MicroManager PolScope (plugin) User Manual
Contents
1. Processing Mode Enabled Description multiplied by a correction factor 0 Birefringence Calculates Birefringence Orientation using c alcul ated based on the 1 DemoTEST Plugin A DEMO TEST Pobplugin using 5 frames and 2 Dichroism Dichroism calculates the optical anisotropy background Images Multiple 3 Fluorescence Fuorescence calculates Fluorescence Pol _ Preprocessors can be selected 4 RT_ Pol E Real Time Quad Fluorescence calculates Note The raw image data that are stored in the raw image channels are never altered during any processing steps including Post Processing Enabled Description Options O0 ColorMap E ColorMap pseudo color _ preprocessing and post processing Processing Mode This is the main processor which controls the acquisition and processing algorithms used It defines the number of raw images acquired the number of computed images which are also called virtual channels the computation algorithm generating the virtual channel images along with other parameters Only one Processor can be selected A Processor has its own options tab Birefringence Post Processing This applies some post processing correction to the images It can also be used to generate additional images Multiple Post processors can be selected Note All the OpenPolScope processors that are currently installed will be displayed in this tab Note Acquisition cannot be performed w2. a oh SETS SE Plot Retardance Value Plot the Retardance Value profile during Calibration Use Sequence Acquisition for Calibration A Use Sequence Acquisition for Calibration faster mode for Calibration and is required Extinction Search Coarse Factor 2 3 4 enabled for certain cameras eg Andor If Brent Optimizer Absolute Accuracy Coarse 0 0100000 Calibration using this mode causes error during Calibration you may disable it De Extinction Search Fine Factor 3 fault is enabled Brent Optimizer Absolute Accuracy Fine 0 0000100 gt Extinction Search When performing a Cal ibration this defines th rch range from State 1 4 Swing Search Factor bration this de el cian ue ae 1 2 the current value The final search range is the multiple of swing defined Default is 4 for Coarse and 2 for Fine Pol Acquisition User Manual Birefringence Imaging 14 Page The Brent Optimizer defines the tolerance or accuracy of the search Default is 0 01 for Coarse and 0 00001 for Fine Thus with a swing of 0 03 and a factor of 4 The LC will scan a range of 0 12 from the current LC value with its respective tolerance Save amp Load Calibration Options Save amp Load Save VariLC Settings Filename Save save palette settings in text file Comments Options Device Serial No W 10 00 2400 0 7000 70017 VarilC Range Low 0 0 High 0 84 V Use VariLC Mapped Values Export
3. Birefringence Imaging 7 Page A Walk Through of Basic Operations While performing this sequence you may wish to refer to the section Overview of Functionality and Settings Tabs Start the Micro Manager software After the camera and VariLC controller have been turned on you may launch the Micro Manager software by double clicking the MM icon on the Windows desktop In the startup query window choose the MM config file that was prepared beforehand see Installation and includes the camera and VariLC controller Setup your sample Prepare your microscope for Koehler illumination Place your sample in the field of view select appropriate objective and direct the majority of the light to the camera Focus on your sample using the live camera window For calibration move the sample sideways out of the live camera window to image a clear non birefringent sample area also called background Set acquisition and processing algorithms Select the Processing tab in the Pol Acquisition window and set Birefringence as the acquisition and processing algorithm For starters deselect pre and post processing algorithm s A Note on Setting the Swing and Retardance Ceiling The swing setting should be adjusted based on the sample specimen you wish to analyze Thin biological samples typically require a swing of 0 03 and a retardance ceiling of 5 nm or higher Thicker samples or samples containing crystals should have a higher swing
4. Mapped Values Sheet Import Mapped Values Sheet Only values in green should be edited in the Excel sheet bers in LC settings are sent directly to VariLC controller Save VariLC Settings A user can save the current LC settings to a text file with comments or Load a saved set tings file polset This is optional as the LC settings are also routinely saved in the IJ_Prefs txt file Device VariLC Range These upper and lower limits need to be determined for the li quid crystal devices installed and need to be set once during installation Use VariLC Mapped Values If checked a device specific calibration table for li quid crystal settings is used Default is enabled checked If unchecked num Export Import Mapped Values Sheet The currently used Mapped values can be exported as an ex cel file A default template is exported in the case of Non Profiled devices This is particularly useful for Non Profiled LC Devices where the default values can be edited on the excel file and imported back in the software Imported values are not retained if Micro Manager is restarted and the excel file needs to be imported again Pol Acquisition User Manual Birefringence Imaging 15 Page Parameters tab Data VariLc Parameters Birefringence Processing Options Logger The Parameters tab includes parameters that are used during Background No Background gt open acquisition and
5. NariLC otal Number of Palette Ele ces Pegin adding new thenever you re ready d button If you help with deciding Port Properties RS 232 settings vices to add highlight ar device and click ally helps Device COM3 Property nswerTimeout COM3 audRate COM3 elayBetweenCharsMs COM3 andshaking COM3 arity COM3 topBits of the subdevices D ry erbose Name DWheel Adapter DWheel DStateDevice DStateDevice VariLC VariLC Setting Device Delays Some devices will execute a command but don t signal to Micro Manager when Note The VariLC is synchronized with the camera or other hardware through a delay time setting since the VariLC controller does not provide a state change callback The delay time to be used depends on the physical properties of the liquid crystal devices A typical delay time is 100 ms but can be shorter For Abrio use 100 ms Before using the system we recommend that you go through the process described in the section A Walk Through of Basic Operations to configure and calibrate the system correctly Pol Acquisition User Manual Birefringence Imaging 6 Page Starting the Pol Acquisition plugin J Systems CAMicro Manager TAA The Pol Acquisition plugin can be E EEE Hepes accessed from the Micro Manager Clojure ed
6. Web Page Bugs and Feature Requests Marine Laboratory 2009 2013 26 Page Pol Acquisition User Manual Birefringence Imaging Appendix A Multi Mode Acquisition The following steps demonstrate how to setup Micro Manager to acquire data using other modalities Brightfield Darkfield Fluorescence etc along with PolScope data using the OpenPolScope Pol Acquisition software This guide assumes the necessary hardware has been configured using the Hardware configuration wizard Step 1 Start by creating a new Group using the button and then naming eg Multi D Group and selecting all hardware that require to be changed between acquiring PolScope dataset and other type of datasets Configuration settings Save Preset Startup preset at Step 2 In this example we change the Nikon TI Filter Block In our setup the TIFilterBlock1 Label 1 TIFilterBlock1 State 0 houses the PolScope Analyzer Here you can specify the properties included Show OK ina configuration group cameras Cancel W shutters E Show read only properties r stages r Wheels turrets etc ther devi Group name Multi D Group other devices Property Mame Current Property Value T1FilterBlack1 Label oF Oe TiFiterBlocki State BO o ooo IZDrive 5peed Step 3 We then proceed to define a Preset using Filter Block Label 2 or State 1 not Label 1 which is being used for PolScope Here you can specifiy the propert
7. are saved in the image metadata Image Path Mirror No Background The selected Black Level 40 background that is used for background correction when Averaging 5 computing the specimen Sample 8 l am retardance and slow axis W Background 3 orientation Note Background data can be stored in a file located on disk or in an open window Background data should be recorded at least once at the beginning of a session Image Path Mirror Used for an Inverted microscope or if there is a mirror in the optical path Default is No Setting the mirror parameter inappropriately leads to incorrectly computed orientation values Retardance values are not affected by the mirror setting Black Level The Black Level also called baseline is the average pixel value when the camera is not illuminated The black level primarily depends on the camera offset setting Averaging is available if the FrameAverager plugin has been installed in the Micro Manager plugins folder appears in the MM plugins folder Averaging generates raw image data by averaging N camera frames Averaging reduces image noise at the expense of time resolution Averaging more than 8 frames is usually not effective Default is Sample disabled and Background enabled at 8 Pol Acquisition User Manual Birefringence Imaging 16 Page Birefringence tab Data varic Parameters Birefringence processing Option
8. checked in the Data tab The name of the newly acquired background stack will appear in the Background field of the Parameters tab and will be used for background correction of the samples subsequently acquired It is advisable to acquire an additional background image at the end of a time series as the variable retarders may drift and corrections can be applied later to compensate for this Acquire a sample image Position and focus on your sample in the Live Camera window Proceed to acquire an image stack by clicking the SM sample acquisition button from the Pol Acquisition window The newly acquired image will appear and automatically be saved to disk if Save Acquired Images is checked in the Data tab The resulting sample image should appear in a new viewer window with background correction applied If no background stack is specified the sample retardance and orientation values are computed without background correction Once you have successfully acquired a sample image you are ready to perform Pol Acquisition User Manual Birefringence Imaging 10 Page Multidimensional acquisitions e g z slices time series etc Calibration of calculated retardance and orientation values The calculated retardance and orientation values are best calibrated against a sample for which the retardance and slow axis orientation values are known This is particularly important for the slow axis orientation which depends on physical p
9. made with a thin glass fiber e g soda lime glass diameter 100 um that is taped to a microscope slide When taping the fiber down on both ends induce a slight bend in the fiber The bend causes stress birefringence whose slow axis is oriented in a predictable fashion with respect to the fiber axis The birefringent layer on the outside of the bend where the soda lime glass is stretched has its slow axis parallel to the fiber axis while the birefringence in the inside layer where the glass is compressed has its slow axis perpendicular to the fiber axis For best imaging results the fiber can be imbibed in immersion oil to remove the cylindrical lens effect of a fiber that is surrounded by air refer Appendix B for alternate method using cheek cells Mapping of calculated retardance and orientation values into 8 bit and 16 bit images The calculated images in channel 1 and 2 represent the measured retardance channel 1 and orientation of the slow axis channel 2 values in every pixel of the sample or background image As all images associated with a PolScope stack are either 8 bit or 16 bit images including channel 1 and 2 the calculated retardance and orientation values are mapped into either 8 bit or 16 bit integers The computed retardance values are linearly mapped from 0 to the retardance ceiling value into integer values from 0 to 255 8 bit or 0 to 65 535 16 bit Retardance values that are computed to be higher than the ceiling val
10. setting usually 0 1 Calibrating the variable retarder settings At the beginning of a session and sometimes during a session the five liquid crystal settings need be calibrated Setting 0 generates circularly polarized light and is called extinction setting for which LC A is nominally 0 25 quarter wave and LC B is 0 5 half wave Settings 1 through 4 generate elliptically polarized light and LC A and B deviate from the extinction values by adding or subtracting a swing value The automatic calibration routine optimizes the extinction and the elliptical settings based on the swing value chosen by the user The system should be calibrated while observing the slide near the area where your sample specimen of interest is located as background birefringence can vary across the Slide Before starting calibration make a ROI selection in the camera window that identifies a Specimen area devoid of specimen birefringence Identify calibration parameters 1 Select the VariLC tab in the Pol Acquisition Window 2 Identify appropriate swing value If you are unsure of this value you may begin by entering a median value of 0 1 Once the actual range of retardance Pol Acquisition User Manual Birefringence Imaging 8 Page values in your sample are known you may adjust this number for improved performance 3 Check your lamp power and camera exposure time by selecting the Check Intensities tab If the intensity readings are above 2
11. 1 and 2 Setup Time and Z if required Proceed acquisition by clicking the Acquire button If SM button is used instead of Series the following dialog does not show up and a Single time point acquisition proceeds with the default settings Time points Acquisition order Close Acquire Internal fo ms i Autofocus Stop l Multiple positions XY TSAN TF Edit position list Skip Tamers OEN stacks slices Summary Number of time points 1 Advant e start um 25 Set wend um 2s Set step um 2 relative 2 h e keep shutter open Humber of positions 1 Number of slices 1 Number of channels 7 Total images 7 Total memory 56 ME Duration Oh Om Os Order Channel W Channels Channel group variLC_PolAca m Keep shutter open tee Configuration Epos zotte zs oa m pemes hosp moo b T peges heas bh e o TA WerlC State0 Acquired mage 2 0245 b M 0 TA farl Statet Acqured Image h2 o2a5 p E p The final Acquired dataset should show a total of 9 channels 2 Acq Presets 7 Pol related If you are satisfied with how you have defined your hardware for the Acquisition you may disable the option Always ask for Pol related Hardware Settings on Merge Doing so will from now on only bring up Step 9 amp 11 when using the Series button and only Step 9 when using the SM button Data varil Parameters Birefringence Proces
12. 10 lower the lamp power and or reduce exposure time 4 Set Black Level Block light from reaching the CCD and acquire an image Determine the average pixel value and enter it in the Black Level Recommended is a value between 3 and 7 for 8 bit images If the value is unacceptable change the camera offset setting 5 Press Calibrate Calibration will take roughly a minute to complete 6 Optimize exposure based upon calibrated LC settings With optimized exposure the numbers from your results should be within the following ranges assuming 8 bit images with pixel values between 0 and 255 LC Setting Intensity 0 10 to 80 1 80 to 210 2 80 to 210 equal to setting 2 3 cts 3 80 to 210 equal to setting 2 3 cts 4 80 to 210 equal to setting 2 5 cts If the numbers from your results fall significantly out of the ranges shown above you will have to perform the calibration sequence again Check the microscope to make sure that all OpenPolScope components are present and that there is nothing in the optical path that may interfere with proper operation Be sure to remove common accessories such as filter cubes or linear polarizers that may have been left in place by a previous user During calibration if three successive measurements are too bright the calibration will stop and display a warning You may adjust the lamp and repeat the calibration sequence until all the values are in the desired ranges Nevertheless even if
13. Pol Acquisition User Manual Birefringence Imaging 1 Page Pol Acquisition OpenPolScope software for Micro Manager Last Updated April 11 2013 Revision 1 07 This manual was prepared based on Pol Acquisition software version 2 0 and Birefringence Processing enabled Other versions of this manual are being prepared with Dichroism Processing and with Fluorescence Polarization Processing enabled Copyright license and warranty disclaimer statements at end of document Table of Contents Before installation canna nue NA E E 2 NNW css ser TE A T E A E E T A accu 4 Ei Ei OM E E A A A E E E E EEE 5 Starting the PolAcquisiton plug Mieurwiriiiunitana aaa 6 The PobAcguisiton WINdOW seisseen aiaia eekanna a iaaa a 6 A Walk Through of Basic OperatiOns cccccsccccsssccccssececeececeeseecseneceseueceseeceeseeceseaecessuecesseeetes 7 Calibrating the variable retarder SCttin gs cesessssssssseeseesesessseeseesesesenesesseseneieesseasensneaeaeesaseneneeaeeseseneneaeaees 7 Acquiring birefringence iMages esssesssscssssseseseeseseseseseseeseneseacsesseseneieaeesaseneneieaeeseneneneeaeeseneneneeaeeneneneeieaniniena 9 Calibration of calculated retardance and Orientation valUeSs s ssssssssrssrssrenrrssrssrssnssnrsrsrnnsnsnnnnsnenenns 10 Mapping of calculated retardance and orientation values into 8 bit and 16 bit images 10 Overview of Functionality and Interface Tabs cccccsccccsssccccssecccesececeesceeseneee
14. Rudolf Oldenbourg rudolfo mbl edu Cellular Dynamics Program Marine Biological Lab Woods Hole MA USA 2013
15. arameters of the optical setup including the orientation of the liquid crystal compensator and of the camera The retardance values are already calibrated by virtue of the calibration of the liquid crystal devices The manufacturer of the LCs of the Oldenbourg Lab included a calibration table in the VariLC controller which allows the setting of an LC device to an absolute retardance usually expressed as a fraction of the wavelength Therefore the swing value is given in absolute retardance and the specimen retardance in each image pixel is calculated in absolute retardance Note that the retardance of the LC settings and the swing are given in fraction of wavelength while the retardance of the sample is given in nm The orientation of the slow axis needs to be calibrated using a specimen with known slow axis orientation The calibration involves the correct setting of the orientation reference angle see Birefringence tab and of the mirror setting see Parameters tab A number of test specimens can be used for this purpose Biological specimens such as living or fixed cells with stress fibers or dividing cells with a living spindle or well preserved spindle microtubules are well suited because the morphologically distinct fibers are birefringent with a slow axis orientation parallel to the fiber axis Also collagen fibers have a Slow axis that is oriented parallel to the fiber axis A simple and accurate calibration sample can be
16. d acquire a background stack by pressing BG button on Pol Acquisition plugin Fe BG_2013_0130_1323_1 75 i l a ioj x c 1 7 1024x1024 pixels 16 bit 14MB K c D Es E E w Pi fps 7 3 678 s z 4 848 8501 um Retardance Computed Image Retardance Value ceiling 5 0 Orient Lines zoom for display Figure 3 Background captures effect of uneven illumination The field stop may be considered an uneven illumination at specimen plane Pol Acquisition User Manual Birefringence Imaging Page Step 4 Acquire sample stack and ensure that the slow axis orientation is along the edge of the cell R lolx fe 5M5_2013_0130_1331_1 on disk 150 cl 26 9 1024x1024 pixels 16 bit cha c zb a a D Peres fps 6 5 s z 4952 2751 um Retardance Computed Image Retardance Value ceiling 5 0 M Orient Lines zoom for display Eed Pol Acquisition User Manual Birefringence Imaging 35 Page Copyright license warranty disclaimer 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 copy
17. ded to increase the ImageJ memory Edit gt Options gt Memory to a minimum of 1024MB or more for smooth camera operations Guide to abbreviations VariLC Digital controller for liquid crystal devices The controller which might have a name different from VariLC is connected to the computer through a serial or USB cable Filenaming SM_2012_0904_0213_5 Prefix convention SM Sample Prefix_Year_MonthDate_HourMin_Suffix BG Background SMS Sample Series Multi Dimensional OpenPolScope data directory structure Pol Acquisition organizes the image data according to the following directory tree lt LCPolScopeData gt user chosen top directory lt UserNamel1 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 Acquisition User Manual Birefringence Imaging 4 Page Overview The following schematic summarizes the hardware configuration typically required for birefringence imaging with the OpenPolScope raw LC PolScope images intensity images CCD camera desk top computer ab N gt 5 linear polarizer is quarter wave plate G objective Q Cc specimen 5 O condenser 2 LC B variabl
18. e retarder 4 slow axis 0 o LC A variable retarder S 2 slow axis 45 an ji l D inear polarizer S gt transmission axis 0 Z Cc l O interference filter 2 an arc lamp gt Oo op computed PolScope images The optical design left builds on the traditional polarized light microscope with the conventional compensator replaced by two variable retarders LC A and LC B The polarization analyzer passes circularly polarized light and is typically built from a linear polarizer and a quarter wave plate Images of the specimen top row aster isolated from surf clam egg are captured at five predetermined retarder settings which cause the specimen to be illuminated with circularly polarized light 1st left most image and with elliptically polarized light of different axis orientations 2nd to 5th image The retarder settings are computer controlled through the retarder controller also called VariLC Based on the raw PolScope images the computer calculates the retardance image and the slow axis orientation or azimuth image using specific algorithms Pol Acquisition User Manual Birefringence Imaging 5 Page Installation For setting up the OpenPolScope hardware the user needs to be familiar with Micro Manager and its Hardware Configuration Wizard in Tools menu The wizard is used to add the camera and VariLC hardware to the Micro Manager configuration file xxx cfg These additions are necessary before the Pol Ac
19. easured for separate settings are not reproducible Click Check several times and watch intensity values LC A and LC B columns retardance values of LCs as fraction of wavelength can be changed by either typing value or clicking spinner wheel Intensity column intensity recorded in camera ROI is updated each time LC retardance is changed Current row current retardance values of LCs The remaining buttons and fields in the lower half of the VariLC tab affect the five settings used for OpenPolScope imaging Defaults Populates the Settings with the nominal LC retardance values The Settings are not calibrated Setting Setting 0 is also referred to as the Extinction Setting The other four Settings are the swing applied settings that are calibrated during the calibration procedure Clicking a Setting button sets the LCs to the corresponding retardance values Check Measures intensities for Settings 0 through 4 and calculates the extinction coefficient Pol Acquisition User Manual Birefringence Imaging 13 Page Calibrate Calibrates the five LC settings using intensity readings from the camera ROI Calibrating the Extinction Setting 0 searches for an intensity minimum by varying the retardance values of LC A and LC B The search starts from the values shown in the Current Setting For best results start from a setting that is close to the minimum Once the Extinction values are found the swing is applied to the
20. eeecessescesseeeeaees 11 Bel ee Rac eerenrt tre re tren rere tr caer erect rears eer errr ere eet et rer tre eee rere ree 11 WM I A eg tcc sac ae nc es tte E 12 elaine meee ener cee eee ene eee eee eae ee ee ee eee eee ee eee ee eee 12 Eeo om Gola 6 ok eee een ne nen eer eee na ee ee eee 13 L a E adsense E E E AE sete cane scne dace EE E A EA E A E E EEE 14 OLTE a o NE E EEE E ET et EE I E E O E A E A A E O E eee 14 Pra A E 15 PPG PE TINS INC Sr i a E E E E E E A E eau ene 16 FOC SU aD a E A ee 17 OPON A D E ee eee re 18 Lono ages n E E E EEE E ee 19 ACU On BU ONS on a iSS S are eee ee creer ee 20 FCI eN E E E E E E E E E eeseteeussounte 23 Repor TOO keee aei AaS E E E EE 23 eh EO arn eerie eerste terre ree E ee eee eee 24 ADON Eose E E E esses niesscesoies 25 FN 61 6 Arere ener ne ate en ee ee ee eee ree ere 26 Mu ulir Mode Acguis la l6 ee 26 POG D onana E EE 31 Calibrating the OpenPolScope for measurement of slow axis of birefringence using cheek cells Beats E E E E sts bene keene eee eee eeec eee 31 Pol Acquisition 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 It is used to acquire PolScope images applying PolScope algorithms and controlling the O
21. esults are explained in screenshots that follow 1024x1024 pixels 16 bit 2MB Lag EJ Stop Li B E snap album Figure 2a Calibration was done by choosing a ROI over background as shown The above image was acquired with VariLC set to setting 1 after calibration Pol Acquisition User Manual Birefringence Imaging 32 Page Help lag User Shalin Session 2011 01 30 Ortentation erificationCheekCell Acquire Sm BG Series Preferences sawed Data Yarilt Parameters Birefringence Processing Options Swing 0 03 fraction Reset LE Wavelength 546 0 nm Exercise LC LC Li_ B Intensity Current 0 268 0 481 27654 798 Defaults Check Setting 0 0 2384 0 481 7070 487 Setting 1 0 2684 0 481 25068 726 23496 991 0 51 0 45 Setting 3 oz Setting 4 ozo 041 22654 798 Extinction Ratio P 25a550 0 topa 60 Setting 2 0 238 Figure 2b At the end of calibration the VariLC panel is populated with appropriate retardance values for LC A and LC B as shown above High extinction ratio indicates that calibration was successfull Pol Acquisition User Manual Birefringence Imaging Page Step 3 Acquire background We notice in above image that there is uneven illumination of the specimen This and similar other imperfections in the optics are accounted for by a background Polstack Move to an area of coverslip where no specimen is present an
22. he computed images while acquiring In the case where acquisition speed is extremely fast or the Image resolution is higher than usual and enough computation power is not available to avoid any lag that could affect acquisition this selection can be used This acquired dataset will then need to be Processed using the Pol Analyzer interface Default is enabled Parallelize Processing This option will utilize all available CPU cores for processing the acquired images Default is disabled Pol Acquisition User Manual Birefringence Imaging 19 Page Logger tab Session Log Created 2013 03 28 01 04 18 01 04 18 Session User Rudolf Oldenbourg 01 04 18 Session Name 2013 03 28 Birefringence 01 04 18 Mode Birefringence 01 16 01 Acquired SMIS 2013 0328 1314 1 Image comment Session Exited 2013 05 28 01 24 58 Session Resumed 2013 03 28 01 37 42 Session Exited 2013 03 28 01 38 48 Session Resumed 2013 03 28 01 51 56 Session Exited 2013 03 28 01 34 24 Session Resumed 2013 03 28 01 54 53 Session Exited 2013 03 28 01 35 53 Session Resumed 2013 03 28 02 02 00 Session Exited 2013 03 28 02 02 42 Session Resumed 2013 03 28 02 04 50 Session Exited 2013 05 28 02 05 46 Saved 2013 04 11 04 41 28 This tab creates a chronological event log for the Session The log file session_log txt resides in the User s session directory Other than providing basic User information all images acquired in Micro Manager are logged in th
23. ion window Choose whether or nota mirror is present in the optical path the Black Level and the use of frame averaging are set correctly Please note that recalculation after image acquisition is possible should the settings initially be incorrect refer to Pol Analyzer manual Set pre and post processing algorithms Select the Processing tab in the Pol Acquisition window and select the desired pre and post processing algorithms Set birefringence parameters Select the Birefringence tab in the Pol Acquisition window and designate an appropriate retardance ceiling for computed retardance images For biological samples this is often in the 5 10 nanometer range Acquire a background image If the retardance values in the sample are less than 20 nm as is often found in biological images we recommend that you take a background image Background images are taken without a sample in the field of view but with the microscope Slide in place These images are used to compensate for the birefringence of the microscope optics themselves Position your sample so that the live camera window is free of sample and debris Defocusing slightly can help to reduce the visibility of particles in the area of the Slide where the background is acquired Proceed to take a background stack by clicking the BG acquisition button from the top of the Pol Acquisition window The new background data will be saved to disk if Save to Disk Acquired Images is
24. is log file A User can also add their own comments and notes in the text area regarding their experiments Note It is particularly useful for adding key changes to the system s hardware eg Objective Filter wheel etc that are not motorized and connected to Micro Manager and thus their information is not logged in the image metadata Pol Acquisition User Manual Birefringence Imaging 20 Page Acquisition Buttons aS ERE REC SM Acquire Sample images BG Acquire Background Series Acquire Series Multi Dimensional AutoClear Erases the Acquisition Comment after each acquisition Birefringence Preferences Saved fican Stop During Acquisition the Series fel fe button functions as a Stop button SM BG ct Frames 1 3 Channet 7 7 End Time 13 16 21s These acquisition buttons are added to the TopFrame if it has been started Note During Series acquisition the display of the progress bar changes with each acquired image However the End Time is calculated at the start of series acquisitions and remains fixed If the user pauses the series acquisition the displayed End Time will not change and is therefore incorrect The Series Multi D Acq uses Micro Manager s built in Multi D Acq engine and dialog Pol Acquisition User Manual Birefringence Imaging 21 Page el Note The Channels settings should not a e r Acquisition order sie _ be changed at this stage Number a Time S
25. ithout selection of a Processor plugin Note Pre Post Processor may or may not have additional options that are indicated by a button Pol Acquisition User Manual Birefringence Imaging 18 Page Options tab Multi Mode Acquisition Multi Mode Acquisition For merging PolScope acquisition with another mode of imaging such as fluorescence or bright field defined as acquisition Group Fi Fl Ask to merge with preconfigured Group Display refer Appendix A for more details E Show Computed Image During Acquisition Ask to merge with preconfigured Processing Group allows merging with an acquisition Group that has been W Enable On the fly Processing during Acquisition 3 aia previously defined by a user Parallelize Processing EEE Always ask for PolScope related Hardware Settings Mises ee on Merge If the merge option has been used Always ask for Pol related Hardware Settings on Merge previously the fields Can be populated using the stored values Micro Manager i Configuration settings pre exist eS Would sd sa like to ae them with PolScope for SAn Display Show Computed Image During Acquisition During series acquisition this option will keep the display on the retardance image and not display raw images This can be changed during series acquisitions Processing Enable On the fly Processing during Acquisition This option is available to disable processing for t
26. itor Configuration settings ASI CRISP Control Group Preset Plugins menu Date Browser System Startup i a ii EA If the Pol Acquisition plugin is ocalization icroscopy se 3 p Pease cte Image Flipper i accessed without the VariLC ever ROI Multi Andor Control is Z aa gt Group J Eaux Peeet eu being connected to the computer Image info i z y range 8 bits Onm pix Z 0 00um XY 0 00 0 00 um and configured as a hardware l ol Acquisition g Cowes Live Replay component in Micro Manager an scale Slide Explorer sync channels Siow nis r mente sutoaretch iai SEI too ht information prompt will come up Stage Control VariLC not detected and VariLC functions are disabled The Pol Acquisition window I Pol Acquicition Mira After correctly configuring the hardware Help the Pol Acquisition plugin can be ee ee ues a oe rere eee accessed A window opens that contains Acquire Birefringence action buttons a progress bar and several tabs that let the user set SM BG Series measurement parameters settings Acquisition Comments F Autocicar options data locations etc Data Location Data Location C Users Amitabh Desktop LCPolScopeData User Rudolf Oldenbourg 7 Create New Session 2015 05 28 Brefingence Session Birefringence data spermatocyte Description Set Default to Save Acquired Images Pol Acquisition User Manual
27. lice Channel z as Note The Saving format that is used by O en E Ithe PolScope plugin is Single image i eae ere ce files and should NOT be changed E Edit position list Skip frame s Interval 20 r W Z stacks slices 7 r Summary Number of time points 3 f Number of positions 1 Zenion gt Iaz Number of slices 2 Number of channels 5 aR um 2 Total images 30 7 Total memory 10 MB lative z Z Duration Oh 1m 0s Order Time Slice Channel Z start um 0 E Keep shutter open r W Channels Channel group VaewilC_PolAcg Config Exposure 7 offset 7 stack SkipfFr Color J Save images Directory root CALCPal ScopeDataiRudolf Oldenboungi2012_05_ 02 Name prefix SMS 2012 0914 0227 Saving format Single image files i Multi image files r Acquisition Comments Pol Acquisition User Manual Birefringence Imaging 22 Page ciii 130x130 pixels 86 bit 116k PolScope image stack The information at the bottom ofa PolScope stack includes pixel values in terms of the properties defined by the processor The Birefringence processor will display the Retardance value of a pixel Orient Lines This check box causes an overlay of orientation lines to be drawn inside a specified ROI Parameters such as the line interval are set with the Orie
28. mage info fram camera 2046 2046 amp 2 Intensity range 16 bits Onm pix 2 4910 75um Step 7 At this point it is recommended to Save this hardware configuration using the Save button 28 Page Pol Acquisition User Manual Birefringence Imaging Step 8 Start Pol Acquisition Under Options in the Multi Mode Acquisition panel enable both op tions Data varilC Parameters Birefringence Processing Options Multi Mode Acquisition J Ask to merge with preconfigured Group M Always ask for Pol related Hardware Settings on Merge Step 9 Click on the Series button and the dialog below should pop up Select the group you want merged with PolScope acquisition if you have multiple Groups and hit OK Configuration settings pre exist Would you like bo merge them with PolScope For Acquisition Step 10 The dialog below should pop up The preset name will be populated and the only thing to be selected is the Label Set it to 1 since 1 has the Analyzer cube Hit OK Here you can specifiy the property values OK in a configuration preset Cancel Preset name Varilc Stated Acquired Image Property Mame Preset Value aril C String send to WariLC TFilkerBlock1 Label 1 FiterBlocki State Pol Acquisition User Manual Birefringence Imaging 29 Page Step 11 If Series button was clicked previously the Multi D dialog will show up At this point change the exposure if required for the Acq Preset
29. next settings and calibrated such that their intensities are equal Before starting calibration make a ROI selection in the camera window that identifies a Specimen area devoid of specimen birefringence Typically calibration is done with the camera showing a background region in the specimen and is immediately followed by recording a Background stack Extinction field shows the extrapolated extinction coefficient The extinction coefficient characterizes the quality of the polarization optical train The extinction is defined as the ratio of the recorded intensity when the universal compensator is set to maximum transmission divided by the intensity when the compensator is set for minimum transmission extinction is measured directly Setting 0 while the maximum ext transmission is extrapolated from Setting 1 using the measured intensity and L extrapolating to a virtual setting that would cause maximum transmission de de dh dAd Extinction 12 3 4 la _ 4 I BlackLevel sin Swing 1 Note The Calibration routine can be tweaked by using the Brent Optimizer Accuracy and Low High range limits of the VariLC from the Options panel Note It is recommended to change the VariLC settings through this tab rather than Micro Manager s Device Property Browser Calibration Options Controls Calibration Options Save amp Load Options _ Plot Intensities Plot the Intensity profile during Calibration
30. ntation LinesV3 plugin button on the Viewer Ina series the lines are re calculated for each time point and or z slice 4 wT F ie playback fps T 5 E 1 544 s z 0 um Retardance Computed Image Retardance nm Value ceiling nm 10 0 Birefringence dataset The Pol Acquisition routine writes metadata and comments in the Micro Manager Metadata panel rs Background Processed Using Retardance Ceiling nm Perimage comments The pixel value in the Image represents the Orientation in degrees Pol Acquisition User Manual Birefringence Imaging 23 Page Help Report Tool Report Pol Acquisition icroMa polscopem mbledu avermat mbl edu gharrisi mbledu rudolfog mbl edu 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 Help in the Menu bar to send bug reports or suggestions Pol Acquisition User Manual Birefringence Imaging 24 Page ChangeLog ChangeLog Taled Versin 2 02b 04 11 2013 Latest Version Available 2 02b 04 11 2013 ChangeLog LCPolScope Date Wednesday April 10 2013 9 44 57 PM Message converter for image polstack to Mhgr dataset with additi Date Tuesday April 09 2013 8 44 22 Pi Message converter for image polstack to Mgr dataset using Storage Api make shift implementation to convert Aster image fix for focus change from Q
31. penPolScope hardware attached to the microscope 2 Pol Analyzer plugin after its installation into the plugins folder this plugin appears in the Image Plugins menu providing functions to view and analyze PolScope data that have been previously acquired The OpenPolScope software installer 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 Orientation Lines PolScope Legacy RatioAzimPolStackToRGB_V2 ROI_Averages_With_Lines_V1 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 10 MB Hard Disk Space additional space required for UserData ae amp S Pol Acquisition 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 recommen
32. quisition plugin can be used Once the VariLC has been added its properties have values similar to the ones seen in the image below The Port Properties required for communicating with the VariLC are the default values for COM port communication For the Abrio the baud rate needs to be set to 115200 instead of 9600 the total number of LCs to 3 and active LCs to 2 One can use the Property Browser in the tools menu to test the communication with the VariLC a Hardware Configuration Wizard Configuration settings Step 2 of 6 Add or remove devices E Adapter Library Core IMMCore Default Description Core controller QCamera Available Devices list by vend voreersTangS Thorlabs ThorlabsDCxxxx L ThorlabsFilterWheel ThorlabsSC 10 Tofra TwainCamera Utilities VariLC H VariLC VariLC Vincent XCite 120PC_Exacte XCiteLed D Xcite Yokogawa ZeissCAN b t l Gd a Ga Go Go COM3 QImaging universal OK S Device VariLC Library VariLC Label VariLC Initialization Properties Edit Adding or Removing Devices 1 The list above displays all of the devices that will be handled 9 Manager in this ion file making a new ion file for the first Se visit the Device Property nager website ro manager org and Devices to find NVariLC ort VariLC VariLC umber of Active LCs otal Number of LCs sins for setting up all
33. right 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 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
34. s The main Processing Mode selected in the Processing panel here Birefringence will be displayed as an additional tab Its panel will Retardance Ceiling 20 0 nm h t EEE E E m rae contain options only applicable when this Mode is selected Retardance Ceiling Sets the value of the F Use 4 Frame Acquisition instead of 5 Frame retardance ceiling that is used for storing the E Use 4 Frame Processing instead of S Frame retardance values in its image channel usually first channel For further explanations see section Mapping of calculated retardance and orientation values into 8 bit or 16 bit images Orientation Reference Sets the orientation reference that is used for processing the slow axis orientation image Use 4 Frame Acquisition Option to acquire only 4 frames instead of the default 5 frames dropping the acquisition of the raw image for Setting 4 Use 4 Frame Processing Option to use only 4 frames for processing instead of the default 5 frames Pol Acquisition User Manual Birefringence Imaging 17 Page Processing tab The Processing tab is divided into 3 main segments Pre Processing Enabled Description Options 0 Ratioing E Ratioing use a ROI selection to scal Pre Processing This applies some modifications to the raw image data before they are sent to the main processor An example is Ratioing in which the pixel values of the raw sample images are
35. sing Options Multi Mode Acquisition Iw Ask bo merge with preconfigured Group Always ask For Pol related Hardware Settings on Merge If any of the Multi D Group related hardware is changed eg Filter cube position then one will need to re enable the above option once to redefine the position of the Analyzer Pol Acquisition User Manual Birefringence Imaging 30 Page Appendix B Calibrating the OpenPolScope for measurement of slow axis of birefringence using cheek cells Microscope used for the following Step 1 After swabing cheek cells onto coverslip and immersing it in saliva isotonic immersion setup Kohler illumination Cheek cells do not refract light much so we need to have some type of contrast enhancement Reducing the illumination aperture by stopping down the condenser NA one of the simplest type of cohtrast enhancement works well When Kohler illumination is setup and condenser NA is stopped down we see something like this 1024x1024 pixels 16 bit 2MB B Lill Snap LEN Live gt Album Figure 1 Kohler aligned microscope The hexagonal aperture visible in the specimen plane is the field stop The aperture stop of the condenser was closed down to be smallest sins 31 Page Step 2 Now open both the field stop and aperture stop slightly choose a ROI over background region and calibrate the VariLC Notice that as aperture stop is opened image has better resolution but poorer contrast The r
36. the Average Gray values of Setting 2 through 5 are as low as 40 or as high as 230 the OpenPolScope will still function though not optimally The ratio of Setting 2 to Setting 1 is an indicator of the retardance measurement signal to noise ratio Bigger ratios are better For large retardance values which usually involve a retardance swing of 0 2 the ratio is 10 to 1 or better For small retardance values which usually involve a retardance swing of 0 03 the ratio is typically 2 to 1 or better For microscope applications the diameter of the aperture diaphragm in the condenser has a Significant impact on this ratio and thus the retardance signal to noise ratio If the aperture is decreased this ratio will go up However signal will go down and the exposure time will increase Of course the adjustment of the condenser aperture will also impact Spatial resolution and depth of focus The Extinction number is another indicator of the quality of the polarization optical train An Extinction of at least 100 should be achieved more routine is 200 and better The optical setup will be more sensitive the higher the Extinction Do note that if you change the swing value you will need to recalibrate It is advisable to re calibrate whenever you change the sample specimen slide Pol Acquisition User Manual Birefringence Imaging 9 Page Acquiring birefringence images Set acquisition parameters Select the Parameters tab in the Pol Acquisit
37. the user when a session is created and can be modified later to provide a quick view description This would typically describe the types of datasets or other user notes that describes the session Set Default to Save Acquired Images Pol Acquisition User Manual Birefringence Imaging 12 Page VariLC tab This section applies when using VariLC or Abrio as a liquid crystal device controller Controls varie The VariLC tab has controls for Ithe LC universal compensator Swing 0 03 fraction Reset LC with 2 liquid crystals LC A and Wavelength 546 0 nm Exercise LC LC B Swing Sets the nominal Controls retardance bias as a fraction of LCA LCB the wavelength for the elliptical 02861 0 42212 ee settings of the liquid crystals ee Default is 0 03 Defauts Calibrate Wavelength Sets the wavelength Setting 0 ae eis that will be used when setting the Retardance values Usually equal Setting 1 sini nini a to center wavelength of light used Setting 2 0256 2 0 457 to illuminate specimen Default is 546 nm Setting 3 0 256 0 39 Setting 4 t223 0 422 Reset LC Resets the Error light on the VariLC controller Occasionally this light might Extinction Ratio come on during the calibration process without consequence TEX ercise LC Cycles the LCs between high low values to assure consistent settings Click if LCs have been powered off for a long time and intensities m
38. uadRoiianager image window to another when Rois are defined added not Unit for Retardance display images Date Monday April 08 2013 9 09 10 PM Message Added Birefringence Dichroism User manual Birefringence and Dichroism User manual to installer Date Monday April 08 2013 5 10 39 PM Message refactoring PoLAnalhzer Pol Acquisition User Manual Birefringence Imaging 25 Page About B Pol Acquisition MicroManager About Pol Acquisition MicroManager Version 2 02b build 04 10 2013 API Build 04 11 2013 LC PolScope software for Micro Manager Copyright amp 2009 2013 Marine Lab NOTICE Some of the algorithms herein are protected by US patent 9921705 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 and the following disclaimer in the documentation and or other materials provided with the distribution 3 Nether the name of the Marine Biological Laboratory nor the names of ts contributors may be used to endorse or promote products derived from this software without specific prior written permission
39. ue are converted to 255 or 65 535 respectively A pixel value in channel 1 can be converted into a retardance value by the following formula Pol Acquisition User Manual Birefringence Imaging 11 Page _ pixel value 8 bit image retardance 964 retardance_ ceiling 12 bit image retardance pe ae retardance_ ceiling 16 382 16 bit image retardance pixe vane retardance ceiling The highest possible pixel value 255 for 8 bit 16 383 for 12 bit and 65 535 for 16 bit images is reserved for indicating any value higher than the retardance_ceiling The computed orientation values range between 0 and 180 and are stored as integer values from 0 to 180 8 bit 0 to 1800 12 bit or 0 to 18000 12 and 16 bit Hence an orientation angle stored in channel 2 of a 16 bit image is equal to the pixel value divided by 100 Overview of Functionality and Interface Tabs Data tab The Data tab lets one choose a root Dala Localion folder Data Location where i OpenPolScope data will be saved Siaina neti ini After selecting a root folder User and User Rudolf Oldenbourg Session folders can be recalled in drop down menus or created as Session 2913 03 28 Birefringence m needed Set Default to Save Checking this option will set the default behavior to Save acquired images into the specified Session folder Session Birefringence data spermatocyte Description Session Description This text is set by
40. y values OK In a configuration preset Cancel Preset name 4cq Presetl Property Mame Preset Value FiterBlocki state Pol Acquisition User Manual Birefringence Imaging 27 Page Step 4 The defined Multi D Group will now show up File Tools Plugins Help ji Snap Camera settings Configuration settings Save GP uve Eoswemg i2024 Manj System E Multi D Acg Shutter TiFilterBlock Label 2 TIFilberBlock1 State 1 A Refresh did Auto shutter W Close Please cite Micro Manager so funding will continue ROl zoom Profile Autofocus Kj aal Bl WIP con f come ca Image into from camerar 2048 2048 2 Intensity range 16 bits Onm pix 2 4910 80um Step 5 If there are more than 1 then proceed to add a second hardware state using the button for Preset selecting Label 3 Here you can specifiy the property values in a configuration preset Cancel Preset name 4cq Preset2 Property Mame Preset Value i IFilterBlock1 Label 5 Fiker lockd state a Step 6 We now have defined 2 Presets for acquiring non PolScope images File Tools Plugins Help ji Snap Camera settings Configuration settings Save La te Exposure ms 12 0246 E Binning dc MultiDGroup ae System EEE F multi p Acq Shutter F Refresh Auto shutter fw Close Please cite Micro Manager so funding will continue foom Profile Autofocus Biel raia lat e cme car I
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