MicroManager PolScope (plugin) User Manual
Contents
1. Session Birefringence data spermatocyte a Description Saving V Overwrite Original Processed Images V Show Overwrite Confirmation message When enabled prompts for a confirmati overwritten Default is enabled The top line shows the folder names of the current User Project and Session The Process button initiates the recalculation of the image data as specified by the settings in the tab panels The progress bar gives an estimate of the completion of an active process Status messages are printed below the bar In the Data tab panel the root folder Data Location can be selected where Micro Manager data were saved The lists of existing User and Session folders are then loaded into their corresponding pull down menus Note The PolScope Analyzer only works on existing data Use the Pol Acquisition plugin to acquire new PolScope images Session Description This description is imported when the Session was created and can be updated during Processing Saving Overwrite Original Processed Images Enable this option to overwrite previously computed images Default is enabled Show Overwrite Confirmation message on before previously computed data are Note If overwrite is enabled only processed images are overwritten Raw images are never modified or overwritten Note Newly computed data can only be saved by overwriting previous results at the time of generating the new2. 21 44 57 0400 Wed 10 Apr 2013 1 line converter for imageJ pol stack to MMgr dataset with additional options Pol Analyzer User Manual Birefringence Imaging 13 Page About About Pol Analyzer ImageJ Build 04 15 2013 LC PolScope software for Micro Manager Version 2 02b build 04 15 2013 Copyright 2009 2013 Marine Biological Lab NOTICE Some of the algorithms herein are protected by US patent 5521705 7202950 and other patents applied for or pending LICENSE Berkeley Software Distribution License Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met 1 Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer 2 Redistributions in binary form must reproduce the above copyright Notice this list of conditions and the following disclaimer in the documentation and or other materials provided with the distribution 3 Neither the name of the Marine Biological Laboratory nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND Web Page Bugs and Feature Requests Marine Biological Laboratory 2009 2013 Pol Analyzer User Manual Birefringence Imaging 14 Page Copyright license warranty disc
3. between 0 and the ceiling value into 0 and 255 8 bit or 0 and 4095 16 bit 4095 was chosen to make the converted retardance values similar to the intensity values acquired by a 12 bit camera Retardance values that are computed to be higher than the ceiling value are stored as 255 or 4095 Orientation Reference Sets the orientation reference that is used for processing the slow axis orientation image See Pol Acquisition manual sections Calibration of calculated retardance and orientation values and Mapping of calculated retardance and orientation values into 8 bit and 16 bit images Use 4 Frame Processing Option to use only 4 frames for processing dropping the last raw image channel Convert Non Micro Manager PolStack to Mmgr Dataset This option allows converting PolStack acquired using Psj or any software to be converted as a Micro Manager PolStack Default values might be used to populate metadata field and once converted the new dataset should be re processed with correct parameters eg Background retardance ceiling swing etc Pol Analyzer User Manual Birefringence Imaging 8 Page Processing tab Data Parameters Birefringence Processing Options The Processing tab is divided into 3 main segments i i A A D D i Pre Processing Enabled Description Options PreProcessing This applies some OfRalioing I Ratioing use a RO sele 1 modifications to the raw image data befo
4. ANY THEORY OF LIABILITY WHETHER IN CONTRACT STRICT LIABILITY OR TORT INCLUDING NEGLIGENCE OR OTHERWISE ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE The views and conclusions contained in the software and documentation are those of the authors and should not be interpreted as representing official policies either expressed or implied of any organization From the Laboratory of Rudolf Oldenbourg rudolfo mbl edu Cellular Dynamics Program Marine Biological Lab Woods Hole MA USA 2013
5. Pol Analyzer User Manual Birefringence Imaging 1 Page Pol Analyzer OpenPolScope software for Image Last Updated April 15 2013 Revision 1 07 This manual was prepared with Pol Analyzer software version 2 0 Copyright license and warranty disclaimer statements at end of document Table of Contents B fore E E E H be 1 wT O o EEEE meee rere E AA eae ie DE err REE eee rr rE rar 4 Pol Analyzer VV WENN eege eebe Eet 4 Overview of Functionality and Ta DS aseaicesccsiesscsszessccscessstincexoaacasecsecstacincisosersnsqscevisciesaxcnmtdaceinnsieczenustannsiseia 5 D ta D 5 ere Ug 00 2 E E 6 EECHER ee 7 Processing E 8 OPHONS ADs r Ea N AnA 9 B tchMod Ba settee eles eee iaaa eee eee 10 Reger 11 EE ee 11 RE 12 Eeer ee 13 Pol Analyzer User Manual Birefringence Imaging 2 Page Before Installation OpenPolScope plugins come in two flavors which are installed in different plugin folders inside the Micro Manager folder 1 Pol Acquisition plugin after its installation into the MMplugins folder this plugin appears in the Micro Manager Plugins menu Itis used to acquire PolScope images applying PolScope algorithms and controlling the OpenPolScope hardware attached to the microscope 2 Pol Analyzer plugin after its installation into the plugins folder this plugin appears in the ImageJ Plugins menu providing functions to view and analyze PolScope data that have been previously acquired The OpenPolScope software install
6. Run JavaConsole Micro Manager PolScope Poranayzer e The PolScope Legacy entry contains PolScope Legacy gt plugins that operate only on traditional _Orientation Linesv3 Image PolScope stacks ImageJ stacks Volume Viewer Pol Analyzer Window Pol Analyzer ImageJ The Pol Analyzer window contains Help jaction buttons a progress bar and User Rudolf Oldenbourg Session 2013 03 28 Birefringence SeVeral tabs that let the user set data Process Birefringence locations measurement parameters options etc The following sections 8 give an overview of the window oe components Preferences saved Data Parameters Birefringence Processing Options Data Location Data Location C Users Amitabh Desktop LCPolScope User Rudolf Oldenbourg Session 2013_03_28_Birefringence Session Birefringence data spermatocyte Description Saving Overwrite Original Processed Images V Show Overwrite Confirmation message Pol Analyzer User Manual Birefringence Imaging 5 Page Overview of Functionality and Tabs Help User Rudolf Oldenbourg Session 2012_09_02 Process va E D Process Preferences saved Data tab Data Parameters Birefringence Processing Options S Data Location Data Location C Users Amitabh Desktop LCPolScope e DIS User Rudolf Oldenbourg
7. er installs both plugins Software requirements 1 Micro Manager tested with release 1 4 13 subsequent changes in Micro Manager code might result in unexpected behavior 2 OpenPolScope software Installer for Windows XP 7 and Vista LCPolScopeSetup exe Installer for Mac LCPolScopeSetup pkg OpenPolScope software components Micro Manager Plugins menu Pol Acquisition FrameAverager TopFrame Image J Plugins menu PolScope Pol Analyzer Birefringence Orientation Lines PolScope Legacy RatioAzimPolStackToRGB_V2 ROI_Averages_With_Lines_V1 StackAverage_ Java Console Minimum and recommended system requirements e Hardware 1 OpenPolScope hardware consisting of liquid crystal LC universal compensator and VariLC or equivalent electronic controller Camera supported by Micro Manager Computer recommended 1 5GHz CPU dual core 64 bit 2 GB RAM recommended 4 GB RAM an fF WN 10 MB Hard Disk Space additional space required for UserData Pol Analyzer User Manual Birefringence Imaging 3 Page e Software 1 Windows XP Windows 7 Mac 32 64 bit OpenPolScope software is currently not tested on Mac 2 Java 1 6 update 31 Note It is recommended to increase the ImageJ memory Edit gt Options gt Memory to a minimum of 128MB or more if images are kept in RAM Guide to abbreviations Filenaming SM_2012_0904_0213_5 Prefix convention SM Sample Prefix_Year_MonthDate_HourMin_Suffix BG Background SMS Sam
8. he mirror parameter inappropriately leads to incorrectly computed orientation values Retardance values are not affected by the mirror setting Wavelength Sets the wavelength that will be used when setting the retardance values Default is 546 nm Swing Sets the nominal retardance bias as a fraction of the wavelength for the elliptical settings of the liquid crystals Black Level The blacklevel is the average pixel value that is measured when the camera is not illuminated The black level primarily depends on the camera offset setting Pol Analyzer User Manual Birefringence Imaging 7 Page Birefringence tab Data Parameters Birefringence Processing Options The main processor selected in the Processing panel here Birefringence will be displayed Retardance Ceiling 90 nm 4 as an additional tab Its panel will contain options only applicable when this processor is selected Orientation Reference 0 0 degree IC Use 4 Frame Processing instead of 5 Frame Retardance Ceiling Sets the value of the retardance ceiling that is used for storing the Convert Non Micro Manager PolStack to MMgr Dataset retardance values in its image channel usually first image By way of explanation all image data are stored as 8 bit or 16 bit pixel values including the computed or virtual channels For display and storage computed retardance values are converted to 8 or 16 bit integer values linearly mapping the retardance
9. laimer Copyright 2009 2013 Marine Biological Laboratory NOTICE Some of the algorithms herein are protected by US patent 5521705 7202950 and other patents applied for or pending LICENSE Berkeley Software Distribution License Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met 1 Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer 2 Redistributions in binary form must reproduce the above copyright notice this list of conditions and the following disclaimer in the documentation and or other materials provided with the distribution 3 Neither the name of the Marine Biological Laboratory nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS AS IS AND ANY EXPRESS OR IMPLIED WARRANTIES INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED IN NO EVENT SHALL THE COPYRIGHT HOLDERS OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT INDIRECT INCIDENTAL SPECIAL EXEMPLARY OR CONSEQUENTIAL DAMAGES INCLUDING BUT NOT LIMITED TO PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES LOSS OF USE DATA OR PROFITS OR BUSINESS INTERRUPTION HOWEVER CAUSED AND ON
10. nd Processing at the same time Default is enabled Program Show only Open Windows Modifies the behavior of the Open button under Parameters to List view instead of File dialog opener Default is enabled Batch Processing Mode Selecting this option provides a BatchMode panel for batch processing multi datasets The Process button is transformed for Batch Process and its corresponding panel is added next to Options panel Process Birefringence Batch Process Preferences saved Data Parameters Birefringence Processing BatchMode Options Note During Batch Processing mode Open windows will be ignored and the datasets required for processing can be selected from the panel s table view The currently selected Pol Analyzer User Manual Birefringence Imaging 10 Page Processing Mode is the default method used If a Session consists of different type different modes of acquired data those other datasets will be skipped when processing BatchMode tab Data Parameters Birefringence Processing BatchMode Options System ET Background l E Image Pap Mirror No TI Black Level 5 Birefringence E Retardance Ceiling 25 0 d M j w Orientation Reference 0 0 degree Sample SM_2012_1010_1832_1 Kl S B n IR C SE IS v sw 20i2 10010681 e A s Bam TR e Bam oOo o T SM_2012_1010_2222_1 Batch Processing only allows certain parame
11. ple Series Multi Dimensional OpenPolScope data directory structure Image data is expected to be organized according to the following directory tree lt LCPolScopeData gt user chosen top directory lt UserName1 gt contains directories of a single user project lt SessionDate1 gt contains image and metadata files of a day s session lt SessionDate2 gt lt UserName2 gt contains directories of another user project lt SessionDate1 gt contains image and metadata files of a day s session lt SessionDate2 gt Pol Analyzer User Manual Birefringence Imaging 4 Page Installation For using the Pol Analyzer plugin the user needs to be familiar with ImageJ The plugin is installed into the plugins folder and appears in the Image Plugins menu providing functions to view and analyze PolScope data that have been previously acquired Image data need to be formatted as Micro Manager stacks not Image stacks Therefore in its current release Pol Analyzer requires the co installation of Micro Manager The Pol Analyzer plugin can be accessed Process Analyze GMS Window Help from the ImageJ Plugins menu JAJAN JAJA macros Shortcuts Utilities New The Orientation LinesV3 plugin can be used to set parameters in a single query panel and will operate on an open and active PolScope stack Micro Manager and Image stack format space bar and drag Compile and
12. re they are sent to the main processor An example is Ratioing in which the pixel values of the sample images are multiplied by a correction Processing Mode Enabled Description factor calculated based on the 0 Birefringence EI Calculates Birefringence Orien a 1 DemoTEST Plugin DEMO TEST Pol plugin using 5 background mages Multiple 2 Dichroism Dichroism calculates the optica Preprocessors can be selected 3 Fluorescence Fluorescence calculates Fluor Note The raw image data that are stored in the raw image channels are Post Processing Enabled Description Options never altered during any processing 0 ColorMap E ColorMap pseudo color steps including preprocessing and postprocessing Processing Mode This is the main processor which controls the processing algorithms It defines the number of raw images expected the number of computed images which are also called virtual channels the computation algorithm for the virtual channel images along with other parameters Only one Processing Mode can be selected A Processing Mode has its own options tab eg Birefringence PostProcessing This applies some post processing correction to the images It can also be used to generate additional images Multiple Postprocessors can be selected Note All the OpenPolScope processors that are currently installed will be displayed in this tab Note Pre PostProcessor ma
13. results If Overwriting newly computed data displayed in the stack data stack because Save commands in Micr data Original Processed Images is not checked the can only be saved to disk as a new complete o Manager don t allow overwriting previous Pol Analyzer User Manual Birefringence Imaging 6 Page Parameters tab Data Parameters Birefringence Processing Options This tab lets the user select the image window that holds the sample data Processed Data to be processed and the Sample No Sample si geen associated background data if available Pull down menus let the user choose Data Background a Gackoround M between available datasets Depending on the check box settings in the Options System panel the entries in the sample pull down ege Pap Mirror No M menu vary The Open button lets the user REN swo E open sample data from the disk The tab also shows measurement parameters at the time of acquisition or last processing The information displayed here is stored in the metadata file and parsed when the dataset is Swing 0 03 fraction loaded These parameters should not require any modifications However if background correction was not applied during acquisition or other parameters were not correct the values can be edited and once reprocessed are saved to the image metadata Image Path Mirror Used for an Inverted microscope or if there is a mirror in the optical path Setting t
14. ters that are Batch Processable Once selected by en abling their checkbox those values and or selections will be applied to all the datasets that have been selected for Processing Pol Analyzer User Manual Birefringence Imaging 11 Page Help Report tool polscope mbl edu averma mbl edu gharris mbl edu rudolfo mbl edu E Email address used for replying PolScope plugin Report Note ImageJ MM Core and Error log will be attached with this email at the time of Sending The Report tool can be accessed via the Menu bar to send bug reports or suggestions Pol Analyzer User Manual Birefringence Imaging 12 Page ChangeLog ChangeLog Installed Version 2 02b 04 15 2013 Latest Version Available 2 02b 04 15 2013 r806 amit 2013 04 12 18 53 14 0400 Fri 12 Apr 2013 2 lines Added option under Calibration for searching Maxima under Calibration Options Automated installer building and updating changelog info via Installer build script r805 amit 2013 04 11 21 13 53 0400 Thu 11 Apr 2013 5 lines fixed missing lib matlabcontrol used by PolScope API in installer script Changes to Tuning panel in VariLC moved calibration options from Options and renamed Tuning panel to Calibration Options Changed wordings for selections in Options Updated ChangeLog to use flexible updatable links for displaying content 1st draft of PoLAcquisition manual r804 amit 2013 04 10
15. y or may not have additional options that are indicated by a button Note If a PolScope data set is opened and the associated Processing Mode plugin is not installed the data set will open as a regular Micro Manager data set and not a PolScope data set Pol Analyzer User Manual Birefringence Imaging 9 Page Options tab i Data Parameters Birefringence Processing Options In the Options tab one can set preferences for the Pol Analyzer Processing Processing Run Processing from Slider Process stacks If checked processing occurs for all computed channels of a data set stack if not checked V Parallelize Processing processing occurs only for the currently selected time point and or z position Default is enabled Dont Process Only Walk Through DataSet 250 ms delay Run Processing from Slider When enabled will execute processing whenever a value is changed from the slider eg Retardance ceiling slider Default is disabled Program dl Show only Open Windows in Sample Data List Batch Processing Mode Dont Process This option allows walking the data set through time slice position without processing The currently selected channel is always displayed Parallelize Processing Enables processing using multiple cpu cores that are available This option provides full utilization of processing power when processing a Multi dimentional stack This should not be enabled when acquiring images a
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