key component You ll use the Fiber Optic Module to switch from Kohler to Critical Illumination You ll also use the Fiber Optic Module to align the illumination path with the microscope a Before You Check or Adjust the Illumination Path Alignment lists the settings and filters that you ll need to select before you check or adjust alignment a Checking Illumination Path Alignment shows how to check the alignment in both Kohler and Critical positions To ensure optimal performance check the alignment every two weeks If acquired images seem dim you should also check alignment to verify that the light is reaching the sample m Path Alignment shows how to align the illumination path in both Kohler and Critical positi
103. e in the same room as the system will facilitate communication with Technical Support Air Movement Air movement around the microscope can cause specimen drift on the scale of several microns Two common sources of air movement are window air conditioners and open windows Central air conditioning is recommended However the system should not be placed in the direct path of the incoming air Vibration Isolation The vibration absorbing design of the system minimizes motion artifacts from internal vibration due to shutters filter wheels and stage movement The system is also designed to damp out external vibration as well Avoiding locations near refrigerators elevators ventilation equipment and other sources of vibration will improve image resolution 1 TEC 61010 1 274 ed International Electrical Commission defines POLLUTION DEGREE 2 as follows Normally only non conductive POLLUTION occurs Occasionally however a temporary conductivity caused by condensation must be expected ppliedPrecision Chapter 7 Facility Requirements and Components 95 Ambient Illumination For best results minimize ambient illumination during data collection A light tight room is recommended Ensure that there are no light sources pointed downward into the lens A small desk lamp located near the workstation is recommended for preparing and monitoring experiments You can press the BLANK SCREEN key on the keypad to darken the monitor for
104. e minimum and maximum exposure times allowed for this field depend upon the camera type Find Finds the exposure time necessary to reach the target intensity currently entered in the Target Intensity dialog box This dialog box opens when you click the Find button For dim samples 300 900 counts are acceptable For bright samples 3000 3600 counts provide a high dynamic range without saturation Calibrate Calibrates images when they are collected from the camera If you use this option you must load and activate a calibration table that fits the current imaging parameters wavelength image size bin choice before you acquire images Shutter Control Selects the desired excitation shutter ppliedPrecision Appendix E Resolve3D and Keypad Options 189 Image Size Specifies image size pixels or CCD detector elements for acquired images The pull down list contains predefined sizes for convenience You can enter special sizes by choosing Other from the pull down list Image size must be a multiple of four Lens Specifies the objective lens name The pull down list contains the lenses that are known to be part of the microscope system Info Opens the Lens Information dialog box which displays information about the current objective lens Bin Specifies the number of CCD detector elements to add together to form one image element Binning is applied in both the X and Y directions It increases intensity but it decreas
105. ect the light path focus and select filters It also introduces the keypad and joystick a Turning DeltaVision on shows how to turn on the system a Acquiring an Image shows how to place the slide and the objective find the sample and acquire an image a Saving Image Data describes how to set up a personal data folder and how to save an image file a Turning DeltaVision Off shows how to turn off the system 14 Delta Vison Core and personalDV Users Manual Before you start Before you start make sure that you e Select the proper oil for your objective The immersion oil kit includes 18 oils with refractive indexes that range from 1 500 to 1 534 in increments of 0 002 For personalDV the kit includes 6 oils that range from 1 512 to 1 522 To calculate the best refractive index for your application follow the instructions in Appendix A The Immersion Oil Kit on Page 153 If you are working at standard temperature and pressure the oil with a 1 516 refractive index is generally a good place to start For work at 37 degrees C use the oil with a 1 520 refractive index e Know your login ID and password e Prepare your sample using the recommended practices that are documented in the following references If you are untrained in sample preparation consider attending a course like the one shown below Books on Cell Biology Methods Current Protocols in Cell Biology Bonifacino Dasso Lippincott Schwartz Harford Yamada
106. elopment a What Can You Use DeltaVision for lists the supported imaging modes and summarizes the data acquisition options that are supported by DeltaVision a What should you know to use DeltaVision summarizes the background and experience required to run the system What is DeltaVision The DeltaVision Restoration Microscopy System can be used to collect and analyze three dimensional microscope images acquired over long periods of time and on multiple samples With the sophisticated softWoRx image analysis and model building software the system is a comprehensive package for biological image data collection interpretation and display DeltaVision Core and personalDV User s Manual History The original restoration microscopes were designed and developed in the laboratories of Dr John W Sedat and Dr David A Agard at the University of California San Francisco Their first working system actively collected images as early as 1983 At that time a small deconvolution 128x128x64 required overnight processing on a million dollar mainframe computer During the evolution of the UCSF microscope it became clear that micropositioning was a critical part of the optical sectioning process In particular controlled movement of the focal plane relative to the specimen the Z axis was identified as a key to reliable deconvolution To accomplish adequate Z scans Dr Sedat built a microscope stage using Applied Precision s Nanomotion
107. ely 2000 counts Use the following instructions to find the best exposure time for each excitation wavelength This procedure must be performed for each excitation wavelength used in the experiment To find exposure time 1 Set the neutral density filter to 100 2 Set the exposure time to a low value e g 0 1 sec 3 Set dZ to the desired value to sample different images throughout the sample 4 Click the dZ textbox 5 Enter the desired value for the Z step 6 Move through the area of interest by pressing U4 and E in the Stage dialog to move the stage in the Z direction 7 Click Acquire If performing a 2D experiment go to Step 13 8 Continue performing steps 6 and 7 to obtain a sampling of Z sections Find the maximum intensity in these sample images by observing the value of the Max field in the Resolve3D window The focal plane with the highest maximum intensity value is the plane of optimal focus Note There could be a higher intensity value in the image data that you did not sample Therefore do not set the exposure time to the limits of the sampled data because a higher intensity un sampled plane may saturate the camera 9 Move to the plane of optimal focus This is the Z section with the highest intensity value 10 From the Resolve3D menu choose View Point List 11 Click Mark Point The Z value is the third coordinate listed for the point 12 Document the Z value with the brightest intensity
108. en choosing a Move Threshold it is important to select one that is small enough to keep the cell in the field of view yet large enough to buffer out needless stage movement that could result from numerous small cell movements 04 720104 Rev D 1008 72 Delta Vision Core and personalDV Users Manual Field of View at Times 1 and 2 Field of View at Time 3 Cell tracking lags cell movement Cell movement exceeds the threshold between Time Points 1 and 2 left In response DeltaVison movesthe stage so that it iscentered on the cell position at Time Point 2 The stage is at this position when Time Point 3 is acquired nght ROI Percent With rare exceptions few live cells are isolated on the substrate Most are genetically programmed to seek attachment and communication with other cells When performing live cell microscopy not only is it important to filter out unnecessary stage movement it is also important to define an appropriate region of interest within the field of view This region of interest must be large enough to include the entire cell or structure of interest yet small enough to exclude other cells or structures that may tend to wander in and out of the field of view throughout the experiment The ROI Percent parameter defines how much of the field of view the software will use for image recognition In the software ROI units are specified as a percentage of the width of the field of view For example a 50 ROI has
109. ensemble of software developed by Applied Precision in collaboration with Bitplane AG It provides sophisticated multi dimensional data visualization analysis image restoration image correction and image viewing management all within an easy to use streamlined browser interface softVWoRx Suite Advanced Option The Advanced Option for softWoRx Suite includes two sophisticated analysis features 4 D Particle Tracking and ImarisColoc softWoRx Suite Advanced Option is offered by Applied Precision in collaboration with Bitplane AG 4 D Particle Tracking allows users to observe temporal changes of objects This tracking module offers a choice of methods for both detection and tracking and allows analysis and measurement of various object properties The Colocalization feature enables users to easily isolate visualize and quantify regional overlap in 3D and 4D images Results can be presented in two ways as a new 3D or 4D channel or as a statistical report softWoRx Data Management Solution DMS Support softWoRx DMS provides a functional infrastructure for the storage of biological images and their associated metadata in a centralized database softWoRx DMS is a standard component included with DeltaVision Core but for personalDV this software is optional Consumable Parts Common to 100 120 V and 220 240 V Systems Component Fuse for Component API Part Number IC MIC 6 3A 250V UL High Break 19 170045 000 Capacity Microscope TS
110. ensions Methods in Cell Biology 30 353 377 Aikens R S Agard D A Sedat J W 1989 Solid state imagers for microscopy Methods in Cell Biology 29 219 313 Anderson J T Paddy M R Swanson M S 1993 PUB1 is a major nuclear and cytoplasmic polyadenylated RNA binding protein in Saccharomyces cerevisiae Molecular amp Cell Biology 13 6102 6113 Asada T Kuriyama R Shibaoka H 1997 TKRP125 a kinesin related polypeptide involved in the centrosome independent organization of the cytokinetic apparatus of tobacco BY 2 cells Journal of Cell Science 110 in press Babcock D F Herrington J Goodwin P C Park Y B Hille B 1997 Mitochondrial Participation in the Intracellular Ca Network Journal of Cell Biology in press 12 96 Bass H W Marshall W F Sedat J W Agard D A Cande W Z 1997 Telomeres cluster de novo before the initiation of synapsis A three dimensional spatial analysis of telomere positions before and during meiotic prophase Journal of Cell Biology 137 1 5 18 Belmont A S Braunfeld M B Sedat J W Agard D A 1989 Large scale chromatin domains within mitotic and interphase chromosomes in vivo and in vitro Chromosoma 98 129 143 Belmont A S Sedat J W Agard D A 1987 A three dimensional approach to mitotic chromosome structure evidence for a complex hierarchical organization Journal of Cell Biology 105 77 92 04 720104 Rev D 1008 174 Delta Vision Core and personalDV Users Manu
111. epiece filter wheel belongs to and update accordingly Note that it is not necessary to remove the oculars before plugging or unplugging the Eyepiece filter wheel To change the Excitation Filter Wheel if necessary 1 Ensure that the IC MIC is turned off and make sure that the xenon arc lamp has been off and allowed it to cool 2 Loosen the two silver thumb screws at the top and bottom of the filter wheel housing ppliedPrecision Chapter 8 Changing Camerasand Filters 123 Thumb screws should loosen without the use of tools but can also be removed with a flat blade sc rewdniver if necessary i Loosening top thumb screw from filter wheel housing Loosening bottom thumb screw from filter wheel housing 3 Loosen the Focusing Lens housing thumb screw to disengage it from the beveled support on the Excitation filter wheel housing and gently slide the filter wheel housing outward from its mounted position 04 720104 Rev D 1008 124 DeltaVision Core and personalDV User s Manual Removing the Excitation Filter Wheel housing from its mounted position 4 Disconnect the cable to the IC MIC from the Excitation filter wheel and connect it to the alternate Excitation filter wheel Disc onnecting the IC MIC cable from the Excitation Filter Wheel 5 Slide the alternate Excitation filter wheel housing back into its mounting position by pulling back on the Focusing Lens assembly as shown A Pullin
112. era or photo bleach your sample High Speed CCD Camera saturation occurs when the intensity values reach 4145 counts The 12 bit High Speed camera has an intensity range of 0 to 4095 counts The EM CCD 16 bit High Speed camera has an intensity range of 0 to 65535 counts For successful deconvolution a minimum intensity of 50 counts above background is recommended Note If you are using 0 5X Gain the saturation is less than 4095 counts For the EM CCD camera a 16 bit camera saturation is over 50 000 counts Photo bleaching occurs when the fluorescent dyes lose their emission intensity as they are exposed to illumination Certain dyes are more susceptible to photo bleaching than others The potential for excessive photo bleaching is increased with increased exposure time and decreased by use of a higher value neutral density filter There are two ways to decrease illumination and therefore the likelihood of photo bleaching Decrease exposure time Increase neutral density value Note For live cell imaging cells tolerate shorter exposures better than longer exposures even at increased illumination intensity ppliedPrecision Chapter 6 Data Collection Techniques 83 The response of increasing the exposure time is roughly linear Therefore if an exposure time of 0 2 sec results in a maximum intensity level of 1000 counts then an exposure time of 0 4 sec will result in a maximum intensity level of approximat
113. era window using low pressure air Do not use canned air See Page 151 for further recommendations regarding cleaning system components Clean camera window using low pressure air Do not use canned air See Page 151 for further recommendations regarding cleaning system components Check camera temperature in Resolve3D Consult camera documentation for proper setting Disconnect the Photo sensorcable and the EX module cable Connect the Photo sensorcable to the EX module Set the Excitation filter to FITC orsome other visible light Open the EX shutter Bend the cable and examine it forlight leaks If you observe a light leak replace the cable Direct the light to a wall If you observe Inconsistencies in the light output as you bend the cable replace the Photosensor cable Delta Vison Core and personalDV Users Manual Table B 3 Image Quality Troubleshooting Chart Cont d Indication Image hasa traveling light or bubble Interference in image data Very bnght image or camera saturation message Zsenes shows uneven oroff center illumination No image when Acquire is pressed Zsenes out of focus and incomplete Cause Air bubble in immersion oil Possibly dirt dust oil orairbubble Camera saturation Poorly aligned illumination Knob at base of microscope Is directing light to the eyepiece Stage wasnot centered within sample at start of expenment Conection Clean f
114. ersonalDV 22in x 54 to 62in depending on whether or not the keyboard is kept in a slide out tray Include 18 in 45 cm space behind instrument rack Maximum System Weight 940 lbs 425 kg Service Indoor use only 04 720104 Rev D 1008 Delta Vision Core and personalDV Users Manual Temperature 65 77 F 18 25 C daily variation of no more than 3 F 1 8 C The actual room temperature should be stable to within 1 degree Fahrenheit or Celsius per hour Fluctuations in temperature will affect microscope optics which can cause the specimen to drift approximately 1 um per 0 1 degree Celsius Humidity Stable humidity levels under 50 with daily variations of less than 10 High humidity can result in condensation on the CCD camera window that obscures image formation Excessive humidity may also reduce filter life and may result in chromatic aberrations in the images Altitude up to 6550 ft 2000 m Pollution POLLUTION DEGREE 2 in accordance with IEC 664 Ingress Protection Level IP20 Communication Recommendations Connect the workstation to a local area network for data storage To connect to a network you will need an IP address domain name server address and network mask A connection to the Internet will provide access to Applied Precision s web site CAUTION Applied Precison INC isnot responsible for damage orham to the workstation or scanner due to network secunty breaches Having a telephon
115. erthan 0 75 N A a measured OTF will generally give you better results For information about calculating an OTF see the softWoRx online Help A well measured PSF is a key to successful deconvolution For this reason make sure that you a Thoroughly check all imaging conditions m Take the time you need to get a good signal to noise ratio in the image Completely scan the bead Tools This procedure requires the following tools A clean and aligned 3 D microscopy system a A bead slide with 0 1um or smaller fluorescent beads A grid slide or other microscopic ruler An immersion oil set if appropriate see Selecting the Correct Immersion Oil on page 165 The following steps describe how to calculate pixel size measure the PSF and obtain the corresponding OTF To calculate pixel size 1 Place the new objective in the Objective Turret and set the Max image size best camera speed and magnification slider at 1X 2 Use the Eyepiece Filter Wheel to select the FITC eyepiece filter 3 In the Resolve3D window select the following filters In this Field Select Excitation TRITC Emission FITC AT 0 1 4 Place the silicon target grid on the stage and focus it 9 995 um square Align the grid image to the vertical and horizontal axis and maximize the image ppliedPrecision Appendix C Acquinng a PSF 163 10 11 Switch the beam selector to SP or SPL Then adjust the exposure time and T filter and
116. es resolution Pixel size is a function of binning Aux Mag Specifies to use the microscope s 1 6x manual auxiliary magnification On IX70 stands the manual auxiliary magnification is 1 5x On IX71 stands that have a Cascade II camera the auxiliary magnification is 2 0x Pixel size Displays pixel dimensions in microns pixel The value is calculated from the Lens Bin and Auxiliary magnification settings Stage Position Control Relds and Buttons The Resolve3D Stage Position Control area allows you to control the microscope s X Y and Z stage positions The Center Mark and Visit tools and the Stage Motion Controls are shown below 04 720104 Rev D 1008 190 Delta Vison Core and personalDV Users Manual Figure E 9 The Resolve3D Stage Position Contol v Pe ele File View Options Calibration Help AHH Hix a apoo aile apo El Z d 10 00 112 Max 159 Mean 133 8 lo Saved fred05_R3D dv 118 Saved fred05_R3D dv 119 Saved fred04_R3D_REF dv 7 Stage Control and Display Tools Center Object Centers the stage on an object selected in an Image Window The pixel size must be correct in order for object centering to work properly which means that the correct lens and auxiliary magnification setting must be selected An image will be acquired after the object is centered Mark Point Marks the current X Y and Z stage coordinates as points to be visited later Mark Top of Sample Marks the cu
117. esign Experiment Design PK Experiment Run Experiment Experiment name Resolvead Estimated File Size 668 05 Mb _ Enable Fast Acquisition fast Acquisition PHOS Sectioning Channels Time lapse Points Panels Actions Conventional Multiplexed Exp EX Filter EM Filter ST utter m 1o00 atock pLock 100 TRANS Multiplexed ERRO w WTR ALE EILE Z PPS Sep arra w rgan PN wee OS TPZ w WR ARE Fe ry DRO SHONE H REREN Bes Se SH eat See See WS TE Refresh exposure conditions The Multiplexed tab of the Design Run Experiment window is displayed as shown 04 720104 Rev D 1008 62 Delta Vison Core and personalDV Users Manual Select Checkbox F WY Design Run Experiment Resolve3D_Startup exp File Help agu Oe Design Experiment Design PK Experiment Run Experiment Experiment name RESSAM Estimated File Size 668 05 Mb _ Enable Fast Acquisition fast Acquistion TIPRO Sectioning Channels Time lapse Points Panels Actions Conventional Multiplexed ie Do Multiplexed Channel Imaging Settings Fe ryg BHON DHONE oe BALNE Best hae WH eass See See WH 8 eed Refresh exposure conditions 3 From the Multiplexed tab select the Do Multiplexed Channel Imaging checkbox If the currently active filter set is not Multiplex capable the following window is displayed The currently selected filter set is not Multi
118. ettings dialog box Imaging options allow you to select camera settings Figure E 20 The Settings Dialog Box Imaging Options Display imaging Fites Autofocus Mise Camera COOLSNAP_ESZ ICX285 Frames to average m Gain 100 or Transfer speed 20000 Target temperature 0 00 Current temperature 0 00 Refresh se Lamp bulb age hours 58 7 Refresh Reset F Use photosensor Settings change based on Excitation Filter Done Save Settings Help Camera Specifies which camera to use For the EM CCD camera this list also specifies whether to use Conventional or Electron Multiplication mode Frames to average Specifies the number of successive camera images to average into a result that is displayed in the window This is similar to the AVG macro command Gain Specifies a gain value for the selected camera Transfer speed Specifies a transfer rate for the selected camera in kHz Higher speeds may boost performance at the expense of image noise Target temperature Displays the cooled CCD camera s target temperature This can be changed only by modifying the appropriate configuration file on the Instrument Controller computer ppliedPrecision Appendix E Resolve3D and Keypad Options 207 Current temperature Displays the current temperature Resolve3D monitors the temperature and updates this value occasionally If you want to force a request for the current temperature
119. ex of the immersion oil Actually experimenting with oils with refractive indexes very close to this value is the best way to select the optimal oil Resolution Ratio Displays the ratio between the Z resolution and the XY ratio This serves as a reference to the degree of Z elongation Maximum XY Pixel Size Displays the maximum recommended XY pixel size for deconvolution Recommended ZStep Displays the smallest possible Z step for this objective Choosing a smaller Z step will add to the size of the image file but will not improve image quality ppliedPrecision Appendix B Troubleshooting This appendix was designed to help you diagnose and correct the most common problems encountered on the DeltaVision system It covers two types of troubleshooting tasks m Diagnosing System Problems a Analyzing Reasons for Poor Image Quality If you are unable to correct a problem fill out the DeltaVision Problem Report Form at the end of this appendix and either e mail it to hotline api com or fax it to 425 557 1055 attn Bio Service Hotline Diagnosing System Problems Troubleshooting the Contoller The following table shows the most common Instrument Controller problems and their resolutions Table B 1 Contoller Troubleshooting Chart DeltaVision Core and personalDV Users Manual Indication Cause Conection Encoder Eror when Poorcable Power down system including IC MIC Initializing stage connection Resat Y andizmotores bles
120. eypad SLOW MEDIUM or FAST Buttons Medium is typically the best speed to start with These buttons control the stage speed when you are using the joystick or keypad to move the stage ppliedPrecision Chapter 6 Data Collection Techniques 81 8 10 11 Open the EX shutter and find the desired focal plane optically using either the focus knobs on the microscope or the Resolve3D Z stage controls Note If you are using an Olympus or Nikon microscope rotate the top of the focus knob toward you to move the objective up and away from you to move it down when you are sitting in front of the microscope Use one of the following methods to maneuver the slide and find an area of interest Keypad The buttons on the keypad are used for movement in the X Y and Z directions Using the arrow keys on the keypad causes the stage to move by steps The size of each step is doubled each time the STEP INC REASE button is pressed and halved each time the STEP DEC REASE button is pressed By adjusting the step size to the frame size you can create a condition where each press of a step arrow will move the sample one frame This is a convenient way to scan a large area for rare events e g mitosis Since the step movements are rapid using the arrow keys in this way can be much less fatiguing than using the joystick Joystick The joystick controls stage movement in the X and Y directions Workstation The Resolve3D module a
121. ference Channel should not be confused with Reference Image which issomething very different Reference imagesare described later in this chapter in Acquinng a Reference Image ppliedPrecision Chapter 5 Acquinng Data From Live Specimens Channels are numbered as they appear in the Experiment Designer window In the following example DAPI is Channel 1 and FITC is Channel 2 QJ Design Run Experiment Resolve3D exp modified st titi OSt S co g Oe aT Design Experiment Design PK Experiment Bun Experiment Experiment name Resolves Estimated file size 64 02 Mb 22 28 Gb Available Use Fast Acquisition fast Acauisiien intens J Lamps Off when finished Sectioning Channels Time lapse Points Fanels Actions Conventional d Muitipiexed Exp EX Filter EM Filter T Ex Shutter efo ene fefee from fox y ar sa iii Bes 3 A A See NA ETT Py stat PN sted Ky ded 3 3 bgi fo ts HHE i Fen 4 AMG E aire A Nf _ Reference Image Refresh exposure conditions Move Threshold The Move Threshold is the distance the center of the cell as defined by the tracking method must travel before the system resets the stage When the cell moves beyond this threshold the stage moves so that the center of the cell retains its original position in relationship to the center of the field of view Live cells by their very nature are constantly on the move Wh
122. fies the number of the active display wave channel as it is listed on the left side of the Data Collection window Select anew wave to change which display window channel to use Calculate statistics Calculates image intensity statistics The typical reason for disabling this feature is to improve readout speed Calculate histogram Calculates and displays the image intensity histogram in the Resolve3D window during image collection This option does not set scaling Auto histogram range Provides automatic scaling of the histogram width for each image that is analyzed This option provides automatic histogram ranging from the minimum to the maximum intensity When off the histogram range remains at its current settings Display images Displays images when the Acquire button is clicked or an experiment is running Deselecting this option can increase performance Deconvolve preview images Displays instantly processed 2D image previews that closely resemble images processed with advanced 3D image restoration techniques 04 720104 Rev D 1008 206 Delta Vison Core and personalDV Users Manual Auto intensity scale Provides automatic scaling of the image intensity between the minimum and maximum brightness This switch applies only to the appearance of the image not the actual data Acquire after point visit Automatically acquires an image when the Visit Point option is selected Settings Dialog Box Imaging Options The S
123. g back the Focusing Lens housing and re engaging the filter wheel ppliedPrecision Chapter 8 Changing Camerasand Filters 125 6 Re engage the Focusing Lens housing into the beveled support on the new Excitation filter wheel housing and align and tighten the two thumb screws at the top and bottom of the Excitation filter wheel housing 7 Tighten the thumb screw on the Focusing Lens housing Lt Note If you still need to change the EP or EM filter wheels do so now before continuing with this procedure 8 When you are finished changing filter wheels restart the IC MIC 9 If Resolve3D does not automatically display the correct filter module there are two ways to expressly let the system know you have a new filter set e From the Resolve3D main menu select Settings change to the Misc tab and select the current filter set e Unplug and then re plug in the Eyepiece filter wheel position sensor cable see Step 8 in the previous procedure The system will automatically recognize which filter set the Eyepiece filter wheel belongs to and update accordingly Calibrating the Filter Wheels Calibration initializes the filter positions and ensures that the filters are centered in the filter wheel openings If you notice poor light transmittance or poor image quality one possible cause is a misaligned filter wheel The filter wheel calibration re establishes the zero position of the filter wheel for the Instrument Controller
124. he filename conventions used by DeltaVision a Standard Fluorescence Filters shows the excitation and emission peaks of the standard filters included with DeltaVision a Live Cell Filter Sets shows the excitation and emission peaks of the filters included in the optional Live Cell filter wheel module Reference List includes references for microscopy Linux image processing optics microscopy and sample preparation Standard Rlename Extensions The following is a list of filename conventions used by DeltaVision Filename Extension Type of File dv Standard DeltaVision image otf Optical Transfer Function R3D dv Resolve3D image D3D dv Deconvolved image VOLdv Volume Rendered image DeltaVision Core and personalDV User s Manual Standard Huorescence Filters DeltaVision provides a four color filter set a polarizer and multiple sets of optional filters These filters are designed to be used with many common fluorescent probes If you are using fluorescent probes that are not well matched with the standard DeltaVision filters contact Applied Precision for assistance The excitation and emission peaks of the DeltaVision filters are provided in the following table Filter Name DAPI FITC TRITC CY 5 CFP optional YFP optional Excitation UV 350nm Blue Green 490nm Green 555nm Red 645nm Deep Blue 436 nm Blue Green 500 nm Live Cell Filter Sets Emission Blue 455nm
125. hen they are set in position a The Fluorescence Illuminator Slider on the back of the microscope is in an open position a The standard Sedat color set polychroic filter usually labeled is selected and in position on the Filter Cube Turret Alignment may vary between polychroic mirrors Factory alignment is performed on the standard color set a The shutter on the Filter Cube Turret is open O a The Field Stop Aperture lever is pulled out all the way to close the aperture a The Magnification Changer is completely in the 1X position a The 60X objective is in its lowest position a The Beam Selector is on the lt eye position m The Eyepiece Lens Selection Wheel is in the open O position Eyepiece Focus Eyepiece Lens Selection Wheel enema Telescope _ Focus Fluorescence Illuminator Slider back view Field Stop Aperture And Adjustment Screws Eyepiece Filter Wheel ee Beam Selector J ZFocus Filter Cube Turret Auxiliary Magnification Changer ppliedPrecision Chapter 9 Maintenance 143 Checking Illumination Path Alignment To provide an optimum light source the tilt and position of the Fiber Optic Module must be aligned with the microscope This procedure shows how to perform an inside outside focus test to make sure that the module is properly aligned It also shows how to perform in focus tests to make sure that the position of the module
126. ht 5 Inthe Resolve3D window click H to open the Point List dialog box Mark Paint Visit Point Delete Point Replace Paint Open List save List Clear List Refresh List Done Help 6 Click on a point to select it and then click Visit Point to move the stage to that point 7 Use the XY Stage Controls on the Stage View to move the stage to the exact XY ry Ele XY Stage Controls position Use the Fine Z Focus knob to focus while looking at the sample through the eyepieces Then acquire an image to execute an LMC move and verify that the 04 720104 Rev D 1008 50 Delta Vison Core and personalDV Users Manual image is in the best focal plane Repeat this step if necessary until the image is in focus l Important Do not use the microscope Focus knob after you complete Step 8 Instead use the Resolve3D Zcontrols the Zbuttons on the Resolve3D window or the Zslider or the AF autofocus button 9 On the Point List dialog box click Replace Point to replace the old coordinates with the new exact coordinates for the point 10 Repeat Steps 6 9 to replace all of the points in the list with the exact X Y and Z coordinates Editing a Point List To delete a point using Resolve3D 1 Inthe Point List dialog box select the point that you want to delete 2 Click Delete Point To save the point list 1 In the Point List dialog box click Save List 2 Inthe Prompt dialog enter the desired name
127. i icicle Gain 1 00 Bulb age Fial al arl a dx iaon a Transfer speed 10000 nE e dz 0 50 Target temperature 25 00 nee o Re eSh Refresh reset i M Use photosensor Reset bulb Current temperature on age se Lamp 1 bulb age hours Settings change based on Excitation Filter save Settings Help Min 150 Max 188 Mean 1684 Io D Scale min 150 Scale max N86 Saved FRU Shutter Rm dy 29997 Saved FM Simtter_ AID dy 29998 Saved FAU Shutter _ RWM dy t 29993 fal P Note For DeltaVision systems with the Multiplexed Wavelength option installed the procedure for changing the xenon bulb in the secondary lamp housing is identical to the procedure described above Replacing the Transmitted Light To replace the LED transmitted light assembly 1 If the DeltaVision system is on exit Resolve3D and ensure that the IC MIC is off prior to proceeding 2 Loosen the two hex screws on the top and right sides of the transmitted light housing as shown ppliedPrecision Chapter 9 Maintenance 139 3 Carefully remove the transmitted light assembly from the housing 4 Disconnect the other end of the attached cable from the Transmitted Illumination Source connection on the back of the IC MIC upper left hand corner when facing the back of the unit shown below 04 720104 Rev D 1008 140 DeltaVision Cor
128. ialized Hold the EX or ND filter wheels to stabilize them and help ensure that motor torque does not cause them to fall off of the work surface You are then prompted with Calibrate the EX filter wheel y n 8 If you are calibrating the Excitation Filter Wheel press y Otherwise press n to move to the next filter wheel ND or EM When you press y the following message is displayed bed rdesktop 159 159 159 2 er hy Computer b i Delbavision Instrum T Jeltavision Irstrument yUse the right left arrow keys to move the EX filter The down up arrow keys vill move one filter position A Deltavision Instrumen Pressing the up or down arrow keys advances the filter wheel one complete position The left and right arrow keys move the filter wheel in small increments 9 Find the Home filter position on the filter wheel The Home filter position is marked on each of the filter wheels as follows e EX 1 e ND 1 e EM O The position is stamped on the surface of the filter wheel and may be viewed through the filter wheel hole 04 720104 Rev D 1008 Filter position not centered Delta Vison Core and personalDV Users Manual Filter position centered 10 Once the filter wheel is centered press Enter After all necessary filter wheels have been centered you are prompted with Save the new filter offsets y n yy 11 If you made a mistake during calibration you can press n and sta
129. ifiable Laser Module QLM adds a laser beam into the back aperture of the microscope objective to provide a focused illumination spot in the center of the optical field The lasers are mounted in a closed system in the cabinet Light is directed from the lasers to the light path through the Laser Optic module that mounts on the back of the Fiber Optic module Note The QLM isnot an available option for personalDV If your system has the QLM hardware module you can use softWoRx to analyze Photokinetic photo bleaching and photo activation experiments QLM Laser Optics Module The QLM module provides software to control the lasers and to analyze the data obtained from these experiments For more about QLM see the DeltaVision QLM Getting Started Guide Total Intemal Reflection Huorescence TIRF Module The TIRF module includes the Olympus TIRF component and special adapters required to install it on a DeltaVision system TIRF requires a system with a QLM hardware module ppliedPrecision Chapter 7 Facility Requirements and Components 111 O Note TRF isnot an available option for personalDV _ IRF Component Lt Note TIRF is an optical sectioning technique that limits fluorescence imaging to a thin area at the surface of a specimen typically 100 nm or less resulting in an enhanced signal to noise ratio and higher imaging contrast EM CCD Camera The EM CCD Camera is an optional com
130. ility between cells within an experiment can be assayed eliminating uncertainty as to the behavior of cells in a single experiment Cell 1 X Y Point 1 2 etc SATE Cel 4 X4 Y4 8 Point Visiting monitors points that are in different fields of view To set up an experiment that visits points you must a Mark the points to visit with the Keypad or with Resolve3D and save a point list a Edit the point list if necessary Load the point list and specify which points to visit Notes 1 If you are placing your Side directly on the microscope stage without the Repeatable Slide Holder you can store the X Y and Zcoordinates of the stage positions you mark however if the slide is removed from the stage orthe Zfocus knob is adjusted these coordinatesno longerapply 2 If you are using Applied Precision s Repeatable Slide Holder you can remove the cide and then place it in the same position when you want to revisit the points To do this you must record the position A G of the slide before you remove it Then 04 720104 Rev D 1008 48 Delta Vison Core and personalDV Users Manual put the slide in the same position when you retum it to the slide holderand update the Zcoordinates Marking Points There are two ways to mark points within a sample using the keypad or using Resolve3D An efficient way to mark points is to use both methods together First mark the points using the keypad which is quick an
131. ime lapse experiment as shown in Setting up Time lapse Experiments on Page 42 2 On the Time lapse tab on the Design Run Experiment window select the Enable Cell Tracking option ppliedPrecision Chapter 5 Acquinng Data From Live Specimens QJ design Run Experiment modified aa g Ae Design Experiment Design PK Experiment Run Experiment Experiment name Resolves Estimated file size 64 02 Mb 22 29 Gb Available Use Fast Acquisition Fasi Acuulsiion tintions Lamps Off when finished Sectioning Channels Time lapse e Points Fanels Actions Time lapse Hours Minutes Seconds Milliseconds Time lapse e e fo fo Total Time fo lo fo fo Time Points f W Enable Cell Tracking Cell Tracking Options W Autofocus before imaging Autofocus Options 3 Click Cell Tracking Options A cell Tracking Options Tracking Method Center of Intensity Reference Channel i v Move Threshold microns nooo 4 Manually acquire an image for each channel and determine which single channel best identifies the features of your specimen In the Reference Channel list specify that channel The channels are numbered in the order in which they are listed on the Design Experiment Channels tab Note DeltaVision uses the reference channel forimage recognition 5 Inthe Move Threshold field enter the distance in microns that the cell must move to trigger stage movement
132. improved image quality Pressing any key on the keyboard restores monitor function Dust It is important to minimize dust on the microscope components because dust on components can cause spots on microscope images Minimize contamination by maintaining a clean room and covering the microscope when it is not in use Overview of Components The main standard DeltaVision components for both DeltaVision Core and personalDV are shown below Detailed descriptions of these components and descriptions of optional components are included in the following sections Standard DeltaVision Core Components Optical Desktop Components Components Cabinet Components IC MIC Workstation Vibration ane eee a leolation optional components Table 04 720104 Rev D 1008 96 DeltaVision Core and personalDV User s Manual personalIDV Components Optical Components la IC MIC and Workstation Desktop Components Optical Components Xenon Arc Lamp Py White LED Trans Light Exc ita tion Filters Neutral Density Filters Eyepiece Filters quam ae Camera P Emission Filters Note Optical components are shown fora typical installation which includes the Olympus IX71 Microscope Other microscope configurations vary slightly The Olympus 1X71 is currently the only microscope available ppliedPrecision Chapter 7 Facility Requirements and Components 97 Huorescence Microscope The microscope is an advanced research gr
133. in microns Stage Trails Window a YZ 0 00 0 00 0 00 The blue box represents the current stage location Drag the box to move the stage in X and Y dZ Specifies the Z step size in microns ppliedPrecision Appendix E Resolve3D and Keypad Options 193 Image Intensity and Scale Values The Resolve3D Intensity values show the statistics for the intensity values of the image in numerical and graphical formats The scale of the image is also shown Figure E 10 The Resolve3D Image and Intensity and Scale Values Min Max Mean Displays the minimum maximum and mean intensity values of the most recently acquired image For a 12 bit CCD camera these values range between 0 and 4095 A value of 4095 indicates camera saturation unless image calibration is in effect If you are using 0 5X Gain the saturation is less than 4095 counts Histogram Shows the intensity distribution for the most recently acquired image The vertical blue bars indicate the Scale min and Scale max and can be dragged interactively with the mouse to change how the display is scaled The X axis represents intensity and the Y axis represents the number of pixels Scale min Scale max Specifies the settings for the minimum and maximum display values These numbers can be changed manually or by moving the histogram threshold bars lo Indicates valid or invalid photo sensor values or saturation The indicator is green if the photo
134. ing Channels Time lapse Points Fanella Actions W Z Sectioning Z Scan Options Focus point when scan starts Middle of Sample Optical section spacing ozo Number of optical sections B4 Sample thickness 12 80 Get thickness Enable OAI Scan 3 Inthe Experiment Name field enter a name for the macro Note By default softWoRx uses Resolve3D asthe experiment name Thisisa special disposable name that doesnot require confirmation to overwrte If you do not intend to reuse an expenment using Resolve3D forthe expenment name isthe simplest method Refer to Sectioning Specimens for 3D Images immediately following this section for the specific components of designing an experiment 4 From the File menu choose Save The macro name appears in the Design Run Experiment window title bar 5 Click the Run Experiment tab ppliedPrecision Chapter 4 Setting Up and Running Expenments 35 w Design Run Experiment g Zoe Design Experiment Design PK Experiment Run Experiment Image file name MoE Settings DMS Setup Image title pooo Add note to log a Do It Change next time lapse ooo Dod W Show images during acquisition W Show PK progress graph W Launch viewer after experiment J Deconvolve during experiment Options Images acquired requested 0 0 Disk space required 0 00 Mb Current command Start Time Current Time Elapsed Time Estimated Finish 6 Inthe
135. ing instructions to turn off DeltaVision on a daily basis For instructions that show how to shut down the system see Shutting Down and Starting the System on Page 131 To tum off DeltaVision 1 Turn off the xenon light source using the bulb F icon on the Resolve3D main menu Clicking the icon will switch it to the off F state ppliedPrecision Chapter 3 Getting Started 31 2 Save all data on the workstation 3 On the softWoRx menu bar select File Exit Then exit all other workstation applications 4 Log out of the workstation account 5 Press the IC MIC power switch to turn off the IC MIC Wait 30 60 seconds for the IC MIC to power down LL Note Ifthe IC MIC failsto tum off within 30 60 seconds after pressing the power switch pressand hold the IC MIC power switch for 5 seconds to tum off the device 6 Clean the objective with a clean unused cotton swab or lens paper to remove all of the oil on the objective Then apply chloroform to a cotton swab gently roll it over the objective lens once and discard it CAUTION After you finish imaging Always clean the objective with a clean unused swab orlens paper Never reuse swabsor lens paper 7 Lower the objective by turning the Coarse Focus knob 04 720104 000 Rev D 1008 32 Delta Vision Core and personalDV Users Manual ppliedPrecision Setting Up and Running Expenments A DeltaVision experiment is a macro that runs a set of
136. ion selected DeltaVision automatically keeps cells in the field of view Cell Tracking Options Opens the Cell Tracking Options dialog box that allows you to set the parameters for cell tracking For more information on using this dialog box see Tracking Cells on Page 67 wd celi Tracking Options Tracking Method Center of Intensity yl Reference Channel 1 v Move Threshold microns i 000 50 ROI Percent r m Autofocus before imaging Automatically focus the camera before each time point For point visiting experiments the camera is automatically focused every time that a point is visited Point Visiting Setup Use the Design Experiment Point Visiting tab to specify a list of marked points to visit during the experiment Q Tip Before you specify these points make sure that the point list is open in the Point List dialog You cannot specify the points when the point list is closed Figure E 16 Expenment Designer Point Visiting Setup Options bg Design Run Experiment x Eile Help Design Experiment Design PK Experiment Run Experiment Experiment name Resolve30 Estimated File Size 96 03 Mb Enable Fast Acquisition ast Acquisition Uistions Sectioning Channels l Time lapse Points Panels Actions F Visit Point List f Autofocus before imaging AUOIGCUS Oplons ppliedPrecision Appendix E Resolve3D and Keypad Options 201 Visit Point List
137. ired Contact Applied Precision for more information Note The vibration isolation table is not included with personalDV Instead personalDV includesa bench top isolation platform to provide similar stability for the microscope The DeltaVision vibration isolation table is available for personalDV as an optional upgrade Cabinet Components Note The component cabinet is included with DeltaVision Core only The cabinet is not available for personalDV The cabinet contains all of the electronic control equipment and provides surfaces for the keyboard and the Flat Panel Display The front two wheels should remain locked and the cabinet left in place There are many cables connecting the microscope that can be damaged if they are pulled The standard cabinet components are shown below ppliedPrecision Chapter 7 Facility Requirements and Components 103 Cabinet Components Keyboard Mouse Tray IC MIC Workstation QLM optional Note Configuration of cabinet components may vary slightly Instrument Contoller Microscope Interface Chassis IC MIC The Instrument Controller IC is the portion of this computer that interfaces with all of the microscope hardware including the microscope stage motors filter wheel motors and cameras It coordinates all activities related to positioning the stage and collecting images Data from the camera feeds through the controller to the workstation The controller also
138. ivation experiments The QLM supports up to three lasers and provides software to control and to analyze the data that is obtained from these experiments Note The QLM hardware isnot an available option for personalDV TIRF Total Intemal Reflection Huorescence If your system has the TIRF hardware module you can use DeltaVision to run and analyze TIRF experiments TIRF is used in a number of research applications such as cellular protein and vesicle trafficking focal cellular adhesions single biomolecule dynamics studies in neuroscience and cell to cell communications The ability to excite molecules on the surface of a specimen while eliminating the fluorescence from the depth of the sample makes TIRF a valuable tool for examining cell surface structure and protein dynamics Note The TRF hardware module isnot an available option for personalDV What should you know to use DeltaVision This document assumes that you are familiar with the basics of microscopy Correct operation of the microscope is fundamental to obtaining quality images with DeltaVision In addition an understanding of image processing basics will help you use the system to its full potential To manage the computer systems some familiarity with Linux workstations and IBM type personal computers is helpful We have taken care to ensure that DeltaVision is straightforward to use reliable and complete Please report errors and problems with DeltaVision to Applied P
139. l hesa Bs e ET 2 E ecrosoh Chuttook w i sofia 1 7 0 Flu ke Piler Mir fe ResokeD a S E jl W 7 Ba Window Dara 7 ET The Resolve3D window includes acquisition parameters and controls for moving the stage The Data Collection window displays images as they are acquired and the Filter Monitor displays the filters that are selected Note You will use the Resolve3D window throughout the data collection process For more about this window see The Resolve3D Window on Page 182 Acquiring and Saving Data To acquire an image of your sample you will need to e Set up DeltaVision by placing the slide and selecting the appropriate filters e Find the sample so that it is visible in the eyepieces e Acquire an image with the camera Setting Up the Sample for Image Acquisition Setting up DeltaVision for imaging includes placing the sample on the stage so that it is in contact with the immersion oil and selecting the appropriate filter set for the fluorescent probe used to label your sample 04 720104 000 Rev D 1008 22 DeltaVision Core and personalDV User s Manual To prepare for image acquisition 1 Rotate the Coarse Focus knob so that the top of the knob rotates away from you while you are sitting in front of the microscope to move the objective all the way down 2 Rotate the objective turret to select an objective Use the objective turret to select an objective Q Tip Use a high power objectiv
140. lamp 109 Consumable parts 123 24 Contact Applied Precision xii Continuous Acquire 196 Contrast calculation polarity 223 Control Mode button 226 Controlling the stage 202 6 Conventions document x Converting PSF to OTF 178 81 Counter space for system 101 Country specific requirements 101 Critical illumination 5 89 91 150 155 position 159 Critical illumination spring 89 Critical spring 151 Current rating of electric supply 100 Current temperature display 220 Customer Service Hotline xii Customer support 7 Damage prevention 11 Data collection techniques 84 98 monitoring acquisition of 91 96 saving 30 32 Data Collection window 93 95 Deconvolve preview images 219 DeltaVision capabilities 3 4 components 99 124 damage prevention 11 prerequisites 15 turning off r 32 Depth of field 42 Design Experiment tab 36 38 40 207 8 Design Run Experiment dialog box 215 17 Desktop components 110 12 DIC 80 82 DIC imaging 4 Dichroic filter wheel 24 Differential Interference Contrast See DIC Digital Microscopy 4 Dim illumination problems 168 Dim image problem 168 Disable Motion keys 227 Display modes 93 95 Displaying histogram 96 Document conventions x Editing a point list 53 Editing macros 96 98 Electrical requirements 100 101 EM CCD camera 121 22 EM Filter field 212 Emission filters 18 26 47 changing 129 32 specifications 184 Enable fast acquisition 209 Encoder error 167 End
141. lder Adapter also supports Petri dishes from 25 40 mm in diameter Calibration Kit The Calibration Kit is provided for calibration operations on the DeltaVision system The kit includes the following six slides e Three plastic fluorescent calibration slides for flat field calibration 04 720104 Rev D 1008 106 DeltaVision Core and personalDV User s Manual e Blue plastic EX 408nm EM 440 nm Good for DAPI Hoechst etc e Orange plastic EX 488nm EM 519nm Good for FITC GFP TRITC CY 3 etc e Red plastic EX 590nm EM 650nm Good for CY 5 etc e Silicon mirror slide for light path alignment and troubleshooting e Silicon grid slide for pixel size measurement e 100nm Rhodamine bead slide for PSF measurement The Fiber Optic Module Use the Fiber Optic Module to align the light path from the fiber optic cable to the Fluorescent Illuminator This module allows you to adjust the tilt horizontal and vertical orientation of the light path Fiber Optic Module The Tool Kit A tool kit is provided that includes tools for maintaining the system This kit includes e Ties and belts for suspending cables screws and other fasteners e Cleaning material Q tips and lens paper e A hex wrench set 5 64 3 16 e A set of metric L keys 1mm 5mm e A 1 Phillips screwdriver e A5mm T Handle hex key e An acrylic bulls eye level ppliedPrecision Chapter 7 Facili
142. le binding protein in yeast Molecular Biology of the Cell 8 2677 2691 Segal E D Lange C Covacci A Tompkins L S Falkow S 1997 Induction of host signal transduction pathways by Helicobacter pylori PNAS USA 94 7595 7599 Shaw P 1994 Deconvolution in 3 D optical microscopy Histochemical Journal 26 687 694 Shaw P J Agard D A Hiraoka Y Sedat J W 1988 Tilted view reconstruction in optical microscopy Three dimensional reconstruction of Drosophila melanogaster embryo nuclei Biophysical Journal 55 101 110 Shimanuki M Miki F Ding D Q Chikashige Y Hiraoka Y Horio T Niwa O 1997 A novel fission yeast gene kms1 is required for the formation of meiotic prophase specific nuclear architecture Molecular and General Genetics 254 238 249 Stevenson R 1996 Bioapplications and instrumentation for light microscopy in the 1990s American Laboratory April 1996 ppliedPrecision Appendix D Reference Information 179 Straight A F Marshall W F Sedat J W Murray A W 1997 Mitosis in living budding yeast Anaphase A but no metaphase plate Science 277 574 578 Sullivan W Minden J S Alberts B M 1990 daughterless abolike a Drosophila maternal effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions Development 110 311 323 Swedlow J R Agard D A Sedat J W 1993 Chromosome structure inside the nucleus Current Opinions in Cell Biology 5 412 41
143. llows you to finely control stage motion The arrow buttons move the stage in discrete increments that are indicated by the values in the dX dY and dZ text boxes Resolve3D provides the only mechanism from which to obtain discrete step sizes Focus Knob The manual focus knob on the microscope base moves the nosepiece objective in the Z direction CAUTION To record and maintain accurate stage coordinates focus by moving the stage with the Resolve3D controls instead of using the focus knobsto move the objective Resolve3D cannot track the movement of the objective Tighten the Focus Lock on the left side of the focusing knob see Page 15 Use the keypad to find the approximate center of the specimen and place it in the center of the field of view using stage controls 12 Direct light to the camera port using the Beam Selector knob on the microscope base 04 720104 Rev D 1008 DeltaVision Core and personalDV User s Manual 13 Record the position of the slide on the Position Indicator the letter scale at the bottom of the Repeatable Slide Holder Position Indicator Finding Exposure Time The exposure time correlates with the signal intensity level for the image acquisition Finding the appropriate exposure time for each filter in an experiment is crucial to acquiring the best image data Many factors must be considered Although you want to see the maximum intensity in each wavelength you must not saturate the cam
144. ls Q Tip You can also click the Find button on the Resolve3D window to find a good exposure time Use Find carefully Overuse of this option can photo bleach the specimen You need to be particulary careful when determining exposure times for live cells When using Find for live cells start with lower exposure times by changing the Target Intensity Value to around 200 counts Click iif to acquire an image Q Tip You can also use Auto Focusto focus the sample as follows On the Resolve3D window click AFto auto focusthe sample If yourimage Is far out of focus you may need to click AFmore than once To focus use the mouse to slide the Stage Z Control bar up or down 04 720104 000 Rev D 1008 27 28 Delta Vision Core and personalDV Users Manual ZControl i T 7 ae Pot Pal Pot E a n aa ga sze 0 00 0 00 0 06 10 KT MAKE 6 To center the image click and then click on the object that you want to center in the Image window X Tip You can also acquire an image by clicking the ACQUIRE IMAGE button right clicking on the stage view orchoosing Fle Acquire on the Resolve3D menu 7 To enlarge the thumbnail image displayed in the stage view drag the mouse down over the zoom wheel OJ Tale Hal al l apon aele apm El pan A dY 10 00 K ee The Zoom Wheel Note Thumbnails appear only when the Show Stage Thumbnails option is selected on the Misc tab in the Resolve3D Setting
145. ls for 55 57 Exposure time 88 marking points for 50 52 monitoring 91 96 point visiting 49 55 troubleshooting 168 70 with continuous Z sweep 78 80 Image control fields 200 202 Image Display and Analysis 4 Image Fusion tool 82 Image intensity 206 Images 2D in 3 wavelengths 17 19 3D 38 44 acquiring 22 27 28 30 and optical section spacing 41 44 calibrate 201 collecting panels for 55 57 display mode 218 distortion 168 intensity values 206 live cell 5 68 82 modes for viewing 93 95 reference 80 82 running experiments for 35 57 saving 30 32 scale values 206 setting size 202 symmetric flare 177 viewing previews 93 Immersion oil flare from 178 selecting 177 78 Immersion oil kit 85 164 65 Purchasing from Applied Precision 177 Instrument controller 113 troubleshooting 166 67 Intensity values 87 Internet Address Applied Precision xii Joystick 19 20 111 225 29 227 Keylight button 227 Keypad 19 20 111 225 29 Kohler illumination 5 89 91 150 155 Kohler spring 151 position 157 58 LCD monitor 111 Lens ID 180 setting with Resolve3D 202 Lens information window 25 Light path 16 Line requirements 100 Listing points 49 55 Live cell filter module 119 Live cell filter sets 184 Live cell imaging 5 68 82 Live specimens tracking 71 77 LMC Reset button 226 lo setting 206 Loading a point list 53 Locating a sample 84 87 Locking knob 151 Long exposure times 168 Macros 36
146. lumination position Then tighten the Locking Knob Critical Illumination Spring In Critical Illumination position the Focus Control is extended so that the Cnitical Spring isin the groove CAUTION When you are adjusting the Fiber Optic Module be careful not to disconnect the FiberOptic cable orto bend it in a diameter that is less than 24 ppliedPrecision Chapter 6 Data Collection Techniques 85 To switch to Kohler Illumination gt Loosen the Locking Knob and push the Focus Control toward the stand until it snaps into place in the Kohler Illumination position Then tighten the Locking Knob Kohler Illumination Spring Locking Knob In the Kohler Illumination position the Fiber Optic Module is pushed in so that the Kohler Illumination Spring isin the groove Monitoring Data Acquisition You can set options that control how images are displayed as they are acquired Viewing Deconvolved Image Previews You can use 2D deconvolution to get a more accurate representation of what data will look like after deconvolution These imagesshow how imagesare displayed dunng data acquistion with and without Real Time 2D Deconvolution 04 720104 Rev D 1008 86 DeltaVision Core and personalDV User s Manual To view previews of deconvolved images 1 From the Resolve3D window click Settings to open the Resolve3D Settings dialog box Then click the Display tab ba hesolvesD Settings Display i
147. ly the fuse types listed on the component or in the manual ppliedPrecision Chapter 2 Safety 11 Leaving a camera out of the tray when it is not in use presents an opportunity for the camera to fall to the floor and break Always place the camera in the camera tray when it is not in use Waming Labels Warning labels have been applied to the components of the system that pose a potential hazard to the user The labels have been duplicated here and carefully explained Please read this section carefully Note Fora description of DeltaVision components see Chapter 7 Facility Requirements and Components DeltaVision Waming Labels 04 720104 000 Rev D 1008 Hazardous Voltage Waming Label This label indicates the danger of electnc shock This label is found on the xenon arc lamp housing Caution or Waming Label This label indicates a danger of personal injury orpossible damage to equipment It is accompanied by an explanation of the specific danger Thislabel may be found on the microscope the High Rescamera the Fast Camera or the workstation 12 Delta Vision Core and personalDV Users Manual ppliedPrecision Getting Started This chapter shows how to get started with DeltaVision Before you start includes a checklist of things that you need to have before you can acquire images for your sample a Getting Familiar with DeltaVision describes the key controls that you will use to dir
148. maging Files Autofocus Mise GLM Image display mode None bd Window zi Wave Le W Calculate statistics Calculate histogram _ Auto histogram range Display images W Auto intensity scale Acquire after point visit Done save Settings 2 Select the Deconvolve preview images option L Note Thisoption doesnot provide a full iterative deconvolution but it allows you to preview images as you collect them Selecting Viewing Modes You can select from several modes for displaying images in the Data Collection window or windows as they are collected You can choose other modes to display images in color display each point in a point visiting experiment in a separate window or to display each channel in a separate window as follows fay 2 Window test fa RG Ooo dy SE ERE T aj zZ Auto Grayscale mode displaysa separate window foreach channel ppliedPrecision Chapter 6 Data Collection Techniques To select the display mode l From the Resolve3D window click Settings to open the Resolve3D Settings dialog box Then click the Display tab On the Image display mode list select a display mode w Reso We3D Settings Display imaging Files Autofocus MISC l QLN Wave Suto Grayscale FIO Calculate statistic Point Track Calculate histogram Auto histogram range Display images Deconvolve preview images Wo Auto intensity scale Acquire after point visit D
149. metric flare 177 Target temperature display 220 Technical support xii 103 Temperature requirements 103 Theoretical OTF See Optical transfer function Thickness of sample 43 44 Tilt screws 151 Time lapse 4 5 setting up experiments 44 49 Time lapse tab 48 212 13 TIRF module 121 Tool kit 116 Toolbar Resolve3D 199 200 ppliedPrecision Appendix E Resolve3D and Keypad Options Top of sample 41 Top of Sample setting 203 Total Internal Reflection Fluoresence Module See TIRF module Tracking cells 71 77 Tracking methods 74 Trans Shutter button 19 229 Transfer speed setting 220 Transmitted light source 19 plugging in 150 replacing 149 50 Troubleshooting 166 70 encoder error 167 image quality 135 168 70 instrument controller 166 67 light transmittance 135 softWoRkx Workstation 167 stalled image sequence 167 Turning off DeltaVision 32 33 Ultraviolet light 9 University of California San Francisco 2 URL Applied Precision xii Use photosensor setting 221 Viewing image previews 93 Viewing modes 93 95 Viewing statitics 96 04 720104 Rev D 1008 221 Visit Bottom setting 204 Visit Middle setting 204 Visit Top setting 204 Visiting points See Point visiting Voxels 56 180 Warning labels 12 Workstation switch 142 X adjustment screw 151 X and Y arrows keys 228 X step size setting 205 Xenon bulb replacement 144 48 Xenon light source 109 and bulb replacement 144 48 Y adjustme
150. n Customer Service See Customer Service Hotline on page xii ppliedPrecision Chapter 2 Safety Xenon Lamp Safety Ceramic xenon lamps are under high pressure and emit high levels of radiation Proper handling procedures and safety precautions should be observed to assure the safety of the users of this product Only operate this lamp within the recommended operating specifications as detailed in this manual Refer to Replacing the Xenon Lamp in Chapter 9 of this manual for details on the proper procedures for lamp replacement Explosion These lamps are under high pressure Use of face shields or safety glasses during handling is recommended Avoid applying excessive shock or stress to the lamp during handling High Voltage The ignition voltage of the xenon lamp presents a very high voltage hazard Do not touch the lamp during operation To avoid the risk of electrical shock the input power should be disconnected prior to servicing the lamp UV Visible and IR Radiation Xenon lamps emit high levels of radiation that can cause severe skin burns and permanent eye damage Avoid direct exposure to the emitted or reflected beams Thermal Hazards Xenon lamps can get very hot during and after operation up to several hundred degrees C To avoid potential for serious burns do not touch the lamp during operation Do not touch the lamp after operation until the lamp has adequately cooled Disposal It is recommended th
151. n M 1991 Drosophila gastrulation analysis of cell shape changes in living embryos by three dimensional fluorescence microscopy Development 112 365 70 Kam Z Volberg T Geiger B 1995 Mapping of adherens junction components using microscopic resonance energy transfer imaging Journal of Cell Science 108 1051 1062 Kaplan K B Swedlow J S Varmus H E Morgan D O 1992 Association of p60 s with endosomal membranes in mammalian fibroblasts Journal of Cell Biology 118 321 33 cover article Kaplan K B Bibbins K B Swedlow J R Arnaud M Morgan D O Varmus H E 1994 Association of the amino terminal half of c Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527 EMBO Journal 13 4745 4756 Kaplan K B Swedlow J R Morgan D O Varmus H E 1995 c Src enhances the spreading of src fibroblasts on fibronectin by a kinase independent mechanism Genes Devel 9 1505 1517 Kirk K E Harmon B P Reichardt I K Sedat J W Blackburn E H 1997 Block in Anaphase Chromosome Separation Caused by Telomerase Template Mutation Science 275 1478 1481 Lauer S A P K Rathod N Ghori K Haldar 1997 A Membrane Network for Nutrient Import in Red Cells Infected with the Malaria Parasite Science 276 Lieb J D Capowski E E Meneely P Meyer B J 1996 DP Y a Link Between Dosage Compensation and Meiotic Chromosome Segregation in the Nematode Science
152. n a large area with a relatively high magnification lens You can use the panels as a means of reviewing a large area of a slide or as data that you want to stitch together to form a single large image A set of panel images left can be stitched togetherto create a single image nght Image files that are collected with Panel Collection Setup enabled have a pn1 extension These images must be stitched to create DeltaVision image files See Stitching in the softWoRx Imaging Workstation User s manual CAUTION Do not try to reuse panel collection experiment macros These macrosare sensitive to microscope settings such as image size and magnification because the number of panels required depends upon many factors Determining Border Rolloff Voxels Before you collect panel images you must first determine the number of voxels in the Border Rolloff that is used in Deconvolution Note Conceptually a voxel isa three dimensional pixel So while a pixel generally refersto XY data a voxel includes information from the Z ordepth plane To determine Border Rolloff Voxels 1 Determine the size of the panels that you plan to collect Smaller panels work best because they have the flattest intensity distribution Y From the main softWoRx window choose Process Deconvolve to open the Deconvolve window In the Input field enter the file name of an image that is 04 720104 Rev D 1008 54 Delta Vison Core and pe
153. n easily switch between Critical and Kohler Illumination Kohler Illumination provides very even specimen illumination across the field of view You will typically use Kohler Illumination for most of your data collection Critical Illumination directs the entire light source to the size of the detected area It is useful for low abundant probes that require more light Live Cell Imaging You can set up a controlled environment to acquire data from live specimens DeltaVision supports an optional environmental chamber that you can use to control temperature and inject carbon dioxide DeltaVision also includes software that is specially designed to acquire data from live specimens m Cell tracking is used for point visiting time lapse experiments It automatically changes the coordinates of a point to follow a cell as it moves Real Time Z Sweep Acquisition or Optical Axis Projection allows you to quickly acquire 2D projections of specimens Real Time Deconvolution provides previews of deconvolved images as they are acquired 04 720104 000 Rev D 1008 DeltaVision Core and personalDV User s Manual m Reference Imaging is useful for acquiring reference images that can be used for Differential Interference Contrast DIC and other techniques Laser Photo bleaching and Photo activation If your system has the Quantifiable Laser Module QLM hardware module you can use DeltaVision to run and analyze laser photo bleaching and photo act
154. n is automatically updated Save and run the experiment For instructions that show how to use softWokRx to stitch panels together see the softWoRx Imaging Workstation User s Manual Note You cannot easily use Panel Collection with time lapse or point visiting expenments It is possible only by editing the header ppliedPrecision Chapter 4 Setting Up and Running Expenments 55 Using the Multplexed Wavelength Option The optional Multiplexed Wavelength module for the DeltaVision system allows you to perform nearly simultaneous two channel imaging without the drawbacks associated with true simultaneous two channel imaging This option uses two shuttered illumination sources and a dual band emission filter to eliminate filter wheel movement between channels and therefore greatly reduces the time required for the DeltaVision system to acquire a set of two channel images The combined light path ensures no registration artifacts are introduced and independent excitation of probes helps to ensure minimal crosstalk 4 position EX 2 Filter Filter Wumination 1 100 mirror Shutter Slot 1 Slot 2 Beam Com nie 2 50 50 beam combiner 3 450 long pass filter 4 500 long pass filter TATATA Taran ae on es Sen ee ay Sea a 3 z S Combined Light Path E 100 Fiber optic cable EX 1 ND EX Mirror Shutter Filter Wheel Stage sample Combined Light Path Fiber optic cable lia Objective 1 Q
155. n module ppliedPrecision Chapter 9 Maintenance 135 AN enrt374 i s a Femeeeeeen 6 Remove the center clip from the internal lamp mechanism as shown 04 720104 Rev D 1008 136 DeltaVision Core and personalDV User s Manual 7 Lift the bulb assembly small black box from the two supporting pins in the lamp mechanism 9 Replace the clip around the internal lamp assembly and gently slide the internal lamp mechanism into place within the lamp housing ppliedPrecision Chapter 9 Maintenance 137 11 Place the open end of the lamp housing over the flange on the DeltaVision and tighten the three hex screws as shown 04 720104 Rev D 1008 138 Delta Vision Core and personalDV Users Manual 12 Turn the DeltaVision on as usual and start Resolve3D 13 Before resetting the bulb age write down the age of the bulb you just replaced This will help you to keep track of when you may need to replace the next one 14 Open the Imaging tab on the Settings window and reset the bulb age Fie View Options Calibration Help Acquire Experiment Settings Excitation RD TR PE w ss Emission FITC sT 0 1 EX Shutter TRANS e Bese oreo Find 1 Calibrate Display Imaging Files Autofocus Misc Image size 10241024 Se Camera CoolSNAP_H 2 ICX285 Lens 60 int an Bin 1x1 Aux Mag Frames to average E Pixel size 0 1075
156. n of oils with refractive indexes that range from 1 500 to 1 534 Use of the correct immersion oil decreases the spherical aberration in the image data e For DeltaVision Core the immersion oil kit includes eighteen oils that range from 1 500 to 1 534 in increments of 0 002 e For personalDV the immersion oil kit includes six oils that range from 1 512 to 1 522 in increments of 0 002 Many factors influence the optimum refractive index of the immersion oil including specimen preparation temperature humidity and atmospheric pressure The Oil Calculator In order to calculate the desired refractive index softWoRx is equipped with the Lens Information function This function is located in the Utilities menu in softWoRx It can also be accessed from Resolve 3D by clicking the Info button The following parameters are explained here to help you enter the appropriate information and use the resulting calculations 154 DeltaVision Core and personalDV User s Manual Distance from Coverslip to Specimen microns Establishes the distance from the surface of the coverslip to the desired focal plane Temperature Defines the temperature of the specimen and the immersion medium Specimen Refractive Index Defines the refractive index of the specimen which is usually that of the mounting medium In some cases the specimen itself contributes significant refraction Recommended Refractive Index Displays the resulting optimal refractive ind
157. n peaks 184 eyepiece 18 eyepiece wheel change 129 30 fluorescense 184 live cell sets 184 neutral density 18 optional modules for 119 Sedat polychroic 152 sets of 17 19 specifications 184 standard optical 106 usuable life span 106 wheel calibration 135 39 Find exposure 201 Finding a sample 23 27 28 84 87 Finding exposure time 87 89 Fine focus 17 Flare 178 Flare from immersion oil 178 Flat panel monitor 111 Fluorescence 3 Fluorescence microscope 106 Fluorochromes 17 19 Focal planes 41 42 Focus 17 Focus knob 86 Focus lock 17 04 720104 Rev D 1008 217 Focus point 40 41 Fourier transform 178 Frames to average setting 220 Frequency operating 100 Fuses 12 100 Gain setting 220 Get Thickness setting 210 Goodwin Paul 3 Guidelines for 3D experiments 41 Hi Res Camera 122 Histogram of intensity values 96 Histograms 206 History 2 3 Humidity requirements 103 IC MIC replacing fuses 161 view of back 150 IC MIC switch 142 IC MIC switches 143 Illumination adjust fiber optic module tilt 156 57 aligning the path 150 61 ambient 104 center field stop aperture 155 56 checking alignment 153 55 critical position adjustment 158 exposure time 87 89 Kohler sping position 157 58 switching between methods 89 91 testing path alignment 159 types of 89 91 Image acquisition 22 27 28 30 2D in 3 wavelengths 17 19 and exposure time 87 89 and live specimens 68 82 collecting pane
158. n this example into Filter Slot 1 of the secondary light path and click Next to continue yp Activate Filter Set Wizard Move the MPs beam combiner to position 4 500 LP se Skip ta immediately activate the selected sets Next gt gt Skip Cancel The system gathers the information for this window in this case position 4 500 LP from the MXWSetup ini file not from the Instrument Controller 7 Move the beam combiner to the appropriate position and click Next to continue ppliedPrecision Chapter 4 Setting Up and Running Expenments 59 Activate Filter Set Wizard Move the polychroic turret to position z GFP mCherry Qual MIP A Push the Finish button to activate the new sets Finish Cancel Again softWoRx gets the information for this window in this case position 2 GFP mCherry from the MXWSetup ini file not from the Instrument Controller 8 Click Finish to complete the Multiplexed Wavelength filter activation process After the selected filter set is activated the Design Run Experiment window will look similar to the following J Design Run Experiment Resolve3D exp modified ja 7oeu Design Experiment Design PK Experiment j Run Experiment Experiment name Resolve30 Estimated file size 64 02 Mb 22 29 Gb Available Use Fast Acquisition ast Acaulsition tintons _ Lamps Off when finished Sectioning Channels Time lapse Points
159. nareyuiatedsyoulsed 4 Live Cel IMAZEN OA OE EERO 5 Laser Photo bleaching and Photo activation essessessessessesresesrersersersersrrsreeresresressesseee 6 TIRF Total Internal Reflection Fluorescence seseeeessssseeesssssreessssseessssssreesssssreess 6 What should you know to use Delta Vistort sissisota A EN 6 7 E AO E E A AEE MOLY EX OSU O morina a aan mouaalncahe as emtmramen tema tintaen mews asiaren aaa eae 8 Bight Light EX posure eassa or aea E tees ements aay 8 DUTIES ceca sila a eR ak al S le tact aed it aA de Nile se cease lds cl Saal tedaes 8 NOC Kanasar e a a a a a e 8 DSTO Lanp SAE Ey sa ses esses asa sta i eg Wo cae spss sa eats A esate 9 FE FO SIO sate ect nase gees actete Gone estas eens senses A motel asae cea toa T eat TAAA 9 Mien Voldg eesin a eiuesneaaseuntanes 9 UV Visible and IR Radiation 200 eee ceceeccccsecccccscccccsscccccsssccccssccseesesccecsecsceeesenecs 9 Thermal Hazards acininein e th sinet acelensedetehesn sauna unos east cuiosdee 9 VTS OOS EN E E E E A A sas ess E A T ie etm E sa teaeee eee 9 Damage Preventi oisin nin irin a E EOT ON 10 WV et LADES ariere T A T E ee eanomee malariae 11 3 GSU SANEA iiini a LO Belore yous tir Eriga a E A T 14 Gettin Familjar Wi Deka Viston sesersi iari eean e Sonedaanhaclewab ton tinngasiteet 15 Controlling the Licht pach nroa ie aa a E T 15 POCUS TING risasi iR S E E ned decison ieaeee ae 15 TOO SIS FIETS airin ea a a a NEA 16 Usine the keypad and JOy
160. ndow choose File Open Reference Macro In the Open Experiment Macro File dialog box select a macro file and click OK to open it Reference Macro Use the mouse to select the part of the reference macro that you want to copy On the Reference Macro menu choose Edit Copy On the Resolve3D Experiment Macro Editor choose Edit Paste to copy the macro into the macro editor Edit and save the file as a new experiment macro ppliedPrecision Facilty Requirements amp Components This chapter describes the main components of the DeltaVision System It includes the following sections Electrical and Environmental Requirements describes the DeltaVision operating and facility requirements Overview of Components shows the location of the key system components Optical Components describes the system light sources cameras and filters Desktop Components provides information about the monitor the keypad and joystick the vibration isolation table that supports the microscope and other components Cabinet Components describes the combined Instrument Controller and Microscope Interface Chassis IC MIC and the Workstation Other Standard Components describes the Repeatable Slide Holder the Fiber Optic Module the tool kit and standard software Optional Components describes the Quantifiable Laser Module QLM the EM CCD camera The Rainbow option dual light sources for DIC and the softWoRx
161. neutral density filter to one of a lower or higher gt value When the neutral density filter is at the minimum value and you push the decrease arrow the wheel moves to engage the maximum value filter When the ppliedPrecision Appendix E Resolve3D and Keypad Options 213 Neutral Density filter is at the maximum value and you push the increase arrow gt the wheel moves to engage the minimum value filter RETRACE ARROWS Retrace moves the stage one position back in the stage movement history buffer Retrace gt moves the stage one position forward in the stage movement history buffer EX ARROWS Changes the excitation filter to the previous or next gt filter on the filter wheel This button changes the excitation filter but not any other settings such as exposure time and shutter configuration PANEL 1 2 MARK Defines the area of the specimen to be stitched as shown in the following diagram Figure E 24 Panels Diagram Pandit oo eo nnn E b dob be te te eee ee ee ee a M o 4 b doe be b bd Saat aaah Caan ee ann er ra ala a enc a es a ear ors ACQUIRE IMAGE Commands the system to collect image data This key function is identical to clicking Acquire in Resolve3D Use this key when you are scanning through your sample and using the eyepiece to find a region of interest or when you want to get a quick look at the specimen on the monitor SAVE IMAGE
162. ng which filters to use Your choice of filters will be determined by the types of fluorescent probes with which you have labeled your sample and the filters available on your system The excitation and emission spectrum of your filters should match the spectra for the absorption and emission of the fluorescent probes you want to use The following two tables show which DeltaVision filters match each particular probe The interchangeable Standard and Live Cell filter modules are available for both DeltaVision Core and personalDV The Standard filter module filter sets are typically used for fixed specimens while the Live Cell filter module sets are commonly used for live specimens Figure 3 Sandard DeltaVision Module Alter Sets Probes Filter Name Exc itation Emission DAPI Hoechst DAPI UV 350nm Blue 455nm Coumanin Fluorescein FITC Blue 490nm Green 525nm GFP CY2 Al488 Rhodamine TRITC Green 555nm Orange 605nm Texas Red Cy3 CY 5 CY 5 Red 645nm Infrared 705nm Figure 4 Live cell Module Filter Sets Probes Filter Name Exc tation Emission Cyan GFP CFP 430nm 470nm Yellow GFP YFP 500nm 535nm mC heny mCheny 572nm 632nm EGFP sgGFP EG FP 470nm 525nm You can select other combinations of excitation and emission filters You can also add your own custom filter sets To determine the optimal filters for your application compare the excitation and emission spectrum of your filters to the spectra for the absorption and emission of
163. ngs are Activate Filter set amp lt PERRA REMEN 7 changed Done save Settings Help 4 Click Activate Filter Sets The following confirmation window is displayed A A Note Ifyou select filter sets forthe Excitation filter wheel and Emission filter wheel fields and then click Done in this window your selections are retained until you either activate the filter sets or exit Resolve3D 04 720104 Rev D 1008 58 DeltaVision Core and personalDV Users Manual WY Activate Filter Set Wizard Are the selected filter wheels currently installed Use Skip to immediately activate the selected sets i Next gt Skip Cancel Note For the filtersetsto be activated the selected multiplexed filter set filters must exist in the currently installed excitation and emission filter wheels To determine the proper filter sets referto the Changing Filter Wheel Modules section in Chapter 8 5 If the selected filter wheels are installed on your DeltaVision click Next to continue Note Ifyou click Skip from this window the selected filter wheels are activated immediately and the remainder of the activation wizard is skipped Activate Filter Set Wizard Insert the mCherry filter into the appropriate slot of the secondary excitation tube se Skip ta immediately activate the selected sets Next Skip Cancel 6 Insert the selected secondary filter insert mCherry is used i
164. nitialized or to move the objective up to the slide until the oil is touching the slide Choosing Filters Choosing and controlling filters is a key for any fluorescent probe experiment When a fluorescent probe is excited by a specific wavelength it emits light at another wavelength Choosing the right filters for the dyes in your sample allows you to obtain a complete set of data specifically from your probe without interference from other wavelengths ppliedPrecision Chapter 3 Getting Started 17 Eyepiece Filter Wheel Neutral Emission Density Filter Filter Exc itation Filter DeltaVision provides five different types of filters for controlling the fluorescent light path e Neutral Density filters reduce the amount of light that illuminates your sample when you are using fluorescence DeltaVision provides six filters that block from 0 to 99 9 of all light e Excitation filters block all but one band of wavelengths to provide a specified range of light to excite the fluorescent probes in the sample e Polychromatic Beam Splitter reflects the excitation wavelengths to the sample and transmits the emission wavelengths from the sample DeltaVision ships with a standard polychromatic beam splitter for DAPI FITC TRITC and Cy5 Other beam splitters ship with optional live cell sets e Emission filters allow only a single band of light from the excited probe to reach the camera e Eyepiece filters are emission fil
165. nly if you have configured your system for alternate filter wheels Reinitialize Filter Wheels Reinitializes the filter wheels Action Buttons Done Closes the Resolve3D Settings dialog box Save Settings Preserves the current options for your next Resolve3D session In addition to options in the Resolve3D Settings dialog box current state information such as current filters and exposure time is saved Help Opens the Help for the Settings dialog box ppliedPrecision Appendix E Resolve3D and Keypad Options 211 Keypad J oystck Operation Many of the functions accessible through Resolve3D are also available on the keypad joystick This section describes each key on the keypad joystick Note Some buttonson your keypad may not be active Figure E 23 Keypad J oystick CONTROL REMOVE KEYLIGHT MODE ON OFF ON OFF rf CAMERA DISABLE SHUTTER MOTION Clears communication buffers closes shutters stops all motors and clears encoder errors Use the RESET button when you suspect that the workstation and controller are not synchronized CONTROL MODE Toggles the controller mode between local mode and remote mode Local mode enables control by the keypad and joystick The remote mode disables the joystick and the keypad buttons except for CONTROL MODE and a few other buttons and shifts control to other internal components 04 720104 Rev D 1008 212 Delta Vison Core and pe
166. nsussneeserssnneecsoneesensnnesecsenneseussneesersonaescsenensensnnesecsennes Qed 04 720104 000 Rev D 1008 viii Delta Vision Core and personalDV Users Manual ppliedPrecision Preface This manual shows how to use the DeltaVision system to acquire images It also shows how to maintain the system a About This Manual describes the information in each chapter a Document Conventions explains the typography notes and other conventions used in this manual Contacting Applied Precision Inc provides information about how to contact customer support About This Manual This manual provides instructions for scientists who are using DeltaVision to acquire data It also includes instructions and references for maintaining the system The Introduction provides a brief summary of the DeltaVision system features Safety warnings and guidelines are provided in Chapter 2 Chapters 3 6 show how to use the system to acquire data Delta Vision Core and personalDV User s Manual a Chapter 3 Getting Started describes how to turn the system on acquire an image and run an experiment macro a Chapter 4 Setting Up and Running Experiments shows how to set up experiment macros for 3D sectioning Time lapse Multiple wavelengths Paneling for stitching and Point Visiting a Chapter 5 Acquiring Data From Live Specimens provides information on how to use the DeltaVision Core system to collect images from live specimens a
167. nt screw 151 Y step size setting 205 Yes button 227 Z arrow keys 228 Z focus 17 Z sectioning 38 44 43 methods for 44 Z slider 205 Z step size setting 205 Z sweep acquisition 68 78 80 Z1 Mark and Z2 Mark buttons 228 Zoom tool 205
168. o NA oa Working Distance um 3100 0 Focal Length um 15690 0 Refractive Standard Refractive Index 1 000 Recommended Refractive Index 1 000 Index yE Distance From Coverslip to Specimen tum 0 0 Coverslip Thickness um i 70 0 Optical E Condones Temperature Z 25 0 Specimen Refractive Index 1 470 Glycerol 1470 HeSOMTION Lalculations Resolution Ratio 2 44 a2 Depth of Field urn 3 16 Ci za i Maximum 4 Pixel Size urm 0 48 Recommended lt Step Size um 4 39 6 Inthe Optical Conditions fields enter the conditions for the sample 7 Note the displayed value in the Recommended Refractive Index field and use an oil with that refractive index CAUTION Before you start each data collection session calculate the oil Your oil selection should be the same if the sample and conditions are the same 8 Place a drop of oil on the objective Be sure to use the proper oil see Page 14 AN CAUTION Do not touch the glass dropperto the objective 9 Place a drop of oil on the coverslip Be sure to use the proper oil see Page 14 10 Mount the slide on the Repeatable Slide Holder with the coverslip down 11 Use the Adjustment knob on the Repeatable Slide Holder and the joystick on the Keypad to center the coverslip over the objective Q Tip If you have the Repeatable Slide Holder you can record the position of your Slide This is useful when you are performing a Point Visi
169. o be useful for other types of reference images Only one reference image per Z stack is collected Time lapse Setup Use the Design Experiment Time lapse tab to specify the number of time points the time periods and the total time for a time lapse experiment Figure E 15 Expenment Designer Time lapse Setup Options Ww Design Run Experiment File Help Design Experiment Design PK Experiment Run Experiment Experiment name Resolvea0l Estimated File Size 32 01 Mb Enable Fast Acquisition fast Acuiititien Captions Sectioning Channels Time lapse Points l Panels Actions F Time lapse Hours Minutes Seconds Milliseconds Time lapse gt o fo lo Total Time gt fo fo lo Time Points f F Enable Cell Tracking Cell Tracking Options F Autofocus before imaging Autofocus Options Time lapse option Specifies running a time lapse experiment Time lapse Specifies the hours minutes and seconds for each time period Total Time Displays the total time of the experiment which can also be calculated as follows Total time The number of time points 1 x time lapse 04 720104 Rev D 1008 200 Delta Vison Core and personalDV Users Manual Time Points Specifies the number of time samples to collect in a time lapse experiment Enable Cell Tracking Moves the stage laterally to follow cells as they move during a time lapse experiment With the Enable Cell Tracking opt
170. o enter the PSF file to convert e Drag the appropriate PSF file from the File Manager into the PSF File text box e Click PSF File to display a small version of the File Manager and then choose the PSF file that you wish to convert ppliedPrecision Appendix C Acquiring a PSF 169 e Type the desired path and filename into the PSF File text box 4 Click Do It To place OTF into OTF Library If the objective used is in addition to those already present you ll need to modify softWoRx to use the new objective by adding the file to either usr local softWoRx or dv2 10 config system dvrc as follows 1 Login to Linux as root 2 Navigate to usr local softWoRx config system dvre 3 Find the section labeled Lens to OTF matching and follow the instructions provided for the OTF file The following is an example of this section of the file Lens to OTF matching These are of the form LENS lt lensIDNumber gt OTF and are defined to be the file name in the OTF directory of the OTF that is to be used for this lens ID LENS 2 OTF 60X140 sample otf LENS 10602 OTF 60X140 otf LENS 10003 OTF 100X135 o0tf LENS 10403 OTF 40X135 sample otf LENS 10603 OTF 60Xw_120 otf LENS 10205 OTF 20X00 15620b 04 720104 Rev D 1008 170 DeltaVision Coreand personalDV User s Manual ppliedPrecision Appendix D Reference Information The appendix includes the following topics Standard Filename Extensions lists t
171. oc us in Expenment Macros You can set up your experiment macro to automatically focus before each image is acquired This is especially useful for long time lapse experiments that are susceptible to changes in environmental conditions Autofocus allows you to acquire focused images without closely monitoring the experiment The DeltaVision Autofocus algorithm is a software based method that uses both image contrast and peak image intensity to determine when the specimen is in focus To optimize the focus position for DeltaVision you must find the Z position where the contrast is greatest However this position may not always be at the optimal in focus plane of the sample Autofocus allows you to adjust several parameters within the standard algorithm so that the final Z position is most favorable for the specific sample Limiting the step size and maximum Z range of travel can limit the effects of objects that are not of interest In cases where the contrast outside of the actual middle of the sample is highest you can create an offset so that the middle of the sample is located during the Autofocus process The Autofocus option works the same way as the AF button on the Resolve3D window Use the following instructions to include this option in the experiment macro To setup an expenment that automatically focuses before ac quinng points 1 At the top of the Resolve3D window click the Experiment button to open the Design Run Experiment win
172. of vanous Resolve3D activities see Page 193 Looking for DV controller Initializing DY controller E Initializing Camera wa ee Pzd F ppliedPrecision Appendix E Resolve3D and Keypad Options 183 The Resolve3D Menu The Resolve3D menu has the following menu items The File menu includes commands to acquire images and to open the key dialog boxes for setting up and running experiments The View menu includes commands to manage marked points and to create a blank screen The Options menu includes commands to open the Settings dialog box where you can set display and image options and to save settings The Calibration menu opens the Calibration tool The Help menu provides options to show or hide ToolTips and to get Help The File Menu Use the following Resolve3D File menu commands to acquire images create scratch files and open the Design Run Experiment dialog box Figure E 2 The Resolve3D File Menu Continuous Acquire snapshot acratch File Experiment Ctrl E uit Ctrl G Acquire Image Collects and displays an image from the microscope This image is only displayed in the Data Collection window It is not saved to a disk file for later use Continuous Ac quire Opens the Continuous Acquire dialog box that you can use to collect and display images continuously These images are only displayed they are not saved toa disk file Snapshot Launches a t
173. ollect for each wavelength for the experiment softWoRx automatically calculates and displays this value if the Sample thickness and Optical section spacing are entered If you specify this value the Sample thickness value is changed Sample thickness Displays the Number of optical sections multiplied by the Optical section Spacing Get Thickness Retrieves the Sample thickness based on the locations defined with the and l buttons in the Resolve3D window OAI Acquires a 3D Z projection of the interval defined by the marked top and bottom of the sample The optical section spacing and the number of optical sections are determined automatically based on the depth of field of the objective and the exposure time 04 720104 Rev D 1008 198 Delta Vison Core and personalDV Users Manual Channels Setup Use the Design Experiment Channels tab to select wavelengths filters and to specify an exposure time for each filter Figure E 14 Design Expenment Channels Options J Design Run Experiment File Design Experiment Design PK Experiment Run Experiment Experiment name Resolves Estimated File Size 64 02 Mb F Enable Fast Acquisition Fast Acquisition Options Sectioning Channets Time lapse Points Panels Actions Conventional d Multiplexed Exp Ex Filter EM Filter AT Ex Shutter ie Refresh exposure conditions Refresh exposure conditions Updates the filter and expo
174. on Chapter 7 Facility Requirements and Components 93 Country specific Requirements The following table shows the accredited agency and power cord set requirements for each country Accredited Country Agency Power Cord Set Requirements Australia EA NSW The flexible cord must be lt HAR gt Type HO5VV F 3 conductor 1 0 mm conductor size Power cord set Austra OVE fittings appliance couplerand wall plug must bear Belgium CEBC the certification mark of the agency responsible for evaluation in the country where it will be used Denmark DEM KO Finland SETI France UTE Gemany VDE Italy IMQ Norway NEMKO Sweden SEM KO Switzerland SEV United Kingdom BSI United States UL The flexible cord must be Type SJ Tor equivalent No C d CoA 14 AWG 3 conductor The wall plug must be a two anes pole grounding type with a NEMA 5 15P 15A 125V configuration Japan JIS The appliance coupler flexible cord and wall plug must beara T mark and registration number in accordance with the Japanese Denton Law The flexible cord must be Type VCTF 3 conductor 2 00 mm conductor size Environmental Requirements An important aspect of collecting high quality images is having the proper environment for the system The following important environmental requirements are outlined in this section General Environmental Requirements Floor space DeltaVision Core 3 ft x 6 ft 90 cm x 180 cm Include 18 in 45 cm space behind instrument rack p
175. on Core and personalDV Users Manual ppliedPrecision Appendix C Acquiring a PSF This appendix shows how to acquire a Point Spread Function PSF and convert it to an Optical Transfer Function OTF a Acquiring a PSF shows how to measure a Point Spread Function Converting PSF to OTF shows how to convert the Point Spread Function to the Optical Transfer Function that is required to process images Before You Start Before you attempt to measure a PSF check the OTF library included with softWoRx to find out if the library provides an OTF for your objective Acquinng a PSF To measure the point spread function PSF you need to optically section a fluorescent bead Since the properties of the objective lens are the most important elements of determining a PSF it is necessary to measure the PSF whenever new lenses are added to your microscope The deconvolution software adapts to the PSF actually the OTF wavelength so it is unnecessary to measure the PSF at more than one wavelength 162 DeltaVision Coreand personalDV User s Manual Note If you do not have the tools necessary to acquire a PSF softWoRx includes a utility that allows you to calculate a theoretical OTF based on the numencal aperture of the camera lens index of refraction and emission wavelength If the aperture of the lensis lower than 0 75 N A the calculated OTF may work as well or even better than a measured OTF However if the aperture is great
176. on difficulties and abnormalities in image data Table B 3 Image Quality Troubleshooting Chart Indication Cause Connection Dim imagesor long Poor illumination exposure times Fully open field aperture Align xenon lamp and fiberoptic cable as shown in Replacing the Xenon Bulb ppliedPrecision Appendix B Troubleshooting Indication Dim illumination When fiber optic cable and focusing lens are removed the projected light doesnot fom a circle Dark out of focus spots on image Image is distorted around edgesor throughout Occlusion seems to creep in toward center Bightnessof Z section images vanes greatly 04 720104 Rev D 1008 Cause Filter wheel s out of alignment Dust interference Condensation on camera window possibly due to improper camera temperature A broken Photo sensorcable 157 Corection on Page 134 Ensure that filter cube turret is locked in position on rail mount Ensure shutter on filter cube isopen Ensure slider behind microscope Is seated in an open postition Ensure proper filter cube isin position and seated in detent Shut down and start up asdescnbed on Pages 19 and 30 orunplug and plug in the Eyepiece filter wheel This will reset the home position of the filter wheels and align filter wheel position Calibrate filter wheels following instructions on Page Enor Bookmark not defined Clean polychroic filter emission filter and cam
177. one save Settings Table 1 Image display modes Mode Desc nption None Displays imagesin the current window Scratch Displays all images in the default Data Collection window Window 21 Auto Displays images in a separate window for each emission filter Grayscale Auto Color Displays images in colorasthey are collected When using Point Visiting imagesare automatically displayed in separate windows for each point Thisoption should be used only when you are running an expenment Point Track Opensa separate window foreach visited point in a point visiting expenment 04 720104 Rev D 1008 87 88 Delta Vision Core and personalDV User s Manual Displaying Statistics and the Histogram You can choose to display statistics and a histogram of the intensity values of each image as it is collected These values are displayed at the bottom of the Resolve3D window The Min Max and Mean values are the minimum maximum and mean intensity values The histogram displays the intensity distribution To display statistics and the histogram 1 From the Resolve3D window click Settings to open the Resolve3D Settings dialog box 2 Click the Display tab Then select from the following options To Select Calculate image intensity Calculate Statistics Statistics Note You can improve readout speed by disabling this option Calculate and display an Calculate histogram image intensity histogram Automatically scale the Auto His
178. ons ppliedPrecision Chapter 9 Maintenance 141 The Fiber Optic Module Use the Fiber Optic Module to align the light path from the fiber optic cable to the Fluorescence Illuminator This module allows you to adjust the tilt horizontal and vertical orientation of the light path Figure 7 The ber Optic Module Tilt Sc rews Critical Spring sf Y Adjustment Screw Adjustment or Lock Tilt Screws Photo sensor Port Kohler Spring Critical Spring Locking Knob Y Adjustment Sc rew X Adjustment Sc rew 04 720104 Rev D 1008 Photo sensor Port _ K hler Spring Locking Knob lt X Adjustment Sc rew Use to Adjust the tilt of the Fiber Optic Module body Connect the fiberoptic cable to the photo sensor detector in the IC MIC Set the Kohler illumination position Two locking screws hold it in postion Set the Critical illumination position Two locking screws hold it in postion Temporanly lock the position of the Focus Control Adjust the vertical postion of the cable Adjust the horizontal position of the cable 142 DeltaVision Core and personalDV User s Manual Before You Check or Adjust Illumination Path Alignment Before you check or adjust alignment remove all sources of blockage to the light path Then verify the following settings to make sure that the various lenses and controls are in the correct positions you can feel the controls snap into place w
179. ool to let you collect a single 2 D multi wavelength snapshot image If you need to collect a Z series time lapse image or other complex scheme you will need to design an experiment with the Resolve3D Experiment Designer 04 720104 Rev D 1008 184 Delta Vison Core and personalDV Users Manual Scratch File Creates a scratch file to which you can save individual image frames for later use After a file is opened clicking Save Current Image saves the most recently collected image frame Clicking Close Scratch File or Done closes the file Note that the image size cannot be changed while a scratch file is open Expenment Opens the Design Run Experiment dialog box You can use Design Run Experiment to select a previously created experiment open the Experiment Designer window to design a new experiment or open the Experiment Macro Editor to create or edit an experiment macro Quit Closes the Resolve3D window The Resolve3D View Menu Use the View menu to manage points that you have marked to clear the history of the path that you took while exploring your sample or to create a black screen for light sensitive conditions Figure E 3 The Resolve3D View Menu View Paint List Clear stage Trails Clear Stage Thumbnails Blank Screen Command Line Interface Point List Opens the Points List dialog box This dialog box helps you manage a list of points
180. oretical Z resolution for each objective Number of optical sections This field defines the number of optical sections in the Z series of the experiment Enter the number of sections necessary to span from the lower to the upper boundaries of the sample The required number of Z sections depends upon the thickness of your specimen A typical scan consists of between 16 and 64 sections but there is no set number ppliedPrecision Chapter 4 Setting Up and Running Expenments 41 For best results use at least three optical sections Using one or two optical sections however will also provide acceptable results The maximum number of optical sections is limited by memory and time Applied Precision s benchmark deconvolution consists of 64 optical sections that have XY dimensions of 512x512 If you find it necessary to acquire many Z sections use the following formula to figure out how much random access memory RAM your computer needs to efficiently perform the deconvolution Less RAM results in slower performance Available RAM bytes gt X size x Y size x Z size x 4 Sample thickness This field displays the thickness of the optical section range It is approximately one section thicker than the actual stage movement as shown below Sample thickness R f Stage ange of Stag Optical Section Range Movement dO me For optimal deconvolution the Z sections should extend beyond the volume of interest by 1 3 se
181. p 1 5 or 0 17 mm eae Optical section spacing This parameter indirectly defines the size of the incremental Z stage movement the distance between focal planes When DeltaVision acquires an image the objective collects information within the depth of field above and below the focal plane 04 720104 Rev D 1008 Delta Vison Core and personalDV Users Manual perc cccnrrrreer er ceeer emeent mmen tment emeente emmen Depth of Field Focal Plane Optical Section Spacing The Optical section spacing specifies the upper and lower boundaries for which information is collected It is centered on the focal plane and includes the information above and below that plane When you select an Optical section spacing DeltaVision uses a Z step size that is the same distance as the Optical section spacing The Z step size is the vertical distance that the stage moves between focal planes Figure 2 Optical Section Spacing Focal ZStep Size Planes The standard step size for a 1 40 NA lens is 0 2 um although other step sizes are usually fine Due to the inherent Z resolution of a 1 40 NA lens there is little need to take steps finer than 0 1 um When scanning a PSF use 0 1 um steps For low power lenses the step size should be appropriately larger The Lens Info dialog for each lens contains the suggested Z Step size for each lens derived using Nyquist sampling This value is approximately 1 3 of the the
182. pe Additional controls for moving the stage and acquiring images are provided by the keypad and joystick Contolling the Light path DeltaVision provides a transmitted LED light and a xenon arc lamp The transmitted light works the same as the light source for a traditional microscope with the light path directed on the specimen from above The xenon arc lamp provides excitation light directed through the back of the microscope and focused on the specimen from below You can use the Beam Selector to direct either light path from the specimen to the eyepiece or to the camera Transmitted LED Light Camera i Fx a xenon Arc a a N C Lo La mp J il a e Fiber Optic Cable Bea m Sele ctor Foc using There are three manual focus controls and a Focus Lock on the microscope These controls are similar to those on other systems 04 720104 000 Rev D 1008 DeltaVision Core and personalDV User s Manual Fine Z Eyepiece Focus Focus Coarse Z Focus Not currently used Not currently used Eyepiece Focus Use the Eyepiece Focus on the left ocular to focus the eyepiece Focus Lock Use the Focus Lock to lock or unlock the Z focus Fine Z Focus Use the Fine Z Focus knob to move the objective in very small increments It is used to focus on the focal plane Coarse Z Focus Use the Coarse Z Focus knob to move the objective in large increments It is typically used to lower the objective when the system is i
183. plex capable ok You will need to change the active filter set to continue Press OK to return to the Design Run Experiment window To change the active filter set for Multiplexed Wavelength experiments see the procedure for activating a multiplexed wavelength filter set in Setting Up the Multiplexed Wavelength Option Also see Setting Up Filter Wheels in Chapter 8 for additional information on changing the active filter set If the currently active filter set is Multiplex capable the Design Run Experiment window is displayed and will look similar to the following ppliedPrecision Chapter 4 Setting Up and Running Expenments YW Design Run Experiment Resolve3D_Startup exp a X File aga 0e Design Experiment Design PK Experiment Run Experiment Experiment name RSME Estimated File Size 668 05 Mb Enable Fast Acquisition ast Acuuisifen Usotions Sectioning Channels Time lapse Points Panels Actions Conventional Muttiplexed Do Multiplexed Channel Imaging Settings Exp EX Filter EM Filter YT Ex Shutter 1 lo 775 GFP GFP 100 EX 2 0 775 mCherry mCherry EX2 Place mCherry filter in the secondary Filter Slot Be sure that Beam Combiner is at position 4 500 LP Be sure that the Polychroic is at position 2 GFP mCherry Dual MPX Reference Image _ Refresh exposure conditions At this point you have completed the initial por
184. point spread PSF to an optical transfer function OTF Essentially the OTF is the Fourier transform of the PSF The pixel size of the resulting image is given in cycles um To reduce problems associated with measurement noise the PSF is radially averaged during the conversion and as a result the 3D PSF image becomes a 2D OTF image The horizontal axis of the OTF represents axial Z frequency and the vertical axis represents radial XY frequency The brightness of the OTF image elements on a scale of 0 to 1 represents the frequency response of the microscope system at the corresponding radial and axial frequencies ppliedPrecision Appendix C Acquinng a PSF 167 Figure C 2 Sample OTF Image Image Window 1 Sample100 _a40 oth File View Options Tools rE Ea Radial Resolution XY Zoom 3 0000 d lt 4 Axial Resolution gt Figure C 3 PSF to OTF Conversion gt PSF to OTF Conversion PSF File i X Range i Y Range i T Range Start Wavelengths a of po mi Sub Image Center p 00 Lens ID fo Additional Parameters Done Do It Options Each option in PSF to OTF Conversion is described briefly below For additional information regarding these options refer to the online Help PSF File Defines the name of the PSF image file to be converted to an OTF OTF File sym Displays the name of the resulting axially symmetric OTF For yo
185. ponent ee SS e a aaa e _ lt SS a MON NNN Note The EM CCD Camera isnot an available option for personalDV LL The addition of the Cascade II with Electron Multiplying CCD EM CCD technology further improves the signal to noise ratio SNR of the DeltaVision system Unlike conventional CCD cameras an Electron Multiplying CCD EMCCD is not limited by the readout noise of the output amplifier even when operated at high readout speeds A solid state Electron Multiplying EM register allows weak signals to be multiplied before any readout noise is added by the output amplifier 04 720104 Rev D 1008 112 Delta Vision Core and personalDV Users Manual The EM CCD camera can be operated either as a high performance EMCCD camera for unparalleled low light level sensitivity or as a traditional non electron multiplying CCD camera Applications for the EM CCD camera include e Low light Fluorescence e Total Internal Reflection Fluorescence Microscopy TIRF e Single molecule Fluorescence Analysis Workstations You can purchase two types of Analysis workstations the softWoRx Linux workstation and the softWoRx Suite Windows workstation The softWoRx Linux workstation includes all of the softWoRx Analysis modules including 3D Visualization tools Colocalization FRET Analysis FRAP Analysis Intensity and Distance Measurement and Modeling The softWoRx S
186. proper oil Note An oil kit isincluded with your DeltaVision system To purchase replacement oil please contact Applied Precison To confi that you are selecting the conect immersion oil 1 Collect the image data and generate a 3 D maximum intensity volume projection 2 Rotate the 3 D image to get a view of the XZ or YZ plane For example rotate the image 90 degrees about the X axis or 90 degrees about the Y axis To better see the shape of the PSF it is helpful to do an exponential scaling an exponent of 5 usually works well Note If you are using softWoRx use the Volume Viewerto generate a 3 D rendenng of the bead scan 3 Look for symmetric flare in the resulting image Symmetry indicates that the oil is correct and in virtually all situations the most symmetric PSF along the Z axis is also the smallest and has the highest intensity In other words symmetry corresponds with the highest resolution 4 Repeat the process with different oils until you determine the optimal immersion oil The following figure demonstrates how image flare can be affected by the use of different immersion oils 04 720104 Rev D 1008 166 DeltaVision Coreand personalDV User s Manual Figure C 1 Hare from Immersion Oils 3 D Maximum Intensity Projections e c l Pap 1 Corect Immersion Oil Immersion Oil Index Too Low Immersion Oil Index Too High Converting PSF to OTF The PSF to OTF program converts a measured
187. recision using the e mail address servicehotline api com or alternatively use the problem report form in Appendix D Reference Information ppliedPrecision Safety The precautions detailed in this chapter must be carefully observed to prevent possible personal danger UV Exposure discusses potential for UV exposure from the xenon arc lamp Bright Light Exposure warns about bright light exposure from the transmitted light source installed in the microscope Burn provides guidelines for avoiding burns from the xenon arc lamp The arc lamp reaches very high temperatures Shock includes warnings about potential shock hazards Hazardous voltages are present even when the system is disconnected from the AC main power outlet Damage Prevention describes actions that can damage the system Warning Labels describes the system labels Additional safety guidelines for maintenance and alignment are detailed in Chapter 9 Maintenance DeltaVision Core and personalDV User s Manual UV Exposure Since the xenon arc lamp emits ultraviolet UV light there is a danger of exposing your eyes and skin Loss of eyesight could occur if unfiltered light from the xenon arc lamp reaches your eyes To prevent UV exposure e Open the shutter only when an excitation filter is engaged e Do not open the xenon arc lamp housing during operation See Chapter 9 Maintenance for detailed instructions on changing the xenon arc lamp bulb
188. res in a cell nucleus or other rapidly moving structures Continuous Z Sweep versus Traditional Projections If you are acquiring data objects that you plan to use for 2D projections Continuous Z Sweep provides several advantages over creating 2D projections from multiple optical sections Complete Z data acquisition collects all data in the interval of interest with 2D imaging or Z section sampling some data is lost a Fast data acquisition provides accurate image registration of rapidly moving objects Low total exposure time reduces the risk of damage to the specimen Low total read noise the camera is only read once improves signal to noise ratio 04 720104 Rev D 1008 74 DeltaVision Core and personalDV User s Manual Note Forsamplesthat contain a large amount of fluorescence throughout 3D space forexample a tumor spheroid that hasa lot of fluorescence optical sectioning may provide better results than OAI The following 2D image of endosomes in a HeLa cell left and a 3D Z projection right of the same area were acquired under similar conditions The additional data in the 3D Z projection include objects that moved out of the depth of field of the 2D image during the data acquisition process endosomesin a HeLa cell left and an instant 3D Zprojection nght of the same area Using Continuous Z Sweep To setup a Continuous Z Sweep Expenment 1 Set up your experiment as shown in Creating an
189. ront and back surfaces of objective and coverslip Reapply immersion oil and restart expenment Clean front and back surfaces of objective and coverslip Use lower exposure time and or higher neutral density filter Align xenon lamp and fiber optic cable See Chapter 9 Maintenance Move knob to direct light to camera Position stage in center of the sample and run expenment again ppliedPrecision Appendix B Troubleshooting 159 Delta Vision Problem Report Fom Research Facility System Serial Number Contact Person softWoRx version use Help gt Software Versions Problem Encountered Please write a detailed description answering as many of the following questions as possible 6 Is the failure the same each time or does it show differentsymptoms __ o 8 Does it go away after the workstation isre booted SSS O 9 Does it go away after the instrument controller is re booted How often do you re boot the workstation and instrument controller Additional Comments See Page 2 for additional clarification issues Please supply the following log files Workstation home userName softworx logs softworxlog txt Instrument Controller c de530 lt dyv log IC 530 1log Ofc 16525 dyv tog iC 525 10g 0 c 1ic540 dv log IC_ 540 1og Please e mail this form to hot line api com or Fax to 425 557 1055 atin Bio Service Hotline 04 720104 Rev D 1008 160 Delta Visi
190. rrent stage Z position as the top of your sample Because the scanning process always moves the stage toward the objective this position also represents the point where the stage is closest to the objective the most negative Z value This also represents the focal plane that is the closest to the slide side of a sample Use this along with the Mark Bottom of Sample button to establish the thickness of the sample You can use these marked positions to aid with the Z sectioning setup ppliedPrecision Appendix E Resolve3D and Keypad Options 191 Note All scansthat are set up using the Experiment Designer scan in Z using relative coordinates The Mark Top of Sample and Mark Bottom of Sample buttons are most helpful in determining the thickness of the sample to be scanned When an experiment is started the scan region isdetemmined by the curent focus point and the thickness of the sample So for example if you have marked three points to visit in an expenment and they all have different middle Z positions the experiment will calculate the scan based on these different Zpostions and the fixed thickness cA Visit Top x Moves the stage to the position marked as the top of the sample Mark Bottom of Sample end of scan Marks the current stage Z position as the bottom of your sample for a potential scan This corresponds to the positive stage Z coordinate value or the coverslip side of the sample E Visit Bottom Moves the st
191. rsonalDV Users Manual LMC RESETON OFF Executes a Lost Motion Compensation LMC move Q Tip You can disable LMC on the Resolve3D Settings dialog For more information see the online Help REMOVE TRAIL Clears the stage movement history from memory KEYLIGHT ON OFF Turns the keypad backlight on and off QUIT Quits the controller program when pressed twice To restart the Instrument Controller program double click the Instrument Controller icon on the desktop BLANK SC REEN Suspends or activates the monitor s light display BLANK SCREEN is a toggle button Use this feature when viewing dim samples or performing light sensitive experiments SLO W MEDIUM FAST Adjusts the joystick stage movement speed DISABLE MOTION KEYS Disables the eight keys below the joystick This prevents accidental input from the keys when using the joystick YES NO Allows response to questions prompted on the screen For example during initialization the computer will ask if you want to continue with initialization The Yesand No buttons are an easy way to respond ACQUIRE MODE ARROWS Changes the acquisition mode This includes the Excitation filter exposure time shutter configuration and many of the options defined in the Settings dialog box JOYSTICK Moves the stage in X and Y POINT ARROWS Scrolls through the list of marked points Press AC QUIRE IMAGE to view the image for a selected marked point ND ARROWS Changes the
192. rsonalDV Users Manual similar to the panel size You can acquire and save a blank image of that size for this purpose Click the More Options button to open the More Deconvolution Options window and record the value that is displayed in the Border Rolloff voxels field Collecting Panel Images To collect 3D panel images 1 8 From the Resolve3D window menu choose View Point List In the Point List dialog box choose Mark Point and mark points in two opposite corners to define the area of your final composite image In the Resolve3D window click Experiment to open the Design Run Experiment window Click the Design Experiment tab Then set up the Sectioning and Channels information in the same way that you would for standard data collection In the Point List dialog box select one of the points that you marked in Step 1 and click Visit Point Click the Panels tab under the Design Experiment tab Then select the Collect Panels option Click Get Start under the Panels tab Then visit the other point that you marked in Step 1 and click Get End Alternatively you can enter the coordinates that are displayed in the Point List dialog box In the Overlap pixels field enter at least twice the number of border rolloff voxels as you recorded when you determined border rolloff voxels see Page 53 If you generally adjust the pixel size to lt 1 or use rotation when stitching use a larger overlap The spacing informatio
193. rt the process over otherwise press y to save your offsets 12 Re install the filter wheels in the system 13 To shut down the Instrument Controller go to the Start tab and select Windows Security Then select the Shutdown option as shown F Internet SV Internet Explorer cs E mail ae j Outlook Express S ic540_dy ini072808 txt ic540_dy ini072808 txt S a Deltavision Instrument ay eta Controller Notepad 4o All Programs gt ay My Documents 3 My Recent Documents gt 7 My Pictures fe My Music S My Computer J L Control Pane 7 Set Program Access and amp Defaults OS Printers and Faxes Og gyl Help and Support 7 Search I Run 6 sitions ice points gt n move the EM filter one filter position hole is centered ppliedPrecision Chapter 8 Changing Camerasand Filters 129 The IC MIC will power down 14 Power the IC MIC back on and boot the DeltaVision as normal 04 720104 Rev D 1008 130 Delta Vision Core and personalDV Users Manual ppliedPrecision Maintenance This chapter provides the following instructions for the basic maintenance of the system a Shutting Down and Starting the System Replacing the Xenon Bulb Replacing the Transmitted Light a Aligning the Illumination Path m Replacing IC MIC Fuses m Cleaning Moving the System Shutting Down and Starting the System
194. s Mesto a eater ous ati 112 Multiplexed Wav elenethy Mi OdU 6 cssconssdacecsusesassnaacaecosmpostaseetdureosisavesuspsnedeegadueqouaennies 112 OEE WA Celta neuter adele E T date laden O T EO O ATT 112 PONS UTA Des AT tS nrrainn aan A O 113 8 Changing Cameras and Fite rs cccsssccsssssccsnsssesnsssecsesssesseees LLS STATS GA VOT AS na peers anes a anes E one date sticos E R 115 Using Live Cell or Custom Filter Wheel Modules eeeesseessecseceeseeseeeeseeens 117 04 720104 000 Rev D 1008 vi DeltaVision Core and personalDV User s Manual Chaneine Filter Wheel MOGI CS iio ieisecasa ciei vicar sty seutvistsevinsaia Valve inaateay idesmawsea ties 118 Calibrating the Pitter VW NCE Siera A TEER 125 9 Maintenance ee ee ee ee eO L omitme Down and Star UIs Me Systeemin O E 131 Delia Vision TOWer Swichers ieee eae es 132 Guidelines Tor USINE SW ICES yiii a EE N 133 SUITES Down the System serria esin n E O A E T 133 Strine TREO VS CON a TR tad ae A E en aeeee nats 133 Replacine the Xenon DUll Deroars aana A R 134 Replacing the Transmitted Lio Bioneer saad E eigen alge 138 ANenine the UWumimnatom Patiess E 140 ine Fiber Opie MO dU ae aie eet ee ade ees 141 Before You Check or Adjust Illumination Path Alignment ccc cess eeeeeeees 142 Checking Ttumination Path Alenen esii A iain 143 PAULUS ATA VTE nicest Se hoarse EEEREN ola del opa ta rests aledes use Sit 144 Replace TC MIC TUSE Seii cue smsnietin tess ues grathan R E O 150
195. s Traditional Projections cece esse eeeeseeeeeeeeeees 73 Usmo OMEMNUOUS A WEEP Ann a is eciesdncaidtananes vines O 74 Acouinne a Rererence Mateni ceed toesutaatoaietadabedciaeerinesaaymios tides 76 Data Collection Tec NNnIqUeS ssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 7D Finding a Specimen and Recording its POSItiON ceceeeeeeseeteeteeeeseeseeseteeneeteeeeseeess 79 Padine Exposure TINE ainn E anes jupadedesauneeteneadees ou sestveateds 82 Using Kohler and Critical imine Ons russiine a 84 Monitorin Datt Acquisti orii ia oE E A E 85 ppliedPrecision Contents V Viewing Deconvolved Image Previews xviii wait aioe aces 85 De lS S VIC Wil WOES rrii sctaat Seas ase anta AAA O 86 Displaying Statistics and the Histogram siicisiiincssnaisnuesdssnctecovouseadsiisendestsdacnonaercabsiede 88 Eine Exper mene MATOS i OE Oran TR MATa Poona tom DERN 88 7 Facility Requirements amp COMPONEMMG cccsssesesssesnsssesesseeee ee OL Electrical and Environmental Requirement ccc ceceseessceseeeseeeseeceseceseseseeeseseaeeeseens 92 Electrical REG UIP OMG i Es escorts aces sewadedipaauwiaues E O E OOT ON 92 Environmental RSG UILCTCIIES cocsnineiyi niiet esate creepers tucetasiert em E 93 OVERVIEW OF COMPONEN S ssensceinis Sentenuauie tics ansebateaieis AEE 95 Optical COM POLICIES ai sa Seeger acl ania A Seca sac caad EE 96 PIWOTES CENCE Microscope serey iaa nn E E R eslaanadmannadss 97 Optical PIETS iine E be
196. s window 8 To clear the thumbnail image click x Saving Image Data After you acquire an image use these instructions to set up a personal data folder and use the Snapshot utility to acquire a single image ppliedPrecision Chapter 3 Getting Started 29 Setting up a Personal Data Folder You can set up a personal data folder in which to save your images After you create and save this folder DeltaVision automatically uses it as the data folder when you log on to the system To setup a personal data folder 1 At the top of the Resolve3D window click the Settings button to open the Resolve3D Settings window Then click the Files tab Display imaging Files Autofocus Mise QLM Data folder ata Experiment macros folder momewoi Data folder is temporary F Auto increment file names Convert to 2 byte signed integer Done save Settings 2 In the Data folder field enter the directory e g data1 myData in which you want to save your images To create a new folder type the name of the folder after datal e g datal myNewData and select Save Settings Important Store all filesin the datail directory unless you are instructed to do otherwise by your system administrator Q Tip If you are using an existing folder for your data you can drag and drop the folder from the Linux File Manager to the Data folder field 3 To reset the default Data folder when you log out select
197. s with 5X20mm 6 3A 250V UL high break capacity fuses API P N 19 170045 000 Install the fuse holder Plug in the power cord ppliedPrecision Chapter 9 Maintenance 151 Cleaning Most system surfaces are best cleaned with a lint free cloth or lint free swabs and spectroscopy grade isopropyl alcohol or chloroform Avoid contaminating the cleaning solution by never reusing the cleaning cloth or swabs Operators should be trained in the handling of flammable liquids such as alcohol Material Safety Data Sheets MSDS should be maintained for the cleaning solutions as with any hazardous material The exceptions to this cleaning practice are the polychroic mirror and the optical filters These components should be cleaned with low pressure air For example use a bulb designed for cleaning camera lenses which blows air across the surface Do not use high pressure Do not use canned air as this often leaves a fluorescent residue To clean the microscope follow the instructions in the manufacturers manuals that are provided for these components CAUTION Improper cleaning of the polychroic minor and the optical filters will result in damage Moving the System If you need to move your DeltaVision system call Applied Precision for instructions 04 720104 Rev D 1008 152 DeltaVision Core and personalDV User s Manual ppliedPrecision Appendix A The Immersion Oil Kit The immersion oil kit is a collectio
198. sensor value is valid It is red before the first image is acquired After the first scan a red color indicates that the most recently acquired image has an invalid photo sensor value A red indicator can also signify unreasonable saturation or an improperly functioning photo sensor device The Message Pane The Resolve3D Message pane reports the status of various Resolve3D activities Use the scroll bar to view messages that have scrolled off the top of the pane 04 720104 Rev D 1008 194 Delta Vison Core and personalDV Users Manual Resolve3D Short uts You can right click anywhere in the Resolve3D window to open a shortcut menu that allows you to acquire images mark points and create a blank screen for imaging under very light sensitive conditions Figure E 11 Resolve3D Shortc ut Menu Acquire snapshot Mark Point Blank Screen Acquire Collects and displays an image from the microscope The current settings are used to collect the image This image is only displayed in the Data Collection window It is not saved in a file for later use Snapshot Launches a tool to let you collect a single 2 D multi wavelength Snapshot image The current settings are used to collect the image Mark point Marks the current X Y and Z stage coordinates as a point to be visited later Blank Screen Turns the computer screen black for imaging under very light sensitive conditions Clicking anywhere on the screen restores it
199. shed Sectioning Channels Time lapse Points Panels Actions W Z Sectioning Z Scan Options Focus point when scan starts Middle of Sample Optical section spacing 0 20 Number of optical sections 164 Sample thickness 12 80 Get thickness Enable OAI Scan 4 Specify the focus point of the microscope at the start of the experiment and move the stage to that focus point as follows Choose one of the following focus Then click the corresponding button in the Resolve3D points in the Focus point when scan window to move the stage to that focus point starts list Top of the sample Middle of the sample For most applications the Middle of the sample provides the best results CAUTION Be sure to set the focus consistently with this setting before you start the expenment For example if you specify to use the Middle of Sample option be sure to focus midway between the top and bottom of the sample Delta Vision generates the sectioning commands based on this relative starting point If this isnot done conrectly you can lose important data 5 Specify the separation in microns between each optical section in the Optical section spacing field If you are using a 60X objective start by using the default value 2 um If you are using another objective click the Lens button on the Resolve3D window to open the Lens Information dialog box and use the value in the Recommended Z Step size field at the bottom
200. siest to find beads in a very dark room Bead slides from Applied Precision include 1um beads that fluoresce brightly at 617 nm and 0 1um beads Coarsely focus on the slide by positioning the lens near the slide Scan the slide while looking for fluorescent haze from the 1um beads When you focus on the fluorescence haze from the 1um beads you should also find the 0 1um beads Note Although a replacement bead slide isincluded in the Slide kit bead sides have a limited shelf life To purchase bead slides from Applied Precision contact us atthe appropnate number oraddress listed in Chapter 1 Getting Started ppliedPrecision Appendix C Acquiring a PSF 165 Selecting the Conect Immersion Oil Accurate PSF measurements depend on the selection of the correct immersion oil Our experience has shown that the oils recommended by microscope manufacturers are often not ideal for 3 D microscopy We recommend that PSFs are measured with a minimal amount of spherical aberration Inappropriate immersion oils yield asymmetric PSF measurements as a result of spherical aberration In the case of Olympus and Zeiss microscopes an index of refraction equal to 1 518 is ideal for measuring beads that are mounted in glycerol using 11 2 coverslips Nikon microscopes use an index of refraction equal to 1 512 There are many variables that can affect the selection of the correct immersion oil The softWoRx Lens Information program can help you select the
201. step 165 Recommended Z Step Size field 40 Reference books 185 92 Reference channel 74 75 Reference images 80 82 212 Refraction index of See Refractive Index Refractive index 164 65 Nikon 177 Olympus and Zeiss 177 Refresh exposure conditions 47 Refresh exposure conditions button 211 Remove Trail button 226 Repeatable slide holder 25 50 85 114 16 Replacing MIC fuses 161 Replacing the tran light 149 50 Requirements altitude 103 country specific 101 electrical 100 101 environmental 101 4 humidity 103 line 100 power cord 100 temperature 103 Reset button 226 Resolution ratio 165 Resolve3D T setting 201 Acquire Image 196 Autofocus options 222 23 Aux Mag setting 202 Bin setting 202 bulb icon 32 143 calibrate images 201 Calibration menu 198 99 cell tracking options 213 Channels tab 211 12 Clear Stage Thumbnails 197 Clear Stage Trails 197 Command Line Interface 197 Continuous Acquire 196 Design Experiment tab 207 8 Design Run Experiment dialog box 215 17 Emission setting 201 enable fast acquisition 209 Excitation setting 201 Experiment 197 Exposure setting 201 Exposure time 88 Find exposure 201 Help menu 199 image control fields 200 202 Image Size setting 202 Info setting 202 Lens setting 202 menus 195 200 message pane 207 Options menu 198 Panels tab 214 15 Pixel Size setting 202 Point List 197 Points tab 213 14 Scratch File 196 Sectioning tab 209 1
202. sure settings to those last used in the main Resolve3D window Active Wavelength Toggle Buttons Enable the exposure time filters and display settings for specific wavelengths Select the buttons that activate the wavelengths that you want to collect You must select one button for each wavelength If no wavelengths are selected the exposure and filters that are set in the Resolve3D window are used Exp Specifies the exposure time in seconds to be used when acquiring an image for the selected wavelength If left blank the value specified in Resolve3D will be used when the experiment is run EX Filter Specifies the excitation filter to use for this experiment or image When it is changed the currently paired emission filter is automatically selected EM Filter Specifies the emission filter to use This filter may be selected independently of the EX Filter setting ppliedPrecision Appendix E Resolve3D and Keypad Options 199 T Filter Specifies the Neutral Density filter to use The value indicates light transmittance A value of 100 indicates that no light is blocked or no filtering Ex Shutter Specifies the excitation illumination shutter to use for each channel of the image Reference Image Specifies to use an alternate filter or the transmitted light to acquire a reference image that can be combined with other images This option is useful for Differential Interference Contrast DIC analysis It can als
203. tenance 145 2 Use the Eyepiece filter wheel to select the FITC eyepiece filter In the Resolve3D window set the other filters for alignment as follows In this Field Select Excitation TRITC Emission FITC AT 0 1 3 Press EX SHUTTER on the keypad to open the EX shutter 4 Use the Z focus knob to focus on the scratches on the mirror slide 5 Pull out the Field Stop lever all the way to close the Field Stop aperture 6 Use the Olympus 3mm hex key to adjust the two Field Stop Centering Screws until the Field Stop Aperture is centered on the reticle Field Stop lever with Centering Screws The reticle should appear in the center of the octagon as shown below 7 Fully open the Field Stop aperture 04 720104 Rev D 1008 146 DeltaVision Core and personalDV User s Manual Step Two Adjust the Tilt of the Aber Optic Module Body 1 8 Make sure that the Beam Selector on the microscope base is set to direct the light to the eyepiece and verify that the 60X objective is set in its lowest position The Z focus knob should be turned until it stops with the objective fully lowered Remove the oil from the objective See Cleaning on Page 151 and remove the Repeatable Slide Holder Place a piece of paper on the stage with a piece of glass on top to keep it flat Press EX SHUTTER on the keypad to open the EX shutter In Resolve3D set the neutral density filter to 100 light transmittance Pull the Field Stop lever all
204. ters that allow only a single band of light from the excited probe to reach the eyepiece and your eyes These filters are arranged in sets that are associated with specific dyes For example a dye such as DAPI is typically used with a DAPI Excitation filter a DAPI Emission Filter a DAPI Eyepiece Filter and a 100 Neutral Density filter You can choose filter sets manually by rotating the eyepiece filter wheel The filter sets are synchronized so that when you change an eyepiece filter the neutral density filter excitation filter and emission filter automatically change 04 720104 000 Rev D 1008 Delta Vision Core and personalDV Users Manual Using the Keypad and J oystick The keypad and joystick are used to move the stage open shutters acquire images and control other acquisition options Key controls are shown below ACQUIRE IMAGE Key controls on the Keypad Acquire Image Acquires an image and displays it on the monitor Use this key when you are scanning through your sample and using the eyepiece to find a region of interest or when you want to get a quick look at the specimen on the monitor Ex Shutter Opens or closes the Excitation i e Fluorescence shutter You will use this control frequently to open and close the shutter Because the shutter is designed to protect your eyes from exposure to ultraviolet light it automatically closes each time that a filter wheel is moved It must be reopened with the EX
205. that is much greater than the range of interest For example design an experiment that scans double the expected Z range Previous experience is obviously helpful when using this method When using this method it is recommended that you figure out which Z sections contain the desired information before performing the deconvolution Cut out unnecessary Z sections during the deconvolution step of the image processing by specifying the section numbers of the Z start and Z end This will help to ensure that the sample is imaged Previous Experience This method is essentially the same as the overscan method except that one strives for a more exact scan For example if you are scanning an erythrocyte with 7um diameter you could focus on the center of the cell and then scan 4um above and 4um below the cell Of course if you underscan the object then you might miss important details so it helps to be conservative Selecting Alters From the Channels tab of the Design Run Experiment dialog box you can define which filter sets to use in the experiment for imaging DeltaVision acquires images from each filter set channel and creates a single DeltaVision file that contains all of the channels that were collected There can be up to five channels per Experiment FITC left and DAPI center filtered imagesare combined in the final DeltaVision file right ppliedPrecision Chapter 4 Setting Up and Running Expenments 43 Choosi
206. the Data folder is temporary option Saving a Single Mult C hannel Image Sometimes it is desirable to acquire a single multi channel image The Snapshot utility helps you quickly create a 2 D multi wavelength image without having to run a full experiment 04 720104 000 Rev D 1008 30 Delta Vision Core and personalDV Users Manual To acquire a single Mult channel image 1 From the Resolve3D window choose File Snapshot to open the Snapshot dialog box iw Resolve3D Snapshot Image File Mame peeke Deconvolve 2D Images Do OAI Scan Thickness 5 00 Select Channels maje gt m ejoa m s RoTRPE Aaf oo jsf Q Tip Altematively you can open the Snapshot dialog box by right clicking on the Resolve3D window and choosing Snapshot from the shortcut menu 2 In the Image File Name field enter a file name Then select which channels to save Snapshot uses the exposure conditions that are displayed in the Resolve3D window for those channels Note SoftWoRxaddsthe R3D dv extension to the file name If you enter a file name without a directory path the file will be located in the current data folder You can specify to place the file in another directory by including the path in the file name e g tmp myfile 3 Click Do It The Image window opens and displays the new image The image is saved as a DeltaVision file that you can open in softWoRx Tuming DeltaVision Off Use the follow
207. the Resolve3D window Version Displays version numbers for the softWoRx components The Resolve3D Toolbar Use the buttons on the Resolve3D toolbar to acquire images open the Experiment Designer Run dialog box that allows you to set up and run experiments and open the Display Settings dialog box that allows you to control display imaging and file output options Figure E 7 The Resolve3D Toolbar ResolvesD File View Options Calibration Help Experiment settings Acquire Collects and displays an image from the microscope with the current settings This image is only displayed in the Data Collection window It is not saved in a file ppliedPrecision Appendix E Resolve3D and Keypad Options 187 Expenment Opens the Design Run Experiment dialog box that you can use to select a previously created experiment open the Experiment Designer window to design a new experiment or open the Experiment Macro Editor to create or edit an experiment macro Settings Opens the Settings dialog box that allows you to control display imaging and file output options Image Control FRelds Use the Resolve3D Image Control fields to a Select the Excitation Emission and Neutral Density filters a Select exposure time m Determine whether to calibrate the image a Select the desired shutter m Select the image size Select the lens and get lens information a Select whether to use auxiliary magnification m
208. ting expenment and you need to remove the slide before you are finished with the expenment see Page 104 ppliedPrecision Chapter 3 Getting Started 25 12 Rotate the Coarse Focus knob toward you to move the objective up until the objective is just in contact with the oil From this point on use only the fine focus knob to raise and lower the objective 13 Rotate the eyepiece filter wheel below the oculars on the scope to select the filter for the probe that you used to stain your sample If your sample has more than one probe select the one with the brightest fluorescence typically DAPI The selected filter is displayed on the Filter Monitor window The filter names are displayed in the Filter Monitor dialog box on the right side of the workstation screen As you rotate the filter wheel the filter name next to EP eyepiece changes and the EM and EX emission and excitation filters change automatically to match The displayed colors match the wavelength of the filters If a QLM module is installed not available on personalDV the Filter Monitor also displays the wavelengths of the lasers that are available on your system RA Note You must move the filter wheel to initialize the Filter Monitor Standard Filter Set Appropnate Filter Probes Name Exc itation Emission DAPI Hoechst DAPI UV 350nm Blue 455nm Coumanin Fluorescein GFP FITC Blue Green Green 525nm CY2 Al488 490nm Rhodamine Texas TRITC
209. tion of the Multiplexed Wavelength experiment design setup You should now continue with the standard steps for the remainder of the experiment design such as Sectioning Timelapse and so on Note Assoon asyou activate the Do Multiplexed Channel Imaging checkbox all conventional imaging settings are disabled This is also true of all multiplexed settings when you reactivate conventional imaging 04 720104 Rev D 1008 63 64 Delta Vison Core and personalDV Users Manual ppliedPrecision Acquinng Data from Live Specimens This chapter shows how to use the following features to image live cells with DeltaVision Set up experiment macros to automatically focus before acquiring each image This is useful for cells that move during the experiment Use Cell Tracking to follow cells as they move laterally and move the stage to keep them in the field of view Use Optical Axis Integration also referred to as Z Sweep Acquisition to acquire a 2D projection This method collects and integrates one continuous image through an extended Z movement Z Sweep Acquisition is much faster than the traditional technique of collecting an individual image at each focal plane It also reduces the risk of specimen damage and has less total camera read noise Acquire reference images that can be used for Differential Interference Contrast DIC and other types of analysis 66 DeltaVision Core and personalDV User s Manual Using Autof
210. tive measurement and analysis Multiple Alters You can acquire images through several filters and combine them into one image file Panel Collection Panel collection acquires a series of images with adjacent fields of view You can stitch these images together to create images that are much larger than a single field of view This is especially useful when you want to collect data at a high magnification over a large area ppliedPrecision Chapter 1 Introduction Time Lapse You can run macros to acquire time lapse images and use the data to create time lapse movies This is especially useful for studies of live samples or for experiments that use lasers Point Visiting Point Visiting allows you to acquire data from several areas of interest during a single experiment You can select which points to monitor on your sample and save them in a list that contains the exact stage coordinates of each point When you run the experiment DeltaVision reads the coordinates for the points in the list moves the stage to each point and captures an image This process is repeated at specified time lapse intervals For live specimens this significantly improves the lab efficiency of experiments by allowing you to monitor multiple points of interest in a single session Autofoc us You can use Autofocus to automatically focus when you are viewing a sample or when you are running an experiment Kohler and Cnitical Illumination You ca
211. togram Range histogram width for each image that is analyzed Note This option changes only the display of the histogram It does not change the image data Editing Expenment Macros The Experiment Macro Editor is used to create or edit Resolve3D experiment macros command scripts that control the DeltaVision microscope Most experiment macros can be generated using the Design Run Experiment window but you may need to create custom macros for certain types of experiments The best way to get started is to modify an existing macro with the Experiment Macro Editor One approach is to use the Design Run Experiment window to generate a macro and then edit it Another approach is to use a reference macro To open the Experiment Macro Editor 1 On the Resolve3D window click Experiment 2 In the Design Run Experiment dialog choose File Edit to open the Experiment Macro Editor ppliedPrecision Chapter 6 Data Collection Techniques 89 Status Area e gt v Resolve3D Experiment Macro Editor File Edit Search Help z ACTIVATELASER search Replace ACTIVE A ACTSHUT Loaded file home worx Resolve3D exp AUTOFOCUS BLANKSCREEN Created by DeltaVision Experiment Designer version 3 7 0 Release 6 A CAHTEST Commands list Experiment Hacro Hame Resolve3D CCD CCDSETUP ACTSHUT 17 CLF Open image file CLF id CLF_REF CLF_SETHEADER Starting Scan From Current Focus Middle of sample CMASS Macro Text Area e gt
212. ty Requirements and Components 107 e A micrometer nut wrench e An Applied Precision Hotline sticker Software DeltaVision Core includes softWoRx for Linux 1 copy softWoRx Suite for Windows 1 copy DMS Server 1 copy 3 seat and softWoRx DMS 2 copies personalDV includes softWoRx for Linux 1 copy and softWoRx DMS 1 copy Note Either system can be ordered with a number of different software configurations Talk with your Applied Precision representative to determine the best configuration for your applications softVVWoRx softWokx is the Linux software application that runs the acquisition workstation The software allows you to perform the following tasks Acquire image data Set up and run experiments Deconvolve data Measure point spread functions Calculate optical transform functions a Process 2 D and 3 D images Perform quantitative analysis a Archive data Configure task chains Manage user accounts softVWVoRx Suite for Windows softWoRx Suite is a Windows based ensemble of software developed by Applied Precision in collaboration with Bitplane AG It provides sophisticated multi dimensional data visualization analysis image restoration image correction and image viewing management all within an easy to use streamlined browser interface 04 720104 Rev D 1008 108 Delta Vision Core and personalDV Users Manual softVWVoRx DMS softWoRx DMS Data Management Solution provi
213. uad eh ee ee 6 position 2 EGFP mCherry Illumination Optics f Dichroic 3 CFRIYFP Turret Dual band EM Filter RSE ie Detector Conceptual view of Multiplexed Wavelength functionality Before you use the Multiplexed Wavelength option you must first have it installed and configured correctly Your Applied Precision representative will assist you in setting up this option and help you ensure that all hardware and software to support Multiplexed Wavelength functionality is installed properly After the option has been installed and configured the menus tools and other infrastructure necessary to use the feature will be available on your workstation Setting Up the Multiplexed Wavelength Option Before you begin designing your Multiplexed Wavelength experiment you ll need to perform the steps described in the following procedures to activate a 04 720104 Rev D 1008 56 DeltaVision Core and personalDV Users Manual Multiplexed Wavelength filter set and prepare the DeltaVision system for Multiplexed Wavelength operation To activate the Multiplexed Wavelength filter set 1 To change the active filter set to a filter set that is Multiplex capable select Settings in the Resolve3D main menu ST File View Options Calibration Help Acquire Experiment settings Excitation 430 10 Settings Emission ere 470 30 aro EX Shutter EX ag igi Exposure 1 000 Find Calibrate Image size 512x
214. uite Windows workstation includes the softWoRx Browser softWoRx Explorer Modeling and 3D Visualization powered by Bitplane and Deconvolution tools Multiplexed Wavelength Module The optional Multiplexed Wavelength module for the DeltaVision systems allows you to perform nearly simultaneous two channel imaging without the drawbacks associated with true simultaneous two channel imaging This option uses two shuttered illumination sources and a dual band emission filter to eliminate filter wheel movement between channels and therefore greatly reduce the time required for the DeltaVision system to acquire a set of two channel images The combined light path ensures no registration artifacts are introduced and independent excitation of probes helps to ensure minimal crosstalk Software softWoRx Explorer softWoRx Explorer is a cross platform image viewer that is available for many commonly used operating systems softWoRx Explorer allows you to view and explore DeltaVision images and images from other sources that contain spatial temporal and spectral ranges In addition to displaying data in the X and Y plane you can scroll through Z sections and time lapse data Individual spectra i e channels or fluorescent wavelengths can be hidden or displayed in a variety of colors ppliedPrecision Chapter 7 Facility Requirements and Components 113 softWoRx Suite for Windows Option for personalDV softWoRx Suite is a Windows based
215. ulin containing rings in the centrosome Nature 378 6557 638 640 Moritz M Braunfeld M B Fung J C Sedat J W Alberts B M Agard D A 1995 Three dimensional structural characterization of centrosomes from early Drosophila embryos Journal of Cell Biology 130 1149 1159 N thke I S Hinck L Swedlow J R Papkoff J Nelson W J 1994 Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells Journal of Cell Biology 125 1341 1352 N thke I S Adams C L Polakis P Sellin J H Nelson W J 1996 The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration Journal of Cell Biology 134 165 179 cover article Neugebauer K M Roth M B 1997 Distribution of pre mRNA splicing factors at sites of RNA polymerase II transcription Genes and Development 11 1148 1159 Paddy M R 1998 Determining Nuclear Structure Using the Fluorescence Light Microscope Methods in Cell Biology 53 49 77 Paddy M R Agard D A Sedat J W 1992 An extended view of nuclear lamin structure function and dynamics Seminars in Cell Biology 3 255 266 04 720104 Rev D 1008 178 Delta Vision Core and personalDV User s Manual Paddy M R Belmont A S Saumweber H Agard D A Sedat J W 1990 Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery
216. ur convenience the OTF filename is created by appending _otf to the PSF filename 04 720104 Rev D 1008 168 DeltaVision Coreand personalDV User s Manual X Range Defines the start and end pixel numbers in X Y Range Defines the start and end pixel numbers in Y Z Range Defines the start and end pixel numbers in Z TRange This field is not used for PSF to OTF conversion Wavelengths Determined by PSF wavelength Lens ID Specifies the lens identification number e g 12004 Sub Image Center Specifies the central XYZ coordinates of the point spread Sub Image Size Specifies the XYZ image dimensions about the central coordinates The standard softWoRx point spread measurement is 256x256x128 Additional Parameters Border Rolloff voxels Specifies the number of voxels to roll off at the edge of the image This reduces edge effects resulting from the Fourier Transform used in the PSF to OTF conversion The procedure for converting a PSF to an OTF is very simple After the PSF file has been identified in PSF to OTF Conversion softWoRx assigns default settings to the rest of the options in the dialog box In almost every instance these settings will be appropriate to use for the conversion To converta PSF to an OTF 1 Click Conversions on the main menu bar of softWokRx 2 Click Convert PSF to OTF in the Conversions menu PSF to OTF Conversions will appear 3 Use one of the following options t
217. use for that channel On the EX Filter list for the same line as the check box you selected select the excitation filter The EM Filter emission filter T Filter neutral density filter and eyepiece filters for the selected filter set are automatically specified DeltaVision specifies the most recent filters that were selected for that filter set in the Resolve3D window If no filters have been selected DeltaVision specifies default filters for that filter set w Design Run Experiment modified File co g oe Design Experiment Design PK Experiment Run Experiment Experiment name Resolves Estimated file size 64 02 Mb 22 29 Gb Available W Use Fast Acquisition Fast Acquisition Options Lamps Off when finished Sectioning Channels Time lapse Points Fanels Actions Conventional i Muitipiexed Exp Ex Filter EM Filter YT EX Shutter Refresh exposure conditions ppliedPrecision Chapter 4 Setting Up and Running Expenments 45 4 To change the exposure time enter a value in the Exp field Values in the Exp field are in seconds For example 0 100 is 1 10 of one second If you do not enter a value in this field the most recently selected exposure value is automatically set for each filter 5 To select a different emission filter or to attenuate the intensity choose a filter from the EM Filter or the T lists 6 You can choose which light source to use for each channel by selecting a
218. ust select the proper objective lens in order to get the appropriate Autofocus settings ppliedPrecision Appendix E Resolve3D and Keypad Options 209 Channel for Autofoc us This setting indicates the wavelength to use for Autofocus Contrast calculation method This setting determines the polarity of the contrast calculation There are three choices for image contrast calculation methods e Auto The instrument controller usually can determine which contrast calculation method to use but not always e Brightfield for dark objects on a light background e Fluorescence for light objects on a dark background Autofocus Z test step um This option sets the step size used for Autofocus within the maximum Z range Maximum Ztest range um The setting indicates the maximum range that the Autofocus will search Post autofoc us Z offset um This setting can provide a constant offset after the Autofocus position determines the best plane of focus Often times Autofocus will find a plane that is consistently different from the desired plane Settings Dialog Box Misc Options Use the options on the Resolve3D Misc tab to select or clear Lost Motion Compensation or to select a filter wheel configuration if your system is configured to use alternate filter wheels Figure E 22 The Settings Dialog Box Misc Options eomm snas ee Display imaging Files Autofocus Mise stage Motion F Allow Lost Motion Compensation LM
219. utter automatically closesand must be reopened with the EX SHUTTER button Focus to find the focal plane Turn the Fine Focus knob toward you to slowly raise the objective until you see a cloud of emission color Then use the joystick to move the stage If the cloud moves you have found the sample If not you may be seeing colors from lens effects Continue to focus until the image is sharp and clear Use the joystick to move the stage around Change the speed of the movement with the SLOW MEDIUM and FASTkeys on the Keypad When you find a sample place it in the middle of the field of view ppliedPrecision Chapter 3 Getting Started On the Stage View note the stage trails that show where you have moved the stage in XY O Fale HW la a ol dx o oo a a dy 10 00 aga 5 fpo y v a Acquiring an Image To acquire a DeltaVision image you ll need to direct light to the camera and work with the images that are displayed in the Data Collection window until you are satisfied You can then save the image as a DeltaVision file or create and run experiments as described in Creating and Running an Experiment Macro on Page 33 To acquire an image 1 2 On the keypad press EX Shutter to close the shutter Switch the beam selector to camera In the Resolve3D window Exposure field enter an exposure time in seconds A good starting exposure time is 0 1 second see Finding Exposure Time on Page 82 for detai
220. v D 1008 DeltaVision Core and personalDV User s Manual The following table summarizes the capabilities of the DeltaVision Core system Capability Desc nption Digital Microscopy Fluorescence Imaging Also capable of Bnghtfield Phase Contrast and DIC imaging Automated Optical Optical sectioning filter changes and shutters Sectioning Time lapse are coordinated by the controller Point visiting Quantitative Processing Image processing and 3 D reconstructions of multi dimensional data files Image Display and Analysis 3 D reconstructions can be visualized rotated and enhanced DeltaVision supports a wide array of imaging applications including Cytoskeletal Studies RNAi experiments Live Cell Imaging Cell Cycling Studies Protein Translocation and Protein Pathway Analysis Standard Data Collection Options DeltaVision supports the following types of data acquisition 3D Imaging To acquire 3D data you can set up DeltaVision to acquire a series of images along the Z axis The softWoRx workstation provides a sleek interface that allows you to control the optical sectioning through a specimen Behind the scenes macro language provides automated computer control of sample position optical filters and shutters After image data acquisition a series of image processing algorithms improve image resolution Three dimensional information can be reconstructed and then visualized in a variety of ways that allow quantita
221. vera 4 hour penod the High speed Camera automatically shuts down CAUTION Do not disconnect cablesto the High soeed Camera when the power ison Be sure to leave a 1 in 2 5 cm minimum space around the cooling fan Use only the PCI cards cable and powersupplies that are designated for this system ppliedPrecision Chapter 7 Facility Requirements and Components 99 High speed Camera Power Supply The High speed Camera power supply provides electric power to the camera It is housed on the bottom shelf of the cabinet ES Camera The CoolSNAP ES CCD Camera is basically the same as the CoolSNAP HQ Camera except the ES is not as deeply cooled This makes it more affordable but allows for slightly higher noise However the noise difference is virtually unnoticeable unless you are using long exposure times 8s Light Sources DeltaVision provides two light sources a xenon arc lamp for the main light source and a white LED for transmitted light Xenon Lamp Illumination for the microscope is delivered from the xenon arc lamp to the specimen through a fiber optic cable The light passes through the fiber optic module FOM where approximately 1 of the light is diverted to the Photo sensor The remaining 99 of the light is delivered to the microscope For instructions on how to replace the xenon bulb see Replacing the Xenon Bulb on Page 134 WARNING Do not disconnect the arc lamp powercable when the po
222. werison 04 720104 Rev D 1008 100 DeltaVision Core and personalDV User s Manual WARNING The xenon arc lamp presents potentially harmful risks to the AN user including the possibility of UV exposure to skin and eyes Before operating the microscope consult Chapter 2 Safety for important information regarding arc lamp operation Note The illumination path alignment is critical to acquiring the highest possible image resolution See Page Path on Page 144 fora complete description of the alignment procedure Photo sensor The Photo sensor measures illumination intensity by sampling a small percentage of the light from the arc lamp The photo sensor signal is recorded by the controller and then used to correct for variations in the xenon lamp intensity during an experiment The Corrections tool in softWoRx normalizes each image based on its photo sensor value These corrections are automatically applied during deconvolution This enables quantifiable intensity comparisons between images even if the brightness of the excitation light varies between the images LED Transmitted Light An LED transmitted light source is also provided OLYMPUS LED Transmitted Light Source If at any point you need to replace the transmitted light see the instructions in Replacing the Transmitted Light on Page 138 Note Either light source the xenon lamp orthe LED transmitted light can be used with the eyepiecesorthe cameras
223. xperiment window and click the Design Experiment tab Then click the Point Visiting tab and check the Visit Point List option The Point Visiting dialog box is linked to the point list that is open 3 Enter the points that you would like to visit in the Visit Point List field For example entering 1 3 specifies to visit points 1 2 and 3 Entering 1 3 5 7 9 specifies to visit points 1 9 but not 4 or 8 Monitoring Point Visiting Expenments You can set up DeltaVision to display a separate Data Collection window for each point in your point visiting experiment 04 720104 Rev D 1008 52 Delta Vison Core and personalDV Users Manual With the Point Track Display Option enabled each point ina point visiting expenment is displayed in a separate window To set the Point Track display option 1 On the Resolve3D window click the Settings button to open the Resolve3D Settings dialog box 2 Click the Display tab Window Em Wave aa Calculate statistics W Calculate histogram Wo Auto histogram range Display images Deconvolve preview images Auto intensity scale Wo Acquire after point visit 3 Inthe Image Display mode list select Point Track ppliedPrecision Chapter 4 Setting Up and Running Expenments 53 4 Click the Save Settings button then click Done to close the Resolve3D Settings dialog box Collecting Panel Images over Large Areas Panel collection macros are useful when you want to sca

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