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ctFIRE Beta Version 1.2.1 User`s Manual(August, 2013)

1. After clicking on open the first slice of the stack is displayed and new controls are enabled on the control panel as shown below Ki ny x L TRE I s a E e oe Batch mode Mat Reset Import image data Post processing Parameters Stack image preview slice 1 4 Slices range whole stack 1 Slices Output Figure Control Minimum fiber lengh 30 Maximum fiber number 2999 Fiber line width 0 5 Histogram bins number 10 Select Output J Overlaid fibers F Non overlaid fibers Angle histogram amp values Length histogram amp values Straightness histogram amp values O O g0 Width histogram amp values 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 to preview the slices and select the slice range Drag the slider or click on the slider arrow to preview the slices in the stack Select the 2nd slice to preview select the slices range as slices 1 to 2 and set the minimum fiber length shown in the overlaid image to 50 The following figure is displayed m pee i 2 ctFIRE Control eS ia Original Image atch mode Mat Reset o Parameters Load Run CtFIRE iz Pame 1 Stack image preview slice
2. 2 Update H 4 gt Slices range whole stack Slices Output Figure Control Minimum fiber lengh 30 Maximum fiber number 2999 Fiber line width 0 5 Histogram bins number 10 Select Output J Overlaid fibers Non overlaid fibers al Angle histogram amp values Length histogram amp values Straightness histogram amp values M Width histogram amp values To be noted the default slices range is whole stack which means to process all the slices in the stack Step 3 set fiber extraction parameters click on the Update button In this tutorial for fiber extraction change the thresh_im2 to 10 for curvelet transform based reconstruction use the default values See the step 3 of Tutorial 1 for details Step 4 run fiber extraction click on the 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 slices Step 5 check the results 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_ teststack3_s1 mat and ctFIRE
3. Excel file default setting is only output Overlaid fibers Bottom information label indicate what is expected to do or the current program status 4 Tutorials 4 1 Tutorial 1 fiber extraction and post processing for a single image Step 1 open an image Leave the Batch mode and mat checkboxes unchecked Click on the Import image data button a file selection window opens allowing the user to choose the image In this tutorial testimage1 tif is selected A Select an Image hetim Lookin J FIRE O sts s Recent Places PA Desktop T eA iai tif teststacks tif Libraries L Computer File name ltestimage1 tif v Files of type tif Cancel After clicking on open the image is displayed and new controls are enabled on the control panel as shown below Batch mode Mat Reset Parameters rosa sesere Run ctFIRE Stack image preview slice Slices range whole stack Slices Output Figure Control Minimum fiber lengh 30 Maximum fiber number 2999 Fiber line width 0 5 Histogram bins number 10 Select Output V Overlaid fibers Non overlaid fibers Angle histogram amp values Length histogram amp values Straightness histogram amp values F Width histogram amp values his 3 yi A Ea p Yi WN 4 Sb Step 2 select run mode and set output
4. N As shown in the flow chart besides fiber extraction for a single image in version 1 0 the main added features in beta version 1 2 1 include batch mode fiber extract 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 To be noted batch mode image stack is not supported In addition after fiber extraction for image stack or batch image files the post processing is not immediately available However as shown above since fiber extraction is applied to each image or slice and saved as individual mat file batch mode mat can be imported for post processing of those results if needed Detailed description about the features will be demonstrated in the examples in section 4 3 GUI control panel The GUI in ctFIRE is modular so that the main user interface is in a separate window from the outputs This allows for the users to resize the output windows to their preferred size The main user interface window is shown below Batch mode Mat Import image data Parameters Load Update Run CtFIR
5. mat file Step 1 import mat file check the mat check box then click on the impart image data button and select ctFiIREout_testimage1 mat The associated original image is displayed and output setting controls are enabled allowing to configure the output setting Uncheck the Overlaying not to create new overlaid image and reconstruction image Check the Angle histogram The following figure is displayed z a i m js J gt m ald me A ctFIRE Control E Original Image Batch mode Mat Reset Import image data pwt prceeting Parameters Run ctCFIRE Stack image preview slice Slices range whole stack Slices Output Figure Control Select Output V Overlaid fibers Non overlaid fibers Angle histogram amp values Length histogram amp values Straightness histogram amp values Width histogram amp values step2 Post processing the mat file uncheck the box for Overlaid fiber and check one for Angle histogram amp values then click on the Post processing button The angle histogram is displayed as follows lt p ctFIRE output angle distri Bca aE HOSE s ASA8082 08 Extracted angle hist Frequency 0 0 1636 54 72 9010812644 627180 Angle degree Step 3 rerun post processing Change the Histogram bins number from 10 to 20 Click on the Post processing button The new a
6. network geometry of three dimensional collagen gels Journal of Microscopy 232 3 463 475 2008 doi 10 1111 j 1365 2818 2008 02141 x
7. E Stack image preview slice Slices range whole stack Slices Output Figure Control Minimum fiber lengh ag Maximum fiber number 2999 Fiber line width 0 5 Histogram bins number 10 s 5elect Output Overlaid fibers E Non overlaid fibera Angle histogram amp values E Length histogram amp values E Straightness histogram amp values F Width histogram amp values Analysis is done where the following list describes the function of each of the buttons and controls Batch mode 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 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 if not loaded parameters Detailed description about these parameters can be seen in Tutorial 1 in section 4 of this manual 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 any selected slice in
8. algorithm 2 for extracting individual fibers The output may be displayed on the screen and written to csv files In the ctFIRE program user can choose to run ctFIRE FIRE or both of them Parameters to run FIRE and ctFIRE have default values and are also 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 fiber angle histogram fiber length histogram fiber straightness histogram and fiber width histogram as well as the associated values saved in csv files The detailed information about the parameters and output of ctFIRE is automatically saved in mat binary format for a later post processing 2 Flowchart load image file Flow chart for ctFIRE beta v1 2 1 y N batch 1 a l N image stack get image image stack select run mode and ctFIRE select slices parameters configure output range figures and files files number get mat files number fiber extraction for a single image last slice Save mat results and update load the or last file parameters output for configuration of example overlaid image output figures fiber angle fiber length and files histograms and values load fiber mat file extraction mat image stack or batch loaded results Pee ee errr ccceseseceseeseeseseseseeesesereeeseees post processing
9. ctFIRE Beta Version 1 2 1 User s Manual August 2013 Yuming Liu and Jeremy Bredfeldt Laboratory for Optical and Computational Instrumentation UW Madison L NOUO ee E E E E E once 1 2 WOW CIVANL eian E E A 2 s UO Ik 62 a a E E eee 3 A TUO a A E E A A E AEE E E A ET 6 4 1 Tutorial 1 fiber extraction and post processing for a single IMAGE cccccceeseccceesecceeeeseceeeuseess 6 4 2 Tutorial 2 fiber extraction for an image StACK cccccceeseccceesscccceesececeesececeeeceeeeseeeseusecesseneees 13 4 3 Tutorial 3 fiber extraction for multiple IMAGES cccccesecccceseccccesececeesececeeecceseseeessuseceseuneses 16 4 4 Tutorial 4 post processing for a mat Wil Oysicaucescsaanetnbsaangeseracecceneatuneanntastanoscestesinenataeanedeaeneradanas 17 a VS ee ayaa denouctayenmasnnmvaeanenanuanes 19 BY RSF ICS ier cateet E E E E E E I A E cee ratens tee E 19 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 angle fiber length fiber straightness and fiber width The program reads in image files extracts the individual collagen fibers via ctFIRE 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
10. fibers cos thresh_linka 150 150 pi 180 thresh_flen 15 minimum length of a free fiber If Load button is clicked the parameters saved previously in csv file can be loaded and updated if necessary as displayed below A CEFIRE Control PE PEPETA Load parameters via cv fle iy Look in ctFIRE ft c Ese i Name Type Size Date modifi h ctFIRE_default_parameters csv Microsoft O 1KB 8 7 20135 Batch mode Import image data Post processing Parameters Stack image preview slice ab Recent Places Desktop Libraries a5 Slices range whole stack Computer glices Output Figure Control Minimum fiber lengh 30 Maximum fiber number 2999 Fiber line width 0 5 File name ctFIRE_defaut_parameters csv Histogram bins number 10 Files of type Change the thresh_im2 to 30 ands _xlinkbox to 5 click on 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 Percentile of the remaining curvelet 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 cur
11. he mat file for the fiber extraction results by ctFIRE OL_ctFIRE_ testimage1 tif the tiff overlaid image of ctFIRE CTRimg_ testimagel1 tif the tiff CT reconstruction image ctfParam_testimage1 tif csv parameters for ctFIRE that can be loaded for later use The two displayed images are shown below 1 KB 715 KB 1 064 KE 290 KB 7 N If ctFIRE and FIRE are both used for analyzing this test image in step 2 the results comparison will show that ctFIRE can yield more accurate fiber extraction than FIRE 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 Minimum fiber lengh 30 Maximum fiber number 2999 Fiber line width o 5 Histogram bins number 10 Select Output F Gverlaid fibers Non overlaid fibers Angle histogram amp values Length histogram amp values Straightness histogram amp values Width 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 Fiber width hist Extracted angle hist Frequency Ss 8 8 120 100 ZB 80 3 g 60 2 uw 40 20 0 0 0 0 18 36 54 72 9010812614462180 Angle degree 06 07 08 09 1 Straightness dimensio
12. ngle histogram is displayed as follows ctFIRE output angle distri E L i am S5 File OSGHMs AAOD E08 Extracted angle hist 50 Q9 oO Frequency 5 Tips 1 For the images in batch processing or slices in a stack the images or slices should normalized such as to have the same dynamic range 2 Use Fiji or ImageJ to convert the image type to 8 bit image which is well supported for this version 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 default parameters and corresponding description about each of them can be downloaded at LOCI website but only some of the key parameters can be adjusted via GUI The thresh_im2 to remove the background is often the only parameters 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 during the Post processing 5 Click on the Reset button to start over if there is any error occurred 6 References 1 curvelet toolbox lt http www curvelet org software html gt 19 July 2012 2 A M Stein D A Vader L M Jawerth D A Weitz and L M Sander An algorithm for extracting the
13. nless In the output folder C CAA_x220 github curvelets ctFIRE ctFIREout five additional output files are highlighted as shown below corresponding to the above figures Windows _OS CG CAA X220 github curvelets ctFIRE ctFIREout E mail New folder Mame Type Size Ly HistANG_ ctFIRE_testimagel csv Microsoft Office E 3 KB Ly HistLEN_ctFIRE_testimagel csv Microsoft Office E 3 KB Ly HistSTR_ctFIRE_testimagel csv Microsoft Office E 3 KB EN Hist WID_ctFIRE_testimagel csv Microsoft Office E 3 KB NOL_ctFIRE_testimage tit TIF File 117 KB f amp ctFIREout_testimagel mat MATLAB Data 1 064 KE fsi ctfParam_testimagel tif csv Microsoft Office E 1 KE E QL ctFIRE_testimagel tif TIF File 715 KB CTRimg_testimagel tit TIF File 290 KB Step 7 reset the GUI reset to import new image or data 4 2 Tutorial 2 fiber extraction for an image stack Step 1 open an image Leave the Batch mode and mat checkboxes unchecked Click on the Import image data button a file selection window opens allowing the user to choose the image In this tutorial teststack3 tif is selected A Select an Image s i lle iii s s d l ms Recent Places Look in b aFRE Y Desktop q y E DS aT ctFIREout testimagel tif testimage2 tif teststack tif teststacks tif Tzxmid tif a Libraries A Cor Computer ca File name teststack 3 tif Y Files of type tif am Cancel
14. out_ teststack3_s2 mat respectively The overlaid images of slice2 and slice 3 are named OL_ctFIRE_teststack3_s1 tif and OL_ctFIRE_teststack3_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 4 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 on the Import image data button check the Batch mode allowing to select multiple images As shown below testimage1 tif and testimagez2 tif are selected BB ctFIRE Control V Batch mode T Mat Import image data Post processing aa a s nP Parameters Recent Places odate Run CtFIRE ia Stack image preview slice Desktop a ctFIREout testimageL tif testimage2 tif teststack tif Slices range hole stack Slices Output Figure Control teststack3 tif File name testimage 1 tif testimage2 tif Files of type Selert Ontnnt 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 4 4 Tutorial 4 post processing for a
15. parameters use the default Run mode ctFIRE but could also select to run FIRE or both ctFIRE and FIRE as shown on the right of Run button in the below window The default output control parameters are used as shown in the above figure only choosing the overlaid fibers to be displayed E ctFrE Control ele BERN Original Image Batch mode i Parameters mm CTFILRE Stack image preview slice FIRE CTF amp FIRE Slices range Step 3 set fiber extraction parameters click on the Update button on the panel of parameters the following window is displayed to update or confirm nine default FIRE parameters Eg Update FL LEE ee thresh_im2 S xlinkbox thresh_ext 70 thresh dang L 15 thresh_short_L 15 s fiberdir 4 thresh_linkd 15 thresh_linka 150 thresh_flen The description of the parameters is listed below Name Value Description thresh_im2 5 thresh_im2 is for a hard value main adjustable parameters radius of box in which to check to make sure xlink is a local max of the s_xlinkbox 8 distance function thresh_ext 70 angle similarity required for a fiber to extend to the next point 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 minimum angle between two fiber ends for linking of the two
16. 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 unit is in pixels default value is 30 Maximum fiber number maximum number of fibers for the output figure s or statistic analysis If the number of the extracted fibers is larger than this value only consider the first Maximum number of extracted fibers Default value is 2999 Fiber line width The line width of the extracted fibers in the overlaid image default value is 0 5 Histogram bins number the number of bins used in the angle and or length histogram 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 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 straightness 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
17. velet coefficients for calculating the hard threshold applied to each scale in the inverse transformation The edit box labeled Number of the selected scales is to set the scales to reconstruct the image Specifically if the total number of scales used by the curvelet transform is N and 3 is the number of selected scales in this edit box then the actual scales used for reconstruction is scale N 1 N 2 and N 3 In this tutorial use the default values and click on 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 save to a new subfolder called ctFlREout if not exit the folder will be created by ctFIRE automatically of the folder where the image exists In this case the path for this subfolder is C CAA_x220 github curvelets ctFIRE ctFlREout Six output files are shown Windows OS C CAA 220 github amp curvelets ctFIRE ctFIREout ny Y Share with Slide show New folder Name Type EMI ctfParam_testimagel tif csv Microsoft Office E OL ctFIRE_testimagel tif TIF File E ctFIREout_testimagel mat MATLAB Data CTRimg_testimagel tit TIF File ctFIRE output ctFIREout_ testimage1 mat t

ctFIRE Beta Version 1.2.1 User`s Manual(August, 2013)

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