- BioMed Central
Contents
1. Number of times the Live Producer attempts to load new frames before it stops the analysis 4 3 Spot Finder JAVA CL This component is intended for facilitating the peak finding part of the analysis 4 3 1 Max Spots per Frame This is the maximum number of peaks the finder will try to find inside a single frame If this number is set too high the Java version of the Spot Finder will use up a lot of RAM and the CL version might fail due to insufficient GPU RAM availa ble If this number is set too low not all peaks in a given frame may be found For a typical STORM PALM dataset this number can be set to 1024 4 3 2 Threshold This is the minimum intensity count the brightest pixel of a peak must have to be considered for fitting This value is critical for analysis If this number is chosen too high not all peaks will be identified If it is chosen too low the finder will pick up noise as peaks This will slow down analysis and Max Spots per Frame might be exceeded before all peaks are identified even if the number of actual peaks with in a single frame is low enough GraspJ determines a rough estimate for the thresh old from the first few frames of the data However the estimate is often not accu rate enough to give the best results Therefore the user is recommended to open the data in ImageJ and determine the threshold by considering the dimmest peaks in each frame that should be included in the analysis 4 3 3 Box Radius2. The reading speed will vary depending on whether it is loaded as a virtual stack Non virtual stacks will already be loaded in RAM which makes reading very fast However most STORM PALM stacks are large and will likely be loaded as a virtual stack If that s the case reading speeds will depend on the location of the file If it is located on a solid state disk SSD speeds will be similar to the raw loading method For conventional hard disks HDDs it will be slightly slower and for network locations it will typically be substantially slower In the latter case us ing a raw format should be considered 2 2 2 Analysis Type Config On the second page you have to specify whether your data is 2D or 3D For 3D data GraspJ currently only supports astigmatism Grasp GPU Run Analysis for STORM and PALM in Image os File Views Wizard Analysis Type Config R Dimensionality 2p 7 Render an Image with all Channels Y Render Channels individually v Channels 1 2 In case the data source you specified on the first page is already accessible and con sists of enough frames 210 GraspJ will try to automatically determine a few pa rameters needed for analysis such as threshold In order to determine the imaging sequence cycle that was used during acquisition for further information see Frame Cycle you will be asked for the number of different channels colors GraspJ should expect The
3. 1 Push Receive Actions When activated zooming and panning actions on this rendering window will be pushed to received from all other rendering windows within the same group speci fied by Group Names Group Names Specifies the groups this rendering belongs to as a comma separated list 4 11 2 Auto Contrast This is used to automatically scale the brightness contrast of the rendered image The brightest points correspond to those that contain the highest number of locali zations 4 12 Workflow on Copy This component can hold and run a workflow on a copy of the current re sult current package of a parent workflow For each package delivered to the com ponent a new workflow will be run 4 12 1 Copy current Result Package If selected the workflow uses a copy of the current result package of the parent workflow A copy in this sense refers to a shallow copy which means that without further configuration the spot collection the mask and the drift are still identical to the original ones i e changes made to the result package on this workflow affect the result package on the parent workflow However the result package itself is a copy which means that you can replace the spot collection mask drift with a dif ferent one without affecting the parent workflow result package 4 12 2 New Spot Collection Mask Drift If this option is selected the spot collection mask drift is replaced with a new one 24 Copy or
4. 7 to run see Java 7 for more information 1 1 1 Requirements OpenCL In order to run GraspJ you will need a device that supports OpenCL 1 1 or higher Among many other devices most modern Intel and AMD CPUs as well as most modern Nvidia and AMD GPUs support OpenCL If you have installed a recent GPU driver from Nvidia or AMD you are most likely already good to go Details for the main vendors can be found here e AMD e Nvidia e Intel Java 7 If you have chosen the Fiji package above and are running Windows or Linux you don t need to worry about Java 7 as this package already ships with Java 7 Oth erwise you will have to install Java 7 manually and make sure ImageJ runs on Java 7 The Java version your ImageJ Fiji runs on can be checked by clicking on the status bar of ImageJ Fiji A version number 1 7 X indicates Java 7 while 1 6 X in dicates Java 6 GraspJ needs Java 7 to run If not already installed Java 7 can be downloaded here ImageJ Fiji has to be running on the Java 7 virtual machine VM in order to run GraspJ 1 2 Run GraspJ Start ImageJ Fiji then start GraspJ via Plugins gt GraspJ 1 3 Run Analysis on Sample Dataset Download the sample dataset and the sample workflow from www graspj com Drag amp drop the sample workflow to GraspJ The workflow will be opened in a Work flow View Expand Producers and click on Live Producer On the right locate the field Source File Drag amp drop the downlo
5. AMD Accelerated Parallel Processing v Intel R Core TM i5 2500K CPU 3 30GHz Barts Favorite Workflows Sample Workflow 14 Chapter 4 Workflow Components 4 1 Acquisition Config 4 1 1 Pixel Size This is the size of one projected camera pixel in nm Pixels are assumed to be squares Example physical pixel size 164m x 16um using a 100x objective gt Pixel Size 160nm 4 1 2 Count Conversion This is the factor to convert pixel intensity values to approximate photon counts This conversion factor depends on the gain settings of the EMCCD camera and should be determined from the provided manufacturer specifications 4 1 3 Frame Cycle This is the order of frames inside the source file Enter the repetitive pattern of the acquisition For each frame of this pattern you need to enter one digit representing the color channel that frame belongs to Frames can be marked as activation frames by putting square brackets around them Example For a two color experiment where identical reporter dyes but two differ ent activator dyes are used a typical frame cycle consists of one activation frame of one color e g Alexa405 followed by three acquisition frames e g Alexa647 fol lowed by one activation frame of a second color e g Cy3 and finally three acquisi tion frames e g Alexa647 For such a frame cycle the following pattern would be adequate 1 199 2 299 The numbers in brackets represent the first an
6. View The Results View shows previews of the final rendered images obtained as a result of the analysis as well as additional results such as the amount of drift in x and y 4 Grasp GPU Run Analysis for STORM and PALM in Image 2 eS File Views Result Result gt Meta Data Name Description Date Created Summary Channel 9 Number of Localizations Localizations after Filtering E 100 x El x lt gt E E E 0 5 20000 20000 frame nr frame nr Acquisition Config y Other y 13 3 3 1 Result Commands Built in Workflows Some workflows commonly run on results can be selected from this menu Attached Workflows Workflows attached to this result can be opened from this menu The workflows are also shown and can be edited in Result View after expanding Others Workflows can be attached to results using the Attach to Result command in Workflow View Favorite Workflows Your favorite workflows can be opened from this menu The workflows are also shown and can be edited in Settings View Workflows can be added to your favor ites using the Add to Favorites command in Workflow View 3 4 Settings View In this view you can change settings used by GraspJ like the used OpenCL plat form and device or your favorite workflows Grasp GPU Run Analysis for STORM and PALM in ImageJ a eam File Views Config Settings Devices OpenCL System
7. range of 600 to 600 nm The peaks in this calibration dataset must be identified and the x and y widths of the PSF must be determined in each frame This analysis allows one to plot a calibration curve x and y widths of the PSF as a function of z height This calibration curve 18 can then be fit to a defocusing curve to determine the parameters needed for the 3D analysis For more details on astigmatic 3D super resolution imaging please re fer to Huang et al Science 319 810 2008 More details on the 3D calibration pa rameters used in GraspJ can be found in Smith et al Nature Methods 7 373 2010 4 5 Fit Filterer This component filters bad localizations based on certain criteria 4 5 1 Apply Mask If this option is selected the obtained filter mask is permanently applied to the Spot Collection i e bad localizations are deleted 4 5 2 X y z Intensity Background For each of those parameters one can specify a lower and upper limit of the value itself as well as the uncertainty of the found value If the value or the uncertainty is outside of that range the localization will be filtered out The cut off for the localization uncertainty in x y and z is set in nm e g 30 nm for x and y and 50 nm for z Localizations with low photon counts and hence large uncertainties will therefore be filtered out This cut off can be changed to accept more or less localizations and will affect the final resolu
8. remaining options on that wizard page concern rendering of found localiza tions If you activate Render an Image with all Channels an image with all local izations regardless of their color channel will be rendered together in one window bad renderings are filtered out The option Render Channels individually lets you specify color channel numbers that will be rendered in separate windows Col or channels are assigned to localizations based on the frame in which they occurred see Frame Cycle 2 2 3 Basic Configuration The third page lets you specify Grasp GPU Run Analysis for STOR xs parameters essential for identifi File Views Wizard cation and localization of mole Acquisition Config a cules GraspJ will try to estimate Pixel Size 157 some of the values itself current CountConversion 0 41 ly limited to Threshold and Frame Cycle 1 Frame Cycle Those parameters y Standard Deviation PSF 1 05 typically have a big effect on the performance of GraspJ see Ac Iterations 5 quisition Config Spot Finder JAVA CL MLE Fitter 2D 3Dfor lt Prev Next gt Finist Cancel details on the individual parame ters and how to set them properly 2 2 4 Optional Features S GraspJ GPU Run Analysis for STORM an 28 File Views Wizard Optional Features Trail Generation v Drift Correction y Method intrinsic i lt Prev Ne
9. Continuous Workflow on Copy cceeeeeeeeeeeeeeeeeeeeceeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeres 25 Live Chanje Renderer escien sda dolida 25 Injection Produits 25 Troubleshooting voc cc ceeccoteeeviienee wes ineteaereececinpsaedsenvbieoacesecscunieeie 26 Chapter 1 Getting Started Please note that GraspJ has only been tested on Windows However it should work on Mac OS X and Linux as well if all requirements listed in this chapter are ful filled If you experience any problems please contact technical supportQgraspj com or consider filing an issue on http code google com p graspj issues 1 1 Installation The easiest way to install GraspJ is to use the pre configured Fiji package provided on http code google com p graspj downloads It comes with its own Java ver sion and all required libraries Just download the zip file and unzip it Please make sure your computer fulfills all Requirements It is highly recommended to use a 64bit version and dedicate enough memory to the Java Virtual Machine JVM otherwise the analysis might fail or get stuck see Troubleshooting Alternatively you can download the plugin as a Java archive JAR also available on http code google com p graspj downloads For installation this file needs to be copied to the plugins folder of ImageJ Fiji Afterwards restart ImageJ Fiji This plugin already contains all required dependencies except Java 7 GraspJ needs Java
10. GraspJ User Manual Version 3 Table of Contents Chapter 1 1 1 1 2 1 3 Chapter 2 2 1 2 2 2 3 Chapter 3 3 1 3 2 3 3 3 4 Chapter 4 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 4 10 4 11 4 12 4 13 4 14 4 15 Chapter 5 Getting Started alii a 1 Installation cia daa dias 1 Run Gras Pl arto do tipa 2 Run Analysis on Sample Dataset oooooocococonncnnccocococononncnncnocononnnononnncnnnnanannnnncnno 2 Using Gras pd icici cise de ccsiccivecdcicadeessedetees vase eiae dani snm 3 SSAC cd C6 ee ee 3 Workflow Wizard 4 2 2 areari ste cenda ra txts a eaa soe teste aa Pete ea a eaae 4 Rel viii AA AA E E EEE E 8 VIEWS e aie ti A Ca EE E E E EEES 10 Workflow Views td a ete SE 10 A nal sis Vii A a a a eee eee sddees 12 RSU VW Ana 13 DELI ES V TO Wii a Sees too at welds a eaa e da Da eos 14 Workflow Components sisii soere e rE inet 15 Acquisition Contig aAa a e a eee 15 A a ee ae E E E E 16 Spot Finder JAVA CLis ic essicestiscesssncsecbasassccsssaessccssasssssosedesddeasbacseapdedeacsaatecs 17 MLE A e ae a a e aa ae e aan 18 IEn HT ETE E EAEE A EA E AEN A E AS 19 Brame Filtret men a tar a nea tw ee 20 Trail Generator td 20 Package Drift Correcto uml iia at i e adea a e 21 Rendering Colite a a i eid e a a tated 22 Result Package Merger ccccescceeseseeeeeeeeenceceseeecnneeeceeeeeseeenteeesneeesteeeneeeeeaas 23 Ihive a is etiolated oa a tee ee i 23 Worktlow on Copyie sinned alee ee ee ee ea eed 24
11. This is the Radius of the box that is drawn around the highest intensity pixel of a peak and is used to exclude overlapping peaks and peaks on the edge of a frame 17 The size of the square box used corresponds to2 Box Radius 1 As a rule of thumb the box radius should be set to approximately 3 Ops where Opsp is the Standard Deviation PSF 4 4 MLE Fitter 2D 3D This component performs the peak fitting part of the analysis 4 4 1 Box Radius This is the Radius of the box that is drawn around the highest intensity pixel of a peak and is used for fitting to obtain its center position Usually this value should be chosen identical to the Box Radius of the Spot Finder JAVA CL 4 4 2 Standard Deviation PSF This is the standard deviation of the Gaussian approximation to the Point Spread Function of your microscope in pixels 4 4 3 Iterations This is the number of iterations run while fitting each peak The higher the number the closer the fit result will be to the MLE but the analysis speed will go down 4 4 4 Defocusing Curve X Y 3D only The z position of fluorophores is determined based on the astigmatism of the PSF in 3D data The astigmatism is compared to a calibration curve in order to deter mine the z position The calibration curve must be obtained by immobilizing single fluorophores or fluorescent beads on the surface of a cover glass and scanning a precision piezo stage in z for example for a
12. aded sample dataset to that field Then click on Workflow gt Run Analysis Chapter 2 Using GraspJ In order to analyze your own data you need to create a custom workflow The easi est way to do so is by using the Workflow Wizard Alternatively you can modify the sample workflow or start with an empty workflow 2 1 Workflows The analysis procedure of GraspJ is organized in a modular way that we call a workflow 2 1 1 Create Workflows Workflows can be created in two ways either by using the Workflow Wizard File gt Workflow Wizard or starting from scratch File gt Empty Workflow You can also modify existing workflows 2 1 2 Save Open Workflows Workflows can be saved while in Workflow View also see general description of Views There are three data formats available gjw gjwx and gjwxz Those abbre viations stand for GraspJ Workflow GraspJ Workflow XML and GraspJ Work flow XML zipped respectively Workflows saved in gjw format are in a binary for mat Workflows saved in gjwx format are text files with XML code to describe the workflow The gjwxz format is identical to the gjwx format except that the data is zipped to achieve smaller file sizes Workflows can be opened either by drag amp drop onto GraspJ or using a dialog from the menu File gt 0pen Workflow 2 1 3 Workflow Phases A Workflow consists of three different phases during which the final result is ob tained Those phases are called Production P
13. code Channel If selected the channel number determined from the Frame Cycle will be saved in the filter mask 4 7 Trail Generator In a typical STORM PALM experiment the acquisition laser intensity is chosen such that individual fluorophores switch off or bleach within one frame However 20 some fluorophores may last longer than one frame When the same peak appears in multiple frames it can be trailed together and the position calculated as a weighted average of the positions in each frame Trailing improves the localization accuracy of a given peak The trail generator allows you to set up the parameters for linking peaks together 4 7 1 Location Tolerance This value determines how far the position of two peaks can be separated from one another in subsequent frames to be still considered the same peak The value is set in nm 4 7 2 Frame Skip Tolerance This value determines the number of frames that a peak can disappear in between frames to still be considered as the same peak Typically trailing is done only for peaks that continuously appear in subsequent frames and therefore this value is set to zero 4 7 3 Min Max Trail Length This value determines the minimum and maximum number of frames that can be trailed together Typically this value is set to be equal to the length of the acquisi tion frames in a cycle e g 3 4 8 Package Drift Corrector This component corrects for stage drift in x and y by re
14. d page you have to choose a method to load your data If your data is saved as a single file in a raw format i e no header footer no gap between images you have to choose the first option Load as Raw Data This option provides the highest reading speed and can also be used for live analysis data can still be written to the file it does not even have to exist yet If you choose to open your data this way you will have to specify the width and height of a single frame in pixels as well as the bytes used to represent a single pixel e g 2 bytes for a 16 bit image Grasp GPU Run Analysis for STORM and PALM in Image 2 File Views Wizard gt Data Source 8 Load as Raw Data Source File C Users Norman Browse Frame Width 256 Frame Height 256 Bytes perPixel 2 Load with LOCI Bioformats wo T Source File C Users Norman Use open Stack ImageJ Stack lt Prey Next gt Finish Cancel If your data is saved in a format supported by the LOCI Bio Formats library the second option can be used to load your data Load with LOCI Bio Formats De pending on the file format this option might be substantially slower than the first option Live analysis is also supported for many but not all formats loaded in this way there is no list of which ones are supported If you already opened your data in ImageJ you can use the third option Use open Stack
15. d second activation frames which typically are dark and contain no localizations The 1 and 2 outside of the brackets indicate the first and second acquisition frames immedi ately following the activation frame The 9 identifies non specific localizations from 15 the frames not immediately following the activation frame For a detailed reference on multi color imaging using different activators please refer to Bates et al Sci ence 317 1749 2007 4 2 Live Producer Reads the data source and splits it into packages The analysis procedure is then done package wise 4 2 1 Frames per Package This is the number of frames per one package Setting this number too high might result in the analysis failing as the package will not fit into the GPU RAM any more Setting this number too low on the other hand might result in low analysis speeds or impaired intrinsic drift correction A typical number of frames per pack age is 1024 4 2 2 Data Source Type A detailed description of the options available here can be found in section 2 2 1 Data Source 4 2 3 Detect End of Data Source Decides whether the Live Producer will try to automatically detect the end of the acquisition or in non live mode the end of the file If not selected one has to stop the analysis manually see Running Analysis View Interval The time in ms the Live Producer waits between two consecutive attempts to load new frames 16 Attempts
16. ick on File gt Save As while in Workflow View the current workflow will be saved Similarly if you click on File gt Save As while in Result View the current result will be saved All views and their specific commands are explained in this chapter 3 1 Workflow View Grasp GPU Run Analysis for STORM and PALM in Image as File Views Workflow v 3 Workflow v 3 Producers 5 Live Producer v Processors Find Config MLE Fitter 2D _ Fit Filterer Max Spots perFrame 1 024 5 Frame Filterer D Trail Generator ME Package Drift Corrector BoxRadius 3 Product Merger v 3 Consumers F Live Renderer All Channels gt 3 Live Render Color 1 gt 5 Live Render Color 2 Threshold 900 The Workflow View allows you to view and modify essential parameters of the analysis including the ones that were entered as part of the Workflow Wizard 10 On the left side the workflow is shown as a tree structure see Workflows for more information on what a workflow and its components are The structure of the workflow can be changed via drag amp drop inside the tree More options are availa ble through the context menu right click If you right click on a component you will be presented the option to remove that component If you right click on a folder e g Processors you can add an additional Processor by choosing from a list of a
17. iginal Spots Mask Drift If this option is selected the newly created Spot Collection Mask Drift is filled with a copy of the original data If you select this option for all three parts Spots Mask Drift you will end up with a deep copy of the result package Changes to that copy will not affect the original 4 13 Continuous Workflow on Copy This component is largely identical to Workflow on Copy The main difference is that instead of executing a full workflow for each package it has one workflow running continuously while the parent workflow is running New packages are then injected into this workflow by the Injection Producer One application of this component is the Live Channel Renderer which is based on the Continuous Workflow on Copy component 4 14 Live Channel Renderer The main functionality of this component is similar to the Live Renderer in fact a Live Renderer is built in to this component It adds the functionality of filtering a copy of the spot collection before rendering to allow for rendering of individual col or channels only This component is based on the Continuous Workflow on Copy component 4 15 Injection Producer This component replaces the Live Producer in Continuous Workflow on Copy based components Instead of reading the source file and creating packages for analysis it uses a copy of the parent workflow package and injects it into the workflow 25 Chapter 5 Troubleshooting The a
18. nalysis stops without any error message shown This can be caused by insufficient memory available to ImageJ Fiji If possible increase the memory ImageJ Fiji is allowed to use via Edit gt 0Options gt Memory 4 Threads gt Maximum Memory then restart ImageJ Fiji and try again It is highly rec ommended to use a 64bit version of Java7 and the 64bit version of GraspJ If none of this works you can try setting a smaller number of Frames per Package in the Live Producer configuration The analysis is very slow While the analysis is running check execution times shown in the Analysis View If one of those is especially high check the configuration of that component If execution times for all components are reasonable the low speed is most likely caused by a low reading speed of your source file More information on how to choose the best option for your data source is given in section 2 2 1 Data Source Briefly the fastest reading speeds will be achieved using a raw file format and read ing the source file from a fast hard drive for example a Solid State Drive SSD ImageJ Fiji complains about GraspJ being compiled for a newer Java version This indicates that your ImageJ Fiji is running of a Java version too low to run GraspJ Please check the Installation section of this manual for more information 26
19. ndering STORM PALM images from subsets of frames and correlating them intrinsic drift correction By default it renders all channel images for correlation If you want to restrict the cor relation to a specific channel you can use the Specific Channel setting of the Ren dering Config used by the Package Drift Corrector If you want to use fiduciary 21 markers that appear in a certain frame of your Frame Cycle you can do so by ded icating an individual number to that frame in the cycle setup and then choosing the same channel number for the Specific Channel setting Example Let s consider the same situation as discussed in the example given in section 4 1 3 Frame Cycle where we had a two color data set which led to the following cycle pattern 1 199 2 299 Now let s assume we added fiduciary mark ers that appear in the otherwise dark activation channel for the second color 2 In order to localize the markers in that channel and identify them separately from the single molecules we will have to assign an individual number to that frame we here choose 3 The cycle pattern would then become 1 1993299 Now by setting the Specific Channel option to 3 the Package Drift Corrector will only use the localization from the fiduciary markers for drift correction 4 9 Rendering Config This feature allows to set up the parameters for rendering localizations as an image 4 9 1 Render Width This is the width of the window in pixels
20. rocessing and Consumption and the components working during these phases are called Producers Processors and Con sumers respectively Available components and their configuration are described in Chapter 4 Components During Production phase typically the raw data is read sliced into pieces of suita ble size which we refer to as packages Those packages will later also store the list of localizations filtering results calculated drift and any other information created during Processing The second phase Processing consists of a number of individual components all facilitating parts of the analysis by working with an individual package at a time Each Processor is called once per package and execution of the subsequent Proces sor is delayed until the predecessor is finished maintaining the order of Processing At the end of this phase the analysis of the individual package is finished The last phase is called Consumption Here the package is simultaneously distrib uted to all consumer components Typical examples of consumers are rendering engines providing the user with the obtained image in different ways 2 2 Workflow Wizard To start the Workflow Wizard click on File gt Workflow Wizard The Workflow Wizard allows you to choose the data to be analyzed and enter the specifications for the data file e g 2D or 3D multi or single color and microscope settings e g pixel size 2 2 1 Data Source On the first wizar
21. that should be used for the final render ing 4 9 2 Render Height This is the height of the window in pixels that should be used for the final render ing 4 9 3 Pixel Size This determines the pixel size in nm at which the image will be rendered 22 4 9 4 Offset X Y This determines the shift of the rendering in nm 4 9 5 Intensity This determines the intensity of the rendering of an individual localization It s the sum of all pixel intensities that belong to that localization 4 9 6 Apply Mask If selected the current filter mask is applied during rendering e g localizations that were filtered out are not rendered Specific Channel This option allows to specifically only render localization that belong to a specific channel defined by the Channel Nr option and are not filtered out Hue and z ColorCoded3D only In 3D super resolution images the z height of localizations is color coded using a continuous color scale Hue is calculated from the z location using the following linear transformation formula hue max m hue min hue maxZmin hue minZmax hue z 2 oO max Zmin Zmin Zmax 4 10 Result Package Merger This component appends the current package to the current result of the workflow 4 11 Live Renderer This component continuously renders the current result of the workflow The ren dering is shown in a modified ImageJ Window allowing for zooming panning etc 23 4 11
22. tion of the image accepting localizations with high error will degrade resolution On the other hand accepting only localizations with very low error may lead to filtering out a large number of localizations and degradation of the image resolution by lowering the Nyquist sam pling The minimum and maximum intensity should be set based on the minimum and maximum number of photons expected from individual molecules 19 The maximum background value can be used to reject localizations from very crowded regions where close by fluorophores could have affected localization accu racy It is not straightforward to find a suitable value that fulfills that task and some trial and error will be needed However this value is not very important and if set to a large number no localization will be filtered out because of this number Bad localizations are still filtered out by the other criteria especially uncertainty in x and y 4 6 Frame Filterer This component filters localizations based on the frame in which they appeared 4 6 1 Apply Mask If this option is selected the obtained filter mask is permanently applied to the Spot Collection i e bad localizations are deleted 4 6 2 Filter by Channel If selected all localizations that do not belong to specified channel are filtered out 4 6 3 Filter by Type If selected all localizations that are not of the selected type activation acquisition are filtered out 4 6 4 En
23. vailable Processors On the right side all options for the selected component inside the tree are shown and can be edited 3 1 1 Workflow Commands Run Workflow Starts execution of the current workflow and switches to a new Running Analysis View Add to Favorites This function adds the current workflow to the list of favorite workflows This list is part of the Result Commands To view and modify the list or workflows added to it click on File gt Settings which will lead you to the Settings View Attach to Result This function attaches the current workflow to an open result You will be able to choose the result you want to attach the workflow to in a popup window Attached workflows can be accessed through the Result Commands They are shown and can be edited in Result View after expanding Others 11 3 2 Analysis View Analysis View shows the status of a running analysis such as the number of locali zations found and how many frames have been analyzed Grasp GPU Run Analysis for STORM and P L 2 gt Alldly 3 2 1 Analysis Commands Pause Analysis This command pauses the running analysis Live Renderings can still be inspected zooming panning Resume Analysis This command resumes a paused analysis 12 Stop Analysis This command stops the running analysis the current package will be finished first It then proceeds to show the result in a Result View 3 3 Result
24. xt gt Finish Cancel On the last page of the wizard optional features can be activated Right now avail able features are Trail Generation and Drift Correction Trail Generation links together peaks belonging to the same localization but appearing on subsequent frames for improved localization precision see Trail Generator Drift Correction intrinsic corrects for stage drift in x and y by rendering STORM PALM images from subsets of frames and correlating them see Package Drift Corrector 2 3 Results Data obtained during analysis is saved as results This includes all found localiza tions named Spot Collection a filter Mask i e one number per localization characterizing its channel or whether it was filtered out and Drift i e one number per frame representing the shift in nm 2 3 1 Save Open Results Results can be saved while in Result View also see general description of Views They can be opened either by drag amp drop onto GraspJ or using a dialog from the menu File gt Open Result Chapter 3 Views Views are the central part of GraspJ s user interface Everything shown below the menu bar is a view Each opened view adds an entry to the Views menu which when clicked becomes the active view Each view type also adds an entry to the menu bar offering commands that can be used within this view The Workflow View as well as the Result View also contains data that can be saved For example if you cl
Download Pdf Manuals
Related Search