CT-FIRE V1.3 Beta2 User`s Manual (November 6 2014)
Contents
1. E Create Overlaid Image i ea Fiber line width 0 2 Max fiber width pixels More 15 sure 10 Histogram bins number 5elect Output OQverlaid fibers Non overlaid fibers E Angle histogram amp values Length histogram amp values Straightness histogram amp values E Width histogram amp values 24 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison If the overlaid image has already generated by the advanced output module then click on Yes to display to show this image If the overlaid image doesn t exist clicking on No to create will generate this image based on the output data of advanced output module 3 9 Tutorial 9 post processing for visualizing the advanced output results of multiple images This operation should run after advanced output is applied to the images or stack and both the statistics and raw data for all the fiber properties including width length angle and straightness are generated User can choose to view any of the property histograms of the total selected fibers in all of the images or in the stack The number mean and standard deviation of the extracted fibers in each image can be displayed as well as the image showing the selected fibers overlaid on the original image for each of images or on each slice of the stack In this tutorial check the Batch mat and OUT adv boxes choose all the available output options and then click the Post p2. 3 8 Tutorial 8 post processing for visualizing the advanced output results of a single image This operation should run after advanced output is applied to the image and the statistics for all the fiber properties including width length angle and straightness are generated User can choose to view any of the property histograms of the selected fibers in the image The number mean and standard deviation of the extracted fibers in this image can be displayed as well as the image showing the selected fibers overlaid on the original image In this tutorial check the mat and OUT adv boxes choose all the available output options and then click the Post processing button to select the advanced output results of testimagel tif is that is an excel file in the folder of selectout as shown below mw vr UF By care V1 3 Beta2 e xs A Choose a processed data file Batch v mat v OUT adv Reset ie J seleto O a c Fae AIRE Post processing E L Name Date modified Type Size s testimagel_statistics xlsx 10 4 2014 6 47PM Microso ft Excel W 16 KB Recent Places l Parameters Desktop Libraries A Computer Network rane Run ctFIRE Stack image preview slice a Slices range Output Figure Control __4 Select Output Overlaid fibers V Angle histogram amp values V Length histogram amp values VJ Straightness his
3. 1 Straight statistics Contains the statistics on the straightness data 2 Overlaid images named filename _overlaid_selected_fibers tif showing the overlaid selected fibers on the original image If multiple batch mode analysis are performed the Excel file with the highest suffix in the filename contains the result of the last analysis 3 7 Tutorial 7 advanced output for stack mode analysis The stack mode analysis allows the user to analyze all the slices of a stack for fiber analysis The option of visualizing and removing fibers is not available for stack mode However after a stack mode operation is done on the image stack different slices of the image are saved as filename_s1 filename_s2 etc If needed these files can then be analyzed separately using single image analysis mode so as to use the visualize and remove fiber options Step I Check the Stack Mode checkbox and leave the checkbox of Batch Mode unchecked Click on the Select File button to select one stack image gaa L n e J Stack Mode la Reset fea Stack Mode l Roset _ Select File Filename ab Pla NN Select File Filename d Y ia R e Fibe Save Fibe lesktop Ni al stimagel testimage2 testst sualise Fibers braies ack ctFIREout te Step 2 Check the checkbox if thresholding of fibers is required Use the drop down menu to select the threshold type The thresholds will be applied to
4. More eS ee as 140 ae Histogram bins number F width 4 85 1 52 n 375 aa angle 90 56 39 36 n 375 i i 2 8 120 o 1 Select Output 2s 100 o a D 80 ei F Overlaid fibers 2 60 g Non overlaid fibers lt 40 V Angle histogram amp values 2 1 2 3 1 2 3 V Length histogram amp values Image number Image number Image number Image number 4 Ti 1 For the images in batch mode processing or slices in a stack the images or slices can be normalized to have the similar dynamic range 2 Use Fiji or ImageJ to convert the image type to 8 bit image which is well supported for this version Use Fiji script to pre processing multiple images such as to adjust the image contrast segment the fibers in color images or to locate the region of interests 25 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison 3 Setting fiber extraction parameters should consider image size image contrast fiber features such as width curvature density Image size of 512 pixels by 512 pixels is suggested so as to use most of the default values All the parameters are adjustable via csv file but only some of the key parameters can be adjust via GUI The thresh_im2 to remove the background is the major parameter to be updated 4 Post processing mat fiber extraction results to generate different output files But be careful not to move the original image to other place since its original path will be used duri
5. Unit is in pixels Default value is 30 Image Res resolution of the output overlaid image unit is in dpi Default value is 300 Fiber line width The line width of the extracted fibers in the overlaid image Default value is 0 5 Max fiber width Used to get rid of the outliers on the fiber when calculating the average fiber width Default value is 15 MORE button at the end of Max fiber width provides the option to use all the defined fiber central points to calculate average width or select certain fiber points based on the mean and standard deviation of the width of all of the fiber central points that satisfy the maximum fiber width condition Histogram bins number the number of bins used in the angle and or length histogram Default value is 10 Auto button near the histogram bins number provides the option to estimate the optimal bins number of the histogram based on the fiber number Select Output Overlaid fibers checked to show and save the overlaid image of the extracted fibers on the original image as well as show the CT Curvelet Transform reconstruction image Non overlaid fibers checked to show and save the image of the extracted fibers Angle histogram amp values checked to show the figure of fiber angle histogram save the corresponding values into an Excel file Length histogram amp values checked to show the figure of fiber length histogram and save the corresponding values into an Excel file Straight
6. the statistics in an excel file To be noted generating raw data is optional If only to show the statistics of the fiber properties raw data will not be saved in the excel file which will save time It is recommended that this raw data box is checked for a single image process For a batch mode analysis demonstrated in Tutorial 6 raw data box can be left unchecked to save the processing time However 1f need to save the raw data of the select fibers or to use the post processing features in Tutorials 8 and 9 to visualize the data then the box for Generate sheet for raw data must be checked 18 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Reset Fiename testrmage 1 tif Thresholds in percentages FP Select Stats Select Deselect desired output stats Output Stats Generate stats for Median Mode Mean Length Width Straights Angle i Wariance EJ Std dev E Min Generate stats Max number of Status TIS a Alignment fibers Generate sheet Deselect Seiect Statistics Output of Post Processing GUI A new folder is created named selectout in the same directory as the selected image All the outputs of the advanced output control GUI are saved in this folder The following are the outputs An excel file named filename statistics xlsx Windows or statistics xls MAC containing two sheets 1 Selected Fibers which contains the raw data of the fibers used for a
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8. Import image data Post processing v NI Parameters d Load Update n cCtFIRE ts Stack image preview slice a Slices range whole stack Slices Fyi Output Figure Control Minimum fiber length pixels 30 Image Res dpi 300 Fiber line width 0 2 0 5 Max fiber width pixels more 15 Histogram bins number auto 10 Select Output Overlaid fibers Non overlaid fibers E Angle histogram amp values E Length histogram amp values El Straightness histogram amp values E Width histogram amp values Load and or update parameters Step 2 select run mode and set output parameters Use the default Run mode ctFIRE The default output control parameters are used as shown in the above figure only choosing the overlaid fibers to be displayed Step 3 set fiber extraction parameters Click the Update button on the panel of parameters the following window is displayed to update or confirm eight default FIRE parameters CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison The description of the parameters is listed below Name Value Description thresh_im2 5 thresh_im2 is for a hard value main adjustable parameters s_xlinkbox 8 radius of box in which to check to make sure xlink is a local max of the distance function thresh_ext 70 angle similarity required for a fiber to extend to the next p
9. Step 5 check the box for thresholding Check the box if thresholding is required The threshold type can be selected from the drop down menu among percentage absolute values Top N and Bottom N According to the option chosen the default values are shown in the corresponding text boxes beside the fiber property names Select the properties to which 17 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison thresholds are applied and enter the thresholds in the text box To be noted the boxes in front of each fiber property should contain a value to avoid errors File Edit View Insert Tools Desktop Window Help Reset 420984 8 08 Filename testimage1 tif g Visualise Fibers Remove Fibers Save Fibers Thresholds in percentages Generate stats for Length Width Straight Angle Status Select from Length Width Straight and or Angle Click threshold Now button to check the thresholded fibers This process can be done iteratively with change of thresholds Click Save Fibers to finalize the fiber selection Step 6 check the fiber properties for analysis Check Uncheck to select deselect the properties for which statistical analysis needs to be done Step 7 Generate stats After clicking Generate Stats select deselect the statistics that are desired and then click Ok to generate
10. about the features is demonstrated in the tutorials in section 4 Version 1 2 1 beta 1 2 3 Add batch mode fiber extraction for both multiple image files and mat files image stack fiber extraction post processing for both single image output single fiber extraction results mat file and multiple fiber extraction results mat files Add the features of outputting non overlaid extracted fiber image fiber straightness and fiber width besides fiber length and fiber angle In addition add the button to load parameters in csv files as well as to update and save the updated parameters for ctFIRE in csv format Version 1 0 fiber extraction for a single image CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison 2 GUI control panels 2 1 GUI for the Main Program The GUI in ctFIRE is modular so that the main user interface is in a separate window from the outputs The main user interface window is shown below ctFIRE V1 3 Beta o7 Batch mat OUT adv Reset Import image data Post processing Parameters Load Update Run CtFIRE v Stack image preview slice Slices range whole stack Slices Output Figure Control Minimum fiber length pixels Image Res dpi Fiber line width 0 2 Max fiber width pixels More Histogram bins number AYT9 Select Output J Overlaid fibers Non overlaid fibers Length histogram amp values St
11. age2 teststack sal E PA wy teststack_sl teststack_s2 teststack_s3 After selecting the file two images will be opened One is the original image and the other is the original overlaid 1 aa with labeled fibers as shown below Step 2 visualise fibers Click on Visualise Fibers to open a window to enter the fiber numbers After clicking Ok an image with the mentioned fiber numbers overlaid on the original image opens up Another window opens up with tabulated value of the length width straightness and angle of the selected fibers The two windows are displayed as below 16 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison too m r W Step 3 remove fibers Click on the Remove Button to open a window to enter the annotations of the fibers to be removed In this tutorial enter 215 296 232 247 223 252 203 146 for the over segmented fibers After Ok is clicked a new image with the fibers removed will be displayed as below The Status shows the fibers which are removed from analysis This step is iterative and can be done for any number of times Step 4 click Save Fibers After all the perceived false fibers and or undesired fibers are removed click Save Fibers to save the fibers for analysis The annotation of the fibers to be used for further analysis will not displayed on the overlaid image
12. al teststack tif is selected After clicking open the first slice of the stack is displayed and new controls are enabled on the control panel as shown below f arre V1 3 Beta2 o B 2 E teststack tif stack 3 slices 512 x 512 pixels 8 bit Lo ps Batch mat File Edit View Inset Tools Desktop Window Help x OGMs F ARCV90CL A2 0H ao Parameters Run CctFIRE cS Load Update Stack image preview slice 1 Slices range whole stack _ Slices Output Figure Control inimum fiber length pixels 30 Image Res dpi 300 Fiber line width 0 2 0 5 Max fiber width pixels more 15 Histogram bins number amon 10 Select Output V Overlaid fibers P Non overlaid fibers F Angle histogram amp values E Length histogram amp values Straightness histogram amp values Width histogram amp values Load and or update parameters Step 2 select run mode and set output parameters Leave the run mode on the right of Run button as default value ctFIRE output parameters are default values as shown in the follows Compared to processing one single image the controls for stack analysis are enabled and allowing user to preview the slices and select the slice range Drag the slider or click the slider arrow to preview the slices in the stack Select the 2nd slice to preview select the slice
13. all the slices in the stack Step 3 select the fiber properties to which the thresholds are applied and enter the values in the text boxes To be noted the boxes in front of each fiber property should contain a value to avoid errors as described in Tutorial 5 and 6 Step 4 select fiber properties for which statistics are desired Step 5 click the Generate Stats button A pop up window is displayed for selection deselection of statistics as described in Tutorial 5 and 6 Raw data 22 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Output All of the output files are saved in the selectout folder mentioned in Tutorial 5 1 Individual slices of the image Individual slices of the image stack are saved in the same folder as the image stack These images can then be used for individual analysis if some fibers need to be removed from each slice using single file analysis mode i e with Batch Mode and Stack Mode checkboxes unchecked These files are named as filename_s1 for Slice 1 filename_s2 for slice 2 etc 2 Image with selected overlaid fibers for each slice For each slice the image of slice with the selected overlaid fibers is saved in the selectout folder These files are named as filename_sl_ overlaid _selected_fibers for slice 1 filename _s2 overlaid _selected_fibers for slice 2 etc 3 An Excel file the naming and contents are similar to those in the bacth mode analysis described in Tutorial 6
14. and upper limit in the second box 2 Width Select to enter the width threshold Specify low limit in first box and upper limit in the second box 3 Straight Select to enter the straightness threshold Specify low limit in first box and upper limit in the second box 4 Angle Select to enter the angle threshold Specify low limit in first box and upper limit in the second box Threshold Now use this button to see the fibers satisfying the chosen threshold criteria The process of changing thresholds can be done iteratively Save Fibers use this button to finalise the criterion for thresholding Generate stats for select the fiber properties to be analyzed and saved Generate Stats pops up a window to allow user to choose the statistics as well as the raw data of the selected fibers Status shows the current status and prompts while executing CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison 3 Tutorials 3 1 Tutorial 1 fiber extraction and post processing for a single image Step 1 open an image Leave the Batch mat and OUT adv checkboxes unchecked Click the Import image data button a file selection window opens allowing the user to choose the image In this tutorial testimage1 tif is selected and new controls are enabled on the control panel as shown below he V1 3 Beta co B xs B testimagel tif 600 x 600 pixels 8 bit ESRA mesm Batch mat OUT adv Reset File A OSES ARE 902 08
15. e and Statistics of them as well The fiber extraction parameters and output of ctFIRE are automatically saved in mat binary format for the later post processing Major features of the versions Version 1 3 Beta2 newest The primary change in CT FIRE V1 3 Beta2 is the advanced output control This version provides the following major features and capabilities 2 3 4 5 6 7 8 9 Manually look up the fiber properties and remove the over segmented fibers Automatically select fibers by use of one threshold condition or combined conditions including the absolute or relative limits of fiber width length angle and straightness Output the a variety of statistic measures of the selected fibers obtained from 2 as well as the option to output raw data and overlaid images of them Analyze the selected fibers in a single image stack or in multiple images and visualize the associated statistics Batch mode fiber extraction for stacks Improved width calculation and the option to output the maximum width of each fiber Option to estimate the number of bins for the histogram based on the number of fibers Control the output image resolution and remove the absolute directory requirement when loading the mat file Fix a bug to load and update the existing fiber extraction parameters and 10 Save the fiber properties of selected fibers into different sheets of one excel file Detailed description
16. es is to set the scales to reconstruct the image Specifically if the total number of scales used by the curvelet 8 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison transform is N and 3 is the number of selected scales in this edit box then the actual scales used for the reconstruction is scale N 1 N 2 and N 3 In this tutorial use the default values and click OK to continue Then the button Run is enabled Step 4 run fiber extraction Click on Run button Then the progress of fiber extraction will be listed in the Command Window The overlaid and reconstructed images will also be displayed Step 5 Check the results A mat file containing the fiber extraction results and corresponding parameters is automatically saved to a new subfolder called ctFIREout if it does not exists the folder will be created by ctFIRE automatically of the folder where the image exists Four output files are shown m t CAA testdatasets CTFIRE_1 3a0 ctFIREout r X Open Share with E mail New folder Sen Name C sktop EL ctfParam_testimagel tif csv 1 ywnloads Z OL_ctFIRE_testimagel tif 1 cent Places i ctFIREout_testimagel 1 rogle Drive el CTRimg_testimage tif 1 CT FIRE output 1 ctFIREout_ testimagel mat the mat file for the fiber extraction results by ctFIRE 2 OL_ctFIRE_ testimagel tif the tiff overlaid image of ctFIRE 3 CTRimg_ testimagel tif the tiff CT reconstruction image 4 ctfParam_testimagel
17. hresholds can be selected from the drop down menu as shown in the accompanying figure e Choose the fiber properties for which the threshold values are to be defined by clicking the appropriate radio buttons Type in the thresholds in appropriate text boxes To be noted the boxes in front of each fiber property should contain a value to avoid errors The same thresholds are used for all the images in batch mode processing 20 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison check the box if thresholding is desired percentage v Thresholds in percentages Select File Length 20 to 70 Visualse Fibers Width 20 to 70 Remove Fibers Save Fibers check the box if threshokiing is desired Oo 100 percentage percentage Absolute Values Top N Bottom N Ro PE inresnon now Save Fibers Generate stats for Length Width Straight uoe Generate stats Status Default Lower bound 0 and Upper bound 100 Step 3 Select the fiber properties for which the statistics are to be generated by clicking to select or deselect the accompanying radio boxes Step 4 Click the Generate Stats button to open a selection window Select Deselect the statistics as described in Tutorial 5 In this tutorial raw data box is checked as displayed below Select Deselect desired output stats threshold now Save Fibe
18. if and OL_ctFIRE_teststack_s2 tif respectively and are shown below After processing all the slices the ctFIRE will be reset But post processing can be done by importing the mat results of the fiber extraction as shown in Tutorial 4 3 3 Tutorial 3 fiber extraction for multiple images The procedure of processing multiple images is similar to processing a single image One difference is in step 1 before clicking the Import image data button check the Batch allowing users to select multiple images As shown below testimagel tif and testimage2 tif are selected To be noted multiple files must be selected if batch mode 1s selected The other difference is after processing all the images the post processing is not immediately available and the ctFIRE will be reset But post processing can be done by importing the mat results of the fiber extraction as shown in Tutorial 4 3 4 Tutorial 4 post processing for a mat file Step 1 import mat file Check the mat check box then click import image data button and select ctFIREout_testimagel mat The associated original image is displayed and output setting controls are enabled allowing users to configure the output setting Step2 Post processing the mat file Uncheck the box for Overlaid fiber and check boxes of Angle histogram amp values and Width histogram amp values then click the Post processing button The two histograms are displayed a
19. nalysis 2 Statistics It contains the statistics of each fiber property An image named filename_overlaid_selected_fibers tif showing the selected fibers overlaid on the original image 3 6 Tutorial 6 advanced output for batch mode analysis of multiple images Step I open a file Leave the Stack Mode box unchecked check the Batch Mode box a dialog qusetion window will pop up as displayed below to provide the option of not generating 19 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison overlaid image during the analysis As generating overlaid image usually takes time and can be done anytime when needed using the post processing feature introduced in tutorial 8 NO is recommended But for the purpose of demonstration the YES is selected here BJ Analysis Module Batch Mode StackMode Reset Filename Display Images in Batchmode Click on the Select file button a file selection window opens up The user needs to select multiple images In this tutorial testimage1 tif and testimage2 tif are selected Two images will be opened for each selected image one is the original image and the other is the original overlaid image showing all the fibers extracted from ctFIRE Step 2 e In the current version there is no option of visualizing the fibers and removing fibers in the batch mode e The enable threshold checkbox needs to be checked if thresholding is required The t
20. ness histogram amp values checked to show the figure of fiber straightness histogram and save the corresponding values into an Excel file Width histogram amp values checked to show the figure of fiber width histogram and save the corresponding values into an Excel file CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Default setting is only output Overlaid fibers Bottom information label indicate what is expected to do or the current program status 2 2 GUI for the Advanced Output Control Checking the box of OUT adv and then clicking on the Post processing button will prompt this GUI shown below p EJ Analysis Module gt an n Sl E Batch Mode Stack Mode Reset Select File Filename Visualise Fibers Remove Fibers Save Fibers percentage Thresholds in percentages 0 threshold now Generate stats for pee meen Generate stats Status Select File s Batchmode Not Selected The functions of each button and control are as follows Batch Mode checked to import multiple images Note multiple stacks cannot be processed in this version Checking this box will prompt a question window to allow user to choose whether to generate the overlaid image of the selected or not during the batch mode analysis Stack Mode checked to process a single image stack Select File select image file s for statistical analysis Files should be grayscale images Use Image J to convert color images to gra
21. ng the Post processing 5 Use advanced output control to customize the output of the selected fibers that meets the user defined fiber property condition s 6 Not to create overlaid image in batch mode analysis to save time which can be done anytime needed later 7 There is a images number limit for the advanced output control module for Windows it is set to be 10 000 for MAC it 1s 250 8 In MAC version launch the application from the terminal to view the analysis progress 9 Click Reset button to start over 1f any error occurs 5 References 1 curvelet toolbox Online Available http www curvelet org software html Accessed 19 Jul 2012 2 A M Stein D A Vader L M Jawerth D A Weitz and L M Sander An algorithm for extracting the network geometry of three dimensional collagen gels J Microsc vol 232 no 3 pp 463 475 2008 3 J S Bredfeldt Y Liu C A Pehlke M W Conklin J M Szulczewski D R Inman P J Keely R D Nowak T R Mackie and K W Eliceiri Computational segmentation of collagen fibers from second harmonic generation images of breast cancer J Biomed Opt vol 19 no 1 pp 016007 016007 2014 26
22. nimum points to apply fiber points selection that means if the number of fiber points found on each fiber is less than this value then all of them will be kept for width calculation confidence region is to define the region of the selected points if this value is set to 1 given the mean and standard deviation of the width of all the fiber points that satisfy the maximum fiber width as MEAN and STD respectively then the fiber width is defined as mean width of the fiber points whose width is within the range of Mean STD MEAN STD User can output the maximum width of each fiber by setting the output Maximum fiber width to 1 Here using all the default value the post processing will yield the following two histgrams E amp FIRE output width distr am File Edi Viev Insei Tool Desktc Windo Hel ff File Edr Viev Insei Tool Deskte Windo Hel JOSHA T A o ry C 30 D 3 e 20 mE Width pixels 3 5 Tutorial 5 advanced output for a single image Step 1 open a file Leave the Batch Mode and Stack Mode checkboxes of the advanced processing GUI unchecked Click on the Select File button a file selection window opens up to allow t userto select one image In this tutorial testimage1 tif is selected 15 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Look in int o try X a ey E Ezy T aH i Ns ned E NS ctFIREout selectout testimagel testim
23. oint cos 70 pi 180 thresh_dang_L 15 dangler length threshold thresh_short_L 15 short fiber length threshold s_fiberdir 4 number of nodes used for calculating direction of fiber end thresh_linkd 15 distance for linking same oriented fibers thresh_linka 150 minimum angle between two fiber ends for linking of the two fibers cos 150 pi 180 thresh_flen 15 minimum length of a free fiber If Load button is clicked the parameters saved previously can be loaded and updated if necessary as displayed below lt A Load parameters via csv file Look in ctFIRE e amp Ev P Name r Date modified i a 44 ctFIRE_default_parameters csv 7 23 2014 1 19 PM RecentPlaces i s Change the thresh_im2 to 30 click ok Another window shown below is displayed allowing the user to set two ctFIRE parameters since the ctFIRE was selected to run in step 2 set ctFIRE parame _ iE Percentile of the remaining curwelet coeffs Number of selected scales Curvelet transform denoising is performed by transforming to curvelet space thresholding the curvelets and then performing the inverse transform using only the selected curvelets Here the edit box labeled Percentile of the remaining curvelet coeffs is where the user should input the percentage of the curvelet coefficients for calculating the hard threshold applied to each scale in the inverse transformation The edit box labeled Number of the selected scal
24. raightness histogram amp values Angle histogram amp values Width histogram amp values Load and or update parameters Where the following list describes the function of each of the buttons and controls Batch checked to import multiple images or data mat checked to import the previous fiber extraction results and parameters saved in the format of mat files OUT adv checked to use the advanced output control feature will described later Reset rerun the GUI Import Image data import the information about an image an image stack or mat files or multiple images or mat files when batch mode is selected Parameters 1 Load load parameters saved in csv file 2 Update update the loaded or default 1f not loaded parameters Detailed description about these parameters can be seen in Tutorial 1 in section 4 of this manual CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Run select run mode among ctFIRE FIRE and both FIRE and ctFIRE Default run mode is ctFIRE Post processing after the fiber extraction of a single image or the import of mat file s the output setting can be configured to do post processing Stack image preview when a stack is opened move the slide bar to view slices in the stack Whole stack checked to process the whole stack Slices enter the range of the slices to be processed Minimum fiber length only display fibers which are longer than this value
25. rocessing button to select the corresponding advanced output results that is an excel file in the folder of selectout whose name is start with batchmode A batchmode output file with 3 processed images is selected and the corresponding statistics for the 3 images are displayed below overlaid images are not shown here i H H Ol P Voy Xem strHIST batchmode_statis willl a maS ee E F roa niie Ti aiaa ef xX a7 File Edi Viev Inse Tool Desktc Windo Hel File Edt Viev Inse Tool Deskte Windo Hel File Edi Viev Insei Tool Deskt Windo Hel 4 File Edi Viev Insei Tool Deskt Windo Hell By ctrire V1 3 Beta2 aa 7 l JA 2 Q mM p 4 l ea Qq Q M Z V Batch V mat V OUT adv GMs kA vi a GdMde k Anava A Import image data 100 100 Parameters 80 80 Jpaate Run ctr S 60 S 60 Stack image preview slice 5 gt d 2 4 2 40 ra ra Slices range 20 whole stack 0 0 18 36 54 72 90 108126144162180 Output Figure Control di Angle degree Vie Gee apmate z Width combined batchm II lengthicombined batchm g 28 z angle combined batchmo L cy 28 File Edr Viev Insei Tool Desktc Windo Hel ile Edi Viev Insei Tool Desktc Windo Hell File Edr Viev Insei Tool Desktc Windo Hel PAEA ae n UGES k AAGA SHUA UGWMS Rk AAVA Max fiber idth pixels
26. rs Generate stats for Mode Mean p Length O Width Straight O we Stddev Min Generate stats Status a number of Alignment Deselect Select Statistics e fibers YV Generate sheet for raw data After Ok is clicked two images for each selected input image are displayed one is the orignal image and the other is the orignal image with the overlaid selected fibers Output Files All of the output files are saved in the selectout folder mentioned in Tutorial 5 1 An Excel file named batchmode_statistics number xlsx Windows or xIls MAC containing nine sheets as follows a Combined Raw data This sheet contains all the fiber properties of the fibers satisfying the thresholds if specified 21 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison b Length Data Contains the length of each fiber satisfying the length thresholds in the image with the image name specified in the first row Similarly c Width Data Contains all the width data of selected fibers d Angle Data Contains all the data of angle of selected fibers e Straight Data Contains all the straightness data of selected fibers f Length Statistics Contains the selected statistic values on the data of length of fibers on each selected fiber Similarly g Width statistics Contains the statistics on the width data h Angle statistics Contains the statistics on the angle data
27. s follows 13 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison x Anann P P r s RY FIRE V1 3 Beta2 2 B amp TY Ml testimage1 tit 600 x 600 pixels 8 bit El Batch mat M OUT adv 2 ctFIRE output angle distri C a File Edit View Insert Tools Desktop Window Help a a a OHseas s 4 eodea4 2 08 a0 OG MS RSA A E 80 Import image data Post processing Parameters aoa Sesion Run cCtFIRE 2 Stack image preview slice Frequency 8 Slices range whole stack 8 Slices Output Figure Control is a LR 0 Minimum fiber length pixels 30 F Se A gt n ny 0 18 36 54 72 90 108126144162180 ge Res dpi 300 Ha A 4 at ti Angle degree Ae z Xs Lae 7 width 0 2 0 5 E aS ile Edi Vi j EN et ty K 3 File Edr Viev Insei Tool Deskte Windo Hel pixels 15 ni 2 Kd AN A R mae z F p j a Sd i a of er a 4 Q Ga z S N 4 As s X Histogram bins number AO 10 ie 33 p To Pete i yt ni i ae Select Output F Overlaid fibers Non overlaid fibers V Angle histogram amp values F Length histogram amp values Frequency Straightness histogram amp values V Width histogram amp values Analysis is done 4 8 6 Width pixels Step 3 rerun post processing by changing bin
28. s number and width calculation method Click on the Auto button and then choose Square root method to calculate the bins number the bins number is changed from 10 to 17 as shown below Run CtFIRE Load Update Stack image preview slice Ey Estimate optimal BINs number based on a h x Slices Output Figure Control Minimum fiber length pixels3 30 Image Res dpi 300 Fiber line width 0 2 0 5 Max fiber width pixels 15 Histogram bins number Hr fe ber line width 0 2 0 5 Max fiber width pixels larez 15 Histogram bins number 10 select Output Moe o e Pi ce ee a a l e To change the width calculation click on the More button then choose select points to calculate the width the following windows will be displayed 14 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Enter the paramet p l Minimum maximum fiber width 10 Minimum points to apply fiber points selection 6 Stack image preview slice S Confidence region times of sigma 1 Fiber line width 0 2 0 5 i Max fiber width pixels imez 15 Histogram bins number 17 Histogram bins number Select Outnont The parameters for selecting points to calculate width include Minimum maximum fiber width that is the maximum fiber width that is used to define the artifact fiber width shoulb be larger than this value mi
29. s range as the whole stack To be noted the default slices range is whole stack which means to process all the slices in the stack and set the minimum fiber length shown in the overlaid image to 30 The following figure is displayed g E PERALIN ACK F HORS DIZ ILE POEM F D m Reset fie dt View imet Toob Oesttop Window Help Ndo k Arga a 0i 2D Step 3 set fiber extraction parameters Click on the Update button In this tutorial for fiber extraction change the thresh_im2 to 30 for curvelet transform based reconstruction use the default values See the step 3 of Tutorial 1 for details 12 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Step 4 run fiber extractions Click on Run button Then the progress of fiber extraction will be listed in the Command Window The overlaid and reconstructed images will also be displayed and saved for each slice Step 5 check the result The results are saved in a folder called ctFIREout as described in tutorial 1 Each slice is considered as an image and has separate file names for the results of each slice Compared to the filename in tutorial 1 the file name of the stack results adds the slice information to the end For instance the mat files for fiber extraction results of the slice 1 and Slice 2 are ctFIREout_ teststack_s l mat and ctFIREout_ teststack_s2 mat respectively The overlaid images of slice 1 and slice 2 are named OL_ctFIRE_teststack_s1 t
30. tensed aa saaetaeas mann teeta taewatacarsiamead neat deena 26 1 Introduction The purpose of this standalone MATLAB package is to allow users to automatically extract collagen fibers in an image and quantify fibers with descriptive statistics such as fiber width fiber length fiber angle and fiber straightness The program reads in image files extracts the individual collagen fibers via ctFIRE or CT FIRE curvelet transform plus FIRE algorithm which is an approach combining the advantage of the fast discrete curvelet transform 1 for denoising the image and enhancing the fiber edge features and the advantage of fiber extraction FIRE algorithm 2 for extracting individual fibers The output may be displayed on the screen and or written to csv or excel files The detailed information about ctFIRE can be found in the paper 3 In the ctFIRE program the fiber extraction parameters have default values and are adjustable via GUI or csv file Optional outputs include overlaid image of the segmented fibers on the original image and the CT reconstructed image image of non overlaid extracted fibers histogram and values for the fiber length fiber angle fiber width and fiber straightness Using CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison the advanced output control module users can select and analyze fibers based on the conditions of fiber properties combine the fibers from multiple images and visualize the overlaid imag
31. the above figures 10 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison J t CAA testdatasets CTHRE 1 3a0 ctFIREout pow FA Open Share with E mail New folder a Name esktop E HistANG_ctFIRE_testimagel csv eS E HistLEN_ctFIRE_testimagel csv ecent Places Zh HistSTR_ctFIRE_testimagel csv oogle Drive Hist WID_ctFIRE_testimagel csv NOL_ctFIRE_testimagel tif raries fa ctFIREout_testimagel ocuments ctfParam_testimagel tif csw fusic 2 OL_ctFIRE_testimage tif ictures 2 CTRimg_testimagel tif Step 7 advanced output Uncheck the mat box and check the OUT adv then click on the Post processing button an advanced output control GUI titled Analysis Module is opened Bj Analysis Module Lo E Batch Mode F Stack Mode Reset xs Select File Fiename les 4 Remove Fibers Save Fibers percentage Thresholds in percentages Generate stats for Status Select File s Batchmode Not Selected Tutorials 5 7 show how to use this GUI for advanced analysis Step 8 reset the GUI Reset to import new image or data 11 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison 3 2 Tutorial 2 fiber extraction for an image stack Step 1 open an image Leave the Batch and mat checkboxes unchecked Click the Import image data button a file selection window opens allowing the user to choose the image In this tutori
32. tif csv parameters for ctFIRE that can be loaded in later use The two displayed images are shown below CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Step 6 post processing Let s suppose we are satisfied with the overlaid image now we want to check the non overlaid extracted fibers and all the statistics In the control panel the corresponding settings are Output Figure Control Minimom fiber length pixela a Image Res dpi 300 Fiber line width 0 2 0 5 Max fiber width pixels more a5 Histogram bins number 10 Select Output E Gverlaid fibers Non overlaid fibers angle histogram amp values Length histogram amp values Straightness histogram amp values Click on Post processing non overlaid fibers as well as the histograms for fiber angle fiber length fiber straightness and fiber width are displayed as in the following A File Edr Viev Insei Tool Desktc Windo Hel le Edr Viev Insei Tool Desktc Windo Hel File Edr Viev Insei Tool Desktc Windo Hel File Edr Viev Insei Tool Desktc Windo Hel NEHIL OL SMS R ARGKA WPSMSRIARVA USHLASA S 2 Frequency Frequency E 00 48 36 54 72 90 108126144162180 Angle degree 6 l Width pixels Straightness dimensionless Length pixels In the output folder ctFIREout five additional output files are highlighted as shown below corresponding to
33. togram amp values File name ltestimage 1_statistics xlsx statistics xlsx 23 CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison After clicking the Open button the following figures are displayed File Edi Viev Insei Tool Desktc Windo Hel x File Edi Viev Insei Tool Deskte Windo Hel File Edi Viev Insei Tool Deskte Windo Heli File Edi Viev Insei Tool Desktc Windo Hell a DSGMS R AAGA P NSH Slk AIAST P NSHU AAT QOGES k AAVA 80 200 200 80 60 150 150 60 Ja gt gt E a E S 40 5 100 100 S 40 gt gt 2 2 ra E 50 50 20 0 0 0 4 6 8 10 04 06 08 1 100 200 300 400 0 18 36 54 72 90 108126144162180 i ma Qi C assi pixels Angle dearee 2 Width testimage1 eT zs straigthness testimagel gt e xa 2 angle testimage1 File Edi Viev Insei Tool Deskte Windo Hel Edi Viev Insei Tool Desktc Windo Hel File Edi Viev Insei Tool Desktc Windo Helj File Edi Viev Insei Tool Desktc Windo Hel DGMsRAROK4 PERS RARGK WPaSMsR AARGKA QESMSRIAARDA Angle degree To display the overlaid image of the selected fibers check the checkbox of Overlaid fibers 9 and select the corresponding file a question window will then be popped up as below i z farre V13 Beta Batch Import image data Post processing Run CctFIRE E slice Update tack image preview z
34. yscale images Reset resets the GUI and starts the process over CT FIRE V1 3 Beta2 User s Manual LOCI UW Madison Filename displays the name of the file under consideration Visualise Fibers button used to pop up a new window for seeing only the selected fibers overlaid on the original image The properties of the selected fibers are listed in a table This function can be used repeatedly Remove Fibers used to eliminate the over segmented fibers This option pops up a window similar to visualise fibers This option leads to an overlaid image with all the remaining fibers This function can be used repeatedly The fibers removed in earlier steps remain deselected The fiber numbers removed using this option are shown in status box Save Fibers used to save the fibers for analysis No more removal of fibers can be done after clicking the Save Fibers button Checkbox for Thresholding check the box if thresholding of fibers is desired based on percentage absolute value Dropdown menu select one among four options based on the fiber properties 1 Percentage the faction of the selected fibers 2 Absolute Value the range of the actual threshold 3 Top N the selected number of fibers with the highest values of the specified fiber property 4 Bottom N the selectd fiber number of fibers with the lowest values of the specified fiber prop Thresholds 1 Length Select to enter the length threshold Specify low limit in first box
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