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softWoRx User`s Manual Revision C

1. 3 Choose how to scale the profile in the Intensity Scaling list e Constant Min Max sets the scale range to the minimum and maximum intensity values in the image e Autoscale sets the range to the minimum and maximum intensity values in the profile 4 Tosave a group of profiles in a spreadsheet compatible text file click Save after each profile that you want to save is displayed When you are finished collecting profiles click Write to File enter the file name and other options in the Save as SYLK spreadsheet dialog box and click OK Calculating Statistics Calculating Statistics for Selected Areas Use Data Inspector to calculate data for selected areas To open Data Inspector gt Select Tools Data Inspector on the Image window to open the Data Inspector window ppliedPrecision Chapter 12 Examining Intensity Data iw Data Inspector Current Window E 617 v 528 v 457 Select Box Columns Rows ie fis _ Constrain To Circle Show 30 Graph Show Histogram L ond _ Ratio Statistics MIM M 1092 1250 1181 515 Total 426527 SD 35 14202 1171 1145 1163 1172 1210 1201 1198 1174 ARA 1160 1174 1149 1165 1167 1197 1188 1170 1149 1163 1161 1133 1173 1176 1168 1192 1176 1143 1161 1156 1185 1188 1169 1189 1154 1185 195 1198 1196 1186 1179 1175 1191 1173 1160 1167 1181 1164 1195 1167 1177 11
2. cceccesessecssecseseeseeeeseeeseeesees 114 Viewing Volumetric or Time Lapse Movies At least one Image window must be open in order to apply the movie function There are two ways to access the movie function from the softWoRx main toolbar and from the Image window Accessing the movie function from the softWoRx toolbar applies the movie function to all open windows Opening the movie function from an Image window applies it only to that Image window 04 720103 000 Rev C 1008 Chapter 8 Viewing Movies 115 To view one image data setas a movie 1 2 Open the file that you want to view as a movie in an Image window Click View Click Movie Unselect the Paused check box To view two or more image data sets as movies 1 2 Open the files that you want to view as movies in Image windows Click View on the softWoRx main toolbar Click Movie Unselect the Paused check box To view time lapse Zsenes data 1 2 Open the file that you want to view as a movie in an Image window Click View Click Movie Unselect the Paused check box Select the Time Lapse check box Adjust the Manual Z Control for Time Data slider Tracking Partcle Movement with Trails Movies You can trace the movement of particles in time lapse data with the Trails Movie tool To tace particle movement di Open an image that contains time lapse data in the Image window 04 720103 000 Rev C 1008 116 softWoRx Imaging Wor
3. 7 A A AA AAAA File Edit View Search Tools Documents Help o 8 Save Print 3 New v 9 Undo Nuclear_Porel_r3d 2D txt Nuclear_Pore_r3d 2D txt X wave 1 time 1 sec poly pixs 1 1 2 2 3 3 4 5 5 6 6 7 7 8 8 9 9 1 NeN ENEN EN EN EN EPENEN wave 2 time 1 sec poly pixs ui ON OVUBUNE 1 ee ee 263 6 261 215 217 185 221 184 wave 3 time 1 sec poly pixs integr_int 18150 33576 15996 25218 15340 22233 10615 14149 21250 24669 17030 14156 17069 22388 16953 14748 24188 27705 integr_int 173347 4014 171848 141459 142986 122070 145557 121316 139238 133238 integr_int area 0 coooocoocooocooococoocococeococco area Li ee ee area S p B Y Copy Paste Cut Nuclear_Porel_r3d 2D txt x centroid 2 1 PA P 21 21 21 centroid 9 19 9 19 9 9 19 9 19 9 19 9 9 19 9 19 center_mass 9 19 9 19 9 19 9 19 9 19 9 19 9 19 9 19 9 19 14 20 14 14 ES L3 14 14 14 14 center_mass Ln 32 Col 64 To measure the volume ofan object 1 Select Measurements in the 3D Object Builder menu 2 Select Table of 3D Measurements The Save Measurements File dialog box is displayed 3 Enter the desired name for the 3 D measurement file 4 Click OK 5 Open the folder containing the saved measurement file 6
4. Add note to log 7th sample from set 11 Do It Change next time lapse ooo Doi W Show images during acquisition W Show PK progress graph W Launch viewer after experiment _ 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 4 Click the DMS Setup button The DMS Destination Setup window is displayed pplied Precision Chapter 6 DMS Integration 81 d DMS Destination Setup W Upload files to Database after collection DMS Database Connection lt lt lt Disconnect from the Database Destination Parameters Image Name 1_01_R3D dv Dataset KP_Dataset New Category Group KP TestGroup v New Initial Annotation 7th sample group 11 Optional Done 5 Be sure that the Upload files to Database after collection checkbox at the top of this screen is activated 6 Enter the project and dataset where you want the acquired image data to upload 7 Optionally you can also enter the category group and the category for the uploaded data and include any annotation necessary 8 When you are satisfied with the information entered in the DMS Destination Setup window click Done 9 Run the experiment macro as normal The resulting image acquisition will be added to the DMS database as specified Downloading Riles from D
5. See Calibrating Image Data on Page 20 Align Image corrects single wavelength images that have motion artifacts problems with Z sectioning or problems with time series It allows you to align adjacent images by applying an XY shift with an optional rotation See Aligning Adjacent Images on Page 21 Chromatic Abenatioon Conector allows you to adjust channels relative to each other to correct for shifts in color that result from oil matching objective anomalies and other environmental conditions that use X Z and Y Z image profiles See Correcting Chromatic Aberration on Page 23 Corecting ZSection Image Data Correct Image options are used to correct systematic errors that occur during data collection The three basic systematic errors are caused by photo bleaching inconsistent illumination intensity and CCD defects By default softWoRx automatically applies these options to images during the deconvolution process These options are specified in the More Deconvolution Options dialog box If you are analyzing unprocessed images images that are not deconvolved use the Correct options tool to apply these options to the images before you process them Applying correction options is especially important when you are performing quantitative analysis on images that contain multiple Z sections or Time series 1 Open the Correct Image dialog box by choosing Process Correct from the softWoRx main menu pplied Precision Ch
6. To stitch an image 1 Click View Stitch in the softWoRx main menu The Stitch Image dialog box is displayed m Output Image Reduction Factor ig Dimensions R Pixel Size _ SSS Area More Options Click Input and browse to the file that you want to stitch softWoRx reads the wavelengths from the file header and automatically selects the appropriate wavelengths Change these settings only if your application does not require a stitched file for a particular wavelength in which case you can de select the appropriate wavelengths Click Output and browse to the same file previously selected The file will appear in the Output field with _ STC added to the file name With this pplied Precision Chapter 3 Stitching 27 naming convention the input source file is always associated with the output stitch file Input datat dv_samples Nuclear_Pore_DaD dy Output datat dv_samples Nuclear_Pore_DsD_STC cdf einni Mornar Reset Details Wavelengths 617 W 528 W 457 d Output Image Reduction Factor 00000 Dimensions B20 x312 x48 9 14 Mb wavelength Pixel Size 0 07430 um x 0 07430 um Area 551 um 0 001 mm More Options Done Do It If desired click More Options to display additional stitch options For most applications the default settings should work quite well When finished with this dialog box click Done to return to the main Stitch Image dialog box When
7. a z Ja i ja Done Do It Help 2 Select the file that you want to convert using the TIFF Input File Selection options 3 Type the name of the directory in which to place the TIFF file in the Output Directory field 4 Type a filename for the converted file into the Output File field 5 Enter the X Y and Z spacing of the pixels in the data set in microns into the Pixel Spacing X Y Z fields A Note The deconvolution program and other softWoRx software rely upon the presence of accurate wavelength and pixel spacing Not all TIFF files contain pplied Precision Chapter 4 Importing Data 33 accurate pixel size and wavelength information so it may be necessary to manually enter some of the fields in TIFF conversion 6 Enter the wavelength of the data to be converted in nanometers into the Wavelength field 7 Use the Stack of Images list and buttons to position the images in the desired order 8 Click Do It Converting ISee Files The See conversion tool is used to convert Inovision ISee images to the DeltaVision tormat The deconvolution program and other softWoRx software rely upon the presence of accurate wavelength and pixel spacing Not all See files contain accurate pixel size and wavelength information It may be necessary to manually enter values in the See Conversion fields A Note The image wavelength fora DeltaVision file indicates the wavelength of the light imaged by the came
8. 3D Object Builder Done Do It Meee borri pzizsys Ria ret SN 4 Net ei ee EN ppliedPrecision Chapter 14 Volume Modeling 189 The main options in the 2D Polygon Finder dialog box are summarized below For information regarding the other options and for additional details on these refer to the online Help Window Sets the Image window number of the image data to be processed Select Region Defines the region of interest Reset Erases the selected region Minimum Perimeter Sets a value for the minimum number of points that can define a polygon Polygons with less than this number of points will be discarded Maximum Penmeter Sets a value for the maximum number of points that can define a polygon Polygons with more than this number of points will be discarded Polygon Smoothing Sets the desired number of pixels between points on the polygon The 2D Polygon Finder will use greater detail when able Exclude Edge Objects Specifies not to use those polygons that touch the edge of the image Outer Objects Only When enabled specifies that 2D Object Finder will only find the outermost continuous polygons When disabled specifies that polygons that are fully contained within others will be created if detected Wave Threshold Defines the minimum intensity to be included in the polygon Default Value Minimum Value for the Wavelength 20 of the Dynamic Range Launch Opens either the Polygo
9. 7 Enter the angle to rotate the data on the XY plane in the Angle Between Axes field 8 If you want to maintain the same relative pixel size in the rotated image activate the Keep Cell Dimensions checkbox Input Se Output OS select Region Reset Details Wavelengths 528 W 617 Je J J Z Rotation a30 ahit In ay po 0 0 Angle Between Axes ago Magnification In a nooo 1 000 A Reduction Factor 1 000 1 000 Output Av Size 256256 W Keep Cell Dimensions Done Dio It Enter the angle to rotate the axes 9 Click Do It 04 720103 000 Rev C 1008 110 softWoRx Imaging Workstation User s Manual fle MEL f 7 0 IM Il 9 Zoom 2 01 erea Image after rotation To reorient images Image before reonentation ppliedPrecision Chapter 8 Viewing Image Data 111 1 Open the Rotate3D dialog box by choosing View Rotate3D on the main softWoRx menu 7 ee Input E QGutput select Region Reset Details Wavelengths 617 528 W 457 J Angle foo 0 0 0 0 Options Done Do It 2 Enter the Image window number in the Input field 3 Specify the X Y and Z rotation angles to apply to the data in the Angle field When viewing an image in a window a positive X rotation rotates the top of the image towards you and the bottom away from you A positive Y rotation rotates the right side of the image towards you and the left away from you
10. 8 bit Grey scale generates compressed data with each channel separate e 16 bit Grey scale generates uncompressed data Use this option for quantitation e 24 bit RGB generates compressed data a 3 channel color TIFF with 8 bits per channel 8 Click Do It to export the data to a TIFF file 04 720103 000 Rev C 1008 154 softWoRx Imaging Workstation User s Manual Exporting to PhotoShop Alles You can save the visible contents of an Image window as an Adobe PhotoShop 24 bit RGB color image file You can choose to m Save the image with the overlay graphics scale markers etc merged into a single image a Save the image without the overlay graphics Save only the overlay graphics Exporting to PhotoShop saves only the pixel image data the original intensity data is not preserved To export to a PhotoShop file 1 Open the folder containing the image file that you want to export Then double click the image file name to automatically open that image in a softWoRx Image window 2 From the Image window File menu choose Save As Photoshop to open the Save Photoshop dialog box Window f Output data1 dv_samples tripolar01_d3d psd We Save Image W Save Overlay r Done Do It Help 3 Enter the directory and file name in the Output directory The default is the directory in which the input file is located 4 Select the one or both of the following options for saving the file e To
11. 91 viewing 90 92 viewing as movie 107 12 Zooming 93 Z stack image 10
12. A positive Z rotation indicates counter clockwise rotation 4 To modify the output size that softWoRx creates from the rotation angle and the dimensions of the input image select Options from the Rotate 3D dialog box The Rotate Options dialog box is displayed Enter the new dimensions in the Output Dimensions field Rotate Options Output Dimensions p20 312 48 Pixel Size 00745 0 0743 0 1500 Translation foo 0 0 0 0 Rotation Center 160 0 156 0 24 0 5 Specify an X Y Z vector for translating the image pixels in the Translation field The size of each pixel in real world coordinates usually microns is displayed in the Pixel Size field 6 Specify a center point about which the rotation occurs in the Rotation Center field The Rotation Center is by default the center of the image You can specify a different center point in pixel coordinates 7 Click Do It to generate the image 04 720103 000 Rev C 1008 112 softWoRx Imaging Workstation Users Manual IR le 59 i KEE M Ii D l Image after reonentation Viewing Cross Sections You can view cross sections of the data by creating orthogonal projections The Orthogonal Viewer allows you to interactively view YZ and XZ plane cross sections To view YZ or XZ cross sections 1 Open the image in the desired Image window 2 Open the Orthogonal Viewer by choosing Tools Orthogonal Viewer from the Image window The orthogonal pro
13. ACM To rendera volume Open an image file Click to open the intensity scaling dialog box and subtract background from the image by setting the left control point at the middle of the first intensity peak 2 Choose View Volume Viewer in the softWoRx main menu to open the Volume Viewer dialog box Ba Volume Viewer AE Input aM Output E Select Region Reset Details Subset Z i Wavelengths W 457 W 528 W 617 m Wavelength Scaling Wave 1 Scale 4168 3 16051 9 1 00 Wave 2 Scale 262 0 26606 0 1 00 Wave 3 Scale 55 0 42173 0 1 00 m Viewing Parameters Method Max Intensity olPaeck nuga Quality Best _ Original Size Z Resolution Best Progressive Threshold fzo m Movie Options Start Angles XYZ oo o0 00 Number of Projections ps Rotation 360 Around Status ee i i Done Do It hemut Interactive Use Volume Viewerto render volumes 3 Drag the Image window number into the Input field 04 720103 000 Rev C 1008 128 softWo Rx Imaging Workstation User s Manual Click Select Region and drag the mouse across the region of interest to select a rectangular region Scroll through the Z sections to insure the region of interest includes all the image data that you want to include in the data set 62 3 window DMS 2ByteUnsignedint R3D_D3Ddv0 C i CO BEE File View Options Tools 2 H
14. 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 portion 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 720103 000 Rev C 1008 71 72 softWo Rx Imaging Workstation User s Manual pplied Precision 6 DMS Integration This chapter describes how to use the DMS Data Management System with softWoRx to manage image files for your particular application What is DMS The DMS Data Management Solution product provides a functional infrastructure for the storage of biological images and their associated metadata The DMS Server contains a data management system that centralizes all image data management Once configured the DMS Server becomes a repository for all data generated by a laboratory s image acquisition system s All visualization and analysis processes are performed on client workstations co
15. New kpalmby Initial Annotation Optional we Done Do It angel Upsioad 5 From this menu select the Project and Dataset to which you want to save the data on the DMS database You can optionally select a Category Group and Category for the saved image data and add initial annotation to the file for future reference When you are satisfied with the image data to be uploaded click Do It When the upload is complete Finished is displayed on the status line as shown 04 720103 000 Rev C 1008 152 softWoRx Imaging Workstation User s Manual R Save To Data Management System Eki File data1 dv_samples Nuclear_Pore_d3d dv DMS Database Connection lt lt lt Disconnect from the Database z Destination Parameters piee Maer ise Dataset KP_Dataset New Category Group KP_TestGroup New Initial Annotation Sample 1 Optional Finished Done Do It Cancel Upload 6 Click Done to exit the Save To Data Management System menu Exporting DeltaVision Riles You can export DeltaVision files to TIFF PhotoShop or MPEG Movie files Exporting to PhotoShop and Movie files saves only the pixel image data the intensity data is not preserved Exporting to TIFF Files Exporting to TIFF files allows you to share data sets in a nonproprietary format To exporta DeltaVision file to a TIFF file
16. Wavelengths W 528 W 617 W 457 _I Create Mode Data Wave Threshold A Background foo Wave 2 Threshold la 0g mor Background lo oo Wave 3 Threshold o oo pa Background o oo 2 Enter an image file name or window number in the Input field 3 Ifyou want to include only selected data use the Select Region button and drag the mouse across the portion of the image you want to include Alternatively you can click Details to open the Region Details dialog box Then specify the ranges of data that you want to include in the Output Options fields 4 Select which wavelengths to filter 5 Select one of the following options in the Create Mode list e Choose Data to create an output image of all intensities equal to or above the threshold the cutoff intensity value If a pixel has an intensity value below the threshold the pixel in the output image is set to this value usually 0 e Choose Mask to put zeros in all pixels where the intensity is below the threshold and ones for all pixels equal to or above the threshold 6 Specify a Threshold and Background value for each wavelength 7 Click Do It ppliedPrecision Chapter 11 Saving Exporting and Printing 147 04 720103 000 Rev C 1008 11 Saving Exporting and Pnnting softWoRx provides several options for saving exporting and printing image data You can save complete DeltaVision files or save only selected image data e g selected wavelength
17. summary of process 182 Modes blended color 101 2 color 101 color and grayscale 99 102 switching between 100 Motion artifacts 18 21 Movie files 153 55 Movie players 154 Movies time lapse 113 14 trails 114 18 viewing 107 12 volumetric 113 14 Multi layered images 27 29 Multiple Segment method 177 Multiplexed wavelength experiment design 68 71 04 720103 000 Rev C 1008 227 filter set activation 63 67 option 62 71 overview 63 Multi Point FRAP 205 Navigating Z sections 90 92 Nearest neighbor 16 deconvolution 10 Nonlinear intensity scales 95 99 Nonlinear local contrast enhancement See Local contrast enhancement Normalize time points 18 19 20 Normalized residual 217 Opening files 88 89 Optical File Transfer function 11 Optional components 5 Orthogonal viewer 111 12 OTF 11 invalid 219 Panel collection feature 25 Panels 25 29 Particle movement 114 18 measuring velocity of 177 79 Pearson Coefficient of Correction 194 Pebble grain texture 218 Photo activation 203 Photo bleaching 203 correcting 17 for FRAP 204 Photokinetics 3 203 4 PhotoShop files 153 Point values 162 Polygon Editor 182 85 maximum points 186 minimum points 186 Polygon smoothing 186 Polygons See 2D Polygon Finder 3D Object Builder Modeling Poor Z resolution 218 Presenting data 85 Primary light path 63 Printing 159 screens 156 57 Process chains 54 57 Processing data 1 3 7 Progressive meth
18. 04 027 528 130 64 Kio 5 T 1 Pd ll To copy this region across wavelengths time points or through Z sections choose Edit Propagate Polygons from the Edit Polygon menu and enter the appropriate ranges Q Tip You can also select time points You can select rangesoryou can enter selected points e g 1 3 5 20 25 Click Do It to copy the polygons Then view the range of Z sections to make sure that all of the data is included in the polygon on each section 7 a Q Tip You can change selected points on a polygon using the 2 button You can move a selected polygon by selecting it with the tooland dragging ittoa new location From the main softWoRx menu choose Edit Cut Mask pplied Precision Chapter 5 Data and Task Manipulation 49 Vg Output i Polygon Data Loaded In Window z Details Wavelengths W 528 W 617 J Create Mode Data Inside Threshold foao A Trim Output Done Do It In the Input field enter the Image window number Then click Details to open the Region Details dialog box and enter the Z or T sections to include Click Close fed Region D Details Z StartEnd Inc f lt r a Time Start End Inc i oe In the Cut Mask window choose which wavelengths to include In Create Mode choose one of the following modes and specify whether to act on the inside or the outside of the polygons e Choosing Data cuts all of the data inside o
19. 1 Open the image that you want to export in a softWoRx Image window 2 From the Image window menu select File Save As TIFF to open the Save as TIFF dialog box ppliedPrecision Chapter 11 Saving Exporting and Printing 153 Select Region Reset Details Wavelengths W 457 W 617 W 528 685 _J Output Folder datavdv_samples File Prefix tripolar01_d3d TIFF Options scale Using Min Max Exp Values Below _ Compression _ Short File Names _ Destination Computer Windows PC or Linux Output Size 16 bit Grey Wavelength Scaling Wave 1 Ceier E Min Max Exp po 4543 7 1 000 Wave 2 Color ERE Min Max Exp oo 193788 0 n 000 Wave 3 Color a Min MaxExp laze 2740 9 1 000 Wave 4 Color R a Min MaxExp BE 95 3 1 000 9 SS Done Do It Use Save As TIFF to specify export options 3 Ifyou want to specify a particular time point or a single Z section click Details and set the applicable options in the Region Details dialog box 4 Enter the directory and file name in the Output directory The default is the directory in which the input file is located 5 Enter the prefix of the file name in the File Prefix field 6 Select the TIFF options from the lists on the dialog box The default settings should work well for most applications 7 Under Output Size select one of the three possible TIFF output formats e
20. 15_ Image after interactive rotation To rendera volume using the RGB Opacity Method 1 Open the desired image file iv Window CHO_Mitosis dv File View Options Tools 200 im za 4 Z pg ppliedPrecision Chapter 9 Viewing Projectionsand Volumes 133 2 Choose View Blend Colors in the softWoRx main menu The Blend Colors dialog box is displayed Select REGION a Reset Details Details Wavelengths W 457 528 W 617 2 mie Color color i RGB fo 00 0 00 1 00 Scale fp 00 227 00 1 00 Color Color Ras p 00 1 00 0 00 Scale poo 21400 1 00 Color E RGE 00 0 00 0 00 Scale in OO 240 00 1 00 Color 7 CEEE Sopje EOG GEG fH Color PESE og Lie on E Method Max Opacity W Opacity Lowest Pieri wave i True Colors save Colors Load Colors Done Do It 3 Drag the Image window number to the Input field 4 Select Max Opacity in the Method option and select the Opacity option 5 Click Do It to create a blended color image File View Options Tools ppe Section 23 04 720103 000 Rev C 1008 134 softWoRx Imaging Workstation User s Manual 6 In the softWoRx main menu select View Volume Viewer to open the Volume Viewer dialog box a Volume Viewer Input IE Output q select Region Reset Details subset Z Wavelengths W RW GW BW Wavelength Scaling W
21. Align Image on the softWoRx main menu to open the Align Image dialog box 04 720103 000 Rev C 1008 22 softWoRx Imaging Workstation User s Manual v Image Output Shift Table a tab x Range Start End Y Range Start End Z Range Start End f Start 1 End Increment f T Range Wavelengths T E m Ji zi e J Align Adjacent Z Sections Reference Wavelength f Dark Image on Bright Background J Use Rotation Center of Rotation Rozo Effort Level 1 minimal E Additional Parameters Done Do It Options 2 Click Input and browse to the appropriate file The range fields and wavelengths are filled in automatically when a file is selected Note This dialog box requiresan image name for processing You cannot provide a window number for this field Output data1 dv_samples Nuclear_Pore_D3D_ALN Shift Table x Range azo Start End azo Y Range paz Start End piz Z Range Start End Wavelengths 817 W s528 W s7 a Align Adjacent Z Sections Reference Wavelength f Dark Image on Bright Background Use Rotation J Center of Rotation eoo 1560 Effort Level 1 minimal 2 Additional Parameters Done Do It Options 3 Inthe Align Adjacent list select whether to align adjacent Z Sections Wavelenghs or Time Points 4 Click Do It to align the image ipplied Precision Chapter 2 Corecting Images 23 Conecting Chrom
22. Details 7 Wavelengths 535 _ Deconvolution Options OTF File fusrlocalotNikon_ 0X_140 126010 Method Enhanced Ratio aggressive Number of Cycles fio Moise Filtering Medium W Apply Correction Deconvolve Projections Run Options More Options sa show image when finished Deconvolution Finished Done Do It The deconvolution status is displayed in the Deconvolve Output window and on the bottom of the Deconvolve dialog box When the deconvolution is finished messages appear in each of these windows and the deconvolved image is displayed in the Image window ive Window oocyte R3D_D3Ddv File View Options Tools The deconvolved image pplied Precision Chapter 1 Deconvolving Image Data 13 If you did not have the Show image when finished checkbox selected default on the Deconvolve dialog box choose File Open to open the View Image dialog box Enter the file name for example usr local softWoRx data samples oocyte _R3D_D3D dv into the Input field Note You can use the same methods drag and drop browse ortype to enter this file as you used to enterthe R3D dv file in Step 2 on page 11 7 Click Do It to open the file in the Image window Deconvolving Several Images You can create a queue to deconvolve several images and specify a time to start the deconvolution To deconvolve several images im 2 3 4 5 On the softWoRx main menu bar click Pr
23. Double click the icon of the desired measurement file to view a text file containing the data Volume Modeling Example The following steps show how to use the Polygon Finder and 3D Object Builder in a specific set of image data The image data file Nuclear_Pore_D3D included with your system is used in this tutorial 04 720103 000 Rev C 1008 193 194 10 11 12 13 14 15 16 17 softWo Rx Imaging Workstation User s Manual Open Nuclear Pore D3D Select Model 2D Polygon Finder in the softWoRx main menu Drag the Image window number to the Window field Click Select Region Using your mouse draw a rectangle around the region of interest It may be helpful to scroll through the Z sections to ensure that all of the desired areas are included Select the wavelength 457 in the Wavelengths check box Type 750 in the Wave 3 Threshold field In order to estimate an initial threshold value use Point Values in the Tools menu in the Image window menu to view the image intensity at various points Click Do It Click Save Polygon File Type aname for the polygon file and click OK In the Launch field click 3D Object Builder The 3D Object Builder dialog box is displayed Notice that softWoRx automatically loads the polygon data from the open Image window which was used to make the polygon Select the wavelength 457 in the Wavelengths check box Click Build 3D Objects Click Model Save Solid
24. Model in the 3D Object Builder menu Type the desired name in the File Name field and click OK Select Model View Solid Model to view the model Use the center mouse button to rotate the model for viewing from other angles ppliedPrecision Chapter 14 Volume Modeling 195 04 720103 000 Rev C 1008 15 Detecting and Analyzing Colocalization softWoRx provides two tools that you can use together to detect and analyze colocalization First use the Colocalization tool to examine the entire image and identify areas that appear to have colocalized data You can use the data generated by this tool to create volume views and graphically examine them to find structures or specific areas that appear to be colocalized Then use the ROI Colocalization tool to examine the specific structures or areas that you have identified The data selection features of this tool can be used to select many types of areas In This Chapter Examine te Bre Mag O ernen T E acwusaooranieys 194 lde ntfying Potential Colocalized Areas ssisssissnsicucesnresiesustsiacnavedevinesanieut cubeseurasbovensetnce 197 Detecting Colocalization With ROIS ccsciscisaspaudexdesnviceedeanasnayaesievachansenbernssauederniserounneenys 201 04 720103 000 Rev C 1008 Chapter 15 Detecting and Analyzing Colocalization 197 Examining the Entre Image Use the Colocalization tool to identify possible areas of colocalization throughout the data set This tool generates a produ
25. Reduction Combinations Environment Structural Kinetics Compartmental Analysis Biomolecular Cycling Transport Compartmental Analysis Biomolecular Cycling Transport Structural Visualization Struc tural Visualization Compartmental Analysis Biomolecular Cycling FRAP FRET ela Affinity Biomolecular Cycling Biomolecular Repeat during cell cycle Environment Rapid Repeat FRET Affinity Biomolecular Environment Sensitized emission Donor Photo bleaching Acceptor Depletion Compartmental Analysis Affinity Biomolecular Cycling Biomolecular Environment Transport Cell Fate Struc tural Kinetics Structural Visualization Photo Activation Analyzing Huorescence Recovery After Photo bleaching The Fluorescence Recovery After Photo bleaching FRAP experiment method consists of photo bleaching a point or points of interest and then observing the recovery of fluorescence in the bleached area For detailed instructions see the Fluorescence Recovery After Photo Bleaching Product Note at www appliedprecision com An example of a Single Point FRAP experiment is shown below 04 720103 000 Rev C 1008 208 softWoRx Imaging Workstation User s Manual Before Bleach immediatley After Bleach e O After Bleach Recovery A point of interest is photo bleached and monitored About FRAP Expenments There are two types of FRAP data e Single Point FRAP data is collected in experiments that mo
26. T OORT 119 Creanne Volume ProjecHUonS errero iiinn a AO 122 Creating 2D Projections Use the Quick Projection tool to quickly combine information from multiple Z Sections Averaging all of the sections into one provides an approximation of a volume rendering of the image looking directly down the Z axis 04 720103 000 Rev C 1008 Chapter 9 Viewing Projections and Volumes 121 v 1 Window DMS myUpload_R3D_5_D3D dv File View Options Tools 2 8 8v ou A multiple Zsection image before projection Choose View Quick Projection from the main softWoRx menu to open the Quick Projection dialog box 7 eee Output 7 select Region Reset Details Wavelengths W 528 W 617 _ Number of Sections to Average 1 Method Max Intensity eooo Done Do It Help Enter an image file name or window number in the Input field If you want to include only selected data click Details to open the Region Details dialog box Then specify the ranges of data that you want to include in the Output Options fields 04 720103 000 Rev C 1008 122 softWo Rx Imaging Workstation User s Manual Reston Deals EA m input Specifications Dimensions ZT 480 480 105 F View Header Labels m Output Options ive Width Height i i 480 jaso Z StarvEnd Inc i po a Time StarvEnd inc 1 B 7 Data Type No anaE zl W Display In Color Header Label fs 4 In the Quick Projecti
27. Uploading Images Using Task Builder To upload image data from your local file system to the DMS database from within Task Builder use the following procedure To upload image data to the DMS database using Task Builder 1 From the softWoRx main menu select Process Task Builder The Processing Task Builder menu is displayed a Processing Task Builder File Image Files to Process Remove selected Files Processing Tasks Task Options Deconvolution ie Add submit Tasks to the Gueue 04 720103 000 Rev C 1008 78 softWoRx Imaging Workstation User s Manual 2 In the Processing Task Builder menu select the file s you want to process and upload to the DMS database You can accomplish this by simply dragging and dropping the file or folder icon s into the Image Files to Process area of the menu Alternatively you can use File Add Files from this menu to select the file s to include Processing Task Builder j File Image Files to Pro ess_ AAMHO fdatal dv_sanples Spindle_ Green_r3d dv Remove selected Files Remove All Files Processing Tasks Task Options Deconvolution ii Add Submit To Gueue Submit To Remote Server 3 Next select the processes to perform on the image file s For the final task select Save to DMS as shown Processing Task Builder File m Image Files to Process fdatal dv_sanples Spindle Green_rid dv Remove selected Files Rem
28. all of the options have been specified click Do It Sutc hing Images That Have Multiple Z Sections For images that contain multiple Z sections you will need to collect the images with the Panel tool and crop them before you stitch them Before you start Collect Panel data with the DeltaVision Acquisition workstation Determine the width of the border rolloff in voxels for the images To minimize edge effects the border rolloff is automatically set to about 1 5 of the image dimensions To crop a multilayered Image 1 2 Collect 3D panel images Deconvolve the _R3D dv file that you collected Open the deconvolved file in an Image window this file has a _R3D_D3D dv file extension Choose Edit Copy Region on the main softWorRx menu The Copy Region dialog box is displayed 04 720103 000 Rev C 1008 28 softWo Rx Imaging Workstation User s Manual Output Boo E e SER ME Set Details Drag the deconvolved file into the Input field and click Details The Region Details dialog box is displayed a Region Details Input Specifications Dimensions XYZT 256 256 64 10 View Header Labels Output Options xMviWidth Height 1 i 256 256 Z Start End Inc oa ae Time Start End nc fe lie ob Data Type Mo Change W Display In Color Header Label C O Enter values in the X Y Width Height Z Start End Inc and Time Start End Inc fields under the Output Options secti
29. in the selected region Then set any other output details i Region Details Input Specifications Dimensions Y ZT 256 256 50 1 View Header Labels m Output Options siviWidth Height ji 73 e7 70 a 37 Z StarvEnd inc ji Bo po Time Start End inc a a Data Type Mo Change za W Display In Color Header Label a 6 On the Save File dialog box choose which wavelengths of the input data to process and include in the output data set ppliedPrecision Chapter 11 Saving Exporting and Printing 151 7 If you want to scale the output data according to the current minimum and maximum intensity scale factors click Scale to Display Min Max Scaling only works when the output data type is 16 Bit integer or 8 Bit 8 Click Do It to save the file Saving to the Data Management System DMS You can save DeltaVision image files from your local file system to the DMS database if you have this feature set up See Chapter 6 DMS Integration for details 4 From the Image Window select File Save to DMS The Save To Data Management System menu is displayed as shown ba Save To Data Management System 7 File data1 dv_samples tripolaro1_d3d dv DMS Database Connection lt lt lt Disconnect from the Database Destination Parameters Project NewKP_ Project New kpalmby Dataset KP_Dataset ba New kpalmby Category Group KP_TestGroup x New kpalmby Category KP_TestCat
30. irregular data regions Selecting Rectangular Data Regions Use Select Region to select data for volume rendering Rotate3D tool applications modeling and other applications that require intensive processing You can also use this tool to crop data and save it in a new file see Cropping a Rectangular Region on Page 45 To selecta rectangular data region 1 On any softWoRx process window that includes the Select Region button click Select Region and drag the mouse across the image to select a rectangular area The selected region is indicated by a dotted line 30 T 3 00 10 33 655 Zoom 2 01 617 393 ja E 2 Click Details to open the Region Details dialog box pplied Precision Chapter 5 Data and Task Manipulation 41 Mi Region Details input Specifications Dimensions XY ZT p56 256 64 10 View Header Labels Gutput Options lt heevidth Height as 26 f 0 a7 Z StartEnainc h fea hoo Time Start Ena Inc al fo z Data Type Mo Change i W Display In Color Header Label RE 3 Inthe Z Start End Inc field select a Z section range to include Start and End are the beginning and end points Inc incremental allows you to skip points e g entering an Inc value of 2 skips every other point 4 Inthe Time Start End Inc fields select a time data range to include Then click Close 5 Select Do It on the process window you re using The selected region is displ
31. loaded in to the appropriate fields for FRET calculation 04 720103 000 Rev C 1008 214 softWoRx Imaging Workstation User s Manual Calculating FRET Calculation of Net FRET and FRET Efficiency uses a FRET Experiment image as input along with Donor and Acceptor Crosstalk factors and background values for each channel It generates an image with 2 channels Net FRET and FRET Efficiency E To calculate FRET 1 Choose the Calculate FRET tab on the FRET Analysis dialog box Calculate Crosstalk Calculate FRET Analyze Results FRET Input Image FRET Results Image Channel Box Crosstalk Donor An Acceptor e EREET ite 2 Open the FRET Experiment image you would like to analyze in an Image window 3 Drag the Image window number icon from the FRET Experiment Image window to the FRET Input Image field in the FRET Analysis dialog box An output FRET Results image file name is automatically generated The FRET Results image is saved to the disk and an Image window containing the saved image is displayed after the calculation is completed 4 Validate that the correct channels have been assigned to the Donor Acceptor and FRET channels of the input image 5 Validate that the crosstalk factors are reasonable These factors were calculated when you calculated crosstalk 6 Use the Get function to specify a background value for each of the 3 channels see Step 6 in To calculate crosstalk on page209 7 Onc
32. more complete information on using the K3b CD Kreator tool select Help K3b Handbook on the K3b main window to view the entire user s manual for K3b Printing Images softWoRx allows you to print DeltaVision images from an Image window To printa DeltaVision image 1 Open the dv image that you want to print in the Image window 2 From the Image window menu choose File Print Image Print ppliedPrecision Chapter 11 Saving Exporting and Printing 161 3 In the Image Print dialog box enter the window number of the image in the Window field 4 Click Do It to print the image 04 720103 000 Rev C 1008 Part Three AANALYZING RESULTS Part Three shows how to use softWoRx tools to perform quantitative analysis In Part Three Chapter 12 Examining Intensity Data scsscssssssseesssssssssseesssnssseeseessnsseeeesens 162 Chapter 04 720103 000 Rev C 1008 Chapter 7 Manipulating Image Data 163 13 Measuring Distance and Veloc ity Chapter 14 Volume Modeling ssssssssessee Chapter 04 720103 000 Rev C 1008 15 Detecting and Analyzing ColocalliZation 11ssccssssseeesseseeesseseeeessssenssaas 193 Chapter 16 Oth r Appi AUONS iisi AAA 203 ppliedPrecision Chapter 7 Manipulating Image Data 165 12 Examining Intensity Data This chapter introduces the tools used to examine intensity data In This Chapter Examine Pont V UE Sergo nn ioogstd soem T AO
33. of two or more wavelengths To switc h between Grayscale and Color mode gt On the Image window choose View Color to toggle between Color and Grayscale w 1 Window Nuclear_Pore_r3d dv File View Options Tools L 26 Zoom 1 60 f Grayscale shows more detail ina single wavelength 04 720103 000 Rev C 1008 102 softWoRx Imaging Workstation User s Manual Color Mode If you choose a basic color for each channel red green or blue you can display up to three channels in this mode If you select any other colors e g cyan magenta or yellow the two colors used to create these mixtures are disabled for the other channels and they are turned off Black Colors such as Cyan Magenta or Yellow can be viewed in combination with only one other color To assign a basic colorto a channel 1 Choose View Select Image Colors on the Image window menu to open the Select Image Colors dialog box Ord Select Image Colors sai ER Color Display Mode Color m Wave 1 Display Color Red _i Wave Z Display Color Green Wave 3 Display Color Blue BAe he Toes nines pei Paley SAREE oP RY ae TAT Ske a LS Set channel coloroptions in Select Image Colors 2 Select the wavelength for each channel in the Display Color option lists The new colors are displayed in the Image window as they are selected 3 Click Done to set the colors that you selected Note You can assign basic red blu
34. orientation of the display and view cross sections of the data In This Chapter CPST an ae Ce aeons detraetasiansinsbacurentacectieatst isonet T 88 Thelmagce Window centenar ena vars co T EEO O NEO ONAE 89 VALE WIN OLD Tnd CS aeie a a aa aa 90 Adjusune Brenmess and Contrast ssascsciesussw actors seca aA E NRR 95 Assigning Colorsor Grayscale to Channels esrrorir senie a E 99 Controlling the Image Window Display seeeeesessesseseeseeseesersrrsresrrsresrerresresrerserrereeseesseee 102 Resizme or INCOM CIES an Imap En ann a E A E E 105 Niewino GLOSS HONS oain a E R T E N 111 04 720103 000 Rev C 1008 Chapter8 Viewing Image Data 89 Opening an Image You can open image data files in DeltaVision or TIFF image file formats To open an image data file ti ei menu The 1 Choose Applied Precision Start softWoRx from the CentOS softWoRx main menu is displayed A softWorx 3 7 0 Running On Host Istech apicom File Edit View Process Filter Model Measure Conversions Utilities Help Data Folder Image Windows 1 Scratch Space 0 30 22 Gb free Data Space 90 22 Gb free 2 Click the amp icon on the softWoRx toolbar or File Open from the menu The Open Image dialog box is displayed Click the Files tab DAMS Database Directory fdatatidv_samples aa 40 _155 o0tf AF_testl4 2 R30 03D dv Drosophila Embryo Mowie_d3d dv LiveCells_ GFP _ 2D Mowie d d dy Nikon1O00 _140 ott Created Ac
35. specific images based on the image file s annotation Browsing Image Files using P D I Hierarchy To browse through image files by Project Dataset Image pplied Precision Chapter 6 DMS Integration 83 1 From the softWoRx main menu select File Open When the Open Image window is displayed click the DMS Database tab Preview Files DMS Database aa Disconnect trom the Database Database View Project Dataset Image Froject NewKP_Project kpalmby Dataset KP_Dataset kpalmby Created Acquired oe ne eet a enan Search annotations Far f Search Last Modified Available Images Drosophila Embryo Movie_d3d dv LiveCells GFE 20 Mowie d3d dv File Spe Nuclear Pore d3d dy eer Nuclear Pore _djd_1 dv 7 44 Mib Nuclear Pore d3d_ dv Spindle _Green_d3d dy 0120907 15 26 Image Info 2139 W 3 T 2 Use the drop down menu in the Database View field to select Project Dataset Image 3 Select the specific projects and datasets to browse using the dropdown menus in the Project and Dataset fields Browsing Image Files using CG C I Hierarchy To browse through image files by Category Group Category Image 1 From the softWoRx main menu select File Open When the Open Image Preview Files DMS Database lt lt lt Disconnect from the Database Database View CategoryGroup Category Image Project KP_TestGroup kpalmby Dataset KP_TestCat w kpalmby Created Acquired ante P
36. start the jobs at a specified time To set up process chains with Task Builder 1 Select Process Task Builder from the main softWoRx menu to open the Task Builder dialog box File Image Files to Process Remove Selected Files Remove All Files Processing Tasks Task Options Deconvolution i Add Submit To Queue submit To Remote Server 2 On the dialog box select File Add Files You are presented with a list of files from which you can choose the specific files you want to add to your process chains pplied Precision Chapter 5 Data and Task Manipulation 55 e Aad Files to Process Filter 7 datal dv_sanples Directories OlympusIx70_60x _140 otf Spindle Green_d3d dy Spindle Green_d3d_D30_cmd sh Spindle Green_d3d_process sh Spindle Green_r3d dv oocyte otf oocyte rid oocyte r3d_process sh spoke_prj dv g Spoke_pr _process sh Cancel 3 Select the file s you want to add to the Task Builder dialog box and click OK The files are displayed in the Input Files section of the dialog box Note You can select files from this dialog box using the SHIFT key to select multiple contiguous files orthe CTRL key to select multiple files from vanous parts of the list oa Processing Task Builder File Help Image Files to Process fdatal dy_sanples Nuclear Pore DID dv fdatal dv_sanples Spindle Green_d3d dv datal dv_sanples cell_21003_r3d d Remove Selected Fi
37. 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 04 720103 000 Rev C 1008 66 softWo Rx Imaging Workstation User s Manual 6 W 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 Insert the selected secondary filter insert mCherry is used in this example into Filter Slot 1 of the secondary light path and click Next to continue W Activate Filter Set Wizard Move the MPs beam combiner to position 4 500 LP se Skip ta immediately activate the selected sets Next 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 Move the beam combiner to the appropriate position and click Next to continue Activate Filter Set Wizard Move the polychroic turret to position GFP mCherry Dual MPA Fush the Finish button to activate the new sets Finish Cancel Again softWoRx gets the information for this window in thiscase position 2 GFP mCherry from the MxWSetup ini file not from the Instrument Controller Click Finish to co
38. to view the image data in 3 D This tool allows greater visual understanding of the image data and comparison of features within the image data It also allows quantitative assessment of structures throughout the entire data set on a single image Volume Rendering A brief understanding of how softWokx creates a volume rendering will help you utilize this tool for various image data sets Theoretically a set of parallel rays is sent through the data set at various angles to analyze the data in those paths and collect new data from that perspective Each time a set of rays is passed through the data a projection is created from the resulting data These projections constitute the volume rendering 04 720103 000 Rev C 1008 124 softWoRx Imaging Workstation User s Manual Figure 4 Volume Rendering Projections 3 D Projection Image JUEGOS ALT TLLLELeLe rT v y y uc y Pi y or Image Data yF Pig v i y 2 y y Paraflel Rays aF s Axes of Rotation Volume Viewer enables you to create a movie of the data rotating around an axis This axis of rotation can be any of the three common axes X Y or Z Given a Z series of data the coordinates are defined as shown in the following figure Figure 5 Axes of Rotation Rotations about the axes are as shown in the following figures Figure 6 Rotation About the X Axis rR E ppliedPrecision 125 Chapter 9 Viewing Projectionsand Volu
39. use ROI colocalization 1 Choose Measure Colocalization ROI from the softWoRx main menu to open the Colocalization Analysis dialog box Enter the number of the Image window that you want to analyze in the Input Window field Ef colocalization Analysis Input Window E x JA SI tq Number of ROIs fo Copy gt Time Copy gt Z Input Channels Threshold Box Plot Min Plot Max Pearson Coefficient of Correlation ial 2 Select which channels to analyze in the Input Channels lists 3 To select specific areas of the image use the Create Freehand ROI Polygon button to create polygons on each section that you want to analyze If the position and shape of the structure are consistent through time and Z intervals you can use the Copy Selected Polygon and the Paste Polygon buttons to copy and paste the polygons through time points or Z sections ppliedPrecision Chapter 15 Detecting and Analyzing Colocalization 205 File View Options Tools Help Z 18 Zoom 7 00 528 1264 f 5J 4 Select a background threshold for each channel by clicking Get and then clicking on a background area of the image The background is an averaged value within a box of the size specified Click Get again when you are finished selecting the background 5 Click Do It to plot the Colocalization graph and calculate the Pearson Coefficient of Correlation value for the selected region this value is displayed in the Coloc
40. view a profile of a line of pixels or a band of pixels This data can be saved as a text file 04 720103 000 Rev C 1008 170 softWoRx Imaging Workstation User s Manual Arbitrary Profile displays a plot of intensity values along a line segment that is oriented at any angle in the Image window This profile is displayed in a separate window You can interactively change the orientation of the line This data can be saved in an s1k spreadsheet compatible file Viewing the Line Intensity of a Row or Column 1 Open an image in the Image window and choose Tools Line Profile on the Image window to view the Line Profile dialog box Window T _ All Windows AutoScale 12 1809 Show Min Max Values Direction Horizontal Band p K Y alls 283 AutoSsave Profiles Saved fo SAVE Clear aie fob Done 2 Drag the window number from the Image window to the Window field in the Line Profile dialog box 3 Click on the image to display a horizontal line profile Y bike ee a eddiddiddiddiddddddddddididddidid File View Options Tools be a byt ae nh ta A l ree k A ET it ara oc Alf niin than tL sin ha kal AA i a 1 Wh l N pe ag 4 J ae i fi t ve fae f au i i en 95 0 Yell nl i wise peli prune M saat any F nS Anh ay Raun 528 243 ppliedPrecision Chapter 12 Examining Intensity Data 171 4 To get the average line profile of several rows and columns of pi
41. zala X l a al ax hooo aj f A hi 10 00 ayj ea 7 a 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 mE Design Run Experiment Resolve3D_Startup exp X File Help agda Oe Design Experiment Design PK Experiment Run Experiment Experiment name Resolvead Estimated File Size 668 05 Mb Enable Fast Acquisition ast Acdiuisifion TPOS Sectioning Channels Time lapse Points Panels Actions Conventional Multiplexed Exp EX Filter EM Filter Yo Ex Shutter m 1 000 BLOCK BLOCK 100 v TRANS Apie BLO l B x Multiplexed a BLOW Tab tires THE A TEN at PERLE A PPS Betting TI A TERY PRL A PPS rb RITE on ee E y HEHA y EEC SEs ERRE An ii i IS Le Lele le is Te fH Sih ate Pati Pe be Pe ak a od Beete WH teed See See NH teed Refresh exposure conditions The Multiplexed tab of the Design Run Experiment window is displayed as shown 04 720103 000 Rev C 1008 70 softWo Rx Imaging Workstation User s Manual Select Checkbox aT Do Multiplexed Channel Imaging Settings F WY Design Run Experiment Resolve3D_Startup exp Design Experiment Design PK Experiment Run Experiment Experiment na
42. 00 06 0 8 Click the Start Scan button to begin the imaging process The ratio imaging process will occur in a separate Image viewer similar to the following iw be Window Ratio Image Monitor File View Options Tools ana TEE r IA 10 8 h 10 T 00200705 069 5 00 00 05 068 A 1 49 a The square outline in the center of the image represents the mean value of the image data When the ratio imaging experiment is complete a two channel time lapse image is displayed along with a ratio graph showing the mean value the outlined area in the middle of the image vs time The square outline 04 720103 000 Rev C 1008 61 softWoRx Imaging Workstation User s Manual represents the portion of the image used to calculate the mean for a ratio eraph r W 2 Window ratio imaging R3D dv a O X File View Options Tools Help C PAN Pa T 00 00 00 000 T 1 00 00 00 000 528 179 Graph Point Labels Ratio Imaging Experiment 4 Time Point 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
43. 138 038 4147 315 4065 483 4077 742 Y 1215 97 1188 47 1215 63 1157 66 1150 04 1166 27 1195 09 1215 30 1189 13 1216 63 1216 96 1152 69 1158 98 1166 93 1162 30 1169 25 1210 33 1216 30 spot diamete width 39 39 39 24 24 24 36 36 36 36 36 16 16 16 16 16 16 39 box 39 39 39 24 24 24 36 36 36 36 36 16 16 16 16 16 16 39 softWo Rx Ima ging Workstation Users Manual incl_points min 1070 1070 1070 408 408 408 960 960 960 960 960 176 176 176 176 176 176 190 188 180 175 180 171 592 612 total intensity 230575 249563 215080 77580 81353 82505 191227 179153 907931 778695 645557 94167 89876 110328 91609 110575 92131 544263 SD The Edit Polygon tool can be used to calculate statistical values for the area of data that is inside of a polygon You can draw multiple polygons on different Z sections and view the statistics for each You can also save statistical reports to text files or to SYLK spreadsheet compatible files To select polygon areas 1 Choose Model Edit Polygon on the main softWoRx menu w Edit Polygon File Edit Options Window 1 Polygon Data i x ol statistics ad 0 Blt Active Wave Mode J Guided Mode Template Mode 2 Click a polygon option e g Q freehand on the Edit Polygon window One Viney Ot SHE wA sa a pean a PALIRILY aj Jy griner taper Br TMr ae he MS Hel
44. 185 SD ODJECE BUNA Ch errio ia ita es aoa E AE 187 Volame Modeling Example seernes nc aleee ab sh ahs i ladedl serene tela antes eek niadlacre 190 04 720103 000 Rev C 1008 Chapter 14 Volume Modeling 185 About Volume Modeling The 2D Polygon Finder and the 3D Object Builder are used to create three dimensional models of features within the image data and obtain quantitative information Initially the 2D Polygon Finder is used to specify the object of interest The 3D Object Builder is then used to create a three dimensional model from the two dimensional polygons in each Z section Finally the real space coordinates are saved to an ASCII file which can be viewed from a table of measurements and the 3D Model Display can be viewed In summary this is the process Image Data Find Edit Polygon Polygons Defined in Build 3 D object 3 D Object Z Senes each ZSection 3D Modeling How Edit Polygon Dialog Box When a satisfactory polygon is not obtained using the 2D Polygon Finder you can use the Polygon Editor to define a polygon File Edit Options Statistics Window fl Polygon Data f Bocas e oo dit OOo Active Wave Mode ne a 46 E J Guided Mode _ Template Mode Several controls enable the Polygon Editor to define the intended feature Descriptions of the most useful controls follow For additional information refer to the online Help 04 720103 000 Rev C 1008 186 Window softWo Rx Imaging Workstatio
45. 3D Settings window select the EX and EM 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 pplied Precision Chapter 5 Data and Task Manipulation Resolve3D Settings a amp Display imaging Files Autofocus Mise QLN Stage Motion Allow Lost lotion Compensation LMC Stage View Options W Show stage trails Wo Show stage thumbnails Wo Show point numbers Select the EX and EM filter sets Filter Wheel Sets Excitation filter wheel GFP mCherry lt lt lt Pending Activation s Message _ Activate Filter S fs 2 displayed when We eaveieaceccccccecosaceseees filter wheel Done Save Settings settings are eee changed 4 Click Activate Filter Sets The following confirmation window is displayed 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 Activate Filter Set Wizard Are the selected filter wheels currently installed Use Skip to immediately activate the selected sets Next gt gt gt akip Cancel Note For the filtersetsto be activated the selected multiplexed filter set filters must exist in the currently installed excitation and emission filter wheels 5 If
46. 600a q02 etc you can simply overwnte the last digit and press Do Itfor each file 8 In the Queue Manager dialog box choose one of the following options To perform the deconvolutions immediately click Start Now or To perform the deconvolutions later click Start Later and select a time on the clock that appears in the dialog box As the files are deconvolved the deconvolution status is displayed in the Queue Manager 9 Click Quit to close the Queue Manager dialog box Common Deconvoluton Options You can set options to deconvolve a region of an image deconvolve only data in specified wavelengths or change the deconvolution method pplied Precision Chapter 1 Deconvolving Image Data A 3 os A See Po Ree eee Ha PDN ee Output ainni Mrr ng Wavelengths _ Deconvolution Options 15 OTF File f Method Enhanced Ratio aggressive Number of Cycles ho Noise Filtering Medium pod Wo Apply Correction Deconvolve Projections Run Options More Options W Show image when finished Use the Deconvolve dialog box to specify options for deconvolving To Deconvolve only part of the image Select which wavelengths to include Selecta deconvolution method Display deconvolved images immediately after processing 04 720103 000 Rev C 1008 Do This Click Select Region and use your mouse to define a specific region of the image to deconvolve Th
47. 82 1181 1166 Re Tei HA S F 1175 1173 1168 1177 1220 1198 1190 1172 1205 1179 1180 1188 1194 98 1195 1186 1193 1193 1187 1185 1176 1180 1209 1208 1197 1199 1216 1192 1185 1178 1194 1184 1211 1213 1244 1218 1197 1194 1191 1181 1225 1211 1220 1216 1230 1200 1188 1192 1181 1221 1222 1217 1223 1212 1190 1167 1194 1207 1205 1227 1231 1220 1223 1218 1193 1213 1209 To gather statistics 1 Click on a point of interest in the Image window and press the space bar The statistics for a rectangular area around that point are displayed in the Data Inspector Statistics field 1 Window actin aurb tub dapi midbody 1_02_rodtest dy File View Options Tools dag 2 From the Data Inspector window choose File Save Statistics Record to open the Statistics Record File dialog box 3 To save the statistics file enter a file name in the Statistics Record File dialog box and click OK The file is saved as a text file similar to the following 04 720103 000 Rev C 1008 Ekes 173 174 File Generated By softWoR x Datalnspector datal statisicsRecord sample2 Tue Feb 17 16 55 01 2004 wave 685 685 685 685 685 685 685 685 617 617 617 617 617 617 617 617 617 617 Calculating Statistics for Imegular Areas XxX 4196 678 4193 034 4167 192 4128 099 4136 05 4129 755 4066 146 4075 422 4191 709 4195 022 4165 867 4136 713 4126 442 4129 093 4
48. B v m a pE 7 5g E KO MI i on jo Specify the Viewing Parameters and Movie Options Click Do It to process the image data Volume created with the Max Intensity method ppliedPrecision Chapter 9 Viewing Projections and Volumes 7 Move the Z slider up and down to view the projections or use the Movie tool To rotate the image using interactive image rotation 1 Open the image file and choose View Volume Viewer in the SoftWoRx main menu to open the Volume Viewer dialog box vE Viewer Input E Output fa Select Region Reset Details Subset Z Wavelengths W 528 W 617 W 457 m Wavelength Scaling Wave 1 Scale 183 0 3772 0 1 00 Wave 2 Scale 197 0 1085 0 1 00 Wave 3 Scale 125 0 1834 0 1 00 m Viewing Parameters Method VYolPack VolPack Options VolPack VolPack Options Quality Besi Original Size Z Resolution Gest Progressive Threshold 20 m Movie Options Start Angles XYZ 178 8 7 1 401 Number of Projections fs ooo Rotation 180 Around Y Status pe uM m Done Dolt imterrint Interactive Hel Done Do It _Interactive Help 2 Drag the Image window number into the Input field 04 720103 000 Rev C 1008 129 130 softWoRx Imaging Workstation User s Manual Qi Window joembi03_05_R3D dv File View Options Tools 528 431 fd Ima
49. Click Show Histogram Arrange the windows on the screen Selecting a Region of Interest The region of interest or ROL is a region of the image that you can resize and move to visually examine image details You can select a region of interest ROI in the Image window to display its intensity values in the 3D graph the Histogram and the table in Data Inspector The display in each window changes automatically as you change the ROI in the Image window ppliedPrecision Chapter 12 Examining Intensity Data 169 Rd i Window test_5_R3D_D3Ddv File View Options Tools The ROI isthe area within the box To define an ROI 1 Choose Tools Data Inspector on the Image window to open the Data Inspector window 2 On the Data Inspector window click Select Box 3 In the Image window click the top left corner of the ROI and drag the mouse to enclose a region of interest with the ROI box 4 To make the ROI a circle instead of a square select Constrain to Circle 5 To change the position of the ROI click on any point in the Image window The ROI is centered on the point that you click Viewing Intensity Line Profiles A line profile is a plot of intensity values for pixels along a straight line Two types of line profile tools are available Line Profile displays a plot of intensity values for pixels in a row or column of the Image window This profile is overlain on the image You can
50. Click on the New Data CD Project icon The Current Projects window opens 11 Note The processforarchiving filesto a DVD isthe same except you choose New Data DVD Projectin this step ppliedPrecision Chapter 11 Saving Exporting and Printing 02 k3b The CD Kreator Eile Project Plugins Tools sail Help acces e3 399 eee 40X_135 otf Nuclear_Pore_d3d_D3D dv Nuclear_Pore_r3d D3 Jog t Drosophila_Embryo_Movie_d3d dv Nuclear_Pore_d3d_D3D_log txt Nuclear_Pore_r3d dv LiveCells_GFP_2D_Movie_d3d dv lt Nuclear_Pore_d3d dv i Nuclear_Pore_r3d_process s Nikon100X_140 otf mi Nuclear_Pore_d3d_process sh Olympus X70_60X_140 otf MSW Screens Nuclear_Pore_d3d_CLC_VOL dv 4 Nuclear_Pore_r3d_01_D3D dv lt gt oocyte otf Root Nuclear_Pore_d3d_D3D_cmd sh Nuclear_Pore_r3d_D3D dv lt gt oocyte r3d Current Projects DataCD1 OO OK3b data project Name Type Size Local Path Link Use drag n drop to add files and directories to the project To remove or rename files use the context menu After that press the burn button to write the CD Available 703 0 MB of 703 0 MB lj Temp 25 3 GB 31 3 GB K3b 0 11 14 4 Navigate to the files you want to archive and double click on each file you want to copy to the CD or drag and drop the files into the Current Projects window Q Tip You can select multiple files in the K3b window by holding down the CTRL key while selecting files sy ely Th
51. Dimensions XYZT 512 51211 View Header Labels m Output Options X Y Width Height 219 5 feos zs Z StarvEnd inc h ho po Time Start End inc ff hooo Data Type No Change d Display In Color Header Label p el Help In the Z Start End Inc field select a Z section range to include Start and End are the beginning and end points Inc allows you to skip points e g entering an Inc value of 2 skips every other point In the Time Start End Inc fields select a time data range to include Then click Close In the Save File dialog box Wavelengths field choose which wavelengths of the input data to process and include in the output data set If you don t have the option of which wavelength to include the toggle buttons are dimmed pplied Precision Chapter 5 Data and Task Manipulation 8 Click Do It to save the cropped image file Then open the saved file in another Image window and view the results of the selections 59 KE Cropping an Irregular Data Region You can crop irregular data regions from image files To crop an inegular data region 1 Open the image in the Image window 2 Choose Model Edit Polygon to open the Edit Polygon window 3 Choose a selection tool e g Oh and draw a polygon around the area of interest 04 720103 000 Rev C 1008 47 softWoRx Imaging Workstation User s Manual 15 5 T 00 00204 027 00 00
52. Dio It ayem Preview Drag the Image window number into the Movie File field or click Movie File and select aname and path for the movie At the top of the window select one or both of the following options e To save the image select Save Image e To save the overlay graphics select Save Overlay In the Movie Format list select from Quicktime AVI or MPEG movie formats 04 720103 000 Rev C 1008 156 softWo Rx Imaging Workstation User s Manual 5 Inthe Animation Style list set the movie looping mode by selecting one of the following options e Forward amp Back records a movie that starts on the first frame selected plays to the last frame and then back again to the last frame e Forward records a movie that starts on the last frame plays to the first frame and stops e Backwards records a movie that starts on the last frame selected plays to the first frame and stops 6 Adjust the Compression Quality slider to specify image quality and file size Moving the slider to the left produces better quality and bigger file sizes Moving the slider to the right produces lower quality and smaller file sizes 7 Adjust the Frame Rate slider to specify the playback speed 8 Select whether to animate through Z sections Then select the range of data to include in the movie and the increment between frames 9 Select whether to animate through Time Then select the range of data to include in the movie and the i
53. Image fusion 52 54 Image graphic files 148 Image viewer cross sections 111 12 Image window 102 7 about 89 90 border tools 103 display controls 92 menu 90 scale bar 103 5 status bar 90 toolbar 90 zoom wheel 94 Images adjacent 21 22 analyzing quality 215 19 and colocalization 194 97 and colocalized structures 197 201 and intensities 162 74 and line intensity 167 68 archiving 157 59 arithmetic 142 43 calibrate 20 21 calibrating 18 combining data 52 54 contrast and brightness 95 99 correction 17 25 cropping 27 44 50 cross sections 111 12 deconvolve single 11 13 deconvolving 9 16 enhancing boudaries 139 40 filtering 137 45 graphic files 148 Inovision See 33 intensity threshold 144 45 interpolation 93 94 local contrast enhancement 143 44 opening 88 89 ratio 57 62 reorienting 105 11 repositioning 92 resizing 105 7 RGB opacity method 131 35 rotating 107 9 saving as JPEG 155 saving as movie 153 55 saving as PhotoShop files 153 saving as TIFF files 151 saving volume rendered 135 scaling 95 99 scaling pixel intensity 143 44 selecting 40 44 stitching 25 29 time lapse 19 20 uploading to DMS 74 81 viewing as movies 107 12 viewing data 87 112 visually evaluating 217 19 window 102 7 with bright spots 218 with dark halo issues 218 with deconvolution holes 218 with invalid OTF 219 with multiple Z sections 27 29 with pebble grain texture 218 with poor Z resoluti
54. Image window number 4 Click Details to open the Region Details dialog box PL Sd Input Specifications Dimensions 27 512 512161 View Header Labels Output Options x Width Height a i a 7 st Z StarvEnd nc ei Time Starv End Inc Ee po Data Type Mo Change _ Display In Color Header Label a asf 5 Inthe Time Start End Inc field enter the first and last time points to include and the increment between points For example entering 15 45 1 includes all of the points between 15 and 45 You could skip every other point in this interval by entering 15 45 2 In the example above every third point is included 6 Click Close to quit Region Details 7 Inthe Copy Region dialog box click Do It to create the new image 04 720103 000 Rev C 1008 52 softWoRx Imaging Workstation User s Manual Nd 2 Window LiveCells_ GFP_2D_Movie_d3d_CPY dv unsaved File View Options Tools Er hile _ gE Le e ll 11 00 22 54 000 528 602 F RE Ending image with 11 time points Combining Data of Two Images Use the Image Fusion dialog box to combine time points Z sections or wavelengths from two DeltaVision images into one output file The input images may come from windows or files After selecting the input images you can specify exactly which wavelengths time points and Z sections you want to combine You can either append selected wavelengt
55. MS You can use softWoRx to download image data from the DMS database to your local file system To download a file from the DMS database 1 From the softWoRx main menu select File Open When the Open Image window is displayed click the DMS Database tab and select the file you want to download 04 720103 000 Rev C 1008 82 softWoRx Imaging Workstation User s Manual Preview Files DMS Database aa Disconnect trom the Database Database View Project Dataset Image Project NewKP_Project kpalmby Dataset KP_Dataset gt kpalmby Created Acquired ta AN ete enas Seereh annotations Far f Search Last Modified Available Images 0109 07 15 26 File Size 7 44 Mib Nuclear Pore d3d 2 dv Spindle _Green_d3d dv Image Info fala Misa T spoke prj_ RSP dv test _5_R3D D3D dv 2 Click the Download button The Choose Destination Folder dialog box is displayed a Choose Destination Folder Directory Ydatat dv_samples u amp t Folder name f OK Cancel 3 Select the directory on your local file system where you want the selected image file downloaded and click OK The image file is downloaded from the DMS database to the specified location in your local file system Browsing and Locating Images in a DMS Database The softWoRx software provides the ability to browse images within a DMS database by either the Project Dataset Image or the Category Group Category Image hierarchies You can also search for
56. RAP log Input Image fdatal InSitu2003 Membrane_FRAPO2_R3D_FRAP dv Result Image dv Result File home worx Membrane_FRAPO2_R3D_FRAP log FRAP Best Fit Results Single Component Bleach Point HE Location X Y 16 3 Post Bleach Fraction Mobile Fraction Final Fraction Beam Radii X Y Time Constant Half time Chi squared Avg Error Avg Abs Error 69 388 4 Ble 70 677 91 023 0 500 0 565 secs 0 608 secs 2301 473 0 022 1 435 FRAP Measurement Information First Measurement Last Measurement Experiment Duration Num Pre bleach Pnts Num Post bleach Pnts Time Zero Offset Image Bias Background Intensity 69 435 at 88 465 at 5 434 secs 3 48 0 000 secs 50 000 counts 0 000 counts Photosensor Normalization off Mean Inten Normalization Pre bleach Statistics Pre bleach Statistics Recovery Measurements on 1 000000 357 273 Ts ModelF ModelI RecoveryF Recoveryl 0 482 1 000 357 273 357 273 357 273 0 365 1 000 0 252 1 000 um ached Frac 30 61 K 0 781 B 1 076 0 500 um 1 757 counts 0 991 counts 0 0010 secs 0 002 half times 5 4350 secs 8 94 half times f 0 000141 fractional 0 051 counts 1 000 357 216 1 000 357 292 1 000 357 312 The FRAP Analysis log file Analyzing Huorescence Resonance Energy Transfer FRET Fluorescence Resonance Energy Transfer is a method for determining whether two types of
57. TAEA 162 Examining Intensity Data With Data Inspectort ssessesesserserseesrssessrsreseseesesseseeseesees 163 View Intensity Line an 116 19 exc jemmreamene ersten Perr E EE 166 Calculating Statisti Sunena tania tisweeaw aaa A A 169 Examining Point Values You can examine intensity values for individual pixels in the Image window The wavelength and intensity value of the point under the mouse are displayed at the bottom of the Image window To view point values gt From the main softWoRx toolbar choose Measure Point Values Select a channel Wave and move the mouse across the image to view individual 04 720103 000 Rev C 1008 166 softWoRx Imaging Workstation User s Manual point values for that channel The point coordinates and intensity of the point under the mouse are displayed in the Point Values dialog box F 2i Window TFA 001 R3D fe x File View Options Tools Help 2E E o a on L Window le Wave 1 Image Coords pix Bazz Image Coords um 3708212000 Slide Coords urn 626 29 3961 54 35 20 2 Stage Coords um 4 626 29 3961 54 35 20 Image Intensity 5e SEEP z Z 7 528 R e Tip You can also view the intensities of individual points for the selected channel on the status bar e g 457 1216 in this example Examining Intensity Data with Data Inspector The Data Inspector includes several tools for examining the intensity data in the imag
58. To hide the tools clear the Show Toolbar toggle on the Options menu The Image Window Scale Bar ppliedPrecision Chapter 8 Viewing Image Data 105 You can display or hide a scale bar to show the scale of the image You can also set options to move the scale bar to show the scale of a point of interest in the image or to control how the scale bar is displayed The scale bar is displayed by default Qi Window Nuclear Pore riddv LOL File View Options Tools To change the image scale bar 1 From the Image window menu choose Options Display to open the Display Attributes dialog box Display Attributes Window 1 Wavelengths 617 528 _ 457 Z Section ze Z Step i Image Scaling 208 0 1894 6 1 0 Zoom Factor 1 604 i Interpolate Zoom m scale Bar Attributes Displayed W Vertical _ Show value W Pasition z432 Move Length feo Color Yellow Thickness El Set Scale Bar Attrnbutes 2 Choose whether to display or hide the scale bar 04 720103 000 Rev C 1008 106 softWoRx Imaging Workstation User s Manual To display the scale bar select the Displayed option To hide the scale bar clear the Displayed option 3 To display the scale as a vertical bar select Vertical the default is horizontal 4 Use the Show Value option to select whether or not you want the scale bar value displayed 5 Adjust the Position Length Color and Thickness of the scale bar The scale
59. Tracking Autofocus before imaging 6 Select the Actions tab and select Ratio Image as the action for this experiment The Time Points specification will default to all and the When control will be After Imaging W Design Run Experiment Resolve3D exp cad g Oe Design Experiment Design PK Experiment Run Experiment Experiment name ResolveaUi Estimated file size 5 00 Mb 4777 08 Gb Available W Use Fast Acquisition Fast Acquisition Options Lamps Off when finished Sectioning Channels Time lapse Points Panels Actions Time Visited Points Action When Options Ratio Image After Imaging 7 7 Select the Run Experiment tab and enter the image file name and a title for the ratio image You can also enter text into the Add note to log field to include the text in your image log file ipplied Precision Chapter 5 Data and Task Manipulation Design Run Experiment Resolve3D exp Xx Design Experiment Design PK Experiment Bun Experiment Image file name ratio_imaging Settings DMS Setup Image title Add note to log Do It Change next time lapse oog Doi W Show images during acquisition W Show PK progress graph W Launch viewer after experiment J Geconvoive during experiment Images acquired requested 10 10 Disk space required 5 00 Mb Current command Finished Start Time Current Time Elapsed Time Estimated Finish 0
60. Workstation User s Manual File View Options Calibration Acquire Experiment settings Excitation Emission C 4704 30 Select the EX filter m primary light path Ex Shutter EX ow A Be Exposure ooo Find Calibrate Image size 512x512 Lens o Info Bin jia 1 Aux Mag Pixel size 0 6680 um OA ale A la ae al dx 110 00 d 0 00 al ere Paqa Y a xall Use the EX button on the keypad to open the primary EX shutter and view the primary light path Use the EX2 formerly CAMERA SHUTTER button on the keypad to open the EX2 shutter and view the secondary 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 From the Resolve3D main menu click the Experiment button to open the Design Run Experiment window pplied Precision Chapter 5 Data and Task Manipulation 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
61. a time lapse experiment A Note Thistool isnotintended to conect for photo bleaching overtime To equalize intensities to a time point 1 Choose Process Equalize Time Points from the main softWoRx menu to open the Equalize Time Points dialog box 04 720103 000 Rev C 1008 20 softWoRx Imaging Workstation User s Manual Output E select Region Reset Details Wavelengths W 457 W 528 W 617 e7 e Reference Time Paint A Use Threshold Done Dio It 2 Enter the original _R3D dv image file in the Input field 3 Enter the time point to use as the standard for adjusting intensity values of other time points in the Reference Time Point field 4 To perform equalization based on background and minimize the influence of the variation of higher intensities signal select Use Threshold This specifies to use only values less than the Threshold value for each wavelength when collecting Min Max Mean statistics for equalization 5 If you are using a threshold specify the wavelengths in the Wave fields and specify the threshold for each wavelength A Note The number of Wave fields in the Equalize Time Points dialog box adjusts automatically to the number of wavelengths in the selected image 6 Click Do It to equalize the time points and then click Done Calibrating Image Data Use the following instructions to calibrate an unprocessed image when you have a calibration file and a bad pixel file tha
62. acing can be obtained in two ways it can be measured with a test target or it can be approximated from the CCD detector element size and the total image magnification For example if the CCD detector has 6 7 um pixels and the image was acquired with a 100X lens and a 1 5X optivar then the pixel size is approximately 6 7 um 100 x 1 5 0 045 um The Z pixel spacing is the distance between adjacent optical sections Figure 2 Maco nvert Pic to DeltaVision DY File _Picdy split Adjacent Panels d Wavelength 1 nm fo Wavelength 2 nm fo O 0 0000 Y Spacing um ooo f Step Size um 0000 Lens IO fo Done Dott Options Options in this dialog box are described briefly in the following paragraphs For additional information regarding Pic file conversion refer to your online Help system nm spacing um um um To converta Pic image file to a DV image file 1 Click Conversions Import from Pic in the softWoRx main menu The Convert Pic to DeltaVision dialog box is displayed as shown in Figure 2 04 720103 000 Rev C 1008 36 softWo Rx Imaging Workstation User s Manual 2 Select the file that you want to convert using the Pic File button and data entry field 3 Type a filename for the converted file into the DV File field 4 If the image file consists of two wavelengths that are arranged in two adjacent panels enable the Split Adjacent Panels option 5 Enter the wavelength of the first i
63. aie aaa 33 POS DE GICS COM Y CLS LOM atest nctags soy a uv osiinsivessacneoesatiens O A O 34 Converting Pierie Gaien adi an team A A A 35 Convertino ORB es epeei T ictmenntalecaue icuvatoveadeersSience 36 Data and Task Manipulation csssssccssssccsssccnnsssenssseessseensssennss OO DOC CHINS DAA ecrectntandtnston ties E E S 40 pelecting Rectang ular Data Regions cscs siccsssicaenGauise vsiuedacaestaeasielseaaGastaendtvaveiaadt einaes 40 Selecting Jrreeuilar Data Recon cites ssniestynslonies bigtime Gseahete ceementbessl dace teau beac tiedes 42 CEO POU ATES Dalaran a a a 45 Cropp r a Rectang dlar Region cenean E nan eae eases 45 Cropping an Irrecular Data KES OM niren n E ATA 47 MPU Mme Data seas 50 Combining Dato TWO maces ia a NE OEO 52 Setting Up Process Chains with Task Builder eseeeseeeeeeeeeeeeeereersesrsersersrrsresresresresresseesess 54 Usine Rato Ma i o T N aebassunaaesewsmaseasats 57 Using the Multiplexed Wavelength Opti Nssniironsneai a 62 Setting Up the Multiplexed Wavelength Option eesessessessesrerresrsresrersesrrsresresrersessees 63 Designing a Multiplexed Wavelength Experiment eeeessesrerrerersrerrerrrrrererrrrrersesees 68 DMS INE gra Uo aa eS Wat IS DM usiue n n r 73 Connecting toa DMS Database cenere E EA 74 Uploading Imaces toad DMS Data pase aoisi A AE 74 Uploading Images from the Image WindoOW sesesessesserrrsersersersrrsereresresresresresressesses 74 Uploading Images Using Task Builder saticsnets
64. ak Pe 03 20 08 11 31 Search annotations For f Sanrei Last Modified ean 05 01 08 14 28 Drosophila _Embryo_Movie_d3d dv Details LiveCells_GFP_2D_Movie_d3d dv Nuclear Pore_d3d_2 dv File Size 50 05 Mb spoke_prj_RSP dyv Download Image Info Vi jew Z 10 W 2 T 5 Ee Cancel 2 Use the drop down menu in the Database View field to select CategoryGroup Category Image 04 720103 000 Rev C 1008 84 softWoRx Imaging Workstation Users Manual 3 Select the specific category groups and categories to browse using the drop down menus in the Category Group and Category fields Searching for Image Files Based on Annotation You can locate images in a DMS database by performing a search based on the image file s annotation To search foran image file based on its annotation 1 From the softWoRx main menu select File Open When the Open Image click the DMS Database tab window is displa Fies l OMS Database lt lt lt Disconnect from the Database yed Created Acquired Unknown search Annotations For Fand memories kast Modited Available Images 01 09 07 15 26 Drosophila Embryo Movie_d3d dv File Size 30 63 Mlb Image Info 7 5 We T 49 2 Use the drop down menu in the Database View field to select Search Annotations 3 In the Search Annotations For field enter the annotation for the desired image file and click the Search button No
65. alee Mean Sa ania oo tance teenie tea sbats 35 Converuine OTK PUSS ssania e r 36 04 720103 000 Rev C 1008 softWo Rx Imaging Workstation User s Manual Converting TIFF Images You can convert a TIFF image file or series of TIFF files to a DeltaVision file format When converting a series of TIFF files the files are converted to a Z section stack DeltaVision expects each TIFF file to be 16 bits of grayscale data representing a single wavelength If you have a multiple wavelength data set you will need to create single wavelength DeltaVision files and merge them using the Copy Region or Image Fusion tools When converting TIFF files to the DV format you will need to provide pixel dimensions and a wavelength value for the output file Other information may be added or modified using the Edit Image Header utility You can also use this utility to reorganize the description of the data as a series of time points instead of Z sections To converta TIFF image file to a DV image file 1 Click Conversions Import from TIFF in the softWoRx main menu The Convert TIFF to DeltaVision dialog box is displayed TIFF Input File Selection DeltaVision Output Options Filter Output Directory data1 fr r ie Tem Output File Directories Files Pixel Spacing X Y Z ih 0000 i1 0000 11 0000 Ti ra 5 1 il j ef Wavelength jo ForCarl lost found Stack Of Images Delete l 7 Reverse Clear
66. alization Analysis dialog box File Graph Point Labels Help Image Nuclear Pore d3d C4C dv Pearson Coefficient of Correlation 0 7776 14000 12000 10000 aop 000 Wavelength 328 4000 UU Si a ag ge ag gg 0 000 4000 gog agg 10000 Wavelength 617 04 720103 000 Rev C 1008 16 Other Applications This chapter shows how to analyze experiments that are performed with the optional Quantifiable Laser Module QLM Contents in this Chapter POOUL PP INOLO KU SULCS ni Uda ac nores otal telsa E ie uiea ies avec says uae eaieande asa 203 Analyzing Fluorescence Recovery After Photobleaching 0 cece eeeeeeeees 204 Analyzing Fluorescence Resonance Energy Transfer cceceessceseseseeeseeeseeeseeesees 208 About Photokinetics Photokinetics refers to the reactivity of fluorescent molecules while they are in the excited state Photokinetic reactions can be used to study the interactions of molecules within living cells Photo bleaching FRET and photo activation are examples of photokinetic reactions The following table shows photokinetic experiment methods and which biological applications can be studied with those methods 04 720103 000 Rev C 1008 Chapter16 Other Applications 207 Photokinetic Methods and Applications Photokinetic Methods FRAP Single Point and Multi Biological Applications Affinity Biomolecular Cycling Biomolecular Point Pattem Bleaching FLIP Background
67. annel Probe Channel 2 Open the Donor and Acceptor images in Image windows 3 Drag the Image window number icon from each of the Donor and Acceptor Image windows to the appropriate Donor and Acceptor Window fields in the FRET Analysis dialog box to connect to these windows 4 Validate that the Probe Channel for the Donor Control Crosstalk is set to the correct Donor channel and the Acceptor Channel for the Acceptor Control Crosstalk is set to the correct Acceptor channel 5 Validate that the FRET Channel is set to the correct channel for each control image 6 Specify the background for the Probe and FRET channels of each image using the Get buttons The Get functions average intensity values in a box specified in the Box fields while you drag the cursor around in the Image window with the left mouse button held down Click the Get button a second time to disable the Get function 7 For each image define ROIs using the tools on the toolbar at the top of the FRET Analysis dialog box to define representative areas where FRET would occur if these were Experiment images 8 Click Calculate Crosstalk to analyze the background and defined ROIs to generate a crosstalk factor for both the Donor and Acceptor control images Typical Donor Crosstalk factors are 50 70 typical Acceptor crosstalk factors are 15 30 if using CFP and YFP and the FRET pair The Donor and Acceptor crosstalk factors are
68. apter 2 Corecting Images 19 Correct Image Aar AUEREN Anea e EREA Lae e EEE fe Hes pias Wavelengths A d m Correction Options W Normalize Intensity W Use Photosensor W Correct Bleaching W Replace Lines W Smooth Lines Camera Intensity Offset 50 Pass Waves Unprocessed _ ae Run Options id show image when finished Done Sears 2 Enter the original _R3D dv image file in the Input field 3 Use the Correction Option toggles to select the desired image correction See the softWoRx online help for a description of each of the Correction and Run options available from this dialog box 4 When you are satisfied with your selections select Do It to perform the correction process Details of the process are displayed in a Correct Image Output window Equalizing Intensities in Time lapse Image Data Use Equalize Time Points to choose a reference time point and normalize the intensities of all other time points to it The tool uses the mean image intensity to help generate a more uniform intensity display A Note In general the data generated by the Equalize Time Points tool should be used for display only and not for quantitative pumoses The Correct tool and the Correction options of the Deconvolve tool make corrections to the intensity values of sections within a Z series and with the exception of photo bleaching inconsistencies can also make corrections to intensity values between time points of
69. ata and prepare it for presentations Viewing Image Data Open data files in softWoRx and adjust the way that the image is displayed e g display a scale bar set grayscale or color modes or adjust brightness and contrast You can also rotate or resize image data or view data cross sections Viewing Projections and Volumes Render volumes and create 2D projections to visualize and explore three dimensional data Several methods for rendering volumes that you can 04 720103 000 Rev C 1008 softWoRx Imaging Workstation User s Manual interactively rotate are available The 2D projections quickly combine information from multiple Z Sections into a single section Viewing Movies Create movies of volume rendered data or time lapse data You can also create movies to trace particle movement Filtering Choose from several filters to improve the visual presentation of data prepare data for modeling or for other types of analysis You can use statistical filters that are useful for removing noise from the image threshold filters and convolution filters Saving Exporting and Printing Save and present data in a variety of formats Export images to PhotoShop or JPEG formats or save image data in a DeltaVision file a TIFF file or a tabular format that can be opened in a spreadsheet You can also save time lapse or volume rendered data as MPEG movies All files can be archived to CD or DVD If your system is configured with a printer y
70. atic Abenation Use the Chromatic Aberration Corrector to adjust channels relative to each other This tool allows you to correct for shifts in color that result from oil matching and other environmental and optical conditions To conect Chromatic abenation 1 Choose Measure Chromatic Correction from the softWoRx main menu to open the Chromatic Aberration Corrector Qa chromatic Aberration Corector e e a File Edit Help Window i Image Profile X Z Z shits o o po wi la Lj a iea dk am EE E a v oO p Y Value 157 Use Arrow buttons to shift current channel lt 2 Select a multi channel Image window to reference 3 In the Image Profile field choose X Z or Y Z as the vertical profile to inspect 4 Drag the yellow line in the Image window to adjust the X or Y position of the profile 5 In the Chromatic Aberration Corrector use the colored toggle buttons on the left to specify which channel you wish to adjust relative to the others 6 Click the up and down arrow buttons on the Chromatic Aberration Corrector to move the selected channel up or down relative to the others 7 Select File Save Image with Corrections f Note Forcorecting chromatic aberation introduced by the optics it is recommended that you make the measurement using a multi colored bead so as not to biasthe data Measure the offset using the bead then apply the same corectionsto actual sample fi
71. ation 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 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 04 720103 000 Rev C 1008 softWo Rx Imaging Workstation User s Manual File View Options Calibration Help Acquire Experiment Excitation 430 10 Settings Emission cP 470 30 1 plocki a EX Shutter EX Exposure 1 000 Find Calibrate Image size 512x512 Lens ox Info Bin ia 1 Aux Mag Pixel size 0 6680 um Chee arr da 10 00 a A dZ foso dY no oo id al Y a The Resolve3D Settings window is displayed 2 In the Resolve3D Settings window click on the Misc tab Resolve3D Settings Display imaging l Files l Autofocus Mis Git Stage Motion Misc tab Wo Allow Lost Motion Compensation LMC stage View Options W Show stage trails Wo Show stage thumbnails W Show point numbers Filter Wheel Sets Excitation filter wheel Standard Emission filter wheel Standard Activate Filter Sets lt lt lt Pending Activation Done save Settings 3 In the Resolve
72. ault Scale Help ppliedPrecision Chapters Viewing Image Data 99 op To change the intensity scale distribution click anywhere in the histogram except on the handles and drag the mouse up or down W 1 Window cell_210C3_ r3d dv File View Options Tools Pad Scale Image Window 1 Wave w 457 528 v 617 Display Min Max Exp 485 33 3297 64 0 4d SS Wave Min Max 22 00 4095 00 n section Min Max Mean 23 00 4095 00 304 88 W Show Histogram W Log Apply To Wave L N TN O hatia iaci Copy Scale Paste Scale Done Revert Restore Default Scale Help Dragging the mouse up ordown in the histogram changes the scale distribution Tips 1 You can improve contrast at the low end of the intensity range by reducing the gamma value To improve contrast at the high end of the range increase the gamma value 2 Another way to scale the image isto enter values into the Min Max Exp fields 3 You can restore all of the default values by clicking Restore Default Scale To change the intensity scale forall open image windows 1 04 720103 000 Rev C Select View Scale All Windows from the softWoRx main menu The Scale All Windows dialog box is displayed fea Scale AllWindows Channel ar Display Min o oo Display Max f 0833 00 Display Exp n on Done Restore Channel Restore All 1008 100 so
73. ave 1 Scale oo 255 010 O Wave 2 Scale on 255 0 1 00 Wave 3 Scale joozsso1og OOO Wave 4 Scale o0 255010 O m Yiewing Parameters Method RGB Onpacity 1 VolPaok non Quality Best Original Size Z Resolution Best Progressive Threshold 20 Movie Options Start Angles XYZ fi7eg7 1 401 00 Number of Projections fe Rotation 180 Around status B Sg Done Do It premuti Interactive Help 7 Drag the Image window number of the blended image to the Input field in the Volume Viewer dialog box 8 To select which data to view click Select Region and drag the mouse across the Image window The area inside of the rectangle that is displayed as you drag the mouse is the region of interest Scroll through the Z sections to insure the region of interest includes all the image information you want to include in the data set ppliedPrecision Chapter 9 Viewing Projectionsand Volumes 135 5 Window CHO Mitosis BLN dv unsaved File View Options Tools erie 1 1 5 Z Py j Section 23 Zoom 1 43 9 In the Viewing Parameters section of the Volume Viewer dialog box select RGB Opacity as the Method option and adjust the Movie Options 10 Click Do It to create the RGB opacity volume hia 3 Window CHO_Mitosis_BLN_VOL dv unsaved File View Options Tools j Section 4 Zoom 3 47 04 720103 000 Rev C 1008 136 softWo Rx Imaging Wor
74. ayed in the chosen output window bed 2 Window Echelon01_R3D_PRJ dv unsaved File View Options Tools 10 00 38 40 820 Zoom 4 32 04 720103 000 Rev C 1008 42 softWoRx Imaging Workstation User s Manual Selecting Inegular Data Regions Use the Edit Polygon tool to select irregular data regions This tool is applied differently than the tools for selecting rectangular regions While selecting rectangular regions is usually used within other tools the Polygon Editor stands alone You must use the Polygon Editor tool with Cut Mask if you want to then apply another tool only to the selected region To selectan inegular data region 1 Open the image in the Image window iw FE Window cell_Z10C3_r3d dv File View Options Tools a is i 725 Ms ll on 457 108 ja File Edit Options Statistics Window 1 Polygon e oi oj Oo Active Wave Mode ONE fr d Guided Mode Seipeted Template Mode pplied Precision Chapter 5 Data and Task Manipulation 43 3 Choose a selection tool e g Sh from the Edit Polygon menu Then press and hold the left mouse button to draw a polygon around the area of interest within the Image window W 1 Window cell_Z10C3_ 13d dv wx File View Options Tools Help 2 H B TE a von oo Pd KE 457 105 ja j gt aa MI Il on 4 To copy this region across wavelengths tim
75. bar changes interactively as you set these properties 6 Select the Move button and use the mouse to drag the scale bar to any position within the Image window Resizing or Reonenting an Image You can use the Resample2D tool to resize an image or to reorient an image in X Y and Z directions Resizing an Image When Resample2D magnifies an image it interpolates values to add pixels to the image When it reduces an image it combines or eliminates pixels to create a subset of the original pixel data To resize an image 1 Choose View Resample2D from the softWoRx main menu to open the Resample2D dialog box W Resample 2D X f Output 3 ha MWesigacsh fSesesiveys ener ret et ae ASF INANEM t x lt Wavelengths J J Z Rotation 0 0 Shift In xY 0 0 0 0 Angle Between Axes 90 0 Magnification In XY 1 000 1 000 xY Reduction Factor 1 000 1 000 Output XY Size 0 0 Keep Cell Dimensions Dore 2 Enter the window number in the Input field ppliedPrecision Chapter 8 Viewing Image Data 107 3 To resize part of the image click Select Region and then drag the mouse across the area in the Image window 4 1 Window DMS imageTest_R3D_NND dv File View Options Tools 2H 6 v a E 528 148 Selecta region in the Image window 4 To magnify the image enter a magnification factor in the Magnification in XY field 5 To reduce an image enter a
76. base 74 integration 73 84 04 720103 000 Rev C 1008 225 uploading images 74 81 Document conventions ii DVD discs 157 59 Edge enhance 139 40 Edge objects excluding in 2D Polygon Finder 186 Edit polygon tools 182 85 Edit Polygon tools for gathering statistics 41 44 Enhance contrast 143 44 Equalize intensities See Equalize time points Equalize time points 18 19 20 Experiments FRAP 204 8 FRET 208 13 Explorer 5 Exporting DeltaVision files 151 56 to JPEG files 155 to Movie files 153 55 to PhotoShop 153 to TIFF 151 Files archiving 157 59 data 148 exporting DeltaVision 151 56 formats of 31 graphic 148 saving as JPEG 155 saving as movie 153 55 saving as TIFF 151 saving DeltaVision 148 51 Filter2D 140 42 Filtering images 137 45 convolution 138 39 enhancing boundaries 139 40 removing noise 140 42 softWokx filters 138 Filters activating multiplexed sets 63 67 Fluctuation of intensity 219 Fluorescence recovery after photo bleaching See FRAP Fluorescence resonance energy transfer See FRET Fluorescence punctate 15 FRAP 204 8 FRET 203 208 13 analysis tool 208 13 analysis workflow 209 analyzing results 212 calculating 211 calculating crosstalk 209 efficiency statistics 212 prerequisites 209 Graph ratio 62 Grayscale 100 about 99 assigning from blended color mode 102 colormap 100 Hide border tools 103 Hide channel 95 Histogram 163 65 Image arithmetic 142 43
77. cal file system to the DMS database in a number of ways You can upload image data e Directly from an Image Window e As part of a Task Builder processing chain e As part of an experiment in which the data is auto uploaded at the conclusion of the experiment Each of these methods is discussed in the following sections Uploading Images from the Image Window The simplest method for uploading image data to the DMS database from your local file system is to upload the data directly from the Image Window To upload image data from the Image Window 1 From the softWoRx main menu select File Open When the Open Image window is displayed click the Files tab and select the appropriate directory pplied Precision Chapter 6 DMS Integration Created Acquired 01 09 07 15 26 Last Modified 01 09 07 15 26 File Size 27 45 Mb Image Info 7 48 W 3 T 1 75 Files DMS Database Directory fdatai dvsamples ea 40 _135 ott AF RID dv AF testid 23 RD DJD dr Drosophila Embryo Movie _d3d dv LiveCells GFP_20 Movie d d dyr Nikom 000Z 140 ott Nuclear Pore _D3D dv Nuclear Pore DID FRET dr Nuclear Pore daa OO O O OO O OOOO O Nuclear Pore d3d CLC VOL dy Nuclear Pore_d3d_CLC_VOL_COR_log txt Nuclear Pore d3d DID dw Nuclear Pore_djd_D30_COR_log txt Nuclear Pore _d3d_D30_cmd sh Nuclear Pore d3d DID log txt Nuclear Pore_d3d_process sh l Nuclear Pore d d task sh File Name Nuclear Por
78. cccccccsssssssscccccccesesssececcscsssseessessssseseeeeeseseesess 5 sof WORX EXPIOTE E ganini EA E OAO ORRE 5 Part One Processing Data sessssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 7 1 Deconvolving Image Data sssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn D About DecOnv olin TrocesSBINE sessidaciirrre ina a E A 10 DPeconvolunon l oo aseran nE E E E Gia endeared 10 POCO VOLS an Ima eei E ET O 11 Deconvolvine Several VG OCS susan vas e E eines de acer engi aura ranean 13 Common DECONVOUTOMO DUO MS ixixesined25205 aah T provera nosbrendsedeesnecsa yes 14 More about the softWoRx Deconvolution Tools cece ceseesseesecsseceseseseseseeeseseaeeeseens 16 2 COMECUNG Mage Saanaa aaa LA About Corrects Image S aneii a ri T E A T E A A 17 Correcine Z Secon limar e Daliesin N O E 18 Equalizing Intensities in Time lapse Image Data seeseeeeesesseeseereererseseereereererseserseeseeseee 19 Calibrating Mace Dalir E N TONG 20 Alcnne Adjacent Images scx ciaacicr ea e Gawd A TA N E ee 21 Correcting ChromaUcADernrahON sirene e E N N S 23 04 720103 000 Rev C 1008 softWoRx Imaging Workstation Users Manual 3 Stitching Images Ihat Have aSingle Z SCCH Om sii eisiaa a ES 26 Stitching Images That Have Multiple Z Sections sre eee eee cee eeseeeeeeseeeseesesseeeseeeees 27 Importing Dat ssssrmiiinnnuisannanninnnnnanaan anaa L Conver tie TIEF Ma CS aasan N A E E 32 COM VET GSC FICS ies costs tte seawicieed sete euneaetnadat sus anton T
79. cctsityacssniencsavesuersunasacsuetedasbaepueccsiessosaes 77 Auto uploading Images after Acguisilons sunrc A 80 Downloade Files Tom DMS euie e aE E T O uaeaseatads 81 Browsing and Locating Images in a DMS Database sessessessesssessesrsseseesrsreseeseseeseeseeses 82 Browsing Image Files using Py D L Hierarchy enasna ie e R issues 82 Browsing Image Files using CG C I Hierarchy sessesseeeeeeererererisesrrrrresrrrrsrrrrrrresess 83 Searching for Image Files Based on Annotation cece cee eeseeeseceeceseeeeeeseeeseeesees 84 pplied Precision Contents Part Two Visualizing amp Presenting Data 00s 0010001 8D 7 Viewing Image Datad ssccsssssssssscsssssesnsssennssecnsssennsssensssesnsssessesO7 CPT AS Geaen desea sess sages so aisaat vce E ioe voocn sane T O 88 THe lmnage W ndower earners cries wee Barer mr E 89 VIEWING OL TIM SCS e aR EN TTE E OEE 90 Viewing Z Sections anid Mme PolMSresrrirra t in aa E ages 90 WIC WINNS AICI S nura T E O E OE 92 Display ine or Hidinge Channie busnes e a a Aa 94 Adjusuno Brie htiess and Con aS oen a n E E OE 95 Assigning Colors or Grayscale to Channels ssessessessseeseereerseeseseeseserseesreseeseeseseeseeseeses 99 FAY SCA VOC ios sot n E N Sowemamastamhiateenelt 100 Color VOC ontrack al anctasalieb oud laa e a staan tae loacloanvaebod dance 101 P ended Clor Mode iis5 Sires Bese tas san tt E sata vaca tieateaane tte tease 101 Controlling the Image Window Display vices sce
80. ce eeeeseeeeeeeeeeeeeeeeees 19 Calibrating mace Da taycistcai nici a cashes screen cusiaae taeda cseetessneasan eases 20 AMMI Adac n NA ES E E R 21 Corecine Chromatic A Derr at ON sisi tecscn oes T E E E eoanmiacranies 23 About Correcting Images Conect image is used to correct errors that are caused by photo bleaching inconsistent illumination intensity or CCD defects The Correct Image tool corrects the intensity values of sections within a Z series With the exception of photo bleaching the tool can also correct intensity values between time points of a time lapse experiment By default softWoRx automatically applies this tool during the deconvolution process See Correcting Z Section Image Data on Page 18 04 720103 000 Rev C 1008 18 softWo Rx Imaging Workstation User s Manual Equalize Time Points equalizes intensities of all time points to a reference time point that you select The tool uses the mean image intensity to help generate a more uniform intensity display You can use this tool to normalize time lapse data for display purposes In general the data generated by this tool should be used for display only and should not be used for quantitative purposes See Equalizing Intensities in Time lapse Image Data on Page 19 Calibrate calibrates a raw image when you have a calibration file and a bad pixel file that applies to the camera and conditions array size pixel size and wavelength that were used to collect the image
81. ct image of two channels after subtracting a threshold value for each Then a scatter plot of the results is created and the Pearson Coefficient of Correction is measured To use Image Colocalization 1 Open the image to analyze in the Image window File View Options Tools Help v Oly 7 Z 16 Zoom 2 00 528 411 f gj The Nuclear Pore image displays two proteins channel 528 istagged to a protein that regulates the gateway to the cell Channel 617 isVOM an HIV protein 2 Choose Measure Colocalization from the softWoRx main menu to open the Colocalization dialog box and enter the number of the Image window that you want to analyze in the Input field fi Output 2 Select Region Reset Details Current Section Only Input Channels Threshold Box Plot Min Plot Max 617 ooo Get p ooo 10833 00 528 fono Get p 0 00 f 4641 00 Pearson Coefficient of Correlation Done Do It 04 720103 000 Rev C 1008 198 softWo Rx Imaging Workstation User s Manual 3 To analyze a region of a window click Select Region and select an area in the Image window by dragging the mouse across the area Adjust the rectangle you ve created until it contains the desired area Then click outside the Image window with the mouse File View Options Tools Help 2 2 2 al Z xila Z 14 Zoom 2 00 617 646 pe Pe Click Details to open the Region Details dialog box Specify the ranges o
82. d 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 Acknowledgements Applied Precision would like to thank Adrian Quintanilla and Dave McDonald of the Fred Hutchinson Cancer Research Center for providing a data acquisition workflow and other content that is used in this manual 04 720103 000 Rev C 1008 Intoduction This introduction provides an overview of how you can use softWoRx to process visualize and analyze multidimensional microscopy data It also introduces optional softWoRx components In the Intoduction NWN TVA TS SO WORN SEE AE TE T sae an Saat tog a bane EE E T 1 What Cam You Use sop WORS IOT x sects supa iataicsal it aew eta E O EE E 2 15 0 ave UE G0 bale 6 ates gi cclemremer meant E N ater em Ey EN PEE oy nem eran 5 What is softVWoRx softWoRx is a comprehensive software package designed for the analysis of multi dimensional microscopy data Although originally developed for use as a component of Applied Precision s DeltaVision Restoration Microscope System softWoRx is now also available on a stand alone analysis workstation giving you a powerful yet friendly environ
83. drop this point to change the Shape of the polygon Connects the last point selected with the first point in orderto close the polygon Allows you to define a circulararea Allows you to define a rectangular area Deletes the selected or most recently added point Deletes the selected polygon ppliedPrecision Chapter14 Volume Modeling 187 polygon Fi Delete all polygons Deletes all polygons Copy polygon for Allows you to selecta polygon to copy pasting Paste polygon Pastes the polygon that is selected for copying Undo last action Restores the last deleted object or moves the object to its former position To create polygons using Snap Selected in Polygon Editor 1 Open the desired image data file 2 Click Model Edit Polygon in the softWoRx main menu 3 Drag the Image window number to the Window field 4 Click the Add Polygon Q button 5 Click points to form the polygon To connect the last point with the first click the Close Polygon E button 6 Select Guided Mode 7 Select Options Guided Mode Options on the Edit Polygon dialog box The Guided Polygon Select Parameters dialog box is displayed fed Guided Polygon Select Parameters Guide Method Maximum Gradient Guide Box 7 Weight Center of Box More W Guide Thresholds 04 720103 000 Rev C 1008 188 softWoRx Imaging Workstation User s Manual 8 Adjust the values in the Guided Polygon Select Parameters dialog box For more
84. e CCD camera elements Deconvolution Holes A bright ringed hole in background areas of the image is probably a result of subtracting too much background intensity during deconvolution The deconvolution hole is the edge of the region that has reached the minimum possible intensity typically 0 Deconvolve the image again using a background subtraction of 0 rather than the default value Increasing the Z Transform Size can also help control deconvolution holes In some situations the measured data may actually have a low intensity threshold caused by the loss of detector response In this case it is not possible to simply add intensity to the image The edge of the low intensity threshold is indistinguishable from the edges being sought by the deconvolution process Poor Z Resolution Elongated blurring in the Z directions is characteristic of spherical aberration Also note that the football shape along the Z axis is normal for an optical microscope The conical extensions above and below an object represent the uncertainty involved with measuring light through a lens with less than a 90 degree cone angle At present the best available lenses have a cone angle of approximately 68 degrees Information between 68 and 90 degrees is not measured by the lens 04 720103 000 Rev C 1008 222 softWo Rx Imaging Workstation User s Manual Section Intensity Huc tuaton Since each optical section is measured separately it is common for the
85. e CD Kreator Eile Project Plugins Tools settings Help aeu en 99 ie Nuclear_Pore_d3d_D3D dv Nuclear_Pore_r3d D3D_Jog t bom ea Movie_d3d dv Nuclear_Pore_d3d_D3D_log txt Nuclear_Pore_r3d dv gt LiveCells_GFP_2D_Movie_d3d dv lt Nuclear_Pore_d3d dv Nuclear_Pore_r3d_process sl Nikon100X_140 otf mi Nuclear_Pore_d3d_process sh Olympus X70_60X_140 otf amp Nuclear_Pore_d3d_CLC_VOL dv Nuclear_Pore_r3d_01_D3D dv oocyte otf Nuclear_Pore_d3d_D3D_cmd sh Nuclear_Pore_r3d_D3D dv lt gt oocyte r3d 9K3b data project gt Spindle_Green_r3d dv DeltaVision Image 4 0MB_ datal dv_samples Spindle_Gre y oocyte r3d DeltaVision Image 32 0 MB datal dv_samples oocyte r3d lt Nuclear_Pore_d3d dv DeltaVision Image 27 4 MB datal dv_samples Nuclear_Po vailable 639 5 MB of 7 RENE Temp 25 3 GB 31 3 GB K3b 0 11 14 5 When you are satisfied with your selections click the Burn button in the bottom right corner of the window The Data Project K3b dialog box is displayed 04 720103 000 Rev C 1008 159 160 softWoRx Imaging Workstation User s Manual iv pete Project K3b Data Project size 59 4 MB Burning Device sat As Options C Simulate On the fly C Only create image Dis C Verify written data K3b Defaults User Defaults Save User Defaults 6 Click the Burn button at the top right corner of the window to begin the CD creation process For
86. e and green colorsto asmany asthree channels You can assign other colorsto two channels Blended Color Mode In Blended Color Mode you can assign any color to each channel You can view up to five channels as separate colors You can also assign grayscale as a color this is useful for DIC data You can select an arbitrary color for each wavelength or you can specify to use the true color that is normally associated with each wavelength in the color spectrum To set Colors in Blended Color Mode 1 On the Image window menu choose View Blended Color to set Blended Color mode 2 Choose View Select Blended Colors to open the Blended Colors dialog box ppliedPrecision Chapter 8 Viewing Image Data 103 fed Blended Colors Channel 1 Z d ctr N Copy Paste True Colors Done 3 To specify the colors normally associated with a wavelength click True Colors 4 To specify a custom color for a channel click the color under the channel to open the Choose Color for dialog box and use the Red Green and Blue sliders to choose the color ac hoosecolorforChannel3 Current Color stored Calor Green 0 68 Blue 0 00 Apply Cancel Choose Color forthe selected channel To assign grayscale to a channel in Blended Color Mode 1 On the Image window menu choose View Blended Color to set Blended Color mode 2 Choose View Select Blended Colors to open the Blended Colors dial
87. e d3d du OK File Types p Cancel C A 2 Select the file you want to upload and click OK The Image Window is displayed with the file you selected wv i Window Nuclear Pore d3d dv File View Options Tools Ea ps 3 From the Image Window select File Save to DMS The Save To Data Management System menu is displayed as shown 04 720103 000 Rev C 1008 76 softWoRx Imaging Workstation User s Manual f Save To Data Management System eee E File data1 dv_samples Nuclear_Pore_d3d dv DMS Database Connection lt lt lt Disconnect from the Database Destination Parameters Category Group NONE o New Category NONE New Initial Annotation Optional From this menu select the Project and Dataset to which you want to save the data on the DMS database You can optionally select a Category Group and Category for the saved image data and add initial annotation to the file for future reference When you are satisfied with the image data to be uploaded click Do It When the upload is complete Finished is displayed on the status line as shown vE To Data Management System File datat dv_samples Nuclear_Pore_d3d dv DMS Database Connection lt lt lt Disconnect from the Database Destination Parameters Dataset KP_Dataset New Category Group KP_TestGroup New I
88. e file You can simultaneously view a graphical image a table of intensity values a 3 D graph and a histogram of intensity values With these views open you can select various regions of interest ROIs to explore the data As you select an ROI the data in each view is updated for that ROI ppliedPrecision Chapter 12 Examining Intensity Data 167 1100 1200 To open the Data Inspector tools 1 Open an image file in the Image window 2 Choose Tools Data Inspector on the Image window to open the Data Inspector window 04 720103 000 Rev C 1008 168 3 4 oF softWoRx Imaging Workstation User s Manual File View Options Current Window E 528 w 617 w 685 select Box Columns Rows 19 Ing iE Constrain To Circle Ratio show 30 Graph Histogram Mati Gap Statistics 84 109 197 391 4 23016 n 361 Total 35158 moa 97 101 100 99 98 101 102 9 X 103 103 100 102 102 102 101 103 10 97 103 102 102 98 93 94 102 10 97 94 100 95 93 95 94 O a 93 95 33 101 94 95 97 o 94 y 96 96 98 95 98 95 90 95 96 95 93 99 98 99 97 95 95 97 95 95 90 100 94 93 94 a 95 94 94 98 91 96 i 90 92 96 95 94 94 102 91 92 92 90 91 95 94 977 94 92 92 90 92 94 97 92 g2 93 93 92 93 3i 33 89 In Data Inspector click Show 3D Graph
89. e microscope a DeltaVision RT microscope or a microscope that is upgraded to the DeltaVision 3 9 level If your system has the QLM hardware module you can use softWoRx to analyze Photokinetic photo bleaching and photo activation experiments The softWoRx FRAP analysis module discussed in Chapter 16 is only available for systems that have OLM hardware softVWVoRx Explorer softWoRx Explorer is a cross platform image viewer 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 spectrum i e channels or fluorescent wavelengths can be hidden or displayed in a variety of colors 04 720103 000 Rev C 1008 softWo Rx Imaging Workstation User s Manual pplied Precision Part One PROCESSING DATA You will typically need to process data before you view or analyze it Part one includes instructions for processing and importing data In Part One Chapter 1 Deconvolving Image Data ssssssssccssssssseeeesssessseseessnnssseeeessnesseesenens 9 Chapter 2 Conec ng IMAGES ssssssnnnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 17 Chapter 3 SME NNO seica a aaa a aaan iiaa aana 25 Chapter 4 Impor ng Data isis ssiscisdisvtennnuciaassvastistevucv
90. e name 003 my file name 004 Any of the above files could be entered into the See File option as the file to MU convert The use of wildcards such as is not supported To convertan ISee image file to a DV image file 1 Click Conversions Import from See in the softWoRx main menu The convert Inovision See to DeltaVision dialog box is displayed as shown in Figure 1 2 Select the file that you want to convert using the See File button and data entry field 3 Type a filename for the converted file into the DV File field 4 Enter the wavelength of the data to be converted usually in nanometers into the Wavelength field 5 Enter the X Y and Z spacing of the pixels in the data set usually in microns into the appropriate fields 6 Enter the correct lens number in the Lens ID field 7 Click Do It pplied Precision Chapter 4 Importing Data 35 Converting Pic Riles The Pic conversion tool is used to convert BioRad MRC 600 Pic images to DeltaVision tormat The deconvolution program and other soffWoRx software rely upon the presence of accurate wavelength and pixel spacing Not all Pic files contain accurate pixel size and wavelength information so it may be necessary to manually enter values in the Pic Conversion fields Note The image wavelength fora DeltaVision file indicates the wavelength of the light imaged by the camera rather than the illumination wavelength X Y pixel sp
91. e points or through Z sections choose Edit Propagate Polygons from the Edit Polygon menu and enter the appropriate ranges Pro pogate Po hyqons Polygon Source Current Section From a To set All E is gt W Zi 10 Time i gt W Time i 1 Wave i gt W Wave Fr JJ Close Dot Help Q Tip you can also select time points You can select ranges or you can enter selected points e g 1 3 5 20 25 5 Click Set All if you want to copy the polygons through all of the Z sections time points and wavelengths Click Do It to copy the polygons Then view the range of Z sections or time points to make sure that all of the data is included 04 720103 000 Rev C 1008 44 fm W 1 Wind softWoRx Imaging Workstation User s Manual in the polygon for each section Use the channel selectors to view the polygons for each of the selected wavelengths ow cell_Z10C3_r3d dv File View Options Tools Help EREE KEA J0 R7 N mN E en DX lN Tee D W i Window cell Z10C3_r3d dv File View Options Tools 2H E a a UT io io Ko o Zofi Kn IMs EE de 617 500 ja In this example all of the data foreach ofthe selected wavelengths and between the selected cross sections is within the polygons foreach Zsection a Q Tip You can chan
92. e you are satisfied that all parameters are set up correctly click Generate FRET Results Image When this process is finished the image opens ppliedPrecision Chapter16 Other Applications 215 Analyzing Results Analyzing Net FRET and FRET Efficiency statistics involves specifying a FRET Results Image and one or more ROI polygons to generate a table and graph of statistics To analyze FRET results 1 Choose the Analyze Results tab of the FRET Analysis dialog box Calculate Crosstatk Calculate FRET Analyze Results FRET Results Statistics FRET Results Window f Pigi Results Number of ROIs lp Copy Through Tine ID Z Time Pt Time RET Min RET Max Specify a FRET Results Image Window 2 Ifitisn t already being viewed open the FRET Results image that you would like to analyze in an Image window 3 Ifitisn t already specified drag the window number of FRET Results Image window to the appropriate Input field in the FRET Results Statistics area 4 Use the ROI creation tool icons at the top of the FRET Analysis dialog box to specify one or more regions of interest 5 If you have an image with multiple time points or multiple Z sections you may want to propagate ROI polygons through time or Z To do this create a polygon and make sure it is selected Then choose Copy Through Time or Copy Through Z to propagate the polygons As change
93. ection Deconvolve Projections Run Options More Options W Show image when finished Done 2 Enter the original R3D dv image file for example usr local softWoRx data samples oocyte R3D dv in the Input field You can do this in the following ways e From the Linux file Manager drag and drop the file in the Input field e Click Input and browse to the file e Type the path and file name into the Input field softWoRx creates an output file name by appending the _D3D extension to the input file name The new name for example oocyte _R3D_D3D dv is displayed in the Output field 3 Enter the otf file for example the oocyte otf file in the usr local softWoRx data samples directory into the OTF File field You can use the same methods drag and drop browse or type to enter this file as you used in Step 2 For DeltaVision files this is done automatically Note The otf file isan Optical Transfer Function OTF file In many microscopy systems there is only one OTF per objective lensand the conect OTF is simply the one that comespondsto the lens used for measunng the optical 04 720103 000 Rev C 1008 12 softWoRx Imaging Workstation User s Manual sections Refer to the lens identification number in the measured data and the OTF to venfy that the corect OTF is being used Click Do It Input EOS samplesfoocyte RaD dv Output idatatidy_ samples oocyte RED Osb dv select Region esej De
94. ential number is added to the prefix to create a unique name for each file 4 To set the delay between when the image is selected and captured move the Capture Delay slider 5 Set the JPEG slider to the right to increase the quality of the screen shot Higher quality results in larger file sizes 6 Choose whether to save a window or the entire screen 04 720103 000 Rev C 1008 158 softWo Rx Imaging Workstation User s Manual 7 Click the window or screen to capture the image 8 To view the screen shot click Display Last re Tip You can also open Snapshot from the Image window the 3DModel window the Chromatic Correction dialog box and the Orthogonal Viewer window Archiving Files to CD DVD You can open the Linux K3b CD Kreator tool directly from the softWoRx main menu to archive your softWoRx files to CDROM or DVD discs This tool supports 650 or 700 MB capacity CD RW discs and 4 3 GB DVD discs To copy files to a CD using K3b 1 Place a blank CD into the CD drive 2 On the softWoRx main menu choose Utilities Archive Data to CD to open the K3b CD DVD creation window iw Pets The CD Kreator Eile Project Plugins Tools Settings Help OO 9 SONY CD RW g Desktop f Home softworx logs g Desktop SW ScreenShots _ jsoftwomtogs Resolve3D exp o EBSW ScreenS softwon ogs txt Root jj Temp 25 3 GB 31 3 GB K3b 0 11 14 3
95. er points Leap Frog Measure the distance between two consecutive selected points Multiple Measure the sum of the distance between consecutive selected Segment points The following procedures describe the most common measurement tasks performed using softWoRx To measure distance using the Standard Two Point method 1 Open an image in the Image window 2 On the Image window menu click Tools Measure Distances to open the Measure Distances dialog box 3 Enter the desired window number in the Window field 4 Inthe Measure Method list select Standard Two Point 5 Select the appropriate unit of measurement from the Units option 04 720103 000 Rev C 1008 179 180 softWo Rx Imaging Workstation User s Manual 6 Select two points using the mouse The distance will be displayed in the gray information window To measure distance using the Multiple Segment method 1 Open an Image in the Image window 2 On the Image window menu click Tools Measure Distances to open the Measure Distances dialog box 3 Enter the desired window number in the Window field 4 Inthe Measure Method list select Multiple Segment 5 Select the appropriate unit of measurement from the Units option 6 Drag the mouse across the image to select consecutive points 7 Click Get Distance to display the coordinates of all the selected points and the sum of the distance in the gray information window Note If an eroroccurs click Delete Las
96. es an approximate deconvolution approach for removing blur from optical sections If you wish to learn more about Nearest Neighbor Deconvolution refer to the online Help The standard Deconvolve tool uses the Constrained Iterative Deconvolution algorithm to remove the out of focus blur in fluorescence optical sections This algorithm calculates a result using the following four steps 1 The algorithm estimates what the object looks like 2 The estimate is mathematically blurred to simulate the effects of the microscope s limited aperture 3 The blurred estimate is compared to the actual image The difference between the images is then used to modify the estimate 4 The modified estimate is constrained to be non negative by setting pixels with negative intensity to 0 The algorithm repeats this sequence of steps until the estimate convolved with the point spread closely approximates the actual image see Agard 2 See Agard D A 1984 Optical Sectioning Microscopy Cellular Architecture in Three Dimensions Ann Rev Biophys Bioeng 13 191 219 pplied Precision 2 Correcting Images softWoRx provides several tools for correcting image data You may need to correct data to prepare it for visualization and analysis In This Chapter POUT C OFC CHINS VAC SS enr nn EE docaeets beuenstedsanetatunar dundee saanensaanavaites 17 C orreching A Secon lima ee Dalies a OE 18 Equalizing Intensities in Time lapse Image Data ce
97. ess sh WOT Gueued spoke_prj_process sh rart ich At Ete i cae Pl Change t Help 7 To start the process chain e Immediately click Start Now e Ata later time click Start Later When you choose this option a set of time option buttons is displayed Set the time at which you want the process chain to start You can use the Change It button if you decide you want to change the time to begin the process chain 8 Select Quit to exit the Queue Manager dialog box 04 720103 000 Rev C 1008 80 softWoRx Imaging Workstation User s Manual Auto uploading Images after Ac quisition To auto upload image data to the DMS database after an experiment is completed use the following procedure To auto upload image data immediately following an acquisition 1 Set up your experiment as normal by clicking Experiment from the Resolve3D window to open the Design Run Experiment window 2 On the Design Experiment tab enter the experiment name and configure the experiment For experiment setup details see the Setting Up and Running Experiments Chapter in your DeltaVision System User s Manual 3 Select the Run Experiment tab and enter the image file name or drag and drop from another location the image title and any annotation you want to add gt Design Run Experiment g g a Zoe Design Experiment Design PK Experiment Run Experiment Image file name 101 Settings DMS Setup Image title Sample
98. esusnsssusanwetinanenucutiseuspussaeanen 31 Chapter 5 Data and Task Manipulation sssccssssssscssnssseesnsssseesensssessensssees 39 Chapter 6 DMS Integration evisssesiscwctitsteccsictastiewaewicvssrawdsnnndushaanabiasatienstactonnceians 39 04 720103 000 Rev C 1008 softWo Rx Imaging Workstation User s Manual pplied Precision 1 Deconvolving Image Data This chapter shows how to use the softWoRx Deconvolve tool to remove blur in fluorescence optical sections You can deconvolve and view a single image or you can set up a queue to deconvolve several images You can also set options to deconvolve a region of an image select which wavelengths to include or select the deconvolution method In This Chapter Converune Wl EPP ide Sen cay ness an teatenat anne ease eeu 32 OTS T SS cre case ects E anaes san oso E oases aos 33 Convertino Pic Files ssxeseicxsccetsa yo arstoentiarmsaesanteaetonem E N E oes 35 Convertino PS isopen E trent comnts 36 04 720103 000 Rev C 1008 softWoRx Imaging Workstation User s Manual About Deconvolution Processing After you acquire images you ll need to process them to remove blurred data The process of relocating signal scatter and out of focus information present in digital images is known as Deconvolution Applied Precision s proprietary deconvolution algorithms preserve the amount of light throughout the entire Z stack Blurred data is not dropped out It is reassigned to its original l
99. f the X Y Z and time data to analyze in the selected region Region Details KAAN idth Height nm iao a2 3s Z Start End pa fa Time Start End Inc i i i Header Label Forexample in this case we are interested in colocalization that isoccuming below the surface of the cell Select which channels to analyze in the Input Channels lists Select a background threshold for each channel by clicking Get and then clicking on a background area of the image The background is an averaged value within a box of the size specified Click Get again when you are finished selecting the background ppliedPrecision Chapter 15 Detecting and Analyzing Colocalization 199 Z 16 Zoom 9 00 528 125 Q Tip You can change the size of the box 7 Click Do It to run the colocalization analysis for the selected data Colocalization Analysis p File Graph Point Labels Help Image Nuclear_Pore_d3d dv Pearson Coefficient of Correlation 0 5737 14000 12000 10000 a000 6000 oO OJ ua ma D T Ti 4000 2000 0 2000 4000 6000 a000 10000 wavelength 617 The Colocalization Graph and a new window with the colocalization data are displayed The Colocalization graph is a plot of the two intensities on a pixel by pixel basis each spot is a pixel The Pearson Coefficient of Correlation indicates how closely the two intensities are colocalized full colocalization is 1 0 and calculates the Pearson Coefficie
100. f you are analyzing a Multi point FRAP data set enter the number of laser sites in the Number of Laser Sites field 5 If you are using background subtraction enter a background value in the Background Intensity field 6 To determine a number to enter in the Spot Radius field use the Measure Distances tool to make an approximate measurement of the bleach spot 7 Click Do It to run the analysis The following files are generated The three channel output image file includes the original time lapse image the ratio of the current time point to average pre bleach time points and the ratio data at the location used for analysis 04 720103 000 Rev C 1008 210 softWoRx Imaging Workstation User s Manual The three channel image generated by the Photokinetic Data Analysis tool A JPEG file contains a recovery graph that is a plot of the fluorescence intensity before and after the event Membrane_FRAPO2_R3D_FRAP dy Fri Dec 19 16 08 52 2003 F 0 0 69 MF 0 71 T 1 2 0 61 I 357 3 Recovery Fraction co 50 0 7 l 0 J 4 J 6 Recovery Time seconds The Fluorescence Recovery Graph ppliedPrecision Chapter 16 OtherApplications A log file contains the analysis results File Edit Plugins Settings Documents Help Ovex Gi el Ff amp SB a 4 New Open Save Close Print Undo Redo Cut Copy Paste Find Exit Membrane_FRAPO2_R3D_F
101. ftWo Rx Imaging Workstation User s Manual Similar to the Image Scaling dialog box the right side of the Scale All Windows dialog box includes a line with three nodes or handles that graphically represent the intensity scale Use the mouse to click and drag the white handles on the line as follows e To change the minimum or maximum scale value click and drag on the left or right handle e To slide the scale range to the left or right click and drag on the center handle As you move the handles the images displayed in all of the currently open Image windows change accordingly Changing the minimum and maximum intensity values changes brightness and contrast by mapping all 256 color shade values to a larger or smaller range of data Data values higher than the maximum value are assigned the brightest shade Data values lower than the minimum value are assigned the dimmest shade To change the intensity scale distribution change the shape of the curve by clicking anywhere on the histogram except on one of the handles and dragging the mouse up or down e Toimprove contrast at the low end of the intensity range increase the slope of the curve at the left side of the graph e To improve contrast at the high end of the range increase the slope of the curve at the right side of the graph Changing the scale distribution increases the contrast at one end of the data range and decreases it at the other end Use the Restore Channel butt
102. g Cancel Job Pause After Job 0 Done Giueued Jobs ID Owner Status Conmand A WOT s queued Huclear_Pore_D3Sb_task sh WOT s Queued Spind le_Green_d3d_task sh T WOT s Queued cell_ 10C3_r3d_task sh x Start Mow Start Later Gluit Delete My Gueued Jobs 6 To start the process chain e Immediately click Start Now e Ata later time click Start Later When you choose this option a set of time option buttons is displayed as shown in the example below Set the time at which you want the process chain to start You can use the Change It button if you decide you want to change the time to begin the process chain pplied Precision Chapter 5 Data and Task Manipulation 57 i Dargai imi Fause After Job O34 Gone Giueued Jobs Job ID Ovner Status command E Delete wor Queued Nuclear_Pore_D3b_task sh ae WwOrS Queued Spind le_Green_d3d_task sh kas WOTE ueued cel _210C3_r3d_task sh k ANT ING At e kie Ph Change g Guit Delete My Gueued Jobs Help 7 Select Quit to exit the Queue Manager dialog box Using Ratio Imaging softWoRx provides a ratio imaging acquisition function that allows you to view a eraphic representation of the ratio of two channels as the images are being collected In addition a ratio graph displays the mean value of an area in the middle of the image vs time Both the ratio image and the ratio graph are for monitoring purposes
103. ge before interactive rotation 3 Click Select Region and drag the mouse across the region of interest 241 Window joembl03_05_R3D dv File View Options Tools 3 bee rSa om J cn HE 59 E Ko mal li co 528 473 4 Click Interactive to open the Interactive Volume Viewer Parameters dialog box ppliedPrecision Chapter 9 Viewing Projectionsand Volumes 131 Oran interactive Volume Viewer Parameters Wavelengths W 528 617 W 457 Current Angles AZN 31 7 26 0 130 0 Reset Resolution During Move Low Done Help 5 Choose which wavelengths to display in the window in the Wavelengths options Q Tip VolPack isthe preferred method for interactive viewing With VolPack you can view up to three wavelengths at a time To use VolPack select this option under Viewing Parameters in the Volume Viewer dialog box 6 Select Low in the Resolution During Move option 7 Drag the cursor on the image to rotate the image to the desired orientation ve Window Interactive Rendering RE 8 Click Done in the Interactive Volume Viewer Parameters dialog box 9 Click Do It in the Volume Viewer to render the volume with the new orientation 04 720103 000 Rev C 1008 132 softWoRx Imaging Workstation Users Manual v 3 Window joembl03_05_R3D_VOL dv unsaved File View Options Tools Tee Section
104. ge selected pointson a polygon using the Z button You can move a selected polygon by selecting it with the tooland dragging itto a new location pplied Precision Chapter 5 Data and Task Manipulation 45 Cropping and Tnimming Data You can selectively crop areas Z sections and channels from data files You can also trim time points from time lapse data Cropping and trimming are useful for presenting data It also helps prepare data for volume rendering 3D rotation modeling and other types of visualization Cropping a Rectangular Region To Crop a rectangular region to a new file 1 Open an Image window From the Image window menu choose File Save Output EEEF of RsD dd select Region Reset Details Wavelengths 525 W 632 _J Scale to Display Min Max Done Do It 2 Inthe Input field enter either a window number or an image file name In the Output field specify an image file or window as output 3 If your input is a window you can select a region To do this click Select Region and drag the mouse across the area to select it Adjust the rectangle that you ve created until it contains the desired area Then click outside the Image window with the mouse 04 720103 000 Rev C 1008 46 5 softWoRx Ima ging Workstation Users Manual 22 Window DMS uploadAfterExp R3D dV File View Options Tools 2 H B Baye __ Input Specifications
105. gonal viewer 111 12 Point values 162 Polygon Editor 182 85 projections 119 35 queue manager 56 79 quick projection 119 22 removing data below a threshold 144 45 reorienting 105 11 Resample2D 105 7 resizing 105 7 ROI colocalization 193 201 2 rotating 107 9 scale bar 103 5 scale image 95 99 switching border on off 103 time lapse movies 113 14 trails movie 114 18 viewing movies 107 12 volume rendering 122 24 volume viewer 122 35 volumes 119 35 Tracking particle movement 114 18 Trimming time data 50 52 True color 101 Tutorials Measuring distances 176 modeling 190 04 720103 000 Rev C 1008 229 UIC MetaMorph STK 36 Undo Last icon tool 182 Uploading images to DMS 74 81 URL Applied Precision iii Velocity 177 79 measuring 175 Viewing data in 3D See Volume Viewer Viewing image data 87 112 Viewing movies See Movies Viewing time points 90 92 Viewing Z sections 90 92 Visualizing data 3 VolPack 126 Volume models 181 92 Volume of object measuring 188 Volume projections 122 35 Volume rendering 122 24 Volume viewer 122 35 axes of rotation 123 24 interactive image rotation 128 31 limiting size of data set 124 rendering 122 24 RGB opacity method 131 35 using with colocalization 199 Volumes 119 35 Volumetric movies 113 14 Voxels 27 Weak convergence 217 Web site Applied Precision iii Z sections and blurring 218 data projections 119 35 measuring points in 175 77 slider
106. 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 pplied Precision Chapter 5 Data and Task Manipulation 63 a _ _ _ m rs e a _ _ 4 position EX2 Filter Filter Illumination 1 100 mirror Shutter Slot 1 Slot 2 Beam Combiner 2 50 50 beam combiner i 3 460 long pass filter 4 500 long pass filter avaPavay aaa Combined Light Path 100 Fiber optic cable it Mirror m EX 1 ND Shutter Filter Filter Combined Light Path Fiber optic cable Turret 1 Quad RR enn ae 6 position 2 EGFP mCherry ilumination Optics a Dichroic 3 CFRIYFP f Dual band EM Filter 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 workst
107. he box size in pixels that determines the size of the region used for the local mean and contrast calculations Only odd values are valid To reposition the intensity scaling curves based on the minimum maximum values of the local mean and contrast select AutoRange Specify the minimum and maximum values of the intensity scaling curve in the Min Max fields To modify the intensity scaling curves displayed on the LM weight function and LC weight function histograms select one of the three points on the curves with the mouse and move the point to the left or right Choose one of the following options for applying the contrast enhancement in the Apply To option list e All Sections creates a regular output image file or window e Current Section stores the result in a temporary scratch window 10 Click Do It to create the filtered output Setting an intensity Threshold Use the Threshold dialog box to remove all data in an image that is below a certain intensity cutoff value You can choose to output either the image values above the threshold or a binary mask where all pixels below the cutoff are 0 and all above are set to 1 To remove data below an intensity threshold 1 Choose Filter Threshold from the softWoRx main menu to open the Threshold dialog box 04 720103 000 Rev C 1008 146 softWoRx Imaging Workstation User s Manual Input Qutput a a Select Region Reset Details
108. he movie The Save As Movie dialog box is displayed v Eat oie Window i4 W Save Image W Save Overlay Movie File datal dv_samples LiveCells GFP_2D_Movie_d3d_TRL Movie Format Quicktime Ea Animation Style Forward d Compression Quality m HM 5 00 Frame Rate FPS r Movie Duration 12 2 sec P ient ONDA EEE Pat et rt FAEN iij AE SE X X i ie i AS W Animate Through Time 61 Time StartvEnd Increment f fe ft SOOO Done Do It imterrupt Preview 04 720103 000 Rev C 1008 118 softWo Rx Imaging Workstation User s Manual From this dialog box you can use the appropriate fields to select the movie format Quicktime AVI or MPEG the animation style Forward Backward or Forward and Back the compression quality the frame rate and the time increment to use You can also select whether to animate the movie through Z sections or time ppliedPrecision Chapter 8 Viewing Movies 119 04 720103 000 Rev C 1008 9 Viewing Projections and Volumes You can create two types of data projections of multiple Z sections Two dimensional projections can help you to visualize how the data are oriented in the XY direction These projections allow you to view the paths of individual fibers chromosomes or other types of linear data Volume projections can help you to understand the three dimensional nature of the data In This Chapter reat 2D Proje cion Sna
109. he volume of an object 188 viewing the object 188 3D viewing data in See Volume Viewer 5D image data files 148 Acquisition data 2 workstation 2 Additive method of data collection 125 Adjacent images 21 22 Adjusting thumbnail images 88 Aligning adjacent images 18 Analysis workstation 2 Analyzing data 4 Applied Precision LLC contacting iii Arbitrary profile 166 Arbitrary profiles 168 69 Archiving 157 59 Area of object measuring 188 ASCII file saving coordinates to 182 Assigning colors 101 Auto Polygon Creation icon tool 182 Axes of rotation 123 24 Background value 145 Bad pixel file 18 20 21 BioRad MRC 600 Pic 35 Blended color mode 100 101 2 Border rolloff 27 Bright spot problem 218 Brightness 95 99 04 720103 000 Rev C 1008 Burning discs 157 59 Calibration 20 21 Capturing screens 156 57 CCD defects 17 CD Kreator tool 157 59 CDROM discs 157 59 Channels assigning colors to 101 Chromatic aberration 21 22 Chromatic Aberration Corrector 18 Close Polygon icon tool 182 Colocalization graph 196 indentifying potential areas of 197 201 Pearson Coefficient of Correction 194 ROIs 201 2 tool 194 97 Color assigning to channels 99 102 blended 101 2 true 101 Color mode 100 Colormap 95 99 changing 100 Combining data 119 22 with image arithmetic 142 43 Combining data files 52 54 Combining multiple tasks 54 57 Connecting to a DMS database 74 Constrained iterative algo
110. hs to a single file or fuse time points or Z sections of the same wavelength creating a single data set for each output wavelength To combine data of two image files 1 Choose Edit Image Fusion from the main softWoRx menu to open the Image Fusion dialog box pplied Precision Chapter 5 Data and Task Manipulation Image 1 pa Image 2 Doo Output eE Image 1 Selections Wavelengths Timepoints Num StartEnd Inc Z Sections Num Start End Inc Image 2 Selections Wavelengths Timepoints Num Start End Inc Z Sections Num Start End Inc Fusion Options Append Wavelengths _ Combine timepoints for like wavelengths Combine 2 sections for like wavelengths 2 Enter image file names or window numbers for the two files that you want to combine in the Image 1 and Image 2 boxes 3 Select which wavelengths time points and Z sections to combine from the first image in Image 1 Selections 4 Select which wavelengths time points and Z sections to combine from the second image in Image 2 Selections 5 Specify how to combine the data under Fusion Options as follows e To append all selected wavelengths to the output data set choose Append Wavelengths With this option if Image 1 had wave 490 selected and Image 2 had wave 490 selected the output data set would have two separate 490 wavelengths e To combine timepoints from all selected match
111. i as New Time Point RA 2 3 FSR bs Geer 2 6 T 2 00 01 46 074 The distance and velocity of the particle movement are displayed at the bottom of the Measure Distances dialog box Window 1 Measure Method Standard Two Point Units Micrometers Draw Lines W Search For Nearest iz LastPoint jg 15 42 8 63 1 00 Next Point ai Total Points E renee aataty eee ei nae e Mt eth AERATED Plea Penson fhe X E aai Jegra r gN 15 42 8 95 1 00 13 806000 15 42 8 63 1 00 106 073997 Rot 180 00 0 00 930 00 deg 1 gt 2 0 32 fun Velocity 0 003507 um sec p A Done save Options Help ppliedPrecision Chapter 13 Measunng Distance and Velocity 183 04 720103 000 Rev C 1008 14 Volume Modeling Modeling features of your image data can help you to understand the three dimensional nature of your data Volume models have been used to study objects such as nuclei and cell boundaries LX Note softWoRxalso provides Line modeling tools that have proven to be useful for studying chromosomes neurons and other complex three dimensional structures For information about Line modeling see the online Help In this Chapter PDOUE Volume MOJCE reiissi ia E icant decane cumiors eee hice eimnariceraedd 182 Edit Poly son Dialog BOX an ututinicinniutecaintiininsns aan nies 182 PAW POYON i big ho 23 naar e ner entre peters eeroN ney eer tween a E
112. image intensity to be slightly different To view this experimental situation flip the optical sections on their side and look at the sections in the XZ plane Striations perpendicular to the Z axis indicate that the relative intensity of the sections varies The striations are typically caused by arc lamp fluctuations during data acquisition Use the image correction program before deconvolution to compensate for arc lamp fluctuation In some cases the striations are visible after the image correction process This is a serious problem that needs to be addressed Such images will yield disastrous deconvolutions since the striations will be enhanced by the image processing Invalid Optical Transfer Function There are at least two ways that an optical transfer function OTF can be invalid e The corresponding PSF may not have been well measured e Incorrect OTF may have been applied for deconvolution Check that the lens ID number of the measured image corresponds to the lens ID of the OTF ppliedPrecision AppendixA Image Quality 223 04 720103 000 Rev C 1008 Index 2D Polygon Finder 182 85 Dialog options 186 Excluding edge objects 186 introduction 182 Minimum perimeter 186 Smoothing pixel distance 186 Specifying outer object only 186 3D graph 163 65 3D Object Builder 187 90 creating the object 188 example 190 introduction 182 loading the input image 188 measuring the area of an object 188 measuring t
113. information on this topic consult the online Help 9 Click Close 10 In the Edit Polygon dialog box click Snap Selected 11 Repeat this process for each Z section that will be used in the 3 D model 12 In the Edit Polygon dialog box select File Save 13 Enter a name for the polygon file and click OK 14 Use the polygon file to create a 3 D model using the 3D Object Builder 2D Polygon Finder The first task that must be performed is to isolate the object that you want to study This is accomplished by finding the 2 D representation in each Z section and combining them into a 3 D object Setting a threshold value in the wavelength intensity and allowing 2D Polygon Finder to create a polygon in each Z section can often isolate two dimensional features in a Z series A simple adjustment of the threshold value allows you to modify the polygon created The softWoRx Polygon Editor includes additional tools for more complex adjustments The 2D Polygon Finder Dialog Box v ES Polygon Finder Window ip Select Region Reset Details Wavelengths W 528 W 457 W 617 J Options Border Width pixels i Polygon Smoothing pixels aoo Minimum Perimeter pixels fs 0 39 um Maximum Perimeter pixels 16383 2132 83 um Exclude Edge Objects _ Outer Objects Only Threshold Values Wave 1 I 661 1 Wave 2 ii 141 4 Wave 3 I 669 2 Number of Polygons 0 Pogon Stalisucs Launo Porygon Editor 8D
114. ing wavelengths into a single final wavelength data set choose Combine timepoints for like wavelengths e To combine Z sections from all selected matching wavelengths into a single final wavelength data set choose Combine z sections for like wavelengths 6 Click Do It Notes 1 If error messages are displayed refering to differences in file types that include image size pixel size lens info data type etc use Copy Region and Edit Header to manipulate these items 04 720103 000 Rev C 1008 53 54 softWoRx Imaging Workstation User s Manual 2 If the number of sections vanes from wavelength to wavelength during an Operation Blank Z sections have been added to the image Isdisplayed These blank sections are added to balance the numberof sections between each wavelength of the output image Each one isa zero intensity image added to the end of the appropnate wavelength Setting Up Process Chains with Task Builder A process chain is a series of tasks that are predefined for a given collection of data Task Builder is a unique feature of softWoRx that allows you to set up process chains for one or several tasks to be performed on a single set or multiple sets of data You can use the Task Builder dialog box to select files and define multiple operations to be performed When you ve finished providing the data information and the processes you want to accomplish you can choose to either start the jobs immediately or
115. is option is only available when the input data comesfrom a window Select the Wavelengths options Choose one of the following deconvolution methods from the Method list Ratio conservative method usesa more conservative algonthm that generally finds an accurate solution Images with punctate fluorescence may deconvolve better using this method Enhanced Ratio the default method is quicker because the residuals stabilize in fewer iterations typically 10 or less Additive usesa more conservative algonthm that generally finds an accurate solution Enhanced Additive is faster than Additive because it requires fewer iterations typically ten or fewer The Additive and Enhanced Additive options are the preferred deconvolution methods fordata acquired with the EMCCD electron multiplication camera These methods are more tolerant of noisy data images with higher noise levels Select the Show image when finished checkbox softWoRx Imaging Workstation User s Manual You can typically use the default settings for the rest of the options in the Deconvolve dialog box including the options displayed when you click More Options More about the softVWoRx Deconvolution Tools softWoRx provides two tools for deconvolving images the standard Deconvolve tool described in this section and the Nearest Neighbor Deconvolution tool In most instances the standard Deconvolve tool provides the best results The Nearest Neighbor tool provid
116. issscsatestasiesstasuebansevaies seeeaataectatze stoncselboasabnes 102 Hiding or Displaying Image Window Border Tools cee eeeeseeeeeeeeeeeeeeees 103 The mace Window pcde Dalein ae E E 103 Resizing or INCONICMUING an Mace siarce ae n E E nee 105 IN OSI ZIV An MACE srei n E E E E O 105 Konono an aE E ae nen te one et Oe ee Ear en Or PEST 107 NAC W IIE ROSS OCCU ONS cora us natin E E O NEO 111 Viewing MOVIES sccsssscnsssccnsssennsssennssecnsssennsssensessensssesnsssensssenns LLS Viewing Volumetric or Time Lapse MOvViI6S scsscsscssssscesecesssessasssessseseseseseeenes 113 Tracking Particle Movement with Trails Movies essessessssssesssseseesesersreseeseesessesreseesees 114 Viewing Projections and VOlIUMES csssssscssssscsnnsseesnsssesnsss L19 Credhne 21 ProjecuOns consonan ora a arenes TE 119 Ceann Volume Troe CE OMS nestori a e inlelao tetova Gate 122 Volume Rendet seian a a aR R 122 AXESOR ON as aa EE EE ante Sareea hide a teat 123 Methods tor Projecting Volume Sesera A a a S 125 10 Filtering Image Data ccsssscssssscsnssscnnssecnsssenssseesssesssssesssees L37 PID OUE SON VOR PIETS o i E R E E OE 138 Usine Convolution FETS ona aera ui yessa ten ctors siete davon ose eeaceeteaa es 138 Enhameine Object DOUG ARIS S ooir E E EONO 139 Usine 2D Statistical FiOS eui sabobseustuaychuarbecanosannustanateeeoueeraees 141 Uone XS AE O E OEE O E OA 142 Scaling Pixel Intensity to Enhance Local Contira stessinereoin
117. jection is displayed The new Image window displays the original image It also displays an XZ projection at the bottom of the window and a YZ projection on the right side of the window Projection lines show the areas of the image that are displayed on the cross sections ppliedPrecision Chapter 8 Viewing Image Data 113 File Options YZProjection Projection Lines XZ Projection M 149 Y 175 2 24 Zoom 1 00 3 To orient the image in real world coordinates select Options on the Orthogonal Viewer and then select the Cover Slip at Bottom Right toggle This orients the image so that the display in the window represents the orientation of the sample when the data was collected e g on the XZ projection the cover slip is down 4 To change the cross sections that are displayed on the projections use the mouse to drag the projection lines across the Image window 04 720103 000 Rev C 1008 8 Viewing Movies Movies greatly enhance the analysis of certain types of image data When used on a volume rendering a movie shows the relationships between objects in 3 D space When used with a time lapse data file a movie allows you to visualize the course of events captured in the study You can also use movies to trace particles in time lapse data In This Chapter Viewing Volumetric or Timie Lapse MOVies sicziiisceyscloinesvieniesotsssantasestvnnessVanmvenedaiaban 113 Tracking Particle Movement with Trails MOvieS
118. kstation User s Manual 11 To view the volume move the vertical Z scroll bar up and down or use the Movie tool To save the volume rendered images 1 Click File Save in the Image window menu to open the Save to File window 2 Examine the Output field to ensure that the desired name is used The softWoRx software automatically adds a _VOL tag near the end of the filename 3 Click Do It to save the image ppliedPrecision Chapter 9 Viewing Projectionsand Volumes 137 04 720103 000 Rev C 1008 10 Filtering Image Data Use the softWoRx filters to prepare data for modeling and other types of analysis In This Chapter About WORY FIETS socsatierasioncanssiel seuicansdebllovull E NS 138 Usne CONVOLUTION PULSES surec A hemeeeene nay tanes 138 Enhancing Object DOUG AICS seuran iien anan A O ENEN 139 Vane 2P Statistical FIE S cici ssc oats ase aueasaveneads attests eects 141 Usine Imaze Arie UC Eiana NE A T E 142 Scaling Pixel Intensity to Enhance Local Contrast eeeeeeeseeseererrereerrerersersrrrerrereese 143 Setin an tensity TNrESNOldnoninea n E E 144 04 720103 000 Rev C 1008 Chapter 11 Saving Exporting and Printing 139 About softWoRx Filters softWoRx provides the following types of filters Convolution filters perform high pass low pass and other digital filtering The Edge Enhancement filter enhances object boundaries 2D Filter limits noise like intensity The Image Arithmetic filter scales images c
119. kstation User s Manual 24 2 Window LiveCelis_GFP_2D_Movie_d3d dv File View Options Tools ees 1 00 00 03 000 2 From the main softWoRx window choose View Trails Movie The Trails Movie dialog box is displayed Qutout a select Region eset De Details aa Wavelengths W 524 J Trails Movie Options Window fo Enhancement Factor i 00 3 Drag the Image window number into the Input field 4 Click Select Region to select a region of interest Then select which wavelengths to include in the movie 5 Enter the length of the trace history in the Window field e g for a value of 5 the trace includes the previous 4 time frames and the current time frame 6 To emphasize the display of the current point enter a value greater than 1 0 in the Enhancement Factor field typical values for this field are 1 0 2 0 ppliedPrecision Chapter 8 Viewing Movies 117 A Note The displayed intensity value of each point isa weighted average of the comesponding points in the previous time frames The previous points all have a weight of 1 0 The Enhancement factor is assigned asthe weight for the intensity of the curent value 7 Click Do It to create the trails display 0 4 Window LiveCelis_GFP_2D_Movie_d3d TRL dv unsaved File View Options Tools 528 1570 fed To create a Trails movie choose File Save As Movie on the Image window 8 and save t
120. l open windows simultaneously Only the windows that are open at the time that you activate the Section slider are affected To scroll Zsections simultaneously 1 Choose View Slider from the softWoRx main menu to open the Section Slider tool ppliedPrecision Chapters Viewing Image Data 93 section Slider n pa Down section lt Up Section Help The Section Slider 2 Move the slider to the left to decrease the current Z Section or to the right to increase it Viewing Areas You can view different areas of a section by sliding the vertical and horizontal scroll bars and the tool buttons to reposition the image iw FE Window Nuclear_Pore_d3d dv File View Options Tools 2 H B v i ae Tool Buttons Zoom Use tool buttons scroll bars and zoom to view different image areas You can also use the following tool buttons to change display characteristics or reposition the image 04 720103 000 Rev C 1008 94 Center Image Zoom Wheel softWo Rx Imaging Workstation User s Manual Use this tool To G Adjust the intensity scale of the image K Pan across the image bed Center the image on a point Select the tool and click the point that you want to center Position the image on its original center Zoom in or out Move the Zoom wheel up to zoom out or down to zoom in Return to a 1 1 zoom level Zoom to fit image to window Q Tip From the Image wi
121. le as a spreadsheet compatible file 04 720103 000 Rev C 1008 13 Measunng Distance and Velocity This chapter shows how to measure distance and velocity In This Chapter IVC AS WUT ION Distineo S sarae cea stats teh asia a ea diese sede as dat 175 IVICAS UTS y CLO CIE a ty sisca teas cig cicasen sheds aeS vas aan daa E E OEA E 177 Measunng Distances The Distance tool allows you to measure distances between points in one Z plane or between points in many different Z planes The measurement data can be saved to a file for off line analysis The Measure Distances dialog box contains the options for measuring distances This section briefly describes these options and contains step by step instructions for completing the most common procedures 04 720103 000 Rev C 1008 Chapter 13 Measunng Distance and Velocity Od Measure Distances 7 Measure Method Standard Two Point nits Micrameters 1 Draw Lines W Search For Nearest os foun Lega phi bape fete AE PoE Last Point i fono 0 00 0 00 a ATT de Sere e ere ete MAIn A Dae ret NPO har th oo Next Point El Total Points Rice e e E Ea E ak ml Done save Options Options in Measure Distances are summarized below and are described in further detail in the softWoRx online Help Select To Standard Two Measure the distance between two points Point Single Reference Measure the distance from a single reference point to oth
122. le color map from the previous example 1 While viewing only the channel choose View Select Image Colors on the output window menu The Select Image Colors dialog box is displayed GJsclectimageCoors PEB Color oa des Mode Color j Wave 1 Display Color Red j Wave 2 Display Color Green ak Wave 3 Display Color Blue afer 2 Inthe Color Display Mode field select Grayscale The Modify Grayscale Colormap button is activated oe Pepay Mode Grayscale Wave 1 Display Color Pe Wave 2 Display Color Wave 3 Display Color Modify Grayscale Colormap pene og 04 720103 000 Rev C 1008 204 softWo Rx Imaging Workstation User s Manual 3 Click the Modify Grayscale Colormap button The Select Image Colormap dialog box is displayed fra Select Image Colormap Current Colormap Grayscale Default _ Apply To All Windows _ Zero always Black 4 In the Current Colormap field change the color map from Grayscale Default to one of the rainbow or temperature color maps for example Rainbow 1 or Cold To Hot Detecting Colocalization with ROIs After you use the Colocalization tool to identity possible areas of colocalization you can use Region Of Interest ROI Colocalization to selectively analyze those areas ROI Colocalization allows you to create ROI polygons from which a scatter plot and Pearson Coefficient of Correlation are derived To
123. les 04 720103 000 Rev C 1008 24 softWo Rx Imaging Workstation User s Manual pplied Precision 3 Sutching You can stitch images together to display images that are 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 You can also use it to display a sequence of time points in a time lapse image If you are using a DeltaVision Acquisition workstation you can create stitched images that are organized as either a series of time points or Z sections Each time point or Z section is treated as a panel of the stitched image LL Note Image stitching is only possible with certain types of Delta Vision image files In particular the image file must contain a senesof images along witha corresponding set of XY coordinates To obtain images suitable for stitching use the Panel Collection feature of Resolve3D In this Chapter Stitching Images That Have a Single Z Section eee eee ese eeeese ina 26 Stitching Images That Have Multiple Z Sections cece ceeseeseeeeeeeeeseeeseeeees 27 04 720103 000 Rev C 1008 26 softWoRx Imaging Workstation User s Manual Sutc hing Images That Have a Single Z Section You can use stitching for simple 2D images or for time lapse images Individual panels were stitched to create the final image on the nght Before you start Collect Panel data with the DeltaVision Acquisition workstation
124. les Remove All Files Processing Tasks Task Options Deconvolution A x Quick Projection 2 x Export As Pr m Add submit To Queue submit To Remote Server 4 Next use the Task options in the Processing Tasks section of the dialog box to select the tasks and the order in which you want these processes performed on the selected file s The Task options to choose from are Deconvolution Correction Crop Image Quick Projection Volume Rendering and Export As Use the Add button to include additional tasks and the X buttons to 04 720103 000 Rev C 1008 56 softWoRx Imaging Workstation User s Manual remove tasks from the chain Use the Options buttons next to each task to view a dialog box of available options for the specific task The task options you specify will be performed on each file in the exact order they appear in the Task Builder dialog box Each of the selected files is run through the entire list of tasks before the Queue Manager moves on to the next file in the list Q Tip You can use the left mouse button to drag image file icon a group of file icons or folder iconsto the Task Builder orthe Queue Manager When you are satisfied with your selections and the order in which the tasks will occur click Submit Tasks to the Queue The softWoRx Queue Manager dialog box is displayed Red softworx Queue Manager Host Istechapicom i WO Current Job lt queue not runnin
125. ls provide a convenient method by which you can adjust the intensity scale of each wavelength in the image Viewing 5D Images After opening an image file use the softWoRx Image window controls for Viewing Z sections and time points in the data Viewing different areas of a section Zooming in on a selected area Displaying or hiding channels Viewing Z Sections and Time Points Use the Z and T sliders on the left side of the Image window to display different Z sections and time points If images contain both Z and T data you can use the Z 04 720103 000 Rev C 1008 92 The Z Slider softWoRx Imaging Workstation Users Manual slider to show all of the sections at a time point or use the T slider to show how a section changes with time To navigate through Z sections and time points gt Move the Z slider down to show deeper Z sections the top section is displayed when the slider is at the top Move the T slider down to show data acquired at later time points delta time increases as the slider is moved down The TSlider 59 i KO PN ll Move the Zand Tslidersto display Zsections and time points X Tip You can also use the right and middle mouse buttons to scroll through Z sections To scroll through time points hold down the CTRLkey and pressthe nght or middle mouse buttons Viewing Z Sections in Several Image Windows The Section slider allows you to scroll through Z sections of al
126. m User s Manual for details on image acquisition If your system has the QLM module you can use it to acquire photokinetic data for a variety of experiments softWoRx photokinetics data includes photo bleaching or photo activation that results from a laser pulse See the QLM Getting Started Guide Processing Data Process image data to prepare it for visual examination and analysis softWoRx provides several types of modules for processing image data Deconvolving Image Data Deconvolve image data acquired with the DeltaVision system Deconvolving improves contrast by relocating signal scatter and out of focus data Conecting Images Correct image data for chromatic aberration color shift that results from oil matching and other environmental conditions You can also correct data collection errors and equalize intensity values across Z sections Sutc hing Stitch panel images collected with DeltaVision to generate a larger overall field of view Stitched images are organized as either a series of time points or Z sections Importing Import data from the TIFF format 16 bit grayscale BioRad s Pic format InoVision s See format or MetaMorph s STK format Selecting Cropping and Combining data Select data to crop it or to combine it with other data Visualizing and Presenting Data After processing data you can view and present it in a variety of ways softWoRx provides several tools that you can use to visualize d
127. mage or panel into the Wavelength 1 nm field 6 If necessary enter the wavelength of the second image or panel into the Wavelength 2 nm field 7 Enter the X Y and Z spacing of the pixels in the data set usually in microns into the appropriate fields 8 Enter the correct lens number in the Lens ID field 9 Click Do It Converting SIK Files The STK conversion tool is used to convert MetaMorph STK images to DeltaVision format Unlike the See and Pic converters the STK converter attempts to read the wavelength and pixel size values from the STK file s header immediately after you specify the name of the input file After reading these values from the header the converter enters this data into the fields in STK Conversion If necessary you may change this information manually before you click Do It The See and Pic converters do not read the input file until you click Do It The deconvolution program and other softWoRx software rely upon the presence of accurate wavelength and pixel spacing As with ISee and Pic files not all STK files contain accurate pixel size and wavelength information and it may be necessary to manually enter some of the fields in STK Conversion A Note The image wavelength fora DeltaVision file indicates the wavelength of the light imaged by the camera ratherthan the illumination wavelength X Y pixel spacing can be obtained in two ways it can be measured with a test target or it can be a
128. maging Workstation User s Manual File View Options Tools Z 5 Zoom 0 50 As you move the handles the image displayed in the Image window changes interactively Data values higher than the maximum value are assigned the lightest shade Values lower than the minimum value are assigned the darkest shade ww Scale image Window 1 LL Wave wv 457 526 v 617 Display Min Max Exp 22 00 1961 52 1 00 E Wave Min Max 22 00 4095 00 Section Min Max Mean 23 00 4095 00 304 88 Apply To Wave W Show Histogram W Log Right Handle Left Handle Copy Scale Paste Scale Done Revert Restore Default Scale Help The Left and Right handles change the scale range 4 To slide the range to the left or right click and drag on the Center handle W 1 Window cell_Z10C3_r3d idv 2 1 J i KEE R a File View Options Tools ea peer A Use the Center handle to side the scale back and forth across the histogram ww Scale Image Windowl Wave wv 457 528 v 617 Display Min Max Exp 1293 47 3232 99 1 0q Wave Min Max 22 00 4095 00 Section Min Max Mean 23 00 4095 00 304 88 W Show Histogram W Log Apply To Wave at Center Handle mal li on Copy Scale Paste Scale Done Revert Restore Def
129. me MRs Estimated File Size 668 05 Mb _ Enable Fast Acquisition fast Acquistion TIPRO Sectioning Channels Time lapse Points Panels Actions Conventional Multiplexed mam oao BEO 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 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 If the currently active filter set is Multiplex capable the Design Run Experiment window is displayed and will look similar to the following pplied Precision Chapter 5 Data and Task Manipulation 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
130. ment for exploring and refining your understanding of specimen structure The flexibility of the software makes it ideal for the study of 04 720103 000 Rev C 1008 softWo Rx Imaging Workstation User s Manual images from fluorescence brightfield Differential Interference Contrast DIC and electron microscopy softWoRx is available on two types of Linux workstations e The Acquisition workstation is part of the DeltaVision data acquisition system and is used to control the system You can also use it to process analyze and visualize data Refer to the DeltaVision Core and personalDV Restoration Microscopy System User s Manual for details on image acquisition e The Analysis workstation is a stand alone workstation You can use it only to process visualize and analyze data The DeltaVision Core data acquisition system What Can You Use softWoRx for You can use softWoRx to acquire process visualize and analyze multi dimensional renderings of a fluorescent specimen You can also use it to save data in a variety of formats Acquiring Data If you are using an acquisition workstation you can acquire images with the DeltaVision Restoration Microscope System The softWoRx Resolve3D module provides various options for acquiring time lapse data data with multiple Z sections and data from multiple channels Refer to the DeltaVision Core and pplied Precision Introduction personalDV Restoration Microscopy Syste
131. mes Figure 7 Rotation About the Y Axis Y E a EF ae i a on al p Figure 8 Rotation About the Z Axis Leif ie According to the parameter settings softWoRx enables you to see the desired portion of the Z section in the desired wavelength using a variety of methods Options in Volume Viewer help you to perform a volume rendering The most important options are Select Region Details and Method By limiting the size of the data set using Select Region and Details the time needed to create projection images can be drastically reduced For detailed information about each of Volume Viewer options refer to the online Help 04 720103 000 Rev C 1008 126 softWoRx Imaging Workstation User s Manual Methods for Projecting Volumes The following table summarizes the six methods of data collection Method Maximum Intensity Additive RG B O pacity Mixed Func tionality Each ray collects the data from the voxel with maximum intensity Each ray collects and sums data from all the voxels in its path and scalesit down to an appropnate intensity between 0 255 Each ray collects the data in the voxel that is closest to the front of the image If the image data hasbeen processed using Blend Colors with the Opacity option toggled on then thisdata can be used to fom a volume rendenng in multiple wavelengths The resulting image captures the positional inf
132. molecules are in close proximity FRET occurs when there is a quantum physical exchange of energy between dipoles The presence of FRET indicates that the molecules are within 60 A 6 nm FRET is orientation specific Negative FRET does not mean the molecules are not interacting Positive FRET means that they are close but does not necessarily mean that the molecules are interacting There are many ways to measure FRET Experiments are simple but controls are essential Q Tip Before you conduct a FRETexperiment consider using the Colocalization tool to determine whether the molecules that you are studying are close to each other Colocalization expenments provide more approximate results than FRET but they are much simplerto prepare and analyze Using the FRET Analysis Tool The FRET analysis tool provided assists in analysis of sensitized emission FRET experiments Acceptor photo bleaching can also be done with Deltavision Use the Ratio Analysis tool or Polygon Editor to analysis acceptor photo bleaching experiments 04 720103 000 Rev C 1008 211 212 softWo Rx Imaging Workstation User s Manual Before You Begin Be sure to follow the conventions described in the online Help topic Acquiring and Preparing FRET Data Before you begin make sure that you have the following data You must have the following three image files m A Donor Control data file that contains data from a slide prepared for the Donor probe o
133. mplete the Multiplexed Wavelength filter activation process After the selected filter set is activated the Design Run Experiment window will look similar to the following pplied Precision Chapter 5 Data and Task Manipulation YW Design Run Experiment Resolve3D_Startup exp agda Oe Design Experiment Design PK Experiment Run Experiment l Experiment name M Estimated File Size 668 05 Mb _ Enable Fast Acquisition fast Acquisition pRO Sectioning Channels Time lapse Points Panels Actions Conventional Multiplexed Do Multiplexed Channel Imaging Settings YT Ex Shutter 1 o 775 GF P GFP 100 yi 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 Exp EX Filter EM Filter _ Reference Image _ 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 option 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 720103 000 Rev C 1008 67 68 softWo Rx Imaging
134. n Editor to enable more sophisticated selection of a polygon or 3D Object Builder to continue creating the three dimensional model Done Closes the 2D Polygon Finder Do It Begins the process of creating polygons 04 720103 000 Rev C 1008 190 softWo Rx Imaging Workstation User s Manual Save Polygon File Allows you to specify a name for the polygon file and save the new data To create polygons using 2D Polygon Finder 1 2 10 Open the desired image data file Select Model 2D Polygon Finder in the softWoRx main menu The 2D Polygon Finder dialog box is displayed Drag the Image window number to the Window field Click Select Region Using your mouse draw a box around the region of interest It may be helpful to scroll through the Z sections to ensure that all of the desired areas are included Select the desired wavelengths in the Wavelengths check boxes Enter the desired values in the Threshold fields In order to estimate an initial threshold value use Point Values located in the Tools menu of the Image window to view the image intensity at various points Click Do It Click Save Polygon File Then enter a name for the polygon file and click OK By default softWoRx uses the previous file name and replaces the file extension with POL Create a 3 D model using the 3D Model Builder or use Polygon Editor to create new polygons or modify existing ones 3D Object Builder The 3D Object Builder j
135. n User s Manual Sets the Image window number of the image data to be processed Polygon Data Defines the Image window to be used by a file name Snap Selected Determines the boundaries of the polygon after you have manually selected an approximation of the polygon Polygon Editor finds the closest pixel to your manually selected polygon that matches the criteria of the Guide Options Options Guide Mode Options Defines the parameters for the Polygon Editor to evaluate when you use Snap Selected For additional information refer to the online Help Done Closes the Edit Polygon dialog box File Save Allows you to specify a name for the polygon file and save the new data The following table provides a brief description for each of the buttons on the Edit Polygon dialog box Edit Polygon Tools EROESSE A EECA Name Select polygon Add polygon Add polygon freehand Automated polygon creation tools Insert point into polygon Close current polygon Add circle Add box Delete selected polygon point Delete selected Func tionality Selects moves or modifies existing polygons or points within a polygon Connects points which you select using the mouse Allows you to draw the polygons freehand It Is especially useful when used with Guided mode Allows you to define parameters for the automatic detection of polygons Addsa point to a line segmentina selected polygon Drag and
136. n e 143 Seme an Intensity TNEESNO l diees ae A E 144 04 720103 000 Rev C 1008 softWoRx Imaging Workstation Users Manual 11 Saving Exporting and Printingd cssssscsssssssssssessssesnsssesnssees LOZ Image Data Files and Image Graphic Piles sisic5 ccss estes cu diaodavit a 148 DAVIN C16 Vioo TIE Siani E E LOE TOOT 148 Saving to the Data Management System DMS sseeeeeeessessesessrssersrrsrrerrerrsresresresrerrerseses 150 EX PONG DClEAY 151 OCS os scarcisicte ater ir EOR 151 Exporting to TIEF Piles siccastasn canes siouyustostanvaceadnasenseans savegame tues ominous eumeiees 151 EX poruine to PhotoShop Biles as cecsstasatte aa E S 153 Exporting to MIOV1esF eS enahn uiesrpviceovniarna A OS a ees eee 153 Px POMS OJ LEGENE S ell aes ide acetate E aan uate atu dan eeaiec sesaneee 155 Capturing SELECH SOUS riests suns veteistinnd unin nevusei uated eataumieealssdatnetteenadennnuleiedts 156 Archiv ine Fles to GB D1 B emp yrr tenn ry oe retere rset a re er ere a eerr Tr 157 PDE VEN aCe Spann sees sega dette atsnacus von aucdes tou seca ston E A cara coast S 159 Part Three Analyzing ReSuhtS sssssnnnnnnnnnnnnnnnnnnnnnnn LOL 12 Examining Intensity Data ccccsssssssssssccsssssecsnssseensssessssesss LOZ EEO PN OME Vale an secu eaic seen cup sais aetoua io tan E EE 162 Examining Intensity Data with Data Inspect nisen a 163 SelECHING a kesion OF IMSL O SE ies eased a A inventors aaa 165 Viewing Intensity Line Promlesivisccscs
137. n the Method option list e Median uses the image intensities within a box shaped region around each pixel and selects the median value for the resulting image This is useful for removing noise e Mean is similar to the median filter except that the mean value is used instead of the median e Variance calculates the statistical variance of the image about the mean intensity within the local region e Weighted Mean is similar to the mean filter except that mean is weighted by the variance 6 Specify the size pixels of the square box used to calculate the filtered images in the Kernel Size field Kernel sizes of 3 and 5 are most useful 7 Specify the number of times to run the filter in the Iterations field ppliedPrecision Chapter 11 Saving Exporting and Printing 143 8 Click Do It The filtered image is displayed in a new Image window Using Image Anthmetic Use Image Arithmetic to scale images combine information from multiple images and subtract images in order to isolate features Image Arithmetic calculates an output image from one or two input images by applying one of several operations The input images may come from one or two windows or files To create the image specify the input images files or windows the wavelength number of Image 1 the wavelength number of Image 2 the arithmetic operation and any necessary coefficients A Note In order forthe calculation to be processed the input images m
138. name ResolvesLi Estimated file size 5 00 Mb 4777 08 Gb Available W Use Fast Acquisition Fast Acquisition Options Lamps Off when finished Sectioning Channels Time lapse Points Fanels Actions Sesary Cusine RZSEiE NZ ARSIZ Z Sectioning 4 Select the Channels tab and specify the two channels you want to use for this experiment cad g Oe Design Experiment Design PK Experiment Run Experiment Experiment name Resolveat Estimated file size 5 00 Mb 4777 08 Gb Available W Use Fast Acquisition Fast Acquisition Options Lamps Off when finished Sectioning Channels Time lapse Points Panels Actions Conventional Muitiplexead Exp EX Filter EM Filter AT Ex Shutter _ Refresh exposure conditions 5 Select the Time lapse tab and specify the time lapse and total time for this experiment 04 720103 000 Rev C 1008 59 60 softWoRx Imaging Workstation User s Manual WY Design Run Experiment Resolve3D exp File du 2 Oe Design Experiment Design PK Experiment Run Experiment Experiment name Resolve3D Estimated file size 5 00 Mb 4777 08 Gb Available W Use Fast Acquisition Fast Acquisition Options Lamps Off when finished Sectioning Channels Time lapse Points Fanels Actions W Time lapse Hours Minutes Seconds Milliseconds Time lapse fo o g d fo Total Time d fo zo fo Time Points E _ Enable Cell
139. nce in 2D Polygon Finder 186 Snapshots 156 57 Spherical aberration 218 Standard residual 216 Standard Two Point method 176 Statistics calculating for selected areas 169 74 using Data Inspector for 170 with Edit Polygon tools 41 44 Status bar 90 Stitching 25 29 border rolloff 27 example 26 images with multiple Z sections 27 29 Table of 3 D measurements 190 Task Builder 54 57 Task chains 54 57 Technical support iii Threshold default value for 2D Polygon Finder 186 Thumbnails adjusting 88 TIFF output formats 152 TIFF importing 32 Time points reducing 50 52 viewing 90 92 Time lapse image data 19 20 59 Time lapse movies 107 12 113 14 Tools Align Image 18 21 22 calibrate 18 calibration 20 21 chromatic aberration corrector 18 21 22 colocalization 194 97 convolution 138 39 copy region 53 Correct Image 17 18 19 correction 17 25 Cut Mask 49 Data Inspector 163 65 deconvolve 9 16 edge enhance 139 40 edit header 53 Edit Polygon 41 44 equalize time points 18 19 20 Filter2D 140 42 ppliedPrecision AppendixA Image Quality for calculating statistics 169 74 for identifying colocalized structures 197 201 for measuring velocity 177 79 image arithmetic 142 43 image fusion 52 54 image window 90 102 7 intensity threshold 144 45 interactive image rotation 128 31 Line Profile 167 local contrast enhancemnt 143 44 measuring distances 175 77 nearest neighbor 10 ortho
140. ncrement between frames 10 Click Do It to save the movie X Tip Select the Preview button to preview the speed and other settings before sa ving A Note softWoRx Task Builder providesthe capability of exporting Image window contents to AVI and QuickTime movies For details on using the Task Builder see Page 54 Exporting to J PEG Files You can save the contents of any window that includes an image as a JPEG file This includes a The Image window a The 3DModel window The Statistics window To save a J PEG file 1 Ina window that contains graphical data choose File Save JPEG Snapshot to open the JPEG Snapshot dialog box ppliedPrecision Chapter 11 Saving Exporting and Printing 157 i JPEG Snapshot Directory datat dv_samples File Name tripolard1_d3d_o1 Wo Auto Sequence names Include Window Frame 2 Enter a directory and file name and click Do It Captunng Screen Shots You can capture a screen shot of any window and save it as a JPEG image This is a useful way to save images for presentations To capture a screen shot 1 From the main window choose Utilities Image Snapshot to open the JPEG Snapshot window iv faze Snapshot Directory nomeiworx File Name DVSnap_07 Wo Auto sequence names Include Window Frame 2 Select the directory and file name for the file 3 To use the same prefix for a series of screen shots click Auto sequence names A sequ
141. ndow you can also use Alt Mouse Wheel to zoom the view in and out Zooming In or Out on Specific Points One common way to use the tool buttons is to position the image and zoom in on a specific point To zoom in on a specific point 1 On the Image window click the Choose New Window Center HH button and then click the point in the image on which you want to zoom The image is centered on the selected point iw 2 Window cell_Z10C3_r3d dv File View Options Tools Use the Center Image control to re center the image 457 104 IJ ppliedPrecision Chapter 8 Viewing Image Data 95 2 Use the Zoom wheel to zoom in on the point that you selected The new zoom level is displayed in the status bar iw 2 Window cell_Z10C3_r3d dv File View Options Tools ofle KES f a Zoom 2 00 457 105 fd m Ii Use the Zoom wheel to zoom on the point that is selected asthe center point 3 To return the zoom to a 1 1 display click the Reset Zoom to 1 1 button Q Tip You can specify to interpolate smooth images when the zoom level is greater than 1 Interpolated images provide better quality results but take longer to display To interpolate choose Options Display and select the Interpolate zoom option Displaying or Hiding Channels You can display or hide channels with the Wavelength Selectors on the Image window When the channel is displayed the chan
142. nel displays the color that is assigned to it When the channel is not displayed the selector is black 04 720103 000 Rev C 1008 96 softWoRx Imaging Workstation User s Manual Channels not p displayed Er Z 24 Zoom 1 62 457 104 Ri Hiding channels in the Image window To hide a channel gt Double click the Wavelength Selector of a displayed channel The Wavelength Selector turns black to indicate that the channel is not displayed To display a channel gt Click the Wavelength Selector of an undisplayed channel The Wavelength Selector displays the color of the channel and the channel is displayed Adjusting Brightness and Contrast You can improve the contrast of selected data in a channel by changing the channel s intensity scale softWoRx uses shades of the color selected for a channel to display an intensity scale The darkest shade is mapped to the lowest dimmest intensity value in the wavelength and the lightest shade is mapped to the highest brightest intensity value The remaining shades are mapped to values between the lowest and highest values Color shades can be mapped to create linear or nonlinear intensity scales In linear scales the color shades are mapped to values that are distributed evenly from the minimum to the maximum intensity values In nonlinear scales the shades are mapped to values that are distributed unevenly throughout the range ppliedPrecision Cha
143. nitial Annotation AY Sample 1 Optional Finished Done Do It Cancel Upload Click Done to exit the Save To Data Management System menu To confirm that the selected image file has been uploaded to the DMS database select File Open from the softWoRx main menu When the Open Image window is displayed click the DMS Database tab ipplied Precision Chapter 6 DMS Integration 77 Note To activate the DMS Database tab you must be currently connected to a DMS database Referto the previous section Connecting to a DMS Database for details Kl open Image Preview Files DMS Database lt lt lt Disconnect from the Database Database View Project Dataset Image Project NewKP_Project v kpalmby Dataset KP_Dataset x kpalmby Agarri heiinkatioincn bu gry NAAA KRE NARE MNE N SAAS ARENZS E A i izi Created Acquired Aarni NASP E ii Unknown Last Modified Available Images i 01 09 07 15 26 2ch_p Ref_2_R3D dv 2ch_p Ref_2_R3D_1 dv Details ce ens Drosophila_Enbryo_Movie_d3d dv File Size LiveCells GFP_2D Movie _d3d dv 27 45 Mb Nuclear Pore_d3d dv Download Nuclear Pore _d3d_1 dv Image Info Nuclear Pore_d3d_2 dv 3A W 3T Spindle_Green_d3d dv _ View blatz01_R3D dv oocyte_R3D_COR_PRJ dv Cancel spoke prj dv Fi The file you uploaded should appear in the displayed list of files
144. nitor a single location or monitor several locations in a sequential fashion e Multi Point FRAP data is collected in experiments that monitor several locations in the sample at the same time Single and Multi point FRAP experiments can be used for the following types of studies a Affinity Studies Biomolecular Cycling Biomolecular Studies Environment Studies Structural Kinetics To analyze FRAP data 1 Open an image in the Image window From the softWoRx main menu choose Measure PK Analysis The Photokinetic Data Analysis dialog box is displayed ppliedPrecision Chapter 16 Other Applications 209 R Photokinetic Data Analysis ME Output Image Eoo Results File Response Auto Determine Recovery Model smooth Curve pe Recovery Data From Site ROI Site ROI Type Circle 1 00 Number of Laser Sites In Background Intensity 0 0 Spot Radius um 1 e2 0 50 Image Bias 50 0 i 7 Section f Wave Num f Last T Num f Done Do It More Options Help Tip You can use the Photokinetic Data Analysis dialog box to specify a recovery model type of ROI beam profile shape and number of sites You can also use it to remove background intensity select which Zsections and wavelengths to include in the analysis and specify other options 2 Drag the window number into the Input Image field 3 Select the desired Response type Recovery Model and Data Recovery options 4 I
145. nly An Acceptor Control data file that contains data from a slide prepared for the Acceptor probe only a The FRET Experiment file that contains data from a slide prepared for both Donor and Acceptor probes Each image must have the following three channels Channel Description 1 Donor Donor Excitation Donor Emission 2 Acceptor Acceptor Excitation Acceptor Emission 3 FRET Donor Excitation Acceptor Emission Analyzing FRET Data Analysis of Direct FRET data consists of determination of Donor and Acceptor crosstalk factors calculation of Net FRET and FRET efficiency and analyzing the Net FRET and FRET Efficiency statistics Use the FRET analysis tool and the following process to analyze the FRET data macadamia Crosstalk gt Femail o gt Nansen Results Calculating Crosstalk Calculation of Donor and Acceptor crosstalk factors involves creating Region of Interest ROI polygons on Donor and Acceptor Control images and determining representative background values to be subtracted from intensities in ROIs To calculate crosstalk 1 From the softWoRx main menu choose Measure FRET Analysis The FRET Analysis dialog box is displayed Make sure Calculate Crosstalk is the active tab ppliedPrecision Chapter16 Other Applications 213 Calculate Crosstatk Calculate FRET Analyze Results l Donor Control Crosstalk Acceptor Control Crosstalk Donor Window a Acceptor Window a Probe Ch
146. nnected to the DMS Server In This Chapter Conmectinie toa DMS Database eresie aaa E EAE EAEE 74 Uploading Imaces toa DMS Databasenessunennnianiieininn nnne a 74 Downloadine Files TOn OMO reias inina E A IORA EOS 81 Browsing and Locating Images in a DMS Database ssnssesessssesseseesessesessesssseseeseseses 82 04 720103 000 Rev C 1008 softWo Rx Imaging Workstation User s Manual Connecting to a DMS Database Before you attempt to connect to a DMS database you ll need to have a user name and password for the database you want to use Check with your system administrator for this information To connectto a DMS database 1 From the softWoRx main menu select File Connect to DMS Database The Connect to DMS Database login box is displayed softWoRx 3 7 0 Running On Host tstech apicom File Edit View Process Filter Model Measure Conversions Utilities Help ed Connect to DMS Database ee Ix Database Connection Database Host Name batin ice 30 22 Gh free User Password kpamby Connect Cancel 2 Enter the name of host computer in the Database Host Name field 3 Enter your user name and password in the appropriate fields and click Connect A pop up message confirms that you are connected to the database If the connection fails make sure you are using the correct user name and password combination for the specific database Uploading Images to a DMS Database You can upload image data from your lo
147. nt of Correlation This value is displayed in the Colocalization dialog box 04 720103 000 Rev C 1008 200 softWoRx Imaging Workstation User s Manual Nuclear_Pore_d3d_CLC dy unsaved Z 4 Zoom 4 00 The new window contains a third channel that is the product of the two intensities at each data pixel This channel indicates possible areas of colocalization If the intensities of both channels are high for a given pixel the product of the intensities is high If one of the intensities is low or zero the product is much lower Identifying Potental Colocalized Areas To visually identify areas that may be colocalized you can select specific points or groups of points to display them on the three channel image You can also render a volume projection of the three channel image that includes the selected product channel in white To identify colocalized structures in Image Colocalization Tool data 1 Select the points on the colocalization graph that have higher intensities by dragging the mouse across the graph as shown below ppliedPrecision Chapter 15 Detecting and Analyzing Colocalization 201 File Graph Point Labels Image Nuclear Pore_d3d dv Pearson Coefficient of Correlation 0 5737 14000 12000 10000 oog BO00 Wavelength 926 4000 ef UU 2000 4000 gong s000 10000 Wi ave l e gth i 1 T 2 Select additional points by holding the CTRL button as you drag the mouse across addi
148. nter the appropriate values into the rest of the dialog box and complete the desired manipulation 04 720103 000 Rev C 1008 38 softWo Rx Imaging Workstation User s Manual pplied Precision 5 Data and Task Manipulation This chapter describes how to select data crop data from images and combine images In this Chapter PV LCE AL eh satsa iad ts acces A E saccades a weab A assge raises 40 Coppin r ania Tat Dalis a T A N ROEE 45 Cropping am recalar Vati RES On dann a tatananl emia cael 47 Combinne Patro WO ACCS octane scar sacyescagnre ENE ES 52 Setting Up Process Chains with Task Builder s covisssiivitesacutsssessasascaueanascetanetindeiocanlebsuets 54 MSU Rato Tma NE eano TE ices 57 Using the Multiplexed Wavelength Option essesssssseessssesessessssesessessesesnesesnesesseseeseseeses 62 COneCliINng o a DMS Databas Cesena a E E 74 Uploading Images toa DMS Datababesdrssnnscnoniieinnan nean 74 Do wnloadine Files Tom OMS nusoni aE A N 81 Browsing and Locating Images in a DMS Database seessesserseesseesseeserserseseeseserseeseeses 82 04 720103 000 Rev C 1008 40 softWo Rx Imaging Workstation User s Manual Selecting Data You can select either rectangular or irregular data regions m Use the Select Region and Details buttons to select rectangular data regions These buttons are included at the top of softWoRx dialog boxes that allow you to save export or select data Use the Edit Polygon tool to select
149. ocation This process increases contrast and comparative intensities within each Z stack image making for extremely sharp 3D reconstructions Unprocessed data on the left isdeconvolved to create the image on the nght Deconvolution Tools softWoRx provides two tools for deconvolving images Deconvolve and Nearest Neighbor The Deconvolve tool described in this chapter provides the best results for most applications This method uses the iterative constrained algorithm described by Agard This tool should be used for experiments where the quantification of intensities is required The Nearest Neighbor tool described only in the online Help uses an approximate deconvolution approach commonly referred to as deblurring for removing blur from optical sections The Nearest Neighbor tool should not be used in experiments where quantification of intensities is required 1 See Agard D A 1984 Optical Sectioning Microscopy Cellular Architecture in Three Dimensions Ann Rev Biophys Bioeng 13 191 219 pplied Precision Chapter 1 Deconvolving Image Data 11 Deconvolving an Image To deconvolve an image 1 On the softWoRx main menu choose Process Deconvolve to open the Deconvolve dialog box bd Deconvolve E Output Z piran tet Details Wavelengths _ Deconvolution Options 7 72 ANYI ee OTF File SEs Method Enhanced Ratio aggressive Number of Cycles fro Noise Filtering Medium pod Wo Apply Corr
150. ocess Deconvolve to open the Deconvolve dialog box In the Input field enter the R3D dv file for example usr local softWoRx data samples oocyte R3D dv You can use the same methods drag and drop browse or type to enter this file as those used in Step 2 of the previous procedure In the OTF File field enter the otf file for example usr local softWoRx data samples Olympus 60X 142 10612 otf In the Deconvolve dialog box click Run Options to open the Deconvolution Run Options dialog box Log File datatidy samples oocyte R3D DaD logt Command File datat dv_samples oocyte_R3D_D8D_cmd sh Run Options Run Mow W Run at Low Priority i Show Output Log Close In the Run Options pull down list select Add to Queue Then click Close 04 720103 000 Rev C 1008 14 softWoRx Imaging Workstation User s Manual 6 In the Deconvolve dialog box click Do It The file is added to the queue and displayed in the Queue Manager e Softwo Rx Queue Manager Host stech apicom Current Job lt queue not running ence job Pause After Job 0 Done Giueued Jobs Job ID Owner Status Command Delete 4 Worx Queued oocyte _R30_D3D_ecmd sh x start Mow Start Later Ghuit Delete My Gueued Jobs 7 Repeat Steps 2 and 3 and click Do It in the Deconvolve dialog box for each of the remaining files Q Tip If yourfile names are all the same except forthe last digit for example 040600a q 01 040
151. ocument Conventions In order to make the information in this manual as easy as possible for you to locate and use the following conventions are observed 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 Notes Wamings and Cautions Note Indicates information about the previous paragraph orstep in a procedure i Important Indicates important or critical information about the previous paragraph orstep ina procedure X Tip Indicates helpful advice AN WARNING Indicates important infomation regarding potential injury WARNING Indicates risk of explosion AN WARNING Indicates risk of shock 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 pplied Precision Preface Initial Capitals indicate the names of windows dialog boxes and tabs ALL CAPITALS SAN SERIF indicates the name of a key on your keyboard such as ENTER or DELETE Uniform width font indicates text to enter on a command line or in the GUI 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 fin
152. od of data collection 125 Projections 119 35 quick 119 22 volume 122 35 Punctate fluorescence 15 OLM See Quantifiable Laser Module Quality of images 215 19 Quantifiable Laser Module 3 203 Queue for deconvolution 13 14 manager dialog box 14 Queue manager 56 79 Quick projection 119 22 Ratio graph 62 Ratio imaging 57 62 Rectangular data regions cropping 45 47 selecting 40 41 Reducing time data 50 52 Region of interest 165 Removing noise 140 42 Rendering with volume viewer 122 24 Reorienting images 105 11 Repositioning images 92 Resample2D 105 7 Residuals 216 17 weak convergence of 217 Resizing images 105 7 Resolve3D 2 RGB opacity method 131 35 RGB opacity method of data collection 125 ROL See Region of interest ROI colocalization 201 2 Rotating images 107 9 128 31 Rotation angles 107 9 Rotation axes 123 24 Rotation center 110 Save as SYLK spreadsheet dialog box 169 Saving as JPEG 155 as Movie 153 55 as PhotoShop 153 as TIFF 151 DeltaVision files 148 51 statistics records 170 volume rendered images 135 with overlay graphics 153 55 Scale bar 103 5 Scaling pixel intensity 143 44 Screen shots 156 57 Scrolling Z sections 91 Secondary light path 63 Section intensity fluctuation 219 Select Polygon icon tool 182 Selecting irregular regions 41 44 rectangular regions 40 41 Selecting data 40 44 Show channel 95 Single Point FRAP 205 Smoothing pixel dista
153. og box 3 Click the color under the channel you want to modify and move all three color sliders all the way to the right to assign White as the Current Color then press OK Contolling the Image Window Display You can hide the Image window Display controls toolbar and scroll bars You can also display and set a scale on your images This is useful for preparing images for presentations 04 720103 000 Rev C 1008 104 softWo Rx Imaging Workstation User s Manual Hiding or Displaying Image Window Border Tools The border tools are the icons and controls on the left of the Image window The toolbar is the set of icons above the Image window These tools are displayed by default You can hide them to capture a JPEG of the image Hiding border tools and the toolbar can focus the screenshot on your data 1 Window actin aurb tub dapi midbody 1_rdd dyv File View Options Tools The Image window with the bordertools and toolbar hidden To switch border tools on and off gt Choose Options on the Image window menu and display or hide the border tools as follows To display the tools select the Show Border Tools toggle on the Options menu To hide the tools clear the Show Border Tools toggle on the Options menu To switch the toolbar on and off gt Choose Options on the Image window menu and display or hide the toolbar as follows To display the toolbar icons select the Show Toolbar toggle on the Options menu
154. oins the 2 D polygons together to create a three dimensional model This is helpful for quantitative analysis as well as visual understanding of the image data ppliedPrecision Chapter14 Volume Modeling 191 The 3D Object Builder Dialog box VSD oika Biler d n five 5 De ged et fa Fenriri PMSEA ADL RAD IS GA DOP TG PMI AIALL le H Input Image IE Folygon Data Details Wavelengths 528 W 457 W 617 _ Color Objects By Wave status zj i Maximum lt Cap f a a a a A E orn TA Dyed 3 Fan i n E S tga Ts pure Lay ER Information loaded into 3D Object Builder must include the polygon data The two methods of accomplishing this are described in the following procedures To load data from an Image window P Select Model 3D Object Builder from the softWoRx main menu and drag the desired Image window number into the Input Image field To load data from a saved polygon file 1 Load the image file which corresponds to the polygon file into the Input Image field by dragging and dropping the Image window number 2 Click Polygon Data and select the appropriate polygon file You may move up a directory level by clicking on the path bar above the desired directory 3 Click OK Creating and Viewing the 3 D Object Once the polygon file is loaded into the 3D Object Builder you are ready to create the 3 D object Before a 3 D object can be viewed it must be saved as a solid model Before the meas
155. ombines information from images or subtracts images to isolate features The Local Contrast Enhancement filter enhances local contrast around pixels The Threshold filter removes data below an intensity threshold Using Convolution Filters Use the Convolution tool to perform basic digital filtering such as high pass and low pass filtering When you select a filter the fields in this dialog box are updated to indicate the kernel values Q Tip You may add orchange convolution kemels to the list by modifying the CONVOLUTION FILTERS file in the softWoRx configuration directory Use the Revise Convolution Kemels menu item within the softWoRx Utilities menu to access this file To use Convolution filters 1 Choose Filter Convolution from the softWoRx main menu to open the Convolution dialog box 3 For more about Convolution filters see Digital Image Processing by Kenneth R Castleman Prentice Hall 1995 04 720103 000 Rev C 1008 140 softWoRx Imaging Workstation User s Manual select Region Reset Details Wavelengths W 528 617 457 d Filter High pass 1 sharpen contrast 0 000 1 000 0 000 ae p D00 5 000 A goo jeer o oo 1 000 o oo E Done Dio It 2 Enter an image file name or Window number in the Input field 3 If you want to include only selected data use the Select Region button and drag the mouse across the portion of the image you want to include Alternatively
156. on The Dimensions XYZT field displays the dimensions of the panel Use the following equation to determine the Width and Height values under Output Options Width x 2n Height y 2n Where x and y are dimensions X and Y respectively and n is the number of border rolloff voxels Click Close to close the Region Details dialog box Then click Do It in the Copy Region dialog box The cropped panel stack is displayed in a new Image window Choose File Save on the Image window menu to save the new cropped panel stack pplied Precision Chapter 3 Stitching 9 Stitch the cropped deconvolved file as shown in Stitching Images That Have a Single Z Section on Page 27 04 720103 000 Rev C 1008 29 30 softWo Rx Imaging Workstation User s Manual pplied Precision 4 Importing Data You can convert the following image file formats to the DeltaVision file format m TIFF Inovision ISee m BioRad MRC 600 Pic a UIC MetaMorph STK LL Note Since Applied Precision does not own the SKK PIC ISEE or TIFF formats changes to those formats may occurthat could make them incompatible with softWoRx If this occurs try saving the file asan older version of the format In This Chapter On Verte FLEE Mine OCS tise sie teeattasntee tie canes le eter aatd CaP Gat dese fetes Racba Gantt 32 CONVETHNG ec Piles itireeitdicesdaeiesasacccstiw decedent te oats eet 33 CONV Cilio 3 P 1 PCS ros ott eect ale Guia d Sled
157. on 218 Importing BioRad MRC 600 Pic files 35 file formats 31 Inovision See files 33 TIFF images 32 Inconsistent illumination intensity 17 Inovision See 33 Insert Point icon tool 182 Intensity data 162 74 and viewing rows or columns 167 68 Intensity fluctuation 219 Intensity scale 95 99 restore defaults 98 Intensity threshold 144 45 Intensity values plotting 166 Interactive image rotation 128 31 Internet Address Applied Precision iii Interpolated images 93 94 Invalid OTF 219 Irregular data regions cropping 47 50 selecting 41 44 JPEG files 155 K3b 157 59 ppliedPrecision AppendixA Image Quality Large images 25 29 Line intensity 167 69 viewing in any direction 168 69 Line Profile tool 167 Line profiles 167 68 Linear intensity scales 95 99 Live specimen tracking 114 18 Local contrast enhancement 143 44 dialog box 143 Local mean 143 Maximum intensity 125 Maximum parimeter 186 Measuring area of an object 188 chromatic correction 21 22 colocalization 194 97 colocalized ROI 201 2 distance See Distance measuring FRAP data 204 8 FRET data 208 13 residuals 216 17 velocity of particle movement 177 79 volume of an object 188 Methods of data collection See Data collection methods of Minimum perimeter 186 Mixed method of data collection 125 Modeling 181 92 2D Polygon Finder 182 85 3D Object Builder 187 90 Edit Polygon 182 85 Edit Polygon tool 182 85 example 190
158. on dialog box select which wavelengths to include 5 In the Quick Projection dialog box choose how to group sections as follows To group sequential sets of sections into output sections specify how many input sections to average for each output section in the Number of Sections to Average field To group all of the sections into one section select All 6 Choose one of the following ways to combine the sections in the Method list e To add the intensity of each pixel to create the output values choose Sum Be careful when using this option If the output intensity values are too large for the output data type specified the output image will appear to be saturated e To average the input data values to create the output image choose Average e To use the largest intensity value of all the input intensities to create an output value choose Max Intensity This method may give you the most realistic representation of a volume rendered image especially when combining all of the images in the input data set 7 Click Do It The projected image is displayed in a new Image window ppliedPrecision Chapter 9 Viewing Projections and Volumes 123 AZ Window myUpload_R3D_5_D3D_PRJ dv unsaved File View Options Tools 2 H geal __ ofie KT ll 5 00 00 35 217 A single Zsection image after projection Creating Volume Projections The Volume Viewer provides you with the ability
159. on to restore the selected channel to its original intensity scale Use the Restore All button to restore all channels to their original intensity scales Press Done when finished with the Scale All Windows dialog box Assigning Colors or Grayscale to Channels You can view image data in grayscale or in two different color modes Grayscale Mode is useful for studying detail in a single wavelength Because of the way the eye reacts to colors you may be able to see more detail in Grayscale than in a Color mode You can only view one channel at a time in Grayscale mode Color Mode can be used to visually compare intensities of two or three wavelengths It also allows you to use the Volume Viewer with RGB opacity ppliedPrecision Chapter 8 Viewing Image Data 101 improve speed of volume rendered images or save multi channel DeltaVision images as a series of TIFF images Blended Color Mode allows you to overlay nonfluorescent data such as Differential Interference Contrast DIC data sets onto fluorescent data sets or to visually compare intensities of more than three channels simultaneously Grayscale Mode You can switch between Color and Grayscale modes Because of the way the eye reacts to colors you may be able to see more detail in Grayscale than in Color In general Use Grayscale when you want to see more detail in a single wavelength of an image Use Color mode when you want to visually compare intensities
160. only The ratio imaging experiment results in a two channel time lapse image W 21 Window Data Collection w X File View Options Tools Lel N ie be 1S be on M co Sample image for ratio imaging experiment 04 720103 000 Rev C 1008 58 To setup a ratio imaging expenment softWoRx Imaging Workstation User s Manual 1 From the softWoRx main menu select File Acquire Resolve3D to open the Resolve3D window o N eD R File View Options Calibration Help Acquire Experiment Settings E Excitation CFP 430710 Emission CFP ji Yj 470 30 xt BLOCK gt EX Shutter EX ERE Exposure 000 i Find Calibrate Image size 512x512 Bg Lens 10x i Info Bin 1x1 bd J Aux Mag Pixel size 0 6680 um L3 UW OW Wu O hax hilean Scale min o Scale max 65535 d ew ey ere as Spee Looking for DV controller Initializing DV controller Initializing Camera EE 2 On the Resolve3D window click the Experiment button to open the Design Run Experiment dialog box pplied Precision Chapter 5 Data and Task Manipulation 3 Select the Sectioning tab and unselect the Z Sectioning toggle Design Run Experiment Resolve3D exp File Au g Ae Design Experiment Design PK Experiment Run Experiment Experiment
161. ools Tool Buttons Wavelength Selectors 15 iia lt p ee es oe z230 T 00t06t13 763 Status Bar ppliedPrecision Chapter 8 Viewing Image Data 91 The Image Window The Image window provides controls and tools that you can use to view and analyze image data The Menu Bar allows you to open or save images control the display of the data and open tools to analyze intensity data The Toolbar provides buttons as altemativesto the menu barabove The buttons can be used to open or save image data files play movies view intensity line profiles or inspect intensity data The Tool Buttons are used to change the image view These controls allow you to scroll through Z sections and time points zoom pan the image vertically or horizontally select which channels to view and scale intensity to adjust brightness and contrast The Wavelength Selectors show or hide the wavelengths channels in the Image window When the Image is displayed in color each button has the same color as the wavelength that it controls The number on each control button indicates its wavelength When the data is displayed as erayscale use these controls to choose which wavelength is displayed The buttons are white with black numbers when on and black with white numbers when off The Status Bar shows which Z section or time interval is displayed It also displays the intensity of the pixel currently under the mouse pointer The Quick Scale Too
162. ormation and relationship among allthe wavelength data sets Different methods maximum intensity additive and progressive can be assigned to different wavelengths Application Best choice for showing intemal detail of a translucent image Generates quantitative projections Thisdata can be used for companson of intensity in va rious structures within the image data Cleary displays opaque features within an image Works well for objects whose intemal details are not needed such asmetaphase chromosomes Realistically rendersa volume ina multi wavelength image Cleany relatesthe data points of the vanous wavelengths One wavelength contains diffuse cloudy features and isset to the maximum intensity method and another wavelength has Opaque featuresand the method is set to progressive ppliedPrecision Chapter 9 Viewing Projections and Volumes Method Func tionality VolPack Generates images that use lighting techniques to highlight surfacesin the 3D rendered image 127 Application Sub sta ntia lly faster than the other methods supported although the method may not be optimal forall image types A Note VolPack uses libraries obtained from the Stanford Computer Graphics Laboratory It isan implementation of the shearwarp volume rendenng algonthm asdescnbed in Lacroute P and Levoy M Fast Volume Rendering Using a Shear Warp Factonzation of the Viewing Transformation Proc SIG G RAPH 1994
163. ou can print DeltaVision files from softWoRx If you have softWoRx Explorer you can print DeltaVision files from a Mac or PC computer Analyzing Results You can use measuring and modeling tools to perform quantitative analysis Examining Intensity Data Study area and line profiles calculate statistics and display single point values Measuring Distance and Velocity Measure features on an XY plane or across Z sections You can also measure the velocity of particle movement Modeling Use tools to create line models or volume models Detecting Colocalization Use Colocalization modules to create a scatter plots and measure the Pearson Coefficient of Correlation to help determine whether colocalization is occurring Analyzing Huorescence Resonance Energy Transfer Data Use the Fluorescence Resonance Energy Transfer FRET module to analyze FRET data Analyzing Huorescence Recovery After Photo bleac hing Data Use the Analyze Fluorescence Recovery After Photo bleaching FRAP module to analyze FRAP data pplied Precision Introduction Optional Components You can purchase two optional components for softWoRx The Quanttative Laser Module The Quantitative Laser Module QLM is a DeltaVision component that 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 This optional component mounts to the Fiber Optic Module of a DeltaVision Cor
164. ove All Files m Processing Tasks Task Options Quick Projection a is Crop Image mj o save ta DMS a Add submit To Gueye submit To Remote Server 4 Click the Options button next to the Save to DMS selection The Save to DMS Options menu is displayed pplied Precision Chapter 6 DMS Integration 79 fed Save to DMS Options DMS Database Connection Disconnect from the Database m Destination Parameters Category Group KP_TestGroup x New kpalmby Category KP_TestCat x New kpalmby Initial Annotatian sample 5 Optional OK Cancel 5 From this menu select the Project and Dataset to which you want to save the data on the DMS database You can optionally select the Category Groups and Category for the saved image data and add initial annotation to the file for future reference When you are satisfied with the image data to be uploaded click OK You are returned to the Processing Task Builder menu 6 From the Processing Task Builder menu click Submit to Queue to open the softWoRx Queue Manager dialog box e E Rx Queue Manager Host istech apis 1 00 m Current Job lt queue stopped gt Cancel Joh Pause After Job 0 Done Gueued Jobs Job ID Owner Status Command Delete Delete Wore Gueued Nuclear_Pore_d3d_process sh WwOrH Queued Nuclear _Pore_rsd_process sh wor Gueued Soind le_Green_d3d_process sh WOT Gueued oocyte r3d_proc
165. ox Then specify the ranges of data that you want to include in the Output Options fields 4 Select which wavelengths to filter 5 If you want to use the arc tangent method to calculate the gradient select Atan To use a linear method unselect this option 6 Specify the relative contribution of the original image and the gradient image by entering a value between 0 and 1 in the Fraction field Increasing the Fraction value increases the relative contribution of the original image 7 Click Do It to apply the filter 04 720103 000 Rev C 1008 142 softWo Rx Imaging Workstation User s Manual Using 2D Statistic al Filters Use 2D filters to remove pixels with noise like intensity or to achieve other effects To use statistical filters 1 Choose Filter Filter2D from the softWoRx main menu to open the 2D Filter dialog box Output E Select Region Reset Details Wavelengths 526 W 617 W 457 _ Method Median a Kernel Size 5 Iterations i Done Lio It 2 Enter an image file name or window number in the Input field 3 If you want to include only selected data use the Select Region button and drag the mouse across the portion of the image you want to include Alternatively you can click Details to open the Region Details dialog box Then specify the ranges of data that you want to include in the Output Options fields 4 Select which wavelengths to filter 5 Select one of the following filters i
166. p a e oo ppliedPrecision Chapter 12 Examining Intensity Data 175 3 To automatically find the borders around objects based on changes in intensity values select the Guided Mode option 4 Drag the mouse to draw the polygons on the Image window You can draw sets of polygons on the same Z section and on different Z sections 1 Window actin aurb tub dapi midbody 1_02_r3dtest dyv ea gt File View Options Tools Help a Bt ia L a27 457 123 Ea l p 5 To copy a polygon to other sections or wavelengths use the arrow tool in the Edit Ploygon window to select the polygon Then choose Edit Propagate Polygons SEE 457 131 6 Choose Statistics Table to display a table that shows statistical values for each polygon 04 720103 000 Rev C 1008 176 softWo Rx Imaging Workstation User s Manual Polygon Statistics Oa Pior Polygon Graph Main Label Polygon Statistics Optional Label X axis z Dd Show Legend W Done Do It 8 In the Y axis field choose which type of statistical parameter e g SD to plot 9 Choose other options in the Plot Polygon Graph dialog box and click Do It to display a graph of the statistics values ppliedPrecision Chapter 12 Examining Intensity Data 177 P oly qo ns File Graph Point Labels Polygon statistics 10 From the Polygons window File menu choose Save As SYLK to save the fi
167. pproximated from the CCD detector element size and the total image magnification For example if the CCD detector has 6 7 um pixels and the image was acquired with a 100X lens and a 1 5X optivar then the pixel size is approximately 6 7 um 100 x 1 5 0 045 um The Z pixel spacing is the distance between adjacent optical sections pplied Precision Chapter 4 Importing Data 37 Figure 3 ba Convert MetaMorph STK to DY STK File ey DY File SSS stack Type Z OenesS Wavelengths lo b gt fo lb b gt spacing um ooo Y Spacing tum 1 000 Z Step Size um 1 000 Lens ID lo Convert to signed 16 bit _ Do It Options in this dialog box are described briefly in the following paragraphs For additional information regarding STK file conversion refer to your online Help system To convertan SIK image file to a DV image file 1 Click Conversions Import from MetaMorph STK in the softWoRx main menu The Convert MetaMorph STK to DV dialog box is displayed as shown in Figure 3 2 Select the file that you want to convert using the STK File button and data entry field 3 Type a filename for the converted file into the DV File field 4 Enter the wavelengths in nm of the light collected by the camera for each channel into the Wavelengths fields 5 Ifnecessary modify the X Y and Z spacing of the pixels in the data 6 Enter the correct lens number in the Lens ID field 7 Click Do It 8 Proceed to e
168. pter 8 Viewing Image Data 97 You can adjust the intensity scale for an individual image window or for all open image windows simultaneously O Note Changing intensity scale values only adjusts the display of the data It does not alterthe image data To change the intensity scale forthe current image window 1 On the Image window click the Scale Image Intensities ES button to open the Image Scaling dialog box This dialog box shows the image intensity scale W 1 Window cell_Z10C3_ r3d dv File View Options Tools 2 B v m a 2 r ll ale TY v scale Image Windowl Wave 457 v 528 617 Display Min Max Exp 16 00 562 00 1 00 Wave Min Max 16 00 562 00 Section Min Max Mean 17 00 508 00 111 71 rN W Show Histogram W Log Apply To Wave o bd ee Z 5 Zoom 0 50 457 ik Copy Scale Paste Scale Done Revert Restore Default Scale Help The Scale Image dialog box isused to adjust the intensity scale The histogram on this dialog boxisa frequency plot that shows the distribution of pixel intensities in the image file The Y axis shows the numberof pixels fora given intensity 2 Inthe Wave field select which channel to scale 3 To change the minimum or maximum scale value click and drag on the Left or Right handles 04 720103 000 Rev C 1008 98 W 1 Window cell ZI0C3 r3didv softWoRx I
169. ptions in the dialog box as necessary for your calculation 10 Click Do It The results of the calculation are displayed in the file or window defined in the Results field Scaling Pixel Intensity to Enhance Local Contrast Use Local Contrast Enhancement to scale the intensity of each pixel in the image based on the Local Mean and Local Contrast of the pixel The term local refers to the fact that the mean and contrast are calculated from the pixel elements that form a box around the pixel of interest To use nonlinear local contrast enhancement 1 Choose Filter Enhance Contrast from the softWoRx menu to open the Local Contrast Enhancement dialog box a Local Contrast Enhancement ree fi Output a Select Region Reset Details Wavelengths W 528 W 617 W 457 d Box 3 E AutoRange Min Max 0 00 10000 00 LM weight function 7 weight function Apply To All Sections Options Done Do It Help ppliedPrecision Chapter 11 Saving Exporting and Printing 145 2 3 Enter an image file name or window number in the Input field If you want to include only selected data use the Select Region button and drag the mouse across the portion of the image you want to include Alternatively you can click Details to open the Region Details dialog box Then specify the ranges of data that you want to include in the Output Options fields Select which wavelengths to filter Specify t
170. quired Nuclear_Pore_D3D dv Nuclear Pore _ D30 FRET dwr 0170907 15 26 Nuclear Pore_d3d dr Nuclear Pore_djd CLC VOL dy Last Modified Nuclear Pore _d3d_ CLC VOL_COR_log txt 01 09 07 15 26 Nuclear Pore d3d D30 dv Nuclear Pore d3d D3D_COR_log txt File Size Nuclear Pore d3d_D30_ocnd sh Nuclear Pore_d3d_030_log txt 27 45 Mb Nuclear_Pore_d3d_process sh Nuclear Pore djd_task sh F Image Infa i 7 49 Wed T 1 File Name Nuclear Pore d3d dyf OK File Types p Cancel Select and preview DeltaVision images in the Open Image dialog box Q Tip For Delta Vision images you can adjust the thumbnail image in the Preview area Change the brightness by dragging the left mouse acrossthe preview image View different Zsections or time points by pressing the nght and middle mouse buttons 3 Select an image file for example Nuclear _PoreD3D dv and click OK to open the image in the Image window 04 720103 000 Rev C 1008 90 softWoRx Imaging Workstation User s Manual iv FE Window Nuclear_Pore_d3d dv File View Options Tools ag B o Cr a 59 E N jl 5 The image opens in the Image window Q Tip You can also open an image by clicking Data Folder and then double clicking onthe dvfile in the datal directory The Image Window DeltaVision images are displayed in the Image window td 3 Window Echelon0l_ R3D dv Menu Bar File View Options Tools Tool Bar Quick Scale T
171. r outside of each polygon and copies it to the output destination e Choosing Mask creates an output file with only 1 s and 0 s representing either the inside or the outside of the polygons In the Threshold field set a background intensity to remove from the selection For example setting a threshold of 200 selects only data with an intensity value greater than 200 Note With Trim Output selected the smallest area containing all the polygonsis the area wnitten to the output window With Tim Output unselected the size of the file hasthe same x y dimensions asthe orginal file but only the part defined by polygons has intensty 10 Click Do It to crop the image 04 720103 000 Rev C 1008 softWoRx Imaging Workstation User s Manual bd 3 Window 7326_1_R3D_D3D_MSK dv unsaved File View Options Tools oe K 6 T 2 00 00 46 41 ay 528 0 ja of IN a gt Thmming Time Data You can trim time points from time Ae images 291 Window LiveCells GFP_2D Movie d3d dv 00 File View Options Tools el r 61 00 31 12 000 528 236 Starting image with 61 time points To im time points 1 Open the image in the Image window 2 From the main softWoRx menu choose Edit Copy Region pplied Precision Chapter 5 Data and Task Manipulation 51 Output f select Region E Details Wavelengths W 528 d Done Do It 3 Inthe Input field enter the
172. ra ratherthan the illumination wavelength X Y pixel spacing can be obtained in two ways it can be measured with a test target or it can be approximated from the CCD detector element size and the total image magnification For example if the CCD detector has 6 7 um pixels and the image was acquired with a 100X lens and a 1 5X optivar then the pixel size is approximately 6 7 um 100 x 1 5 0 045 um The Z pixel spacing is the distance between adjacent optical sections Figure 1 ISee Converter Convert InoVision See to DeltaVisio n ISee File DY File _ISE dy Convert Series d Wavelength nm 510 x Spacing tum 0 0000 A Y Spacing um ooo Z Step Size um ooo Lens ID gt Done Doit Options 04 720103 000 Rev C 1008 34 softWo Rx Imaging Workstation User s Manual Options in this dialog box are described briefly in the following paragraphs For additional information regarding ISee file conversion refer to your online Help system ISee Senes Conversion The Convert Series option combines all files with a similar name into one DeltaVision file The conversion program looks for files that have the same prefix as the input file The program assumes that a series of Inovision files will be the same except for the sequence numbers in the last 3 characters For example the following files would be automatically combined into a single DeltaVision file my file name 001 my file name 002 my fil
173. reduction factor in the XY Reduction Factor field 6 Select Keep Cell Dimensions to keep the X and Y pixel size constant If unselected a new size is calculated based on the current settings for magnification v Se Output E select Region Reset Details Wavelengths W 528 W 617 J J J Z Rotation im Shift In XY po 0 0 Angle Between Axes 90 0 Magnification In YY xY Reduction Factor 1 000 1 000 Output XY Size 142 118 Keep Cell Dimensions j j Done Do It Help 04 720103 000 Rev C 1008 108 softWo Rx Imaging Workstation User s Manual 7 Click Do It iw 3 Window imageTest_R3D_NND_RSP dv unsaved File View Options Tools 2 H B v m rn EIE Rotating an Image To rotate an image Nd i Window Spindle Green_d3d dv File View Options Tools Z 8 Zoom 2 01 528 115 Image before rotation ppliedPrecision Chapter 8 Viewing Image Data 109 1 Choose View Resample2D from the softWoRx main menu to open the Resample2D dialog box 2 Enter the window number in the Input box 3 If you want to reorient part of the image click Select Region and then drag the mouse across the area in the Image window 4 Select the wavelengths that you are interested in 5 Enter the angle to rotate the image in the Z direction in the Z Rotation field 6 Enter the distance to shift the data in the Shift in XY field
174. rithm 16 Contact Applied Precision iii Contrast 95 99 Conventions document ii Converting file formats 31 Convolution filters 138 39 Correct Image tool 17 18 19 Correcting images 17 25 chromatic aberration 18 21 22 inconsistent illumination intensity 17 motion artifacts 18 photo bleaching 17 Cover slip 112 Create Polygon icon tool 182 Cropping regions 44 50 AppendixA Image Quality Cross sections 111 12 Crosstalk calculating 209 Customer Service Hotline iii Cut Mask 49 Dark halo 218 Data Presenting 85 Visualizing 85 Data collection methods of 125 additive 125 maximum intensity 125 mixed 125 progressive 125 RGB opacity 125 VolPack 125 Data files 148 combining 52 54 defined 148 filtering 137 45 opening 88 89 Data Inspector 163 65 3D graphing with 165 and statistics 169 74 Database integration 73 84 Deconvolution 9 16 about 10 constrained iterative algorithm 16 Deconvolve dialog box 12 holes 218 nearest neighbor method 10 16 options 14 16 residuals 216 17 run options 13 single image 11 13 standard method 10 16 using a queue 13 14 visually assessing 217 Delete Selected Point icon tool 182 Delete Selected Polygon icon tool 182 DeltaVision archiving files from 157 59 exporting files from 151 56 saving files for 148 51 Display border tools 103 Display channel 95 Distance measuring 175 77 options 176 Tutorial 176 DMS about 73 connecting to data
175. ructures The halo problem is often caused by the presence of excessive spherical aberration As always ensure that the image properties are correct before beginning an exhaustive study of this sort of problem ppliedPrecision AppendixA Image Quality 221 Use the Line Profile tool to assess the relative magnitude of the dark intensity region Although very noticeable the intensity of the halo is often quite small e g about 5 below the adjacent background Another source of halos is refractive index changes Bright Spot Problem In situations where the object contains concentrated areas of fluorescent probe it is natural for the deconvolution process to yield even brighter spots in the resulting image For example a region with 2000 counts of fluorescence could deconvolve to a brightness of 8000 counts The intensity of such areas can be so great that low intensity structures present in the 16 bit image are not visible on a standard 8 bit computer screen Although these low intensity data exist these data are simply not visible next to the bright spots To view low intensity areas adjust the image contrast brightness and intensity scaling factor Pebble Grain Texture Low intensity texture patterns are often visible in low intensity images where the signal to noise ratio has dropped below about 10 to 1 Increasing the image intensity is the obvious method of avoiding this problem This can also be caused by problems with th
176. s or an XYZ region You can also export DeltaVision files to TIFF Movie or PhotoShop formats and capture screenshots of images After you save images or data you can archive them to a CD You can also print TIFF or DeltaVision files as images directly from the Image window In This Chapter Image Data Piles and Image Graphic Piles crror sovsaswesassnrseostesuens 148 Savne PeraViston TINGS eresanaei iT a sau aed sui A eessepaantoaae inca eeseetse 148 EXPOrtine Delta VISiOn Tiles cosain E 151 Cap turint oreen OLS ea ranean nar teaelaa a Seaeinasheceanaaties 156 Arvin e aidlecm con iO 8 4 B ieena rrr nn rire rT errrer Rerun ert ree errr here 157 PVG Map e Srina n A A a 159 04 720103 000 Rev C 1008 Chapter 11 Saving Exporting and Printing 149 Image Data Files and Image Graphic Files softWoRx distinguishes between image data files and image graphic files Image data files contain more information than image graphic files image data files can be used for analysis while image graphic files are used as graphics e 5D Image data files include spatial and possibly temporal and spectral data e Image graphic files that are created when you save or copy Image windows include only the 2D pixel data that is displayed inside the window Saving DeltaVision Files You can save complete DeltaVision files or you can choose to save only specific data softWokx allows you to select an area and to specify which Z sections and time points to incl
177. s to ROI polygons are made the table of statistics is updated to reflect the statistics of the chosen ROI set 6 To export the table of numbers in a form that can be used in a spreadsheet choose Save Results As SYLK Symbolic Link format or Save Results As CSV Comma Separated Values from the File menu on the FRET Analysis dialog box 04 720103 000 Rev C 1008 216 softWo Rx Imaging Workstation User s Manual 7 To view the results in a graph choose Plot Results to generate an X Y plot of parameters that you choose If the X axis is time related the software associates ROIs from time point to time point and plots the values as connected sets on the graph Q Tips 1 You can modify the details of how the graph isdisplayed after it is created with the Graph Properties tool 2 You can optionally save the FRET Results graph by selecting Save AsJ PEG from the FRET Results graph s File menu ppliedPrecision Chapter 16 Other Applications 217 04 720103 000 Rev C 1008 Appendix A Image Quality Because many restoration problems can be attributed to problems with data collection you ll find it worthwhile to become familiar with the common problems and their solutions All of these methods are simple to perform on a regular basis Note Although the softWoRx restoration algorithms contain many refinements that improve their ability to handle expenmentally obtained optical sections there are certain types of da
178. save the image select Save Image e To save the overlay graphics select Save Overlay 5 Click Do It to complete the export Exporting to Movie Files You can save the contents of an Image window as an MPEG movie in 24 bit RGB color You can choose to ppliedPrecision Chapter 11 Saving Exporting and Printing 155 Save the image with the overlay graphics e g scale markers merged into a single image save it without overlay graphics or save only the overlay graphics Save movies that include Z sections time lapse data or both types of data Specify a range of data to include in the movie file You can play the MPEG movie format on the QuickTime viewer the Windows Media Player or a variety of Linux or Macintosh movie players You can also import movies into PowerPoint Double clicking on an MEPG file in a file browser opens the PlayMPEG viewer which you can use to vary the speed of the movie To save a movie 1 2 3 From the Image window choose File Save As Movie to open the Save Movie dialog box Oa Save I ed R Window i1 W Save Image W Save Overlay Movie File datal dv_samples Drosophila_Embryo_Movie_d3d rmov Animation Style Forward Compression Quality ee o 00 Frame Rate FPS r Movie Duration 1 0 sec Animate Through Z Sections 5 7 StarvEnd Increment A E a Animate Through Time 49 1 i x i Time StartEndiIncerement i as f Done
179. shnise catia E EN 203 Analyzing Fluorescence Recovery After Photo bleaching 0 00 eee eeeeeeeeeeeeeees 204 About RAF EXPErnMEN Siene E E ie 205 Analyzing Fluorescence Resonance Energy Transfer sesessssessersersrrsresresrserseeseesees 208 Using the FRET Analysis Tool Appendix A Image Quallity ssccssssscsssscssssscsnsssesnsseennsseensssesesees LLS Using Deconvolution Residtials icicsacrantintianiinwitienviad asnetieei AE 216 Vistially EvaluaUne a CS oie acciadsisuvantastiensbicinenstelseieass 217 Index 04 720103 000 Rev C 1008 Preface This manual shows how to use softWoRx to process visualize and analyze image data a About This Manual describes the information in the manual 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 is divided into three parts that contain the following information Part One includes instructions for processing and importing data Part Two shows how to visualize data and prepare it for presentations It also shows how to save or export data in a variety of formats Part Three shows how to use softWoRx tools to perform quantitative analysis The manual also includes an appendix that shows how to analyze image quality 04 720103 000 Rev C 1008 softWo Rx Imaging Workstation User s Manual D
180. sidual may not be as useful as the average count residual Use the standard residual to compare deconvolution performance results with deconvolutions prior to version 2 10 of the DeltaVision softWoRx software Refer to the following table when assessing the standard residual Value Quality Suggested Action gt 0 1 Poor deconvolution Check expenment conditions and data quality quality 0 1 0 05 Marginal May not be appropnate for this data Deconvolution 0 05 0 01 Reasonable Typical fordata with a low signal to noise ratio ora large amount of sohencal abenation lt 0 01 Good Nomalized Residual As a means of watching the deconvolution progress the Normalized Residual is also displayed By definition the normalized residual equals 1 after the first 04 720103 000 Rev C 1008 220 softWo Rx Imaging Workstation User s Manual iteration Subsequent residuals are then scaled in the same way as the first iteration to yield numbers between 0 and 1 A residual larger than 1 indicates that the deconvolution algorithm has encountered serious difficulties The table below gives a guideline for assessing normalized residuals Value Quality Suggested Action gt 1 0 Worse than before the first Review expenmental conditions iteration 1 0 No improvement 0 5 Reasonable improvement 0 25 Substantial improvement 0 10 Excellent improvement Most deconvolutions yield residuals somewhere between 0 1 and 0 3 Consistent results bet
181. softWoky Imaging Workstation User s Manual Revision C built with precisionware ppliedPrecision softWoRx Imaging Workstation Users Manual Legal Notices Revision C of the User s Manual for the softWoRx Imaging Workstation Part number 04 720103 000 Rev C 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 Applied Precision and softWoRx are registered trademarks of Applied Precision Inc 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 Manuals and Guides The following documents are provided for softWoRx Document Online Help Product Notes The Delta Vision RT Restoration Microscopy System User s Manual Getting Started with QLM RedHat Linux Bible Purpose Provides reference information for softWoRx and procedures that show how to use softWoRx tools Provide examples and tips for using softWo Rx Shows how to acquire data and how to maintain the data acquisition system Shows how to acquire photokinetic data with the QLM module Sho
182. t Measunng Velocity You can use the distance tool to measure the velocity of objects in time lapse data Use the Measure Distance tool to measure the velocity of particle movement 1 Open an image in the Image window 2 On the Image window menu click Tools Measure Distances to open the Measure Distances dialog box ppliedPrecision Chapter 13 Measunng Distance and Velocity vie qa DELanCee Window i Measure Method standard Two Point Units Micrometers Draw Lines W Search For Nearest Last Point et aa n 00 0 00 0 00 Next Paint El Total Points p E A ALN Pigrimtrs D oeyant flee Dla bees ees eT a To a BeO MAME Nae ae ee ts ee Done save Options 3 Inthe Measure Method list select the Standard Two Point method 4 Inthe Units list select the appropriate unit of measurement 5 In the Image window click on the particle that you want to measure i Window DMS AF _testi4 2 R3D_D3Ddv File View Options Tools S h z bOd a AHR Ss 457 132 04 720103 000 Rev C 1008 Original Time Point 181 182 softWoRx Imaging Workstation User s Manual 6 Move the T time slider on the Image window to display the particle at a different time point 7 Click the same particle that you selected at the original time point v 1 Window DMS AF_testl4_2_R3D_D3D dv File View Options Tools 1 4 A 528 G
183. t applies to the camera and conditions array size pixel size and wavelength used to collect this image If you do not have a calibration file you must create one before you calibrate the image To Calibrate an image 1 Choose Process Calibrate from the main softWoRx menu to open the Calibrate dialog box pplied Precision Chapter 2 Corecting Images 21 Output a Select Region Reset Details Wavelengths W 457 W 528 W 617 J Calibration Files cia o pus W Calibrate Gain W Calibrate Offset Replace Bad Pixels Intensity Offset 50 00 Done Do It 2 Click Input and browse to the file that you want to calibrate 3 To select a region of the file click Details and use the Region Details dialog box to select the region 4 Click the Cal button for each channel and browse to the calibration file to use for this image 5 Click the Pix button for each channel and browse to the bad pixel file to use for this image 6 Select the types of calibration to perform Calibrate Gain Calibrate Offset or Replace Bad Pixels 7 Click Do It to calibrate the image Aligning Adjacent Images Use Align Image to correct motion artifacts problems with Z sectioning or problems with time series This tool allows you to align adjacent images by applying an XY shift with an optional rotation Use Align Image only for images that have a single wavelength To align images 1 Choose Process
184. ta that simply cannot be properly restored In This Appendix Usine Deconvolution ReSiGU als wes caccsisusirssinsesturtanaemanerannieiaaeinaneaamiaas 216 Visually Ev ala LIE MIA COS east cia dss TOO 217 04 720103 000 Rev C 1008 AppendixA Image Quality 219 Using Deconvolution Residuals What is a Residual The deconvolution residual is a measure of the difference between the measured image and the solution convolved with the point spread function PSF In mathematical terms Residual Measured Image Deconvolved Image PSF where represents convolution In principle the two quantities on the right side of the equation are equal so that the residual should be zero Not surprisingly however the use of experimental data prevents perfectly precise results and the residuals are not zero For the purpose of digital deconvolution the residual is calculated from the average of the residuals measured at each point in the three dimensional image As a general rule a small residual is better than a large residual The most useful form of the residual is the Average Counts Residual which is the average difference between the measured image and the result convolved with the PSF The Standard Residual The standard residual is the sum of all residuals divided by the sum total intensity of the image Images with a large total intensity may therefore yield an unrealistically small residual For this reason the standard re
185. te Holding yourmouse cursorover the Search Annotations For field displays a tooltip window containing specific wildcard use information pplied Precision Part Iwo VISUALIZING amp PRESENTING DATA softWoRx provides several tools that you can use to visualize data and prepare it for presentations You can also save or export data in a variety of formats In Part Two Chapter 7 Viewing Image Data ssssssssccssssssseeccssssssseesesnssseeseesnnssseseensnaseesseses 37 Chapter 8 Viewing MOVIECS ccssssssseccssssssssseessessseeeeesnesseesessnnssseeseennsseeenses 113 Chapter9 Viewing Projections and VOIUMECS sssssssssssssssseesessesseeeessneseeees 119 Chapter 04 720103 000 Rev C 1008 86 10 Filtering Image Data 000 Chapter 11 Saving Exporting and Printing softWo Rx Imaging Workstation User s Manual ipplied Precision Chapter 6 DMS Integration 04 720103 000 Rev C 1008 87 7 Viewing Image Data softWoRx provides several options for viewing data You can open DeltaVision or TIFF files in the Image window and use slide bars and controls to view different Z sections and time points You can also adjust the brightness and contrast of each channel in an image and assign a color or grayscale to each channel To prepare your data for presentations you can display a scale bar on the image and hide the Image window controls You can also resample image data to change the size or
186. tional areas E Ws is Fie Graph Paint Labels Image Nuclear_Pore_d3d dv Pearson Coefficient of Correlation 0 5737 14000 12000 10000 5000 00g oo CJ ua ml T T Ti 4000 000 z000 4000 gogg BO00 10000 Wavelength 617 As you select points they are highlighted in red on the image graph and displayed in white on the three channel image 04 720103 000 Rev C 1008 202 softWoRx Imaging Workstation User s Manual File View Options Tools Help 3 Choose View Volume Viewer on the softWoRx main menu to open the Volume Viewer window and drag the output window number post colocalization into the Input field 4 Select Volume Viewer parameters and click Do It to render the volume File View Options Tools Help 5 Click Interactive on the Volume Viewer and examine the image from several angles to find intense white areas that indicate potential colocalization ppliedPrecision Chapter 15 Detecting and Analyzing Colocalization 203 File View Options Tonts Help File View Options Tools Help a Zoom ani 6170 zi tl Interactive Viewer shows product channel and onginal data 6 To view only the colocalized channel select the channel Alternatively you can view the colocalized channel by changing the grayscale color map for the channel to a rainbow or cold to hot color map so the bright intensity is displayed in a different color To change the graysca
187. ude You can also choose which channels to save To save a dv file 1 From the Image window choose File Save to open the Save File dialog box Gutout datat dv_samplesitripolaro1_01_dad dyj select Region Reset Details Wavelengths 457 W 617 W 528 685 d _ Scale to Display MiniMax Done Do It 2 Enter an input file or window number in the Input field Q Tip You can also specify an existing window by dragging an Image window button from the main softWoRx menu to the Input field or by dragging the window number indicatorin the upper left hand comer of the Image window to the Input field 3 Enter an output file or window in the Output field If the window or file exists you will need to choose whether to overwrite the data or to append the new data as new channels 4 To save a region of a window choose Select Region and select an area in the Image window by dragging the mouse across the area Adjust the rectangle you ve created until it contains the desired area Then click outside the Image window with the mouse 04 720103 000 Rev C 1008 150 softWoRx Imaging Workstation User s Manual 2 KE mM R UE P 25 Zoom 2 01 457 175 The Region of Interest A Note Selecting a region isoptionaland you can do this only when your input is a window 5 Click Details to open the Region Details dialog box Specify the ranges of the X Y Z and time data to save
188. urements can be viewed it must be saved as a measurement file These measurement files are saved as tab delimited text files that can easily be exported to spreadsheet programs such as Microsoft Excel To build and save a 3 D object 1 Select Model 3D Object Builder on the softWoRx main menu 04 720103 000 Rev C 1008 192 8 softWo Rx Imaging Workstation User s Manual Load the data into the 3D Object Builder as described in the previous section Choose the desired wavelengths to be modeled Click Build 3D Objects Click Model in the 3D Object menu Click Save Solid Model Enter the desired name in the field By default softWoRx will use the previous file name and replace the file extension with SOL Click OK To view a 3 D object not available without optional 3D Model 1 2 Click Model in the 3D Object Builder menu Click View Model Note You must have the orginal Image window open in order to view the 3 D model To measure the area of an object f 2 Click Measurements in the 3D Object Builder menu Click Table of 2D Measurements The Save Measurements File dialog box is displayed Type the desired name for the 2 D measurement file Click OK Open the folder containing the saved measurement file Double click the icon of the desired measurement file to view a text file similar to the following ppliedPrecision Chapter 14 Volume Modeling A idataiNuclear Poret 3d 2D xt gedit
189. ust have the same X Y and Zdimensions To perfomm an image anthmetic calculation 1 Click Filter Image Arithmetic in the softWoRx main menu to open the Image Arithmetic dialog box Image 2 l Result fa select Region Select Region Reset Detalls lt Reset Details Image 1 Wave f Image 2 Wave Image1Waveli Image waveft 000000000 Equation A1 Image1 B1 0 A Image B2 Operation o Addition W Process Image 1 Al a oond B 0 nong W Process Image z Az W Process Image 2 A2 10000 Be fooooo nong Bz o noga A Absolute Value Log Transform A Exp Transform Apply Thresholds ioe EOG Auto Scale Done Do It 2 Drag a window number from an Image window into the Image 1 field or click Image 1 and browse to an image file 3 If desired repeat Step 2 for the Image 2 field 4 Define the destination file or window for the calculated result The default is Window 1 04 720103 000 Rev C 1008 144 softWoRx Imaging Workstation User s Manual 5 Enter the appropriate input wavelength from Image 1 into the Image 1 Wave field 6 If you are using a second image enter the appropriate wavelength from Image 2 to use as input into the Image 2 Wave field 7 Select the appropriate operation from the Operation o list 8 Set the Process Image 1 and Process Image 2 options to enable or disable the processing of the appropriate images 9 Enable or disable the other o
190. ween 0 3 and 1 0 should prompt a review of experimental conditions and the deconvolution problems listed in Visually Evaluating Images In particular you should check pixel sizes wavelength and PSF selection Visually Evaluating Images The second method of assessing a deconvolution is to simply study the resulting images Comparison of the measured and deconvolved images is an excellent way of verifying that structures present in the results are a valid representation of the actual object With the aid of the deconvolution image it is usually possible to understand the structures present in the measured image It is expected that certain deconvolutions will be less successful than others due to the dependence upon experimental data It is not always possible to meet the conditions required for deconvolution microscopy Fortunately there are only a few characteristic problems Weak Convergence of the Residual A common cause of poor convergence is that the optical sections were measured in the presence of spherical aberration As a consequence the standard PSF is not appropriate for deconvolution Flip the image on its side using the Flip or Rotate program and study the quality of the image along the optical axis Z Asymmetric blurring and greatly elongated points indicate spherical aberration Use of very low intensity images with a corresponding low signal to noise ratio can also limit convergence Dark Halo Around Brght St
191. wesueviews annei a A T eae 166 Viewing the Line Intensity of a Row or Column eessessesserssesrssrseesersesresesseseeseeses 167 Viewing the Line intensity in Any Direction s sssessesseessessserseesresreseeseseesrssesreseesees 168 Calculating Stalis ECS isos te actus a E a 169 Calculating Statistics tor Selected Areds sss srsieer inser aaa 169 Calculating Statistics tor Irregular Areas nnani E 171 13 Measuring Distance and Velocity sscsssscsssssesnsssennsseenssees LID Neasurine IIS CAN CCS cusin e E E seni ans wee santo an isa aaa aaaanasen art sth uses 175 I ess Kove Wag VEO 9 aoe Re oP PRP ee ne ee Pon 177 14 Volume Modeling c sssscsssssccsnsssccnnssscenessecnssseenssssesnesseeness LOL PADOU EV OLUIIIS MORE INO sieisen e E N T onan tengmiietosanels 182 Edit Poly SO Dialog BOX zssinicrsn a E N 182 2D PONV ZON FINALON rai a E E vata do cam cesausaaaiis 185 9D Object Builder a a R R emir 187 Creatine and Viewing the 3 D OpieCh ntact N RE 188 Volume Mode line Example menier on a S N 190 15 Detecting and Analyzing Colocalization s 1sss000s00002s 193 Examining the Entire mal nann EE 194 Identifying Potential Colocalized Ar ds seareiro Sects N E 197 Detecting Colocalization Wali ROS i cas castxcessedretn Goss vent Ea L ETN 201 pplied Precision Contents 16 Other Applic AtiONns sccsssscssssccnssecnssecnssenssecssecnssennssesnssenss 203 ADUE NOTOKINCUICS 55 6 aise asses caetina a l
192. ws how to use Linux Available for All softWoRx workstations All softWorRx users online at www appliedprecision com Acquisition workstations Acquisition workstations that have the optional QLM module All softWoRx Users This is a third party manual pplied Precision Contents PV Tek ic aaa sicaed euteniec cats lev E E a aa SPO Ee Fd S VIN ell Se he tas E Santee Pedeb abana seusee O i Document COMV EN HONS o stcocnusstined A T ed saunaes alate euleee gent eneaaionets ii E E US E E A T E E E E E eens Eets il Notes Warnings and Cautions iennis iiine r sar anced A ii User Interface Desorption On Ven UONS srair anni E A ii Comtacting Applied Precision IhCireeoceeiiin e E E N EA ili Customer Service lO e iui untied tte anaemia eee ennai iii Corporate OUNCE croar on T E E E T T T TAA iii PNEKMIO WACO CINCUILS giao e E dbacte iauvenstacticelseeaes iii alti e e 0 ek 0 9 conais a aa a E Wal as SO NORN starts taht E teat atest dene Sed acheter ch Seb E carts ecb 1 What Gai You Use so WORM ION sts setesettertsustisrstees E ears neasiacatelans 2 PRC GU UII Dataene ies tases ales cdea eb Mls cates a a ll Manatee aatadad an saubies 2 Processie DAL acca a nanos se aea svansnaseagasnegeusecuaaanalacueseuptaugenaeasaeymetaconsies 3 Misuiali zine and Presentine Dataszeresrirerioi iie n a An A 3 Analy ZIV RESU Seri E T T 4 Optional C mponenls erroei a A E O ecaveeuaca uate ears 5 The Quantitative Laser Module wu ccc
193. xels enter the width in the Band field 5 To display a vertical line profile choose Vertical in the Direction list 6 To change the position of the profile click another point on the image or use the and 4 buttons next to the XY field 7 Create a group of profiles to save in a file as follows e To save only selected profiles unselect AutoSave Then click Save after each profile that you want to save is displayed e To save all profiles select AutoSave before you create the profiles 8 To save a group of profiles click Write to File enter the file name and other options in the Profile Output Options dialog box and click Do It Q Tip You can change the Line Profile colors that are associated with different wavelengths From the Image window choose View gt Select Graphics Colors Viewing the Line intensity in Any Direction To display an arbitrary line profile 1 Open an image in the Image window and choose Tools Arbitrary Profile to open the Arbitrary Line Profile dialog box 2 Click and drag on the image to display a line profile 04 720103 000 Rev C 1008 172 softWoRx Imaging Workstation User s Manual wv Arbitrary Line Profile Intensity Scaling Constant Min Max W 1 Window test_5_R3D_D3D dv File View Options Tools 2 H B v m DE Line length 19 474 um angle 18 74 degrees Done Save Saved fo Clear Write To File o oe 7 z p gt
194. you can click Details to open the Region Details dialog box Then specify the ranges of data that you want to include in the Output Options fields 4 Select which wavelengths to filter 5 Select which filter to use in the Filter option list 6 To customize the filter change the values in the numbered grid 7 Click Do It to run the filter Enhancing Object Boundanes Use Edge Enhance to enhance object boundaries This tool uses the image intensity eradient to calculate boundaries The result is calculated from the following expression Result Input Fraction 1 Fraction gradient F Input where Fraction is a number between 0 and 1 and F is either a linear or an arc tangent function ppliedPrecision Chapter 11 Saving Exporting and Printing 141 To enhance object boundanes 1 Choose Filter Edge Enhance from the softWoRx main menu to open the Edge Enhancement dialog box Edge Enhancement f Output EI select Region Reset Details Wavelengths 528 W 617 W 457 J Atan W Fraction 0 0 Wave 1 Min Max 183 0 p7720 Wave 2 Min Max 197 0 1085 0 Wave 3 Min Max 125 0 i8340 Done Do It 2 Enter an image file name or window number in the Input field 3 If you want to include only selected data use the Select Region button and drag the mouse across the portion of the image you want to include Alternatively you can click Details to open the Region Details dialog b

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