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1. 22 tif My fearon File name Places Transtorrmationh atrices txt All Files ss Cancel Save as type 4 Next the user will need to check the resulting alignment of the imported image sequence The macro will align the image sequence and then create two 3D projections of the aligned image sequence The windows may appear over top of one another so the user may need to move them to separate places on their monitor The user will use the scroll bars on the 3D projection windows and the aligned image sequence window to view the alignment The user will need to determine whether or not the images aligned properly If the images are properly aligned they should appear that they fit in a seamless stack that 23 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill seems to align the adjacent images well to each other Once they have made this deter mination the user will need to click on ok in the Check Alignment window Check Alignment Check alignment then click OK When prompted if want to adjust stack and realign then check Realign box ok 5 Next the Adjust Stack amp Realign window appears If the user had a poor alignment then they will need to place a check in the check box next to Realign otherwise they should leave this box unchecked Once the user has made a selection they will then need to click on ok in the Adjust Stack amp Real
2. N Q Brain Gray or Color amp Brain Section Agee Align with o TurboReg Cells o Saved Transforms Grains B IN Q TurboReg Saved Transforms agra Import Image Sequence Directory Start Number 1 End Number 2 Increment 1 Scale 100 Contains Browse B IN Q Brain Section TurboReg Brain Gray or Color TurboReg 4a B IN Q 5a Pre Alignment Processing 1 Fix Image Imperfections 2 Select most centered Image o Align Images B IN IQ 6a Post Alignment Processing 1 Check Alignment o Realign Images o Save Results amp View 3D Projections o Save Results o Start Over o Exit N Q Import Corresponding Images Grains for Brain Gray or Color Brain Section Save amp View Projections Save Results Brain Gray or Color amp Brain Section TurboReg 7b Saved 3D Projections o Yes o No N Q Brain Gray or Color amp Brain Section TurboReg 8a Saved Results o Import Corresponding Images for Alignment Grains o Start Over o Exit INJIQ Brain Gray or Color amp Brain Section Saved Transforms Corresponding Images Grains 4c Import Transforms amp Masks Browse B IN Q Select Tranform File 5 Pre Alignment Processing 1 Apply any changes made to corresponding images when improving alignme
3. e TempImages folder If you find a folder named TempImages in your original images folder it is okay to delete it It should be automatically deleted if you abort the plugin by clicking Quit or selecting Exit on the last panel but will not be automatically deleted if you abort the plugin by directly closing the wizard window e Importing sequence If you get the wrong image numbers when importing an image sequence it might be because the first image in your folder has an image number other than 1 This is because Start number n in ImageJ means the n image in your folder of images For instance if your folder only contains 3 tif 4 tif 10 tif ImageJ will take Start number 3 to mean 5 tif which is the 3 image contained in your folder You can avoid this problem by renaming your images so that the lowest numbered image in your folder is always 1 and that the image numbers in your folder increment by 1 Faulty imports can also be because you did not specify a File name contains string and your folder contains images with different names but the same numbers such as 1 tif and grains1 tif To avoid this problem if you are going to have two different sequences in the same folder make sure all files have a string identifying them before the image number in their file names For instance do not have the sequences 1 tif 2 0f 10 tif and g
4. Adjust Corresponding Images based on Realignment Adjust stack of Corresponding Images based on rotation translation and other changes recorded and made during realignment then click OK to align 42 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill 15 Next the Load Transformation File window appears and the user clicks ok and then se lects the transformation file from where it is located and clicks open so that it can be used to align the corresponding grains images Note if the user followed our sugges tions the file should be named TransformationMatrices txt and located in the same fold er as the cells images If for some reason the user selects a file that isn t the transforma tion file OR selects the incorrect transformation file one for more images in the sequence or images of different dimensions a warning pops up and the user will only be allowed 3 more chances to select the right transformation file Please not that if the user selects an incorrect file one that isn t a transformation file the ImageJ exception window may popup If this happens it is because exceptions were thrown when the alignment plugin attempted to read a file that wasn t a transformation file Once the user is done using the macro the user will be able to close the exception window Load Transformation file Select where to load the transformation text file Note Cancel exits entire m
5. 49 iF 34 tif My Recent TransformationMatrices txt 48 tif 33 tif Sinesmen alignmentResulks Grains nii 47 tif 32 LiF Desktop My D Perens My me f rl i 60 tif 59 tif 58 tif 57 tif 55 tif 5 54 tif 53 tif 52 tif ie tif 4 im 46 tif i 45 tif im 44 tif im 43 tik im 42 tik im 41 tif 40 tiF i 39 tif i 38 tif im 37 tiF 36 tif i 31 tiF 30 tif 29 tif 28 tif 27 tif 26 tif 25 tif 24 tif i 23 iF a 22 tif hy PRED File name Places 21 tif All Files r Cancel Files of type Import Corresponding Grai Starting number End number 10 Increment Scaler File name contains Note Cancel exits entire macro OK Cancel If the user had re aligned the cells images then the Adjust Corresponding Images based on Realignment window will popup If this happens then the user needs to apply the same rotation and translation changes to the same images that they had applied these to when they performed the re alignment of the cells images If the user only selected a different center most image then the user doesn t need to adjust the corresponding im ages using translation and rotation Once the user is done making adjustments they just click ok If the user did not re align the cells images they can just proceed to the next step
6. l 55 tif l 54 tif l 53 tif E 52 tif im 51 tif File name Save as ype im 46 tif me 45 tik l 44 tif l 43 tif 42 tik me 41 tik im 40 tif i 39 tif l 30 tif im 37 tif im 36 tif algnmentResults nit me 35 tif we 34 tif im 33 tif 32 tif i 31 tif 30 tif 29 tif i 26 tif 27 tif 26 tiF 25 tif 24 tif i 23 tif 22 tif 21 tif E Files x Cancel Version 03 tions of the alignment and another window will popup asking the user if they would like to save the 3D projections If they choose yes these projections will be save if they choose no they won t be saved If the user doesn t select that they would like to view the 3D projections then the user won t be prompted to save the 3D projections 27 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill View 3D Projections Eg View 3D Projections No Mo Mote Cancel mMm _ Projection of align Sele _ Projection of alignm Eg 1 10 224 00 168 00 pixels 224x168 RGE 01 36 224x174 pixels RGB 5 4MB 1 36 228x168 pixels RGB 5 3MB Note Cancel exits OK Cancel 10 Next a window will popup that asks the user would like to Align the corresponding grains images If the user selects no then the macro will start over by preceding back to step 1 If the user selects quit then the macro wi
7. to select where to save the resulting nii file and what to name it We suggest storing it 39 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill in the same folder as the one that contained the images that were imported in for align ment Once the user has made their folder and name selections they need to click Save to save the results Save Results of Alignment Select where to save results of alignment as nii file Note Cancel exits entire macro Cancel Save as Nifti lm 50 tif 35 tif s TransformationMetrices tt E 49 tif i 34 tif My Hecent alignmentResults ni i 48 tif i 33 iF Documents E alignmentResults_Grains nii lie 47 tif E 32 tif E nul E 46 tif 31 tif 60 tiF 45 tif 30 tif Desktop 59 tif a 44 tif 29 tiF E 58 tif im 43 tiF 28 tif PI 57 tif 42 tif E 27 tif 56 tif i 41 tif z6 tif l 55 tif l 40 tif i 25 tif i 54 tif lm 39 tif me 24 tif i 53 tif i 38 tif i 23 tiF i 52 tif 37 tif 22 tiF l 51 tif l 36 tif z1 tif lt i My Network File name aignmentResults ni Places Save as type E Files Cancel My Documents 11 Next a window will popup that asks the user if they would like to view the 3D projections of their resulting alignment If they select yes the user will see their 3D projections of the alignment and another window will pop
8. 2 Please select how to align Select Align with TurboReg if you do not already have a transformation file If you have a transformation file you wish to use select Align with Transformation File If you are working on colored images you will not have a choice and they will be aligned with TurboReg iol xi Please select how to align Align with TurboReg cells 2 Align with Transformation File grains 3 Import image sequence Select the first image in your sequence and then enter the appropriate parameters For instance if your sequence consists of the images cells1 tif cells3 tif cells5 tif cells99 tif and you wish to keep the original image size you would enter Start number 1 End number 99 Increment 2 File name contains cells Scale 100 Importing a sequence can be tricky in ImageJ Please see the troubleshooting section if you encounter any problems You will get an error message in the following cases No file is selected Start number is not a positive integer End number is not a positive integer Increment is not a positive integer Scale is not a positive integer Start number is greater than End number Increment is greater than the difference between Start number and End number Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill If the file you selected is in a folder with no image files you will get an error from ImageJ and will have t
9. 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill 5 Next the Adjust Corresponding Images based on Realignment window will appear If the user had realigned using Translation and or Rotation to adjust the cells images the images that provide the transformation file that the user will select for the alignment pri or to re alignment then the user needs to apply these same changes to the imported corre sponding grains images at this point Once the user has completed this task if the user didn t use translation and rotation with the realignment or 1f the user didn t realign then the user will click ok in the window Adjust Corresponding Images based on Realignment F Cells Images underwent Realignment then Adjust stack of Corresponding Images based on rotation translation and other changes recorded and made during realignment then click OK to align Next the Load Transformation File window will appear The user needs to click ok in this window and then they need to select the location of the transformation file that they would like to use to align the imported corresponding grains images If the user selects a file that is not a transformation file OR if the user selects a transformation file for an alignment with a different number of images or different image dimensions then a warn ing window appears that alerts the user that they failed to align the images The user can eithe
10. Brain olor Brain Brain _ Section Ok Cancel Guit 2 The user selects the type of alignment from a drop down menu a TurboReg aligns with TurboReg b SavedTransforms aligns with saved Transforms file from previous alignment c Quit User chooses to align TurboReg Type of Alignment Align with TuboReg or Saved Transforms Mote Cancel exits entire macro Type of Alignment TurboReg TurboReg SavedTransforms Quit gt Next the user needs to import the cells image sequence The user is prompted to select the location of a single file from the image sequence and then the user is prompted to enter the following fields to allow the user to import the cells image sequence Start Number a positive integer that indicates the first image in the sequence End Number a positive integer that indicates the last image in the sequence Increment a positive integer of value 1 or greater that indicates by what increment images are to be imported in Scale a positive integer that indicates how much to reduce the resolution of the imported images where 100 indicates no reduction Contains a string that is common to all image sequence file names After the user enters these fields and clicks ok the image sequence will be imported into ImageJ and appear in the active image window 34 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Import Cells Image Sequence To
11. Saved Transforms Mote Cancel exits entire macro Type of Alignment TurboReg TurboReg SavedTranstorms uit 3 Next the user needs to import the corresponding grains image sequence The user is prompted to select the location of a single file from the image sequence and then the user is prompted to enter the following fields to allow the user to import the cells image sequence Start Number a positive integer that indicates the first image in the sequence End Number a positive integer that indicates the last image in the sequence Increment a positive integer of value 1 or greater that indicates by what increment images are to be imported in Scale a positive integer that indicates how much to reduce the resolution of the imported images where 100 indicates no reduction Contains a string that is common to all image sequence file names After the user enters these fields and clicks ok the image sequence will be imported into ImageJ and appear in the active image window Note that the error messages will appear if the user enters in float or negative numbers into the numeric fields of the import 46 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Import Grains Image Sequence Toimportthe corresponding image sequence select an image file contained in the desired image sequence Note Cancel exits entire macro My Recent Documents Desktop My Documents My Computer al
12. adjust the threshold and click Apply then close Threshold window amp click OK OK F f5 fx Threshold 1110 13 224x168 pixels 8 bit 368k Tol 2 Upper Threshold Image Window Scrollbar Scrollbar Step 3 Then click ok button on the Convert to Mask window that appears 20 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Step 4 Then close the Threshold window by clicking on the x in the upper right hand corner Then click ok button on the User Pick Threshold to Mask Out Background window Now the macro will mask out the background of the images the user imported Step 3 User Picks Threshold to Mask Out Background Use the Threshold tool to adjust the threshold and click Apply then close Threshold window amp click OK nx Fej 26 Convert all images in stack to binary l Calculate Threshold for Each Image Mask Window Black Background Use the Threshold tool to adjust the threshold and click Apply then close Threshold window amp click OK 1410 13 224x168 pixels 83 bit 368K Red Auta Apply Reset Set 1 Next the user needs to check the masking and fix any imperfections in the masking by using the brush tool in ImageJ The user will use the brush tool to paint the background color over all image imperfections To do this the user will first select the color picker tool in
13. importthe image sequence select an image file contained in the desired image sequence Note Cancel exits entire macro pk FE alignmentResults nii 50 tif 35 tif i E xAlign txt E 49 tif 34 tif My Recent 5 TransformationMatrices txt me 48 tif i 33 tif Documents alignmentResults_Grains nii i 47 tif i 32 tiF E nul 46 tif lm 31 tif me 60 tif i 45 tif 30 tif 59 tif E 44 tif E 29 tif E 55 tif me 43 tif E 28 tif 57 tf lm 42 tif C1127 tif 56 tf me 41 tif i 26 tif 55 1 40 tif i 25 tif 54 tiF 39 tif i 24 tif E 53 tif 38 tif E 23 tif 52 1 37 tif i 22 tif 51 tif i 36 tif ie 21 tif lt i Mi ym File name 27 ti he Files of type all Files Import Cells Image Sequence ES Starting number End number Increment Sc alert File name contains Note Cancel exits entire macro JK Cancel 4 Select the Alignment Start Image for the alignment If you don t want to select the first image as the start image use the scrollbar on the active image window to select the start image as the most centered image 35 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill File Edit Image Process Anakze Plugins Window Help E oaoa Aae Ose a Select Alignment Start Image Pick the start image for alignment If you don t want the first image to be the start image use scrallbar
14. to select the mast centered image prior to clicking OK ok 5 Next the user will need to indicate the location in which they want to save the Transfor mation file that will result from the image sequence alignment The user first clicks ok in the Save Transformation File window Then they will need to select the location and name of the saved transformation file We suggest that the user saves this file with its de fault name TransformationMatrices txt in the same folder that they have stored all the imported cells images 36 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Save Transformation file Select where to save the transformation text file Note Cancel exits entire macro i Cancel Save transformations at My Recent Documents Desktop My Documents My Computer i My Hetwark Places Z seGlign txt RI_temp nii alignmentResults nii E TransformationMatrices txt alignmentResulks_Grains nii null 60 tif lm 59 tiF 53 tif ie 57 tif 56 tif 55 tif im 54 tif me 53 tif ie 52 tik 4 File name Save as type 51 tif i 50 tif 45 tif i 46 tif im 47 tif i 46 tiF i 45 tik i 44 tiF i 43 tif 42 tif 41 tik 40 tif 39 tif 38 tif l 37 tif Transtorrmationh atrices txt Al Files 36 tif i 35 tif i 34 tiF i 33 tif i 3
15. 2 tif me 31 tiF 20 tif 25 tif 28 tif 27 tif 26 tif i 25 tif 24 tif 23 tif 22 tif Next the user will need to check the resulting alignment of the imported image sequence The macro will align the image sequence Windows may appear over top of one another so the user may need to move them to separate places on their monitor The user will use the scroll bars on the aligned image sequence window to view the alignment The user will need to determine whether or not the images aligned properly To do so the user will visual inspect the aligned image sequence using the scrollbar in the active image window to making sure the images appear to match up between adjacent images Once they have made this determination the user will need to click on ok in the Check Alignment window a Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill SBAA File Edit Image Process Analyze Plugins Window Help IB ocea Opss PAPO Polygon selections Check Alignment Check alignment then click OK When prampted if want to adjust stack and realign then check Realign box ok Result of Ri_temp_ EBR TAO 2041204 pixels 58 bit 406K 7 Next the Adjust Stack amp Realign window appears If the user had a poor alignment then they will need to place a check in the check box next to Realign otherwise they should leave this box unchecked Once the user has made a sele
16. 7 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Masks in the Mask folder this step fails and a warning window appears The warning window asks the user if they would like to Start Over start with step 1 quit exit macro or try import masks again If the user choose to import the masks again they re peat this step import masks again up to 3 times before they are forced to start over start with step 1 Find Mask Directory To Align with Saved Transforms select any file within directory where the Mask folder is located Note Cancel exits entire macro i Cancel Find Mask Folder Directory E aligamentResults Grains nii E 14 tiF C presentationR B E align tet 13 tif My Recent alignmentResults nii im 12 ti Documents B TransformationMatrices txt Gl 11 tif lm 25 t i 10 tif ie 09 tif Desktop Aes ti nr cs tif lm 22 im 07 tif co we ziti Bo a ti E 05 tif My Documents 04 tif i 03 tif im 02 tif May Computer My Network File name m v Places Files of type All Files Cancel Dimensions of Image Sequences Dont match The Dimensions orthe Number of Cell s Masks dont match those ofthe Grains Images Calls Mask 1 Height 204 Width 204 Grains 10 Height 102 Width 102 Can Import Masks Againgonly 3 timesiprior to forced StartOver How ta Proceed ImportvasksAgain IimporthasksAgain Mote Cancel 48 Date 04
17. 8 Next a window will popup that asks the user if they would like to view the 3D projections of their resulting alignment If they select yes the user will see their 3D projections of the alignment and another window will popup asking the user if they would like to save the 3D projections If the user chooses yes these projections will be save if they choose no they won t be saved If the user doesn t select that they would like to view the 3D projections then the user won t be prompted to save the 3D projections View 3D Projections Eg View 3D Projections No Mo Note Cancel exit JQuit acro OK Cancel Mote Cancel exits OK Cancel 9 Next the macro will start over so the user will see the window that appeared in step 1 51 Date 04 25 2008 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Flow diagram for both Plugin and Macro Legend a Q Quit Button gt End Program B Back Button N Next Button o Radio Button Exit End Program Aligning o Brain Gray Scale o Brain Section Gray Scale o Brain Color Version 03 Masking Select Gaussia Blur Sigma Radius 4 amp Threshold Window Popup mla Realign Masked Images 1 Fix Images to improve alignment amp record all changes made so that they can be applied to corresponding images if they exist 2 Select most centered Image o Realign Images INJIQ Start Over Start at 1
18. Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill User Manual for 3D Neural Alignment Table of Contents Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Creating a Working Environment The user will need to have ImageJ loaded on their computer Both the macro and plugin were written using ImageJ version 1 39u thus earlier versions of ImageJ may not be able to run the macro and plugin properly The plugin will consist of a single java file named AlignWi1z_ java and a number of class files that start with AlignWiz like AlignWiz_ class AlignWiz 0Panel class etc all of which should be placed in an ImageJ folder under plugins like ImageJ plugins tools The macro will consist of a single text file microBrainAlignMacro txt that should be placed in the ImageJ macros folder ImageJ macros In addition the user will also need to load the ImageJ plugins TurboReg and MultiStackRegFix so that both tools can align image sequences correctly Moreover the user will also need to load the ImageJ plugin NIfT1 so that the final result can be saved in a SPM usable format Below one can find more information about each of the plugins and how to install all of the required software E C Program Files He File Edit Image Process Analyze a Window Help Be Egeo A S ESS instal ctri shittem E m Elliptical or brush sele
19. Hill If you choose to save results a file window will pop up and prompt you to choose where to save your aligned results All results will be saved as nii files even if you select otherwise in the file window If you choose to save and view 3D projections the projections will be generated first and then a file window will pop up to prompt you to choose where to save your aligned results You will not be prompted to save the 3D projections You can view them and close them or save them manually Save aligned image as nii Fe Ss 13 tik Sa 2a i 14 tif 29 My Recent 15 tif 30 Documents 16 tif EJ 3l E 17 tif a 22 18 tif 33 Desktop 19 tif 3 34 201 tif 35 S 21 tif 36 22 tif 337 My Documents 25 tif 32 T 24 tif 39 WE 25 tif 40 My pee eet z 27 tif 42 k 1 biy Network File name result lignedCellz nii Fl ces Save ae hype Jal Files Cancel we 9 Results have been saved You can choose to start over or exit Start over takes you back to the starting panel Step 1 Results have been saved _ Start over O Exit 14 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Troubleshooting e Complex eigenvalues If you are aligning colored images and get a window saying Complex eigenvalues found Ignoring imaginary parts simply click OK Alignment should continue and work as normal
20. Number Increment Scale and Contains Note that all these fields but contains will default to the values entered in when the user imported in the cells sequence in step 3 We feel that none of these fields should change with the excep tion of the contains field if the corresponding images truly correspond to the cells imag es But we allow the user to change these fields if for some reason the dimension of the corresponding images differ from that of the cells one would want to use a different scale value so that both images had the same dimensions when imported into the macro Once this is complete the user clicks ok and the corresponding images are imported in and masked Please note that if the dimensions of the imported images don t match the di mensions of the cells images a error message will appear and the user will be prompted to re import repeat this step the corresponding images again and they will only be allowed to re import 3 times before the macro starts over starts at step 1 Import Grains Image Sequence Toimportthe corresponding image sequence select an image file contained in the desired image sequence Note Cancel exits entire macro i Cancel 4 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill 14 Select an image contained in the image sequence Look ir i gt Cased z alignmentResults nii 50 tir E 35 tif 4 E xalign tet im
21. acro i Cancel 43 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Load transformation file Look in i gt Cased E setlign txt 51 tif im 36 tif RI _temp nii 50 tif 35 tif My Recent alignmentResults nii im 49 tif me 34 tit Documents E E 46 tiF im 33 LiF alignmentResults_Grains nii E 47 tif 32 tif E nul im 46 tiF me 31 tif j i 45 tiF jm 30 tif im 44 tif im 29 tiF 43 tif 26 if me 42 tif E 27 ti My Documents r 41 tif ie 26 tif 40 tif i 25 tif i im 39 tif E 24 tif Teo 53 ti 38 tiF 23 tif 37 tif 22 tif i My Network File name TransformationMatrices txt i Places Files of type All Files Cancel Failed Grains Alignment ES Failed To Aligned Grains Images Try to Align Again Yes Mote Cancel exits entire macro OK Cancel 16 Now that the corresponding images are aligned The user will need to choose where to store the results of the alignment and what they would like to name the resulting n11 file The window Save Results of Alignment will popup and the user will click ok Next the user needs to select where to save the resulting nii file and what to name it We suggest storing it in the same folder as the one that contained the images that were im ported in for alignment Once the user has made their folder and name selections they ne
22. ages will be re masked and the Select Alignment Start Image window pops up and the user will repeat steps 6 gt 10 above and follow the applicable directions for those steps 8 Now that the user has a correct alignment They need to choose where to store the re sults of the alignment and what they would like to name the resulting nui file The win dow Save Results of Alignment will popup and the user will click ok Next the user needs to select where to save the resulting nii file and what to name it We suggest storing it in the same folder as the one that contained the images that were imported in for alignment Once the user has made their folder and name selections they need to click Save to save the results 26 Date 04 25 2008 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Save Results of Alignment Select where to save results of alignment as nii file Mote Cancel exits entire macro Save as Nifti My Recent Documents Desktop My Documents My Computer a My Network Places 9 Next a window will popup that asks the user if they would like to view the 3D projec tions of their resulting alignment If they select yes the user will see their 3D projec P xAlign tt l 50 tif TransformationMatrices txt 49 tif alignmentResults ni i 45 tif alignmentResults Grains nii lm 47 LiF null l 60 iF l 59 iF l 58 tik l 57 tik l 56 tik
23. ality of SPM has not been tested 2 Open MATLAB and change the Current Directory to the spm5 folder the main folder where the SPM files are saved You can do this by clicking the symbol next to Current Directory and browsing through your folders 3 Inthe Command Window type in spm and hit enter A startup window should pop up for SPMS Select fMRI time series to start SPM Two more windows should pop up 4 On the upper left window click Display and an Image to Display window should pop up On the left hand side of the Image to Display window browse through your folders and select the folder where your NIfT1 n11 file is located Select the appropriate file and click Done 5 Your image stack should now open in SPM Choosing the Plugin verses Macro We will have a plugin and a macro with similar functionality They will both go through the set of screens as specified in the flow diagram on the last page of this document The only real difference is that the macro runs faster while the plugin has a more elegant user friendly interface The macro runs faster in masking image import and other features that specifically use the built in macro language features The Plugin provides a much more elegant user interface and allows for wizard like features which allow the user to return to the previous selections using the back button and proceeding forward using the next button This
24. ction they will then need to click on ok in the Adjust Stack amp Realign window The user is only allowed to realign 3 times Adjust Stack amp Realign Realign Limited to 3 Realignments this is will f Note Cancel exits entire macra i Cancel 8 Note that if the user selects to realign they will be able to select a different image as the most centered image for when the alignment is performed Moreover the user is also able to adjust the masked image sequence as an attempt to get a better alignment The user can adjust the masked image sequence by using the Translate and Rotate processes in ImageJ they are found under Image in the tool bar Note that if the user does 38 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill 10 use Translate to re center an image in the sequence or Rotate to rotate an image in the sequence to the left or right they need to record all changes they make so that the exact same changes Translate amp Rotate commands can be made on the same image in the corresponding image sequence the grains images In addition the user also needs to make sure they use they do not use the flood fill tool to fill in the background color in the images that they Translate instead the user should leave any corrections black so that the alignment can process correctly on a brain section If the user decides to rotate the images they
25. ctions Shortcuts gt Run Utilities gt Startup Macros Hi New Record I How to select amp run a Edit 5 ae Compile and Run SS 3 5 i 5 Oa 3D Where to load ABSnake the Macro Analyze Examples gt Filters gt tools i Graphics b E plugins Input Output gt i 3D Macros gt Dems loadthe Howie aeea UNS SEE oe k Plugin AlignWiz zs Filters Plugin Read Me Close All Without Saving im Graphics OO Deriche A a ImageJ Updater acros E Stacks MultistackRegFix E _ StackReg TurboReg The following are ImageJ plugins used in our project Used to allow alignment of the imported image sequences TurboReg http bigwww epfl ch thevenaz turboreg x MultiStackReg http www stanford edu bbusse work downloads html Used to export image sequence in a SPM readable format NIfTi hhttp rsb info nih gov 1j plugins nifti html ImageJ general information downloads and documentation Main information page http rsb info nih gov 1j Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Documentation on ImageJ Developer s Resources API _ http rsb info nih gov 1j developer api index html Built In Macro Functions http rsb info nih gov 1j developer macro functions html Statistical Parametric Mapping Software SPM software http www fil ion ucl ac uk spm MultiStackReg was not directly used It was revised and named MultiStackRegFix and al
26. e E Files ie 35 tif we 34 tif 33 tif 32 tif i 31 tif 30 tiF 29 tif i 28 tif we 27 tif im 26 tif 25 tif im 24 tif ie 23 tif 22 tif 21 tif Version 03 tions of the alignment and another window will popup asking the user 1f they would like to save the 3D projections If the user chooses yes these projections will be save if they choose no they won t be saved If the user doesn t select that they would like to view the 3D projections then the user won t be prompted to save the 3D projections 32 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill 1 10 224 00 168 00 pixels 224x168 RGE Note Cancel exits OK Cancel 16 Next a window the macro will start over so the user will see the window that appeared in step 1 ll Aligning a brain section cells and grains l The user selects the type of alignment from a drop down menu a Grayscale Brain aligning whole brain that is in grayscale b Color Brain aligning whole brain that is in color c Brain Section aligning brain section d Quit User chooses to align Brain Section 33 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Begin Alignment Macro Align Brain in Grayscale Brain in Color or Brain Section Mote Cancel exits entire macra Type of Alignment Grayscale_Brain Grayscale
27. e alignment and what they would like to name the resulting nii file The window Save Results of Alignment will popup and the user will click ok Next the user needs to select where to save the resulting nii file and what to name it We suggest storing it in the same folder as the one that contained the images that were imported in for alignment Once the user has made their folder and name selections they need to click Save to save the results 31 Date 04 25 2008 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Save Results of Alignment Select where to save results of alignment as nii file Mote Cancel exits entire macro Save as Nifti Save in gt Cased T Align kst TransformationMatrices txt im 49 tif My Recent Documents Desktop My D ocuments My Computer My Network Places 15 Next a window will popup that asks the user if they would like to view the 3D projec tions of their resulting alignment If they select yes the user will see their 3D projec alignmenthesults nii l 50 tif 48 tif alignmentResults Grains nii 47 tif null l 60 tiF iF 59 tiF l 56 tik l 57 tik l 56 tif l 55 tif l 54 tif l 53 tif E 52 tif im 51 tif i l 46 tif l 45 tif l 44 tif l 43 tik im 42 tif im 41 tif im 40 tif l 39 tik 38 tif lm 37 tif im 36 tif File name aignmentA esulta nii Save as Ipp
28. e user had re aligned the cells images then the Adjust Corresponding Images based on Realignment window will popup If this happens then the user needs to apply the same rotation and translation changes to the same images that they had applied these to when they performed the re alignment of the cells images If the user only selected a different center most image then the user doesn t need to adjust the corresponding im ages using translation and rotation Once the user is done making adjustments they just click ok If the user did not re align the cells images they can just proceed to the next Step Adjust Corresponding Images based on Realignment Adjust stack of Corresponding Images based on rotation translation and other changes recorded and made during realignment then click OK to align 13 Next the Load Transformation File window appears and the user clicks ok and then se lects the transformation file from where it is located and clicks open so that it can be used to align the corresponding grains images Note if the user followed our sugges tions the file should be named TransformationMatrices txt and located in the same fold er as the cells images If for some reason the user selects a file that isn t the transforma tion file OR selects the incorrect transformation file one for more images in the se quence or images of different dimensions a warning pops up and the user wi
29. ed to click Save to save the results Save Results of Alignment Select where to save results of alignment as nii file Note Cancel exits entire macro Cancel 44 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Save as Nifti 50 tif 35 tif ad TransformationMetrices tt i 49 tif i 34 tif MyHecent Fel alignmentResults nii i 48 tif i 33 tif Documents f alignmentResults_Grains nii E 47 tif 32 tif E nul l 46 tif me 31 tif 60 tif l 45 tif 20 tif Desktop ie 59 tif E 44 tif 29 tif l 58 tik l 43 tik lm 28 tiF l 57 tif lm 42 tif 27 tif l 56 tif 41 tif 26 tif i s r me 40 tif me 25 tif E 54 tif E 39 tif E 24 tif 53 tiF 38 tif 23 tif 52 tif l 37 tif i 22 tif 51 tif l 36 tif i 21 tif lt My Network File name aignmentResults ni Places Save as type E Files Cancel My Computer 17 Next a window will popup that asks the user if they would like to view the 3D projections of their resulting alignment If they select yes the user will see their 3D projections of the alignment and another window will popup asking the user if they would like to save the 3D projections If the user chooses yes these projections will be save if they choose no they won t be saved If the user doesn t select that they would like to view the 3D projections then the u
30. el Ee My Recent Documents Deskto p My Documents bly Network File name 4 The screen should now read Alignment in process Do not click the scrollbar in the image window during this process Alignment in process 7 Post alignment processing You can look at the results and choose to realign save and view 3D projections save only or start over 10 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Post Alignment Processing O Realign Images O Save Results and View 3D Projections Save Results Start Over If you choose to save results a file window will pop up and prompt you to choose where to save your aligned results All results will be saved as nii files even if you select otherwise in the file window If you choose to save and view 3D projections a file window will pop up to prompt you to choose where to save your aligned results then the 3D projections will be generated You will not be prompted to save the 3D projections You can view them and close them or save them manually Saye aligned image as nii Save in So fish2 e Bg FE Fe Sj aligned Ss 13 tik Sa 28 Lsi gt Mask 14 tif 29 My Recent _ Templmages 15 tif EJ 30 Documents 1 tif 16 tif 3 E z tiF 17 tif 22 3 tiF 18 tiF 333 Desktop 4 tif 19 tif S 34 S 5 tif 20 tif 335 S 2 6 tir 21 t
31. ge to be the start image use scrollbar to select the most centered image prior to clicking OK ox RI_temp nii 110 2244168 pixels ROB 1 4MB 22 Date 04 25 2008 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Version 03 3 Next the user will need to indicate the location in which they want to save the Transfor mation file that will result from the image sequence alignment The user first clicks ok in the Save Transformation File window Then they will need to select the location and name of the saved transformation file We suggest that the user saves this file with its default name TransformationMatrices txt in the same folder that they have stored the cells image sequence Save Transformation file Select where to save the transformation text file Note Cancel exits entire macro i Cancel Save transformations at Save in i gt Cased T E x ilign tet 51 tif 36 tif RI_temp nii i 50 tif i 35 tif amp My R ecent Documents E Desktop v My Documents m a T E alignmentResults nii 2 TransformationMatrices txt E alignmentResults_ Grains ni 52 tf i 49 iF i 48 tif im 47 tif i 46 tiF i 45 tik im 44 tiF im 43 tiF im 42 tiF i 41 tiF i 40 tiF i 39 iF i 38 tif i 37 tif i 34 tiF i 33 tif i 32 tif a 31 tif 30 tif 29 tif i 20 tiF i 27 tik i 26 tif 25 tif 24 tif 23 tif
32. he active image window 18 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Import Cells Image Sequence To importthe image sequence select an image file contained in the desired image sequence Note Cancel exits entire macra pk FE alignmentResults nii 50 tif 35 tif i E xAlign txt E 49 tif 34 tif My Recent 5 TransformationMatrices txt me 48 tif i 33 tif Documents alignmentResults_Grains nii i 47 tif i 32 tiF E nul i 46 tif le 31 tif me 60 tif i 45 tif 30 tif 59 tif E 44 tif E 29 tif E 55 tif me 43 tif E 28 tif 57 tf lm 42 tif C1127 tif 56 tf me 41 tif i 26 tif 55 1 40 tif i 25 tif 54 tiF 39 tif i 24 tif E 53 tif 38 tif E 23 tif 52 1 37 tif i 22 tif 51 tif i 36 tif ie 21 tif lt f Fl ym File name 27 ti he Files of type all Files Import Cells Image Sequence x Starting number End number Increment Sc alert File name contains Note Cancel exits entire macro JRK Cancel 4 Next the user needs to create masks for the imported image sequence The user will need to use Gaussian blur and Thresholding ImageJ functions to do this 19 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Step 1 First the user will be asked to set the Gaussian blur the program defaults to a value of 4 Note that the higher the value the
33. icable plugin folder in ImageJ ex ImageJ plugins tools or ImageJ plugins stacks 3 Download NIfTi Input Output plugin http rsb info nih gov 1j plugins nifti html save file and load nifti_10 jar file into Input Output plugin folder in ImageJ ImageJ plugins Input Output 4 Download MultiStackRegFix plugin http www cs unc edu Courses comp523 s08 3D_Neural home html extract files and load java and class files into any applicable plugin folder in ImageJ ex ImageJ plugins tools or ImageJ plugins stacks Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill 5 Download AlignWiz plugin http www cs unc edu Courses comp523 s08 3D_Neural home html extract files and load java and class files into any applicable plugin folder in ImageJ ex ImageJ plugins tools or ImageJ plugins stacks 6 Download alignBrainOrBrainSection txt http www cs unc edu Courses comp523 s08 3D_Neural home html and put the file in ImageJ macros in ImageJ 7 Importing Results from ImageJ Plugin amp Macro into SPM 1 Load MATLAB and SPM SPM can be found at http www fil ion ucl ac uk spm software spm5 You will need to complete the registration form to download it Unlike all other open software for this project MATLAB is a license product that the user will need to purchase in order to use In addition other than opening and viewing the nii file in SPM other function
34. if 336 Sy 7 tiF 22 tif 237 My Documents 5 tif 5 tif E 35 9 tik 5 24 tif Sa 39 Br 10 tif 25 tif 40 d A 11 tif 26 tif 41 o omes ERA B27 tit 42 K T Cancel Ee resulbAlignedCells nii biy Metwork File name Save as Ipe fan Files 8 Results have been saved You can choose to import and align corresponding grains images start over or exit Start over takes you back to the starting panel step 1 11 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill If you choose to import and align corresponding grains images you will be taken to Step 3 and you will go through the same process without Steps 5 and 6 5 x Results have been saved Import corresponding images for alignment O Start over O Exit If you selected to align using Saved Transforms 6 Pre Alignment Processing Fix any image imperfections You should apply any rotations or translations that were applied to the original sequence from which the transformation file was obtained You do not have to scroll to the most centered image to align The alignment will start on the slice indicated by the transformation file iol Pre Alignment Processing Apply any rotations and translations applied to the original sequence 7 Import transformation and mask files Select the transformation file you wish to use as well as the mask file t
35. ign window The user is only allowed to re align 3 times Adjust Stack amp Realign Realign Limited to 3 Realignments this is will f Note Cancel exits entire macro i Cancel 6 Note that if the user selects to realign they will be able to select a different image as the most centered image for when the alignment is performed Moreover the user is also able to adjust the masked image sequence as an attempt to get a better alignment The user can adjust the masked image sequence by using the Translate and Rotate pro cesses in ImageJ they are found under Image in the tool bar Note that if the user does use Translate to re center an image in the sequence or Rotate to rotate an image in the sequence to the left or right they need to record all changes they make so that the exact same changes Translate amp Rotate commands can be made on the same image in the corresponding image sequence the grains images In addition the user also needs to make sure they use the flood fill tool to fill in the background color in the images that they Translate so that the alignment can process correctly only use flood fill of the background color for entire brains for brain sections leave the back ground black If the user decides to rotate the images they can just click on the check 24 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill box Fill with background col
36. ignmentResults mii E Align txt E TransformationMatrices txt alignmentResulks Grains nii null i 60 tif 59 tif 58 tif 57 tif 56 tif i 55 tik S tif 53 tif 52 tif ie tif 4 i 50 tif 49 tif im 48 tiF me 47 tif im 46 tif im 45 iF im 44 tif i 43 iF im 42 tif im 41 tif i 40 tiF i 39 tif im 30 tif im 37 tik 36 tif 35 tir 34 tif 33 tiF 32 tif 31 tif 30 tiF 29 tif 28 tif 27 tif 26 tif 25 tif i 24 tif i 23 iF hy Hetwork File name Places 21 ii Files of type All Files Cancel Import Corresponding Grai Eg Starting number End number 10 Increment Scaler File name contains Note Cancel exits entire macro OK Cancel 4 Next the user will need indicate where the Mask folder is located The user needs to click ok in the Find Mask Directory window Next the user needs to select any file not folder within the folder that contains the Mask folder If the user fails to select the cor rect folder location then the Macro fails and aborts leaving windows open that need to be closed so the user will need to close these windows and restart the Macro If the user se lects the correct location of the mask folder then the imported images are masked and the macro continues to process If the images imported are different dimensions than the 4
37. ing n11 file The window Save Results of Alignment will popup and the user will click ok Next the user needs to select where to save the resulting nii file and what to name it We suggest storing it in the same folder as the one that contained the images that were im ported in for alignment Please note that the default location will be the location of where the stored transformation file was found the user will need to select the appropriate loca tion for the results Once the user has made their folder and name selections they need to click Save to save the results 50 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Save Results of Alignment Select where to save results of alignment as nii file Mote Cancel exits entire macro Save as Nifti a 50 tif i 35 tif 4 49 tif 34 tif My Hecent alignmentResults ni E 48 tiF E 33 tif Lacum alignmentResults_Grains nii im 47 tif 32 tif E rui 46 tif 31 tiF i 60 tiF 45 tif 30 tif Desktop 59 tif E 44 tif 29 tif 58 tif E 43 tif z8 tif me 57 tif E 42 tif 27 tif i l 56 tif me 41 tiF 26 tif i Dae amer 55 tif E 40 tif 25 tif 54 tif 39 tif E 24 Jf 53 tif 38 tif 23 tif 52 tif 37 tif zz tif 51 tif im 36 tif i 21 tif lt My Network File name algnmentAesults ni Places Save as Ippe E Files
38. l be prompted to re import repeat this step the corresponding images again and they will only be allowed to re import 3 times before the macro starts over starts at step 1 Also note that when trying to import with nega tive numbers floats or something of that nature the user will get error messages stating the problems with the inputs Import Grains Image Sequence Toimportthe corresponding image sequence select an image file contained in the desired image sequence Note Cancel exits entire macro E slignmentResults ni 50 tiF i 35 iF Ne E xAlign txt 49 tif 34 tif My Recent E TransformationMatrices txt 48 tif i 33 tif Documents aj alignmentResults_Grains nii E 47 tif i 32 tif hay Network Places E null 60 tif lm 59 tif 58 tif 57 tif lm 56 tif i 55 tik File name Files of type im 46 tif me 45 tif im 44 tif im 43 iF im 42 tik me 41 tif 40 tif i 39 tif me 38 tif 37 tif we 36 tif i 31 tiF 30 tif 29 tif 28 tif l 27 tif im 26 tif 25 tif 24 tif i 23 tif 22 tif 21 tif s Al Files 29 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Import Corresponding Grai fx Starting number End number fo Increment laa fa hz Scalers File name contains Note Cancel exits entire macra OK Cancel 12 If th
39. ll exit Ifthe user selects yes then the macro will allow the user to align corresponding grains images Perform Corresponding Image Aligm Ed Align Corresponding Image Grainsi ves Mote Cancel exits entire macro OK Cancel 11 Next the user needs to import the corresponding grains image sequence The user 1s prompted to select the location of a single file from the image sequence and then the user is prompted to enter the following fields to allow the user to import the cells image se 28 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill quence Start Number End Number Increment Scale and Contains Note that all these fields but contains will default to the values entered in when the user imported in the cells sequence in step 3 We feel that none of these fields should change with the excep tion of the contains field if the corresponding images truly correspond to the cells imag es but we allow the user to change these fields just in case the dimensions of the grains images differ from the cells images thus scale must be set differently so that both images have the same dimensions when imported into the macro Once this is complete the user clicks ok and the corresponding images are imported in and masked Please note that if the dimensions of the imported images don t match the dimensions of the cells images an error message will appear and the user wil
40. ll increasingly blur the image instead of resetting the blur so we suggest starting out with a small sigma if you wish to repeatedly blur your image by repeatedly clicking OK ox Masking image sequence Sigma for Gaussian blur 4 Once you click OK a threshold window will appear Please adjust the upper and lower thresholds so that all portions of the image you wish to retain are covered All other portions will be converted to a uniform background color To apply the threshold you can simply click Next without clicking anything on the Threshold window loj x Masking image sequence Sigma for Gaussian blur 4 Please adjust the threshold so that the desired portions are covered click Apph click OK without checking any boxes then click Next Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill H Threshold k ol x Ioj x 22 2 280x210 pixels 8 bit 115K Fed Red a Auto Apply Reset Set 6 Pre alignment processing 1 Erase any image imperfections with ImageJ s paintbrush tool Do not rotate or translate images at this point All rotations and translations should be performed at step 4 If you are aligning colored images the images will appear as grayscale here You can still erase any imperfections and the resulting aligned stack will be in color 2 Scroll to the most centered image and click Next The most ce
41. ll only be allowed 3 more chances to select the right transformation file Please note that if the user selects an incorrect file one that isn t a transformation file the ImageJ exception win dow may popup This happens because exceptions were thrown when the alignment pl ugin attempted to read a file that wasn t a transformation file Once the user is done us ing the macro the user will be able to close the exception window 30 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Load Transformation file Select where to load the transformation text file Note Cancel exits entire macro i Cancel E xAlign tst 51 tif 36 tif i RI_temp ni 50 tif ie 35 tif My Recent ue en im 49 tiF im 34 tif zie meig on ye 4e tir me 33 tif ra ii me 47 tik 32 tif im 46 tif im 31 tif Desktop 60 me 45 tif lm 30 tif im 44 tif l 29 tif 43 tif z8 tif 42 tif E 27 tif 41 tif z6 tif 40 tif l 25 tif 39 tif im 24 tif i 38 tif 23 iF me 37 tif im 22 tif hy Metwork File name TransformationMatrices txt z Places Files of type All Files Cancel Failed Grains Alignment EW Failed To Aligned Grains Images Try to Align Again ves Y Note Cancel exits entire macro OK Cancel 14 Now that the corresponding images are aligned The user will need to choose where to store the results of th
42. lows the following additional functionality 1 Error checking to guarantee that the alignment plugin MultiStackRegFix ran to completion 2 Error checking to guarantee that if a saved Transformation file is used for alignment the alignment will only occur if the image sequence to be aligned has the same number of images and the same dimensions as the image sequence that originally generated the saved Transformation file 3 Modified to allow only three types of alignments when run in batch mode from a plugin or macro a Rigid body alignments that save a Transformation file b Alignments using a saved Transformation file c Alignment using a saved Transformation file Same as b except the program does not prompt the user to select where the saved Transformation file can be found 4 Modified to prevent warning messages from closing the macro if they appear during execution of the MultiStackRegFix plugin 5 Allows rigid body alignment and alignment from saved transforms for color images previously one could only align grayscale images in this way Loading ImageJ and all Needed Plugins and Macros 1 Download and Load ImageJ Go to http rsb info nih gov 1j download html and select the appropriate download option Download ImageJ bundled with Java if you do not already have Java on your computer 2 Download TurboReg plugin http bigwww epfl ch thevenaz turboreg extract files and load java and class files into any appl
43. more blurred the resulting image will appear Once a value has been selected the user clicks OK Step 2 Next the user is asked to mask out the background using by picking the correct threshold value First the Threshold window titled Threshold may appear underneath the two other windows that are open at the time the active image sequence window and the User Picks Threshold to Mask Out Background window If this is the case the user will need to drag all three windows to separate locations on their monitor Once the user is able to get access to the Threshold window they need to slide the bottom most scrollbar upper threshold towards the right until the area that encompasses the entire brain for each image in the image sequence is entirely filled with red If other areas in the image sequence fill with red in the process that is ok as long as the entire image isn t all red If the entire image is all red one need to move the bottom most scroll bar in the threshold window to the left enough so that the entire images aren t all red Once the images in the image sequence all have the entire brain area covered with red the user needs to click the Apply button in the Threshold window Sel Step 1 1110 01 2241168 pixels 85 bit 3686 Select Gaussian Blur fx Sigma Radiusi 4 Note Cancel exits entire macro OK Cancel User Picks Threshold to Mask Out Background Use the Threshold tool to
44. n Grayscale Brain in Color or Brain Section Note Cancel exits entire macro Type of Alignment Grayscale_Brain Grayscale Brain Color_Brain Brain_ Section Ok Cancel Quit 2 The user selects the type of alignment from a drop down menu a TurboReg aligns with TurboReg b SavedTransforms aligns with saved Transforms file from previous alignment c Quit The user chooses to align TurboReg Type of Alignment Align with TurboReg or Saved Transforms Mote Cancel exits entire macro Type of Alignment TurboReg TurboReg Saved Transforms uit gt Next the user needs to import the cells image sequence The user is prompted to select the location of a single file from the image sequence and then the user is prompted to enter the following fields to allow the user to import the cells image sequence Start Number a positive integer that indicates the first image in the sequence End Number a positive integer that indicates the last image in the sequence Increment a positive integer of value 1 or greater that indicates by what increment images are to be imported in Scale a positive integer that indicates how much to reduce the resolution of the imported images where 100 indicates no reduction Contains a string that is common to all image sequence file names After the user enters these fields and clicks ok the image sequence will be imported into ImageJ and appear in t
45. nt e g translation rotation 2 Fix Image Imperfections o Align amp Mask Images N Q 6c Aligned amp Masked Images o Save Results amp View 3D Projections o Save Results o Start Over o Exit N Q Save amp View Projections 7b Save Results Saved 3D Projections o Yes o No N Q 8c Saved Results o Start Over o Exit IN Q 52
46. ntered image is an image with an orientation which you wish to remain the same after alignment For instance it may be the image with the most even amount of white space on all sides lol x Pre Alignment Processing 1 Erase any image imperfections but do not rotate or translate image 2 Select the most centered image to align on 3 The image window will now scroll through and mask each slice After that is complete a popup window will appear to prompt you to choose where you want to save your transformation file The default folder and file name generally works unless you are doing multiple alignments on images in a single folder in which case you would want to rename the default file name so no files are overwritten The mask images will be saved under the default folder in a folder named Mask Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Save transformations at Save in E fish de t i Ez Sj aligned 5 13 tif Sl 26 gt Mask 14 tif 29 _ TempiImages 15 tif Sa 30 1 tif 16 tif 31 Z z tiF 17 tif 332 3 tif 18 tif 33 4 tiF 19 tif 334 5 tif 20 tif a5 J amp tif 21 tif 36 7 tif 22 tif 37 8 tif 23 tif 33 9 tif 24 tif 2 39 10 tif 2 25 tif 40 J 11 tif 26 tif 41 12 tik By 27 tif By 42 a anstormationtd atrices tat Save ae type jal Files Canc
47. o be applied If you are working on images of an entire brain simply select the mask for the first image 12 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill of your sequence and the rest should be automatically imported Please note the mask images should be saved in a folder named Mask and the images should be numbered starting with the number 1 and the files should contain the word mask For instance if your sequence contains grains3 tif grains4 tif grains5 tif grains20 tif we expect your mask files to be called mask1 tif mask2 tif mask3 tif mask18 tif The dimensions of the mask files and the transformation file should both match the dimensions of the sequence you imported Otherwise you will get an error if the image height image width or number of images is inconsistent with the selected mask or transformation file MEE impor Transformation and Mask files Transformation file First mask image 8 Post alignment processing You can look at the results and choose to realign save and view 3D projections save only or start over 5 x Post Alignment Processing O Realign Images O Save Results and View 3D Projections Save Results O Start Over Back _Nex If you choose to realign you will be asked to import a different transformation file and mask file 13 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel
48. o go back to the previous panel iol xi Import image sequence Start number End number Increment File name contains ScCale t OU 2 100 If you selected to align using TurboReg 4 Fix Images 1 If your slices are all roughly oriented the same way you can go ahead and click Next However if the orientation between two adjacent slices differ greatly for instance if the slices are rotated more than 30 degrees please rotate or translate them directly using ImageJ You can go to Image gt Translate or Image Rotate to perform these operations Make sure the Fill with background color box is checked if you are rotating If you are translating you will have to fill the background color in with the flood fill tool afterwards 2 If you chose to rotate or translate any images and are importing a corresponding image sequence later please remember all changes and apply them to the corresponding image sequence when prompted to iol xi Fix Images 1 Rotate or Translate if orientation between slices differ greatly 2 Remember all changes made and apply them to corresponding sequence later 5 Masking image sequence Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill First select a sigma value for the Gaussian blur The default value is set to 4 Enter a higher value for more blur and a lower value for less blur Please note clicking OK more than once wi
49. or to keep the background a consistent color when they rotate the image only click fill with background color if the images to be aligned are en tire brains don t fill with background color if aligning brain sections Adjust show Info Ctri l Properties Ctrl Shift P Color Stacks Hyperstacks RI_temp nii amp leg 110 224166 pixels RGB 1 4MB Crop Ctri Shitt x Duplicate Ctri Shift D Rename Flip Horizontally Flip Vertically Rotate 90 Degrees Right Rotate 90 Degrees Left f arbitrarily Lookup Tables Fix Images for Realignment Adjust Image Stack far improved alignment using rotation amp translation then click OK to align _ If planning to align Corresponding Images record changes so that the same changes can be applied to the corresponding images 25 Date 04 25 2008 Authors Ping Fu and Jennifer Staab UNC Chapel Hill File Edit Image Process Analyze Plugins Window Help BAGO lt A N A Ale pelseler Pla ol 0 y 0 2 0 we2 d h 166 Rotate Angle degrees 15 0 Grid Lines i interpolate Enlarge Image to Fit Result W Previews Ok Cancel File Edit Image Process Analyze Plugins Window Help BAG Ol aaae Oss ed Translate Offset pixels i 5 Y Offset pixels I 5 Version 03 7 Once the user is done adjusting the images using rotation and translation they will want to click ok Once they do that the im
50. r select that they would like to try to align again repeat this step of importing the transformation file quit exit the macro or select no and they will start over step 1 If they choose to try to align again they can only repeat the attempt 3 times before they are forced to start over Load Transformation file Select where to load the transformation text file Note Cancel exits entire macro 49 Date 04 25 2008 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Load transformation file xAlign tek ERI tempni My Recent alignmentResults ni Documents E Desktop My Documents My Computer Places 51 tif 50 tif im 49 iF 40 tiF me 47 tif 46 tif wm 45 tif i 44 tif E 43 tif 42 tif me 41 tif im 40 tiF 39 tiF 38 tiF im 37 tif im 36 tif im 35 tif me 34 tif im 33 tif im 32 tik im 31 tif me 30 tiF 29 tif 20 tif 27 tiF im 26 tif im 25 tif im 24 tif 23 iF im 22 tif Mu Nehvork File name IT ranshormationM atrices txt Y Files of type All Files x Failed Grains Alignment EW Failed To Aligned Grains Images Try to Align Again Yes Mote Cancel exits entire macro OK Cancel Cancel Now that the corresponding images are aligned The user will need to choose where to Version 03 store the results of the alignment and what they would like to name the result
51. rains1 tif grains2 tif grains10 tif but instead have the sequences named cells 1 tif cells2 tif cells10 tif and grains1 tif srains2 tif grains10 tif This way you can and should always fill out the File name contains field if you have image file names containing the same numbers in your folder 15 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Directions for using the Macro General notes and troubleshooting If running both the Plugin and Macro sequentially close out ImageJ and reopen it prior to running the other tool Plugin or Macro dependent on the one that was used first This prevents ImageJ from incorrectly processing the commands that are issued by the second tool to be used If one chooses only to run a single tool like just the Plugin calling the Plugin multiple times in a single ImageJ session will not cause any problems with the Plugin s functionality If macro fails to begin after selecting Plugins Macros and clicking on alignBrainOrBrainSection on the bottom of the drop down menu then one needs to re install the macro to get it to run This will occasionally happen if one terminates the macro using Quit and is presumably a bug in ImageJ Selecting the Cancel Button any time during the running of the Macro causes the Macro to abort and close immediately with no guarantee of saving the progress
52. ser won t be prompted to save the 3D projections Note that the 3D projections for brain sections for the most part do not provide helpful information like they do for entire brain images ections View 3D Projections x Jisa UU Free du View 30 Projections No Mo Note Cancel exitii aero Mote Cancel exits OK Cancel OK Cancel 18 Next the macro will start over so the user will see the window that appeared in step 1 lll amp IV Aligning an entire brain OR brain section using Saved Transformations L The user selects the type of alignment from a drop down menu a Grayscale Brain aligning whole brain that is in grayscale b Color Brain aligning whole brain that is in color c Brain Section aligning brain section 45 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill d Quit User chooses to align Brain Section or Grayscale Brain Begin Alignment Macro Align Brain in Grayscale Brain in Color or Brain Section Note Cancel exits entire macro Type of Alignment Grayscale_Brain Grayscale Brain Color Brain Brain _ Section Ok Cancel Guit 2 The user selects the type of alignment from a drop down menu a TurboReg aligns with TurboReg b SavedTransforms aligns with saved Transforms file from previous alignment c Quit User chooses to align SavedTransforms Type of Alignment Align with TurboReg or
53. should not click on the check box Fill with background color instead they should leave the background color black to allow for proper alignment IB 2 Z type Freehand se Adjust Show Info Ctri Properties Ctri Shift F RI_temp nii SEE Color TAO 204x204 pixels 58 bit 406K Stacks ee Hyperstacks i oe x Bee AR Crop Ctrl Shiftex atid dal PRI Duplicate Ctrl Shift D ara A Rename ar Ctr E Stale Translate Rotate zoom FOF lip Vertically Rotate 90 Degrees Right Rotate 40 Degrees Left Flip Horizontally Lookup Tables Arbitrarily Fix Images for Realignment Adjust Image Stack for improved alignment using rotation amp translation then click OK ta align If planning to align Carrespanding Imaqes record changes s that the same changes can be applied to the corresponding images OK Once the user is done adjusting the images using rotation and translation they will want to click ok Once they do the Select Alignment Start Image window pops up and the user will repeat steps 438 above and follow the applicable directions for those steps Now that the user has a correct alignment They need to choose where to store the results of the alignment and what they would like to name the resulting n11 file The window Save Results of Alignment will popup and the user will click ok Next the user needs
54. that one has made instead just choose Quit when the opportunity is next offered The Macro closes all windows that it opens as it proceeds through its script do not close any windows or open any new windows 1 e importing another set of images unless instructed to do so by the Macro while the Macro is running Opening new windows OR closing open windows when not instructed to do so by the Macro will cause the Macro to run incorrectly and possibly abort midway through its process The Macro tends to open multiple windows one on top of the other as part of the normal functioning of ImageJ Macro language The user may find it necessary to select the active window and move it to a different location to get access to windows that it may have hidden as they are processing their image sequences Please read and follow the directions provided at each step These directions are provided specifically to help prevent the user from making errors in the process of using the Macro so that they will successfully align their image sequence If for some reason the plugin used to align the image sequence fails and throws an error then the error will appear and the macro will be aborted immediately This alignment error would be due to something innately wrong with the imported image sequence that prevented TurboReg from being able to align the images like Insufficient accuracy If the error indicated something that could not be fixed by the user then
55. the ImageJ tool bar and then click on the background color of their image Next the user will select the brush to in the ImageJ tool bar and use this tool to paint the back ground color over any image imperfections like the gray dots circled in blue in the Re sult of Mask window below Image imperfections are areas where the background was not masked out during the masking process Once the user has completed the task they will need to click ok in the Check Masking amp Stack window to proceed to the next Step ZA Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill File Edit Image Process Analyze Plugins Window Help Te a een ae Flood Fill Tool Result of Mask KB Check Masking amp Stack 1110 maski 224x168 pixels 58 bit invert Check masking and if necessary use tools to correct imperfections prior ta alignment then click OK ok 2 Next the user needs to use the scroll bar in the active image sequence window to select the most centered image in the image sequence as the start image for alignment This will help the macro to better align the images Once they have completed this task they should click ok button of the Select Alignment Start Image window Image File Edit Image Process Analyze Plugins Window Help Dale ol lt 4 N AlQ e7 ea gt Select Alignment Start Image Pick the start image for alignment If you don t want the first ima
56. the user would not be able to align the image sequence using our macro and would need to look for other means to align their images 16 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Step by Step instructions To start load the macro for use 1 Open ImageJ go to Plugins gt Macros Install select the alignBrainOrBrainSection txt file from ImageJ macros and click Open 2 The macro should now be installed Go again to Plugins Macros and click on alignBrainOrBrainSection on the bottom of the drop down menu to use the macro ImageJ E File Edit Image Process Analyze eiiies Window Help DB OG o lt 4 A o ESS insta ctri shittem Scrolling tool or press space bar and drag Shortcuts gt Pun Utilities gt Startup Macros New Record B Compile and Run 3D ABSnake Analyze Examples Filters Graphics Input Output Macros Stacks Yv Yv vr vr v vr Yv Yv vr v Tools Read Me Aligning an entire brain in color or grayscale l The user selects the type of alignment from a drop down menu a Grayscale Brain aligning whole brain that is in grayscale b Color Brain aligning whole brain that is in color c Brain Section aligning brain section d Quit The user chooses to align Grayscale Brain or Color Brain 17 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill Begin Alignment Macro Align Brain i
57. type of Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill functionality is not possible using the macro language thus we recommend new and naive user to use the plugin over the macro The macro should be used by experienced users and running multiple alignments where speed is essential Step by Step directions for both of the Plugin and Macro can be found below Directions for using the Plugin General notes Pressing the Enter key is equivalent to clicking the Next button If the Quit button is highlighted by tabbing however the Enter key is equivalent to clicking the Quit button Please do not open additional images in ImageJ while the plugin runs Please do not click on images during masking and alignment This is when the wizard reads Masking in progress or Alignment in progress Please also do not close image windows while the plugin runs with the exception of 3D projections All other windows should close automatically when they are no longer needed Step by step directions 1 What images are you working on Choose whether you are aligning images of an entire brain in grayscale an entire brain in color or a brain section in grayscale io x What images are you working on Entire brain grayscale 2 Entire brain color C Brain section grayscale or color Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill
58. up asking the user if they would like to save the 3D projections If they choose yes these projections will be saved if they choose no they won t be saved If the user doesn t select that they would like to view the 3D pro jections then the user won t be prompted to save the 3D projections Note that the 3D projections for brain sections for the most part do not provide helpful information like they do for whole brain images View 3D Projections Eg View 3D Projections No Mo Note Cancel M Mote Cancel exits OK Cancel OK Cancel 40 Date 04 25 2008 Version 03 Authors Ping Fu and Jennifer Staab UNC Chapel Hill 12 13 Next a window will popup that asks the user would like to Align the corresponding grains images If the user selects no then the macro will start over by preceding back to step 1 If the user selects quit then the macro will exit If the user selects yes then the macro will allow the user to align corresponding grains images Perform Corresponding Image Aligm Eg Align Corresponding Image Grainsi Yes Mote Cancel exits entire macro OK Cancel Next the user needs to import the corresponding grains image sequence The user is prompted to select the location of a single file from the image sequence and then the user is prompted to enter the following fields to allow the user to import the cells image se quence Start Number End

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