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COSMOS MANUAL - University of Memphis

1. File Help Intensity D 1 1 pr 1 1 4 Ig 0 00060 sme 0 00020 y 4 4 usb hn i patton t b nbn i a o 20 40 eo 80 100 120 sample Intersection XY and XZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane Levalvindow ai i Y 0 X 0 47 Z 128 COSMOS MANUAL 0 9 16 Create the forward image 1 Open the Variant sub tab in COSM Tools 2 Specify the following parameters e Object File The path to the ring object e PSF File The path to the LSF e Output File The path where the forward image will be stored e Number of Strata 8 e Start of Strata 108 e Size of Strata 5 3 Click Execute The forward image can be seen in COSM Viewer File Help Intensity AA eese o 20 40 Intersection XY and XZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane Levavindow a m 0 08 0 0599 oo PIET Z 128 O x c Restore the original image 1 Open COSM Estimation 2 Click the Iterative sub tab and then the Variant sub tab This is chosen for the best estimation 3 Select the EMSV algorithm the DVEM algorithm A penalty is not necessary since the image in this experiment is a phantom Real images are noisy and hence a penalty is used 5 Enter the following information 48 COSMOS MANUAL 0 9 16 e Dat
2. Software Package User Manual Version 0 9 16 Written by Sreya Ghosh Daqi Dong Gregorio Lobo Timothy Mathis and Nurmohammed Patwary based on notes from Einir Valdimarsson and Chrysanthe Preza Edited by Chrysanthe Preza 2014 Computational Imaging Research Laboratory Electrical amp Computer Engineering Dept The University of Memphis Supported by the National Science Foundation CAREER award DBI 0844682 C Preza Pl COSMOS software package copyright and terms of usage Copyright O 2007 Einir Valdimarsson and Chrysanthe Preza The COSMOS package is free software you can redistribute it and or modify it under the terms of the GNU General Public License as published by the Free Software Foundation either version 2 of the License or at your option any later version COSMOS is distributed in the hope that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU General Public License for more details You should have received a copy of the GNU General Public License along with this program if not write to the Free Software Foundation Inc 59 Temple Place Suite 330 Boston MA 02111 1307 USA Any publications with results obtained with COSMOS should cite COSMOS COSMOS citations should be made by referring to the COSMOS web page http cirl memphis edu cosmos COSMOS MANUAL 0 9 16 Table of Contents Chapt
3. The object can be viewed in COSM Viewer 39 COSMOS MANUAL 0 9 16 File Help Intensity r E d Y D s 0204 0 60 4 0404 020 alla TR d i o bol cr MA o 20 40 eo 80 100 120 sample Intersection XY and XZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane Lev lvindow 0 500 1 000 Z o Y 04 xX 0 Creating a forward image 1 Open the variant sub tab of the CosmTools 2 The object and PSF generated using the method in the previous chapter are convolved using the convolution tab in COSMOS Tools The simulated microscope image can be viewed in COSM Viewer File Help Intensity 0 020 4 7 t ST letra ada ada dao dada lore o 20 40 eo a0 100 120 sample Intersection XZ and YZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane Eadie 0 500 1 000 Y e x e _ 40 COSMOS MANUAL 0 9 16 Chapter 6 How to process an image measured or simulated using the COSMOS Estimation module The same PSF generated for the forward image in the previous chapter needs to be used for the estimation process 900 me File Help XML File Browse Open Data Input File Browse PSF Input File Browse Centered PSF v Estimation Output File Browse Linear Iterative Algorithm Linear Least Squares LLS Linear Maximum a Poster
4. DIC Note COSM PSF s DIC support is still under development and is not currently functional The following are parameters for computing a DIC differential inference contrast PSF Detail information about the PSF can be found in 13 This section is only valid for those using DIC Microscopy Shear The shear distance due to the Wollaston prism Bias The constant phase introduced by moving one of the prisms Amplitude Ratio For crossed polars the amplitude is 0 5 for each component of the shear beam Rotation Angle The angle that the shear direction makes with the horizontal axis Sum over Y Used to collapse the PSFs over the Y dimension to create LSFs Uncentered Used to create an uncentered PSF file However centered PSFs are more common PSF Filename The path where the PSF image will be stored in the WASHU wu format Generate Real and Imaginary Used to generate both the real and imaginary PSFs If unchecked only the intensity PSF will be created Automatically import configurations from XML file Normally CosmPsf recalls default configurations when it starts but if there is a file of lastest xml in the same folder of CosmPsf then content of lastest xml will be recalled automatically as customized configurations This function is useful to the user who wants to use special configurations which are not default To use this function copy the xml file to the folder containing CosmPsf and change th
5. Number of Components 0 Inputs Original PSF File Specify the original PSF files PCA Based File The Target of PCA Based files Number of PSF Files Number of original PSF files which are used to calculate PCA Based PSF files Depth interval between PSFs pixels Set as 1 Any value other than one will be considered as 1 Number of Components No of component base PSFs Generate higher no of component PSFs i e more than 50 and any number of component PSFs i e 3 5 7 etc can be used for estimation Higher no of components will increase estimation accuracy Output When executed the new PCA Based PSFs will be created which are components average PSF and coefficient files Three files will be generated name PCA BaseZero average PSF name PCA coefficients component PSF weights and lt name gt base PSFs 18 COSMOS MANUAL 0 9 16 14 Wavefront encoded WFE PSF This tab File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant PCA 1 Conventional PSF Mask File Output File Inputs transforms conventional PSF to WFE based PSF Browse Browse Centered PSF v Browse Execute Conventional PSF The conventional original PSF which needs to be transformed to new PSF Notes that this conventional PSF file it must involve certain parameters of PSF s configuration These parameters have been added
6. a SS Ss 9AM Type Application Application Application Application Application extens Application extens Application extens Size 845 KB 567 KB 570 KB 4 473 KB 1 365 KB 1 350 KB 1 055 KB COSMOS MANUAL 0 9 16 Chapter 2 Building COSMOS from source code Because cmake is used to generate project files or makefiles there should not be any changes made to the project file only to the cmake configuration The process for building COSMOS for any operating system is the same 1 Download cmake from www cmake org and install 2 Download Blitz from sourceforge net projects blitz and install and build 3 Download fftw from www fftw org and install There are some special instructions for windows to fix the libraries www fftw org install windows html 4 Download vtk from www vtk org and install and generate project files with cmake and build 5 Dowload fltk from www fltk org and install and generate project files with cmake and build 6 Download tclap from tclap sourceforge net and install and build 7 Generate the project files for COSMOS with cmake and build Start the cmake gui and specify the installation paths for the previously installed packages 2 6 COSMOS MANUAL 0 9 16 Chapter 3 Description of the applications utilities available in COSMOS Introduction All files to be opened and processed in COSMOS require the WASHU header wu extension The heade
7. Computer Engineering the University of Memphis 2009 Preza C and Myneni V Quantitative depth variant imaging for fluorescence microscopy using the COSMOS software package in Three Dimensional and Multidimensional Microscopy Image Acquisition and Processing XVII BIOS SPIE 7570 2 January 2010 in press Markham J and Conchello J A Tradeoffs in regularized maximum likelihood image restoration Proceedings of SPIE 2984 136 145 1997 Preza C Snyder D L and Conchello J A Theoretical development and experimental evaluation of imaging models for differential interference contrast microscopy Journal of the Optical Society of America A 16 9 2185 2199 1999 53
8. Functionalities of COSMOS 41 Chapter 8 re eee t neo ih e IM e UE 465 An Implemented Example Demonstrating the Depth Variant DVEM Algorithm of the COSMOS O Ne 465 Generated testimage ti ttt tt teet A AA da di 465 Ge nerate an PL 476 Create the forward image sess eene een nnne Draai aiia ne ieia 487 COSMOS MANUAL 0 9 16 Restore the original image oconcococnnncnononononnnnnnnnnnnnnonononnnnnonanononnnnnnnnnnnnnnno nn nana nnn nnne asas nnns 487 Captar a e dedo rd LR t ATE 50 Frequently Asked Question ica 50 E reete ede DE 52 References x ote ttim t need eter et pea toe t E e teta de aee te ders 52 COSMOS MANUAL 0 9 16 Chapter 1 Installation Guide Downloading COSMOS COSMOS can be downloaded at http cirl memphis edu cosmos Download the Package according to system requirements e Cosm 0 9 10 Darwin dmg installation package for Mac Tiger 10 4 executable e Cosm 0 9 10 win32 msi installation package for Windows XP win7 executable e Cosm 0 9 10 win32 zip archive of COSMOS executables for Windows XP win7 e Cosm 0 9 10 zip archive of COSMOS source for Windows XP win7 e Cosm 0 9 10 tar gz archive of COSMOS source for Linux e Cosm 0 9 10 Linux tar gz archive of COSMOS executables for Linux Windows 7 installation The following instructions are for the installation package for Windows 7 1 Click on
9. PCA mode Components Number of components of PCA Based PSFs Start 30 COSMOS MANUAL 0 9 16 Stating plane number of object ROI see Section 3 1 12 The number of PCA Based PSFs which are used to do inversing Convergence The graph can be plotted here for various parameters with the simulations and the reference file Various things can be computed here such as IDIV values MSE maximum minimum mean File Help XML File Data Input File PSF Input File Estimation Output File Linear Iterative Invariant Variant Convergence Reference File Error 1 0e U0U TUTTI 1 0e 003 Reference File aes ee oe ee a aaa 40 eo 80 100 Iterations Stop Progress Browse Open Browse Browse Centered PSF v Browse Browse Measure Table Clear maximum 1 v minimum 000000001 w Iterations 100 Update 10 Write 100 The image with which the estimation will be compared If left blank the previous estimation will be used Measure The values to be computed during the comparison Table The table containing the values computed during the comparison These values can be saved as csv file by clicking Save Clear 31 COSMOS MANUAL 0 9 16 Clears the plot in the measurement graph area maximum The maximum value on the vertical axis minimum The minimum value on the vertical axis Generation and Processing of a 2D XZ image using the
10. Refractivelndex value 1 330000 gt lt Thickness unit mm value 0 000000 gt lt Specimen gt lt OTL unit mm Actual 160 000000 Design 160 000000 gt lt PSF gt 38 COSMOS MANUAL 0 9 16 Chapter 5 How to generate a simulated microscope image using the COSMOS Tools module Introduction In order to test the performance of algorithm the generation of simulations is required We can simulate a microscope image using two sub tabs from the COSM Tools e the Object sub tab and the Variant sub tab Convolution sub tab To simulate a microscope image an object and a PSF are necessary see the previous chapter The image is formed when the object and the PSF are convolved This can be done using the convolution tab or the variant tab in the case of the DVEM Algorithm Creating an object Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File paTa test wu Browse Output File pata test wu Browse Create Ellipsoid Box Point Center xy z 64 64 64 Radius xy z 8 8 8 Value 1 Execute 1 Goto the Object sub tab In Create you can enter the output file the input file can be left blank Input the Dimensions of the image and the Data Type Value represents the color fill 0 indicates a black background while 1 represents white 2 Gotothetab specifying the shape desired Here the ellipsoid tab is chosen with center as 64 64 and 64 and radius 8
11. image s is converted and then stored with the name specified in Output File Compare This tab compares an image to a reference image File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File 1428x128x1281Gibson and Lani GL_PSFO wu Browse Reference File 1428x128x1281Gibson and Lani PSF1 wu Browse Error Measure Execute Maximum 0 0016768 Mean 3 82423e 007 Mean Square 1 55294e 011 Inputs Image File The image to be compared Reference File The reference image to which the image will be compared 14 Output COSMOS MANUAL 0 9 16 When executed the two images will be compared and the maximum mean and mean square error between the two images will be displayed 10 Convolve This tab convolves two images M 3 FLTK File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant 1st Image File pata DVEM object256 wu Browse 2nd Image File pata DVEM PSFitm_bead_159ri_40um_psf0 wu Browse Output File pata forward_image wu Centered PSF v Browse Output Image Size Execute Normal Size Size of 1st Image Size of 2nd Image Inputs Output 11 Object 1 Image File The first of the two images being convolved usually an object image Be Image File The second of the two images being convolved usually a point spread function Centered
12. sub tab in the COSM Tools 1 2 Open the Object sub tab in COSM Tools Open the Create sub tab Enter the following information e Image File Leave blank e Output File The path where the object will be stored e Dimensions 128 1 256 e Data Type Float Click Execute A blank object was created Click on the Ellipsoid tab Enter the following information e Image File The path to the blank object just created Same as Output File e Center 64 1 128 e Radius 20 20 20 e Value 1 This indicates that the circle will be white Click Execute This generated a white circle in plane XZ with radius 16 Change the parameters in the Ellipsoid tab to the following e Center 64 1 128 e Radius 10 10 10 e Value O This indicates black Click Execute This generated a black circle in the middle of the previously generated white circle creating a ring The resultant object can be seen in COSM Viewer 46 COSMOS MANUAL 0 9 16 File Help Intensity oso j 0 60 oso 0207 ow Tipp rn BUSESERESERE o 20 40 eo 80 100 120 sample Intersection XY and XZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane Lev lVindow 0 500 1 000 Z 128 Co X es Generate an LSF An LSF can be created using the steps in Chapter 4 using 256 64 128 for the Dimension parameter The LSF can be seen in COSM Viewer
13. to PSF header file beginning from new version 0 9 7 of COSMOS Users may need to recreate their PSF files by new COSMOS version if the PSF files were created by older version Mask File The Phase Mask file which would be used to produce new WFE PSF All zeros around the phase mask should be cropped Raw phase mask data type should be 32 bit float For generating bigger dimension WFE PSF higher memory more than 32 GB RAM may require If data type or memory requirement does not meet program may stop executing Also the mask must be unwrapped because when the mask size was changed within the process of CPM based PSF engineering the input mask must be a raw one unwrapped otherwise the final result will be impacted So we remind the users that don t do the kind of phase wrapping to your mask for this CPM based PSF engineering process 19 COSMOS MANUAL 0 9 16 Output The new WFE PSF file 3 2 COSM PSF This module is used to generate the PSFs The software uses the xml files to input the settings for PSF generations However PSF files can be created according to desired needs by using the GUI File Help PSF XML Import File Browse Open E Refractive Index Thickness mm Radial ER Coverslip ORI Design 1 522 0 17 Model Gibson Lannit992 Actual 1 522 017 Type Non Confocal o SLT Precision Smile rez Et Design 1 515 0 16 ee M Actual 1 515 0 16 Dimension x y z 64 64 64 Specimen Spacing um x y
14. z 0 16 0 16 1 133 0 Absolute Error 1e 006 Lateral Magnification 40 OTL Angle Interval 0 Numerical Aperture 1 Foams imm Thickness Interval mm 0 Wavelength nm 580 Total Number 1 Sum over Y Uncentered PSF Filename Browse Generate Real and Imaginary Generate Progress Parameters PSF XML import file XML file that contains stored parameters from a previously generated PSF This file can be used to obtain multiple images taken under the same conditions Once the XML file has been selected click Open to import the settings Evaluation PSFs can be generated using one of the following evaluations 20 Model Type COSMOS MANUAL 0 9 16 Radial Interpolation The PSF of discrete points are obtained and interpolated over the system to get the output Exact evaluation The PSF is calculated for each point Although it is very accurate it has a large overhead There are two models by which the PSFs can be generated Gibson_Lanni 1992 This is a model for creation of PSFs that tries to effectively model the effects of aberrations in fluorescence light microscopy For further details refer to 7 Haeberle This model is an improvement on the Gibson and Lanni model This combines the ease of a scalar model with high accuracy For further details refer to 8 The PSF can be computed according to one of the following microscope types 2 Photon It is a fluorescence imaging technique that allows the imagin
15. C cli su pca num 3 start 45 size 46 em 1000 update 100 write 100 CosmEstmation and CosmPsf support CLI because these 2 parts are normally time consuming Benefits of using CLI e Multiple jobs could be maintained and executed together automatically e CLI displays job status timely in running time This is useful to the user who is running a time consuming job more than 4 hours COSM Estimation CLI Usage CLI is enabled using the c1i or c option as shown below CosmEstimation cli other options The following is a typical way of using COSM Estimation CLI a4 COSMOS MANUAL 0 9 16 CosmEstimation cli img PATH AND FILENAME OF FWD FILE psf PATH AND FILENAME OF PSF FILE est PATH AND FILENAME OF OUTPUT FILE update 100 em 1000 write 100 double Options The description of all options e cli or c Switch to use CLI function e img or t Set value of forward image s filename e est or t Set value of estimation file s filename e psf or p Set value of PSF file s main filename e otf or f Set value of OTF file s main filename Default value is otf e pha or y Set value of Phantom file s main filename Default value is pha e suffix or x Set value of above files external filename Default value is wu e update or u Set value of number of iterations between sta
16. Convergence Algorithm Space Variant EM EMSV DV Conjugate Gradient DVCG Ordered Subsets EM EMOS Penalty 0 None Double Z OTF in Memory Strata 1 Start 1 Size 1 OTF Name Strata mode Iterations 100 Update 10 Run Stop Write 100 Progress Data input file Blurred WU image PSF input file First PSF file name PSF number should be removed See section 3 1 12 Space variant EM EMSV This is a depth variant algorithm with the variations in the z dimension assumed to be a minimum 29 COSMOS MANUAL 0 9 16 Ordered Subsets EM EMOS This algorithm is still under development DV Conjugate Gradient DVCG This is a depth variant maximum likelihood restoration using a conjugate gradient algorithm Penalty A value between 0 and 1 used for noisy simulations Simulation can either be done with Intensity or Roughness penalty In variant section current version 0 9 10 only supports Space Variant EM algorithm with strata PCA mode Double Z Double Z dimension must be selected while executing EMSV or EMOS algorithms Strata Number of strata usually the number of PSFs minus one Start The starting depth of the first PSF Size The number of planes in each stratum OTF Name The path to the optical transfer function if OTF on Disk is selected Otherwise OTFs are automatically generated by the program so OTF in Memory is usually selected Strata mode PCA mode Switch between strata and
17. Depth Variant DV Approach 1 Forward Image Formation DV The Depth Variant model assumes that a point source in the object will have a PSF depending on its depth in the object The more PSFs we would use the better but unfortunately by increasing the number of PSFs we also increase the computational complexity of the estimation algorithm A middle ground is to pick a several depths that we will generate a PSF and divide the object into parts that we call strata PSF Generation Line Spread Function The CosmPsf application is started If we want n strata we generate n 1 PSFs 1 each for the boundary of each stratum In the CosmPsf application we can specify how many PSF we will generate and the Thickness Depth intervals The depth interval is determined by the spacing in z and how many pixels in z we have in each stratum Normally the CosmPsf application would generate a 3 D PSF but we can force it to collapse the Y dimension sum over it into a single plane by enabling Sum over Y The resulting PSF is a 2 D image often referred to as Line Spread Function LSF Specify the name of the resulting LSF The application will append a number to the filename before the file extension Press Generate and the LSFs will be generated one after the other Image Formation DV The image formation for the Depth Variant model is a sum of convolutions of each stratum with an interpolation of the PSFs that are on each side of the strata St
18. OR A PARTICULAR PURPOSE See the GNU General Public License for more details How to debug a process if a run time error occurs All compile time error and comments show up in the command prompt so going back the cause of the error can be found out This is particularly important when there are multiple iterations being implemented and an error occurs Can a file in tiff format be read by the COSMOS No but it can be converted to the wu format using the COSMOS Tools which can open several different formats Only wu format files i e images with a 1024 washu header can be read by the other applications which are a part of the COSMOS package Images with no header are usually named with a raw extension which can be read by the add header tab in Cosm Tools This tab adds the washu header and converts the file to wu extension What are the system requirements 1 11 KB of Hard Disk space is required COSMOS can run on a variety of Operating Systems Is it possible to process a 2 D image using the LLS or EM algorithms If so how are the parameters set in the COSM PSF menu Yes the number of planes in Z equal to 1 The rest of the parameters are kept as they are If images are saturated can they still be processed with the COSMOS algorithms Yes the intensities will be scaled down but it can easily be processed with some losses 51 COSMOS MANUAL 0 9 16 What does pixel size in object space in the Generate PSF m
19. PSF Convolve two centered PSFs Output File The path to the file where the result of the convolution will be stored Output Image Size The convolution result s image size normal the two image sizes added together or the size of one of the inputted images When executed the two images will be convolved and the resulting image will be stored with the name specified in Output File This tab creates objects 15 12 COSMOS MANUAL 0 9 16 z 2 FLTK CE File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File Browse Output File pata object wut Browse Create Ellipsoid Box Point Position x y z 128 128 125 Value 1 Execute Inputs Image File The path to the existing image that the object will be written onto Note mage File should be left blank when creating a new image Output File The path to the file where the object will be stored Create Ellipsoid Box Point Dimensions The dimensions of the blank image Data Type The image s data type Value The value with which to fill the image s dimensions Center The point on which to center the ellipsoid Radius The radius of the ellipsoid Value The value with which to fill the ellipsoid Center The point on which to center the box Half size The half size or radius of the box in each dimension Value The valu
20. Scale with Max 0 Scale with Value Image File The image to be scaled Output File The path to the file where the scaled image will be stored Scale with Sum Scale the image by the sum of all the values normalization Scale with Max Scale the image by the maximum value Scale with Value A value by which to scale the image Output When executed the image is scaled and then stored with the name specified in Output File Convert This tab converts an image to a desired data type 13 COSMOS MANUAL 0 9 16 File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File 1428x128x1281Gibson and Lani GL_PSF4 wu Browse Output File 1128x128x1281Gibson and Lani GL_PSF4 wu Browse Execute Output Data Type INT Number of File 5 Inputs Image File The image to be scaled For multiple images all of the images must have the same name with a number starting from 0 before the extension and the base name must be given as the input file For example for the two images Image0 wu and Image1 wu the filename Image wu must be given Output File The path to the file where the scaled image will be stored For multiple files give a base filename and a corresponding number will automatically be appended to the filename Output Data Type The desired data type for the image Number of File Number of images to convert Output When executed the
21. a Input File The path to the forward image e PSF Input File The path to the LSF e Output File The path where the restored image will be stored e Strata 8 e Start 108 e Size 5 pixels 6 Click Run The image improves with increased number of iterations The estimated output after 1000 iterations File Help Intensity ec Tapia ed pida Ladaga do 40 eo 1 1 Intersection XY and XZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane LevaWindow EB The estimated output after 50 000 iterations 49 COSMOS MANUAL 0 9 16 File Help Intensity See ey ae pepe Y FOO p T n gu 10 co valo ee PTS all T AAA A o 20 40 eo 80 100 120 sample Intersection XY and XZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane Levalvindow 0 500 1 000 Z 128 Y 0 a Xx e 50 COSMOS MANUAL 0 9 16 Chapter 9 Frequently Asked Questions Can modify or redistribute COSMOS The COSMOS package is free software you can redistribute it and or modify it under the terms of the GNU General Public License as published by the Free Software Foundation either version 2 of the License or at your option any later version COSMOS is distributed in hopes that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS F
22. and path name gt cpm 36 COSMOS MANUAL 0 9 16 Chapter 4 How to generate a microscope line spread function LSF using the COSMOS PSF module The PSFs can be generated using the COSM PSF steps in this chapter There are default values present in the window that will be used for the simulations File Help PSF XML Import File Browse Open Radial Interpolation Refractive Index Thickness mm Coverslip OS Design 1 522 0 17 Actual 1 522 0 17 Model Gibson Lanni1992 Type Non Confocal Immersion Medium oo 94 Precision Single 32 bit Design 1 515 0 16 adi Sl Actual 1 515 0 16 Dimension x y z 64 64 64 Specimen Spacing um x y z 0 06 0 06 0 06 133 0 Absolute Error 1e 006 Lateral Magnification 40 OTL A Angle Interval 0 Numerical Aperture 1 Resign mm mm Thickness Interval mm 0 0003 Wavelength nm 580 Total Number 9 Sum over Y v Uncentered PSF Filename DATA psf wu Browse Generate Real and Imaginary Generate Progress Open CosmPsf Select Exact Evaluation for better results though only marginally more time consuming 3 The image will be devided into 10 parts The ring is divided into 8 parts with a part above and a part below the ring Therefore 9 PSFs will be generated So 9 is entered in Total Number 4 The size between pixels Spacing is specified as 0 06 um and the number of pixels in each stratum is chosen as 5 5 The depth interval Thickness Interval is cho
23. art the ComsTools application and select the Variant tab We specify the object file name and specify the PSF name without the last number next to the extension The output filename needs to be specified The user needs to specify 3 parameters the number of strata the start of the first strata which is the beginning of the object in z pixels Press Execute and the image will be generated Image Estimation DV Depth Variant Expectation Maximization DV EM algorithm This image and a single LSF can be used to estimate the object using the LLS MAP and EM algorithms but the resulting estimation will not be quite like our original object The 32 COSMOS MANUAL 0 9 16 image can be estimated by using the EM Space Invariant Algorithm The COSM Estimation needs to be started and the Iterative tab and the Variant sub tab need to be selected Enable the EMSV algorithm Specify the image file name the LSF and the output filename Then specify number of iterations and how often to update and write The algorithm will process the image upon pressing run 3 4 COSM Viewer COSM viewer is used to display images in 3D cut view File Help Intensity at 4 1 JIN J a 4 O A o a e C o 20 40 eo 80 100 120 sample Intersection XZ and YZ Plane Graph Scale Linear Clear Reset Size XY Plane ZX Plane YZ Plane Lev Nindow J 5 04e 005 2 54e 005 4736 007 Ze Mj es X 04 3D view rotation
24. e file s name to lastest xml To undo this function simply delete or rename lastest xml 24 COSMOS MANUAL 0 9 16 20 COSMOS MANUAL 0 9 16 3 3 COSM Estimation Using the COSMOS to obtain images using the above algorithms e The image from the microscope is given in Data Input File e The corresponding PSF is given in PSF Input File e Parameter The parameter is a value from 0 to 1 When given the correct parameter we get the output close to the original object This is mainly a trial and error method This helps to remove artifacts e The output gets stored by the name given in Estimated Output File Parameters XML File XML file that contains stored parameters from a previous estimation This file can be used to perform the same or similar estimation multiple times Once the XML file has been selected click Open to import the settings Data Input File The image from the microscope PSF Input File The PSF corresponding to the Data Input File Centered PSF Perform the estimation using a centered PSF Estimation Output File The path where the result of the estimation will be stored Linear Method Parameters 26 COSMOS MANUAL 0 9 16 File Help XML File Data Input File PSF Input File Centered PSF v Estimation Output File Linear Iterative Algorithm Linear Least Squares LLS Linear Maximum a Posteriori MAP Parameter 0 Run Progress Linear Least Square
25. e image without the header will be stored Dimension The image s dimensions Output Data Type The output image s datatype Big Endian The input image s byte order Little Endian if left unchecked Output When executed the header is added to the raw image and is stored with the name specified in Output File Resample This tab is used to down sample or up sample an image File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File 428x128x128 Haeberle HB_PSF6 wu Browse Output File 128x128x128 Haeberle HB_PSF6 1 wu Browse Execute Factor x y z 2 2 2 Downsample Upsample Inputs Image File The image to be resampled Output File The path to the file where the resampled image will be stored Factor The factor by which the image will be resampled Downsample Upsample The desired sampling down or up 11 COSMOS MANUAL 0 9 16 Output When executed the image is resampled and stored with the name specified in Output File Transform This tab is used to shift or change the dimensions of an image File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File 1428x128x128 HaeberlelHB_PSF6 wu Browse Output File 128x128x128 Haeberle HB_PSF6 1 wu Browse Execute Output Dimension x y z 64 64 64 Shift xyz 3 0 0 Value Fill 0 Inputs Image File The imag
26. e to be resized Output File The path to the file where the resized image will be stored Output Dimension The desired new dimensions Shift The number of pixels to shift the image Value Fill The value of the new pixels generated from shifting the image Output When executed the image is resized and shifted and then stored with the name specified in Output File Shift This tab shifts an image File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File 428x128x128 Gibson and Lani GL_PSF8 wu Browse Output File 1428x128x1281Gibson and Lani GL_PSF8 1 wu Browse Execute Shift x y z 0 0 15 Circular Inputs mage File The image to be shifted 12 Output Scale COSMOS MANUAL 0 9 16 Output File The path to the file where the shifted image will be stored Shift The number of pixels to shift the image Circular Perform a circular shift meaning that the pixels that are shifted off of the image are shifted back around to the other side When executed the image is shifted and then stored with the name specified in Output File This tab scales an image File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Inputs Image File 1428x128x1281Gibson and Lani GL_PSF4 wu Browse Output File 1428x128x1281Gibson and Lani GL_PSF4 wu Browse Execute v Scale with Sum
27. e with which to fill the box Position The coordinates where the point will be located Value The value with which to fill the point Output When executed the object will be created and stored with the name specified in Output File Variant This tab forms an image by either a sum of convolutions of each of the strata with an interpolation of the LSFs that are on each side of the strata or a sum of convolution of each plane along Z dimension of object with components of PCA Based PSF The settings will be saved into XML file that has same name as the input file with file extension xml 16 COSMOS MANUAL 0 9 16 File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant PCA Object File Browse PSF File Browse Centered PSF v Browse Output File Strata Execute Strata mode PCA mode Number of Strata 0 Start of Strata 0 Inputs Output Size of Strata 0 Object File The first of the two images being convolved PSF File The second of the two images being convolved PSF number should be deleted i e if there are 50 PSFs psfO wu psf1 wu psf49 wu only first psf should be given as input and O zero should be removed Input in this case will be psf wu For PCA mode PSF file will be number removed first base PSF same as strata mode Other necessary files must be in the same folder and COSMOS will aut
28. enu mean Is it the size of a single element of the CCD camera or the size of a single pixel of the image acquired by the CCD camera Pixel size in object space is equal to the physical CCD pixel size divided by the magnification of all the lenses between the specimen and the camera For most microscopes the only lens between the specimen and the camera is the objective lens For other microscopes there may be an additional relay lens that further magnifies the image to the camera When publish results generated with COSMOS which of your papers should I reference and how do cite the COSMOS package COSMOS citations should be made by referring to the COSMOS web page http cirl memphis edu cosmos In addition depending on which algorithm you used to process your images you should cite the publication describing the algorithm A list of all the algorithms implemented in COSMOS and the references for each algorithm are provided on the COSMOS web page 52 COSMOS MANUAL 0 9 16 Chapter 10 References 10 11 12 13 Preza C and Conchello J A Depth variant maximum likelihood restoration for three dimensional fluorescence microscopy J Opt Soc Am A 21 1593 1601 2004 Preza C Miller M I Thomas Jr L J and McNally J G Regularized linear method for reconstruction of three dimensional microscopic objects from optical sections J Opt Soc Am A 9 2 219 228 1992 Preza C M
29. er 1 tse Rte te stes d edendo O 5 Installation Guide ccoo 5 Downloading COSMOS cccssssssscececeseesenneseeececesseseaaeseeeeecesseseaaeeeeeceseeseaeaeeessesseeseaaeaeeeeseesees 5 Windows 7 installation ota tri 5 Chapter 2 etre Sees ee ni 8 Building COSMOS from source code esses nennen enne nnns sns en teras a nass sse n entia nnns 8 Chapter A teda tee eet uter ese eut leti tee teh des esee metuo T 9 Description of the applications utilities available in COSMOS cccccssccecssecesseecsseeeeseeesseeeeeeees 9 Introducir P RE t ait OR ai ease eee eene 9 3 L COSM rq EE idos 9 3 2 COSM ER 20 3 3 COSM EStirmiath ony i iei e ee tcs 265 IA COSM Vi Wet A E PRAE 32 3 5 Command line interface oooooncccnnccccncccconnnononnnnnrcncnnnnononannnno conan cn cnn neni tnen enne nnn nnne nnne 343 Chapter o eet e aet AUS Sonic me A AT e pA Asa 366 How to generate a microscope line spread function LSF using the COSMOS PSF module 376 Cha Pte tide e M 398 How to generate a simulated microscope image using the COSMOS Tools module 398 INTRODUCTION e 398 Image formations n bee perenniter eui EO eie nns 398 Chapter G ot a ette ln a NO LE ds dh ts tad AT RR e e es LEE 41 How to process an image measured or simulated using the COSMOS Estimation module 41 Chapter go eic MAE b D E T A T PboNi O 40 An Implemented Example Demonstrating the Various
30. f 2nd Image 4 The Estimated microscope image File Help intensity E oe EI _ PS AN IO A IP IO PO IO PA IO toh sample intersection XY and XZ Plane Graph Scale Linear Clear Reset Size 5 The image is estimated using COSM Estimation For example using the EM Algorithm for 1000 Iterations or more 43 COSMOS MANUAL 0 9 16 File Help XML File Data Input File paTa forward wu PSF Input File DATA psf_128 wu Centered PSF v Estimation Output File paTA estimation wu Linear Iterative Invariant Variant Convergence Algorithm Expectation Maximization EM Jansson van Cittert JVC Penalty 0 None v Double Z Iterations 1000 Update 10 Run Stop Write 100 Progress 6 The Image after 1000 iterations File Help Browse Open Browse Browse Browse 7 The image viewed in COSM Viewer after 10000 iterations COSMOS MANUAL 0 9 16 45 COSMOS MANUAL 0 9 16 Chapter 8 An implemented example demonstrating a simulation of the forward and inverse ddepth vvariant imaging COSM problem using COSMOS functionalities Generate a test image Note the following example uses 2D XZ images and LSFs to reduce the dimensionality of the imaging problem to allow quick processing of the image Create a ring object using the object
31. g of living tissue up to a depth of one millimeter The two photon excitation microscope is a special variant of the multiphoton fluorescence microscope Two photon excitation can be superior due to its deeper tissue penetration efficient light detection and reduced phototoxicity Confocal circular By having a confocal pinhole the microscope is very efficient at rejecting out of focus fluorescent light The practical effect of this is that the image comes from a thin section of the sample small depth of field By scanning many thin sections through the sample a very clean three dimensional image of the sample can be built Confocal Line This has the same specifications of a confocal microscope apart from the fact that it has confocal slit rather than a confocal pinhole Non Confocal This is conventional wide field fluorescence microscopy DIC Differential interference contrast microscopy is a phase imaging technique that allows the visualization of transparent unstained objects Precision The data precision of the image can be selected as Single The PSF generated has 32 bit precision This is the lowest precision available 21 COSMOS MANUAL 0 9 16 Double The PSF generated has 64 bit precision Long Double The PSF generated has 80 bit precision This is the highest precision available Dimension The dimensions of the 3D PSF image in pixels Note The image space consists of the object space the pixels occu
32. iller M I and Conchello J A Image reconstruction for 3 D light microscopy with a regularized linear method incorporating a smoothness prior in Biomedical Image Processing and Biomedical Visualization R S Acharya and D B Goldgof Eds Proc SPIE 1905 129 139 1993 Agard D A Optical sectioning microscopy Annu Rev Biophys Bioeng 13 191 219 1984 Conchello J A Super resolution and convergence properties of the expectation maximization algorithm for maximum likelihood deconvolution of incoherent images J Opt Soc Am A 15 2609 2620 1998 Murphy D B Fundamentals of Light Microscopy and Electronic Imaging Wiley LISS 2001 Gibson S F and Lanni F Experimental test of an analytical model of aberration in an oil immersion objective lens used in three dimensional light microscopy J Opt Soc Am A 9 54 66 1992 O Haeberl Focusing of light through a stratified medium a practical approach for computing microscope point spread functions Part Conventional microscopy Opt Commun 216 55 63 2003 Myneni V and Preza C Computational depth variant imaging for quantitative fluorescence microscopy in Computational Optical Sensing and Imaging COSI OSA Technical Digest CD Optical Society of America paper CThC4 2009 Myneni V Performance Analysis of a Depth Variant Expectation Maximization Algorithm for 3D Fluorescence Microscopy MS Thesis in Electrical and
33. iori MAP Parameter 0 Progress Open COSM Estimation Select the Data Input File as the forward image created in the previous chapter Select the PSF Input File as the PSF convolved with the object in the previous chapter Specify where the Estimation Output File will be stored Select an algorithm See 3 3 COSM Estimation for information about the algorithms used in COSMOS e Ifa Linear algorithm is selected the image can be processed immediately by clicking Run e Ifan terative algorithm is selected there are a number of parameters that need to be entered depending on the options that the chosen algorithm requires Click Run 41 COSMOS MANUAL 0 9 16 Chapter 7 An implemented example demonstrating a simulation of the forward and inverse space invariant imaging COSM problem using the COSMOS functionalities 1 The image is chosen as a hollow ring with two intersecting axes 128 128 128 Flle Help 3 The simulated microscope using The COSM Tool convolves 42 COSMOS MANUAL 0 9 16 File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant 1st Image File paTa ring_128 wu Browse 2nd Image File paTa psf_128 wu Browse Centered PSF v S Browse Output File paTA output wu Output Image Size Execute Normal Size 1st Image Size o
34. le or a Setvalue of error information s filename e pca or P Switch to use PCA mode Default is strata mode 35 COSMOS MANUAL 0 9 16 COSM PSF CLI Usage CLI is enabled using the c1i or c option as shown below CosmPsf cli other options The following are typical ways of using COSM PSF CLI CosmPsf cla file lt PATH AND FILENAME OF PSF INPUT XML FILE gt or CosmPsf La file lt PATH AND FILENAME OF PSF INPUT XML FILE gt interval 0 0001 total 5 Options The description of all options e cli or c Switch to use CLI function e file or f Set value of input PSF XML file path Default value is psf xml e double or d Switch to use double precision Not support yet Only supports to using float precision now e long or 1 Switch to use long double precision Not support yet Only supports to using float precision now e interval or i Set value of PSF Thickness Interval Default value is O e total or t Set value of PSF total number Default value is 1 e sum or s Switch to do sum over Y uncentered u Switch to not use centered PSF file e gri g Switch to do generate real and imaginary COSM Tools CLI WFE In current version CLI is activated only for WFE tab CosmTools E input lt conventional PSF file name and path name gt reference lt mask file name and path name gt output lt output WFE PSF file name
35. omatically read all necessary files Centered PSF Convolve two centered PSFs Output File The path to the file where the result of the convolution will be stored Strata mode PCA mode Switch between strata mode and PCA mode Number of Strata The number of strata the object will be divided into Start of Strata The start of the first strata which is the beginning of the object in terms of z pixels Size of Strata The size of each stratum in pixels Number of Components The number of components of PCA Based PSF files Start of PSF File The PSF file for the first PSF defined at the starting depth which is the top of the object in terms of z pixels Number of PSF File No of z planes in region of interest ROI i e if object has dimension 128x128x256 and ROI starts from 100 and ends at 150 then start of PSF file should be 100 and Number of PSF file should be 50 When executed the two images will be convolved and the resulting image will be stored with the name specified in Output File The settings will also be stored in an XML file with the same name as the image 17 COSMOS MANUAL 0 9 16 13 PCA This tab transforms original PSF files to PCA Based PSF files File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant PCA Original PSF File Browse PCA Based PSF File Browse Execute Number of PSF Files 0 Depth interval between PSFs pixels 1
36. pied by the object and the background space Spacing The spacing in um between the pixels of the 3D PSF image Note If the PSF is generated to process an image using a COSMOS algorithm then these pixel sizes should match those of the image For experimental data the XY pixel size is dictated by the camera pixel size and the magnification of the lens The pixel size along Z is the distance between PSF planes along Z and it corresponds to the depth at which the lens is focused into the sample It is the Z step size used when the sequence of 2D optical sections is captured For simulations these pixel sizes should be taken based on the Nyquist sampling rate in order to avoid aliasing Absolute Error The maximum error allowable during calculations Lateral Magnification The objective lens magnification This value is obtained from the lens used Numerical Aperture This is also a direct property of the lens and available on the objective lens Wavelength The wavelength in nm of the emission filter used to detect the emitted fluorescence light from the sample Angle Interval This option is for DIC PSFs only and corresponds to the interval between rotation angles Thickness Interval For the Depth Variant Expectation Maximization Algorithm the Thickness Interval indicates the thickness in mm of each slice of the specimen The interval number is the depth at which the specimen is imaged It should be a whole multiple of the pi
37. r is 1024 bytes long The Add Header Tab available in the COSMOS Tools allows the user to add the WASHU header to a file in the raw format raw and make it a wu file Only raw files can be processed by the Add Header If the image is not a raw image you can convert it to one using the open source software ImageJ http rsbweb nih gov ij download html The 4 modules in COSMOS are e 3 1 COSM Tools e 3 2 COSM Psf e 3 3 COSM Estimation e 3 4 COSM Viewer Also COSMOS now has a command line option e 3 5 Command Line Interface 3 1 COSM Tools The tabs available are 1 Info This tab shows the header information of the inputted file File Help Info Remove Header Add Header Resample Transform Shift Scale Convert Compare Convolve Object Variant Image File ipSFsiHaeberle_PSF7 wu Browse Open Image Information Dimension x y z 256 256 256 Data Type FLOAT Size in Bytes 6 71089e 007 Maximum 4 42027e 005 Minimum 1 20513e 011 Average 5 45554e 008 COSMOS MANUAL 0 9 16 Inputs Image file The desired file Outputs Dimension The dimensions of the image Data Type The image s data type Size in Bytes The image size Maximum The maximum value of the image Minimum The minimum value of the image Average The image s average value Remove header This tab removes the WASHU header from the image s File Help Info Remove Header Add Header Resample Transform Shift Scale Conver
38. s LLS This is a one step algorithm that yields solutions in a few seconds that are optimal in the least squares sense Results from sensitivity studies show that the proposed method is robust to noise and to underestimation of the width of the point spread function The method is particularly useful for applications in which processing speed is critical such as studies of living specimens and time lapse analyses 2 Linear Maximum a Posteriori MAP This approach also yields a regularized linear estimator which reduces noise as well as edge artifacts in the reconstruction The advantage of the linear MAP estimate over the LLS is its ability to regularize the inverse problem by smoothly penalizing components in the image associated with small Eigen values 3 Parameter A value from 0 to 1 When given the correct parameter an output close to the original object can be obtained This is a trial and error method which helps to remove artifacts Iterative Method Parameters Iterations The number of iterations for the algorithm to be executed Update 27 Browse Open Browse Browse Browse COSMOS MANUAL 0 9 16 The number of iterations before the terminal window is updated Write The number of iterations before the maximum minimum and mean values is printed to the terminal Invariant File Help XML File Browse Open Data Input File Browse PSF Input File Browse Centered PSF v Estimation Ou
39. sen as 0 0003 mm This depends on the spacing in z and number of pixels in each stratum 6 Normally the CosmPsf application would generate a 3D PSF but it can be forced to collapse the Y dimension sum over it into a single plane by enabling Sum over Y This 2D PSF is the LSF 7 The rest of it is left as is 37 COSMOS MANUAL 0 9 16 8 The output file PSF Filename is entered The generation of the LSF produces the following XML file lt xml version 1 0 gt lt PSF specification file gt lt model lt Gibson_Lanni1992 Haeberle gt gt lt eval Interpolation Exact gt gt lt type lt Non confocal Circular confocal Line confocal gt gt lt PSF version unit mm spacing 6e 005 6e 005 6e 005 dimension 64 64 64 file C Users timathis Desktop psf0 wu precision Single type Non Confocal eval Exact model Gibson_Lanni1992 gt lt AbsoluteError value 0 000001 gt lt LateralMagnification value 40 000000 gt lt NumericalAperture value 1 000000 gt lt Wavelength unit mm value 0 000580 gt CoverSlip lt Refractivelndex Actualz 1 522000 Design 1 522000 gt Thickness unit mm Actual 0 170000 Design 0 170000 gt lt CoverSlip gt lt ImmersionMedium gt lt Refractivelndex Actual 1 515000 Design 1 515000 gt lt Thickness unit mm Actual 0 160000 Design 0 160000 gt lt ImmersionMedium gt lt Specimen gt lt
40. t Compare Convolve Object Variant Image File 1428x128x1281Gibson and Lani GL_PSF wu Browse Output File 1428x128x1281Gibson and Lani GL_PSF raw Browse Execute Number of File 5 Inputs Image File The image from which the WASHU header is to be removed For multiple images all of the images must have the same name with a number starting from 0 before the extension and the base name must be given as the input file For example for the two images Image0 wu and Image1 wu the filename Image wu must be given Output Fil