Neural Circuit Tracer - Northeastern University
Contents
1. File Image Processing Automated Tracing Trace Editing amp Analysis Manage Plugins View Load Library Open Library Save Library Import SWC Export to SWC Stack 0 06 E fom b gt Macros Settings 4 Bo gear p 2 a I Mi Software for automated tracing of neurites from light microscopy stacks of images eee s 1 3 Contents Mirodu EMO gcse erect ce ece E E cseeecuceeetanteseenacetcssecatecaesccseseecess 2 System requirements and installation ss sssssssesersorssrnssrensnnsnrnsnsnnnnsnnonnnsnnnnnnnnrennnnnnnnnnnnnnnnnnnne nenns 2 File tab loading opening and Saving libraries sssesessessseseeeesesesseteseeseeeseaeeneeaeeteneateneneaeaes 2 Image Processing tab MatLab and ImageJ plugins ssssssssssssssessressrensrensrensrensrensrrrnrnrnrnennnennnsnnnenns 4 1 2 3 4 5 Automated Tracing tab initial trace automated branch merger and online training 5 6 7 8 9 Trace Editing amp Analysis tab trace editing optimization and finding l00ps n s 8 Manage Plugins tab adding and removing MatLab and ImageJ plugins 11 View tab image stack z projection 3D View and CONSOLE s s sssssssssressresrrsrnrsrensneernesneennes 12 Macros tab creating and running MACOS eescsesesesssseeeeseesseseeseesesesesseneseseeseeaesnesaeeseneaeeeeaeas 14 10 Settings tab properties user Manual and website s ssssssressrssrnssnsnrnesrensnnsnnnn2. If Fast Marching is selected the user will need to generate the seed points prior to executing the a a algorithm The seed points can be generated seas automatically by using the Fast Marching dialog box left or manually by right clicking over the Image Minimum Radius 2 Stack window and using the seed point selection menu Step size l right The initial trace will be pais Spi ee generated and displayed in the Remove Point Image Stack window If aa Edit Point Generate Seed Points necessary the user may edit the Remove All Points End initial trace by switching to the Fast Marchi i Trace Editing amp Analysis tab Max Dist No Of Steps is If the initial trace contains multiple topological errors Unisotropy such errors can be automatically corrected by using the online training module of NCTracer To reduce the number of topological errors click on the Calculate SS COStS button to generate cost components for the branch merging scenarios Load the Training History see below and click on the Automated Merger button The initial trace will be broken down to the level of disconnected branches reassembled into tree structures based on the prior training history and redisplayed The above steps can be combined into a macro and run sequentially with no user involvement see the Macros tab section The user may adjust the parameters of the automated tracing algorithms by clicking on the
3. branch merging and online learning algorithms The Settings tab also contains the links to this User Manual and the Neurogeometry group website http www neurogeometry net 11 Examples This section provides examples of automated tracing with NCTracer 4 0 The two datasets used in this section and in illustrations throughout this manual were obtained from http www diademchallenge org 15 Tracing a single arbor File tab e Load an image stack e g any of the Olfactory Projection axons No reduction is necessary Automated Tracing tab e Click on Get Initial Trace and select the Fast Marching method e Generate the seed points automatically or right click over the Image Stack window to open the seed point menu and place the seed points manually e Start Fast Marching and let it run until one of the stopping conditions is encountered You may pause and continue Fast Marching at any time during this process e Click on Complete to generate the initial trace Tracing multiple arbors File tab e Load an image stack e g axons of layer 6 neurons No reduction is necessary Image Processing tab e Apply Multi scale LoG plugin Minimum Radius 2 Step Size 1 Maximum Radius 2 e Use Threshold Background plugin Threshold 10 to 25 e Use Eliminate Small Regions plugin Minimum Region Size 100 Automated Tracing tab e Click on Get Initial Trace and select the Voxel Coding method Click Ok and let the algorith
4. and Rohan Gala and Prof Armen Stepanyants developed and implemented the automated tracing algorithms 17
5. and reduce image background prior to performing automated tracing The current version of NCTracer includes the following image processing plugins MatLab plugins Image plugins Accurate Gaussian Eliminate Small Regions Blur Feature Filters Rank Filters Hessian CP Rolling Ball Measures Background Hybrid 3D Median Filter Multi Scale Gaussian Auto Gamma Multi Scale LoG CLAHE Sigma Filter Plus Contrast amp Brightness Simple Filters Adjuster Image Stabilizer Threshold Adjuster Subtract Background Convolver Mean Shift Variance Filter Threshold Background FFT Bandpass Filter RATS Unsharp Mask New plugins can be created and the existing plugins can be removed in the Manage Plugins tab All image processing operations can be undone redone by pressing Ctrl Z Ctrl Y or by clicking on the icons located at the bottom of the main NCTracer window 5 Automated Tracing tab initial trace automated branch merger and online training File Image Processing Automated Tracing Trace Editing amp Analysis Manage Plugins View Macros Settings Stack 06 E S 8 S e The main steps of the automated tracing algorithm are described in detail in 1 2 Briefly they include the following components e Pre processing Image stack may be filtered to smooth the intensity along the neurites and sharpen the transition between the neurites and the background e Initial trace The centerline
6. wheel All views can be refreshed by clicking on the f icon in the main NCTracer window All three views are equipped with zoom in Zoom out and pan features In the Image Stack and Z Projection windows these features can be accessed by clicking on the Qor ality icons at the bottom of the main NCTracer window and then clicking on the image or dragging the image with the mouse Alternatively these functions can be controlled with Page Up Down and the arrow keys a The user can determine the xyz or1 File Edit Select Transformation View Help coordinates and the intensity of any voxel in the Image Stack window or any pixel in the Z Projection window by pointing the cursor at that location The information will be displayed in the main NCTracer window Positions and calibers of the trace vertices can be displayed in a similar manner In the 3D View window zoom in and zoom out features are controlled with Page Up Page Down or the mouse wheel sfa Panning can be done by pressing the arrow keys while holding down Shift Neural Circuit Tracer started on Tue Apr 02 17 21 55 EDT 2013 The image stack can be rotated in 3D by 1 6 2 62 using the mouse or the arrow keys 3 62 4 62 5 62 6 62 7 62 8 62 9 62 The View tab also displays the Console i5742 window right which records all 76 12 62 13 62 operations performed by the user and 134 the software Sa
7. File tab loading opening and saving libraries File Image Processing Automated Tracing Trace Editing amp Analysis Manage Plugins View Macros Settings OpenLibrary Savelibrary Importswc Exporttoswc sakas E A Q 9 le E E The current version of NCTracer supports image stacks saved in several standard image file formats as well as the MatLab data file format mat Traces must be saved in the mat or the SWC formats of neuron morphology Number of Images starting Image Increment File Name Contains To load a new image stack click on the Load Library button eS located in the File tab of the main NCTracer window After Y Reduction Selecting an image from the stack of images a new Load Z Reduction Library dialog box right will appear The user may select ne ee O S the images to be loaded by providing a starting image epee z number an increment and a characteristic string contained in the file names Image stack can be reduced in the x y and z dimensions during loading The user must specify the come reduction factors positive doubles and chose one of five 512 x 512 x 50 8 bit reduction methods Min Max Mean Median and SD The loaded stack of images will be displayed in the Image Stack window below This window allows the user to scroll through the individual image planes within the stack Multiple image stacks can be loaded sequenti
8. Properties button located in the Settings tab Progression of the automated tracing algorithm can be monitored in the Console window Automated tracing time depends on the image size and the complexity of neurites Training history files can be created in the online training module of the software These files can be generalized on image stacks obtained under similar experimental conditions same class of neurites labeling imaging technique To create a training history file load the image stack obtain its initial trace and calculate the cost components as described above Clicking on the Online Training button will open the Online Training table f Te a Cluster Number No of End Points Confidence oo N o in EN us N pi us us ua wo a us ua N 3 3 3 3 3 4 ro D AMM A tiv tx j ao dants WPerceptron l Load Training History Save Training History WSVM The Online Training table shows branch merging scenarios for different clusters of branch points rows of the table One such cluster is shown in the image on the next page Clicking on different rows of the table or right clicking on different parts of the initial trace in the Image Stack window will move the focus squares from one cluster to another To view different branch merging scenarios within the selected cluster cl
9. ally The dropdown list of open image stacks is located at the bottom of the main ei ieee ace NCTracer window This list can be used j to change the currently selected stack of images All operations performed in the NCTracer will be applied to the currently selected stack only The original image stack the pre processed image stack and the trace are collectively referred to as the Library The Save Library button located in the File tab or icon located in the main NCTracer window _ saves these components in the MatLab mat data file format To open a library click on the Open Library button located in the File tab of the main NCTracer window The user can import export the trace of neurites by clicking on the Import SWC Export to SWC buttons The entire trace as well as individual trees contained in the trace can be exported In the latter case follow the instructions given in the Trace Editing amp Analysis tab section Import export features only support the SWC format of neuron morphology 4 Image Processing tab MatLab and ImageJ plugins Automated Tracing Trace Editing amp Analysis Manage Plugins View Macros Settings ins i ImageJ Filters and Plugins l Change to Original j TEREKE The Image Processing tab contains a number of custom filters written in MatLab and plugins adapted from ImageJ These functions can be used to enhance the intensity of neurites
10. ht the entire tree A subsequent click on one of the vertices will result in the deletion of the tree e Individual vertices of the trace can be moved in x y and z directions Select Move Vertices function and click and drag the vertex in the selected plane To move the vertex to a different plane scroll through the stack to the desired z location and click on the vertex e Individual trees can be moved in x y and z directions Select Move Tree function click and drag a vertex of a selected tree to move the tree in the current plane To move the tree to a different plane click hold on a vertex of the tree and scroll through the stack to the desired z location e To export a single tree in the SWC format the user may select Export Tree to SWC and move the mouse over the tree of interest to highlight it A subsequent click on any of the vertices of the tree will initiate the export process To export the entire trace the user must click on the Export to SWC button located in the File tab of the main NCTracer window e End terminates the previously selected action and disables active vertices During manual tracing the user can scroll through the image planes of the stack by using the slide bar at the bottom of the Image Stack window or the mouse wheel zoom in Zoom out with Page Up Down and pan in the plane of the stack arrow keys Manual tracing operations can be undone redone by pressing Ctrl Z Ctrl Y or by clicking on the i
11. ick on next scenario or previous scenario signs or select a scenario number from the dropdown window in the table Once the correct scenario is selected 27 60 26 512x512 pixels 8 bit 15MB the user must assign to it a positive weight typically 1 Weighted scenarios will be highlighted in yellow see the table above After such training on several clusters of branch points select WPerceptron or WSVM classifier and click on the Apply button to generate the Training History Correctly classified branch mergers will be highlighted with green in the table while erroneous mergers will be highlighted in red Erroneous mergers need to be corrected or their weights must be reduced to zero to ensure that the classifiers are not trained on errors The user may continue training or save training history and move to another training stack New training examples will be appended to the old training history as long as the latter is loaded check the loaded Training History path in the Automated Tracing tab Training on about 100 clusters of branch point is typically sufficient to achieve error rates of less than 5 Ambiguous branch merging examples should not be used for training 6 Trace Editing amp Analysis tab trace editing optimization and finding loops File Image Processing Automated Tracing Trace Editing amp Analysis Manage Plugins View Macros Settings Optimize Trace Optimize Radius Ad
12. icons located at the bottom of the main NCTracer window The user must be in the Trace Editing amp Analysis tab to perform undo redo operations on the trace The intensity and the xyz coordinates of an image voxel at the position of the cursor as well as the caliber radius and the xyz coordinates of a trace vertex closest to that location are displayed in the main NCTracer window Trace segments whose vertices are located in the current plane of view are drawn with slightly thicker lines Trace can be switched on and off viewed on a z projection and in 3D see the View tab section These views can be refreshed by clicking on the 7 icon The trace can be optimized with two optimization functions located in the Trace Editing amp Analysis tab of the main NCTracer window details of these Optimize Branch Points optimization procedures are described in 2 The Optimize Terminal Points Optimize Trace function right optimizes both the positions and calibers of vertices in the trace Branch Density of Points l ES and terminal points can be kept fixed during the mean optimization Default values of optimization Mmm Number of Iterations parameters are displayed in the Optimize Trace Trace Stiffness dialog box The Optimize Radius function below Radius Stiffness calculates the optimal calibers of trace vertices while Optimization Step ie the vertex positions remain unchanged C Optimize Radius m The average density of
13. ion Vector Branch Caliber check boxes Additional input Parameter Name Parameter Type output parameters may be lt Array included by specifying the number and the order of these parameters in the Input Output Selection window Drag the rows to match the order of input and output parameters to that in the MatLab function Newly added plugins which modify the image stack or the trace will appear in the list of MatLab Filters and Plugins located in the Image Processing tab Trace analysis plugins will appear in the list of Custom MatLab Plugins in the Trace Editing amp Analysis Tab Image and MatLab plugins can be removed from NCTracer by selecting the plugin type and clicking on the Remove Plugins button 8 View tab image stack z projection 3D view and console Automated Tracing Trace Editing amp Analysis Manage Plugins View Macros Settings Processed Image Trace E Z Projection 3DView V Console s12 The View tab of the main NCTracer maxop1 512x512 pixels 8 bit 256K window allows the user to display the stack on a z projection and in 3D The user can switch between the Original and Pre processed image stacks radio buttons as well as display hide the trace Trace check box The user can scroll through the individual image planes with the slide bar located at the bottom of the Image Stack window or by using the mouse
14. just Point Density Show Hide Loops Eliminate Small Trees Delete Trace Custom MatLab Plugins i BAAS E The Trace Editing amp Analysis tab makes it possible to trace neurites manually and or edit automated traces Right click over the Image Stack window to open the manual tracing menu image on the next page and enable the following functionalities e Select Start and click at a desired location in the Image Stack Si window to initiate tracing of a new tree structure Continue Cu clicking within the Image Stack window to add vertices Each EE new vertex will be connected to the previous vertex with a Delete Segment straight line segment Delete Tree e Tracing can be continued from any vertex of the tree Select Move Veitices Continue place the cursor in the vicinity of the vertex and click Move Tree on the vertex to activate it Export Tree To SWC e Connect Vertices makes it possible to connect any two vertices End of the trace with a segment The user will be prompted if this operation results in the formation of a loop e Individual segments of the trace can be eliminated by selecting the Delete Segment function and moving the cursor to the vicinity of the segment s vertices Clicking on one of these vertices will activate its neighbors A subsequent click on one of these neighboring vertices will eliminate the connecting segment e Delete Tree followed by mouse over the tree vertices will highlig
15. m generate the initial trace e Load Training History Documents NCTracer training history TrainingHistory_L6 mat e Click on Calculate Costs e Click on Automated Merger The above tracing sequences may be written into macros see the Macros tab section and run without any input from the user Automated tracing of individual stacks from the above two datasets may take several minutes The results can be edited in the Trace Editing amp Analysis tab saved in the mat format and exported as swc files 16 12 About NCTracer Contact information NCTracer is being developed by the Neurogeometry group at the Department of Physics and the Center for Interdisciplinary Research on Complex Systems at Northeastern University in Boston All inquiries should be addressed to Prof Armen Stepanyants a stepanyants neu edu Support NCTracer is supported by the NIH NINDS grant NS063494 Related publications 1 Chothani P Mehta V and Stepanyants A Automated tracing of neurites from light microscopy stacks of images Neuroinformatics 9 2 3 263 278 2011 2 Gala R Chapeton J Jitesh J Bhavsar C Stepanyants A Active learning of neuron morphology for accurate automated tracing of neurites Frontiers in Neuroanatomy 8 37 2014 Individual contributions Graduate students Chintan Bhavsar Paarth Chothani Jayant Jitesh and Vivek Mehta developed the Graphic User Interface of NCTracer Graduate students Julio Chapeton
16. nsnnnnsnnennes 15 MMe ISS E car seu T creas sac AAI E T ase snes A E A 15 1 OUI IN CACC ea E E 17 1 Introduction Neural Circuit Tracer NCTracer is open source software built using Java and MatLab The software is based on the core of ImageJ http rsbweb nih gov ij and the graphic user interface was developed in Java Swings NCTracer combines a number of ImageJ functions with several newly developed algorithms for automated and manual tracing of neurites The software is designed in a way that allows the user to add Java and MatLab based plugins build and run macros These and other functionalities of NCTracer are described in detail below 2 System requirements and installation NCTracer 4 0 is developed for the Windows 7 and 8 64 bit operating systems and requires a minimum of 4 GB of RAM This version of the software does not run on Mac or Linux OS To install NCTracer 4 0 download NCTracer_4_0 zip file located at http www neurogeometry net resources tools and extract its components Right click on Setup exe file and run it as Administrator This will install four components Java jre 7 windows x64 Java3D java3d 1_5_2 windows amd64 MatLab Runtime Compiler MCR_R2013a_win64 and NCTracer NCT_4_0 It is recommended that you install all four components even if some versions of these components are already present on your computer To run NCTracer double click on the NCTracer icon amp created on your desktop 3
17. of all neurites contained in the image is detected with the Fast Marching 2 or Voxel Coding 1 algorithms In the latter case it Ggssagaimepaee eer ene is necessary to eliminate image background by setting a threshold the algorithm will be slow if the number of voxels remaining in the foreground is much higher than 10 Erroneous short terminal branches and small loop clusters are eliminated by the tracing algorithms The resulting trace is optimized as described in 2 and is referred to as the initial trace right e Fixing topological errors Initial traces of complex arbors or arbors of multiple neurons may contain topological errors The number of such errors can be reduced by using the geometrical and topological features of different branch merging scenarios see 2 for details All branch merging scenarios are considered by the algorithm and the weighted features of each scenario are combined into a fitness function The weights are determined during online training and branch merging scenarios with the highest overall fitness are implemented by the algorithm Complex mergers involving more than 12 branch end points are not carried out in this version of the software To perform automated tracing the user must click on the Get Initial Trace button located in the Automated Tracing tab of the main NCTracer window and select the Fast Marching Voxel Coding or the Voxel Coding algorithm right a it Fast Marching
18. time Compiler used to run NCTracer Once the jar file is created it can be imported into NCTracer by checking the MatLab Plugins radio button and clicking on Add Plugins This will open the MatLab Plugin Type selection window right NCTracer 4 0 supports three types of plugins plugins which modify the image stack e g an image processing plugin plugins which generate or modify the trace e g a plugin that finds and eliminates short terminal branches or none of the above e g a plugin which calculates lengths of all trees contained in the trace 0 None of the above Clicking OK will open the Input Output Selection window below in which the user must specify the name type order and default value of every input and output variable of the MatLab function There are four variables which must be formatted in a specific way to 11 work with NCTracer These BranchlengthsAM variables include Image Stack input parameters 3D MatLab array Adjacency number of parameters o Matrix NxN array of doubles V Image Stack F Adjacency Matrix V Position Vector V Branch Caliber Position Vector Nx3 array of E TE Default Value doubles and Branch Caliber Nx1 array of doubles N here denotes the number of vertices in the trace Variables that are not in use must be removed from the Input Output Selection window by un Number of Parameters 0 che ckin g the corre sp on din g Image Stack Adjacency Matrix Posit
19. trace vertices number length can be adjusted with the Adjust Point Typical Radius 2 Density function located in the Trace Editing amp E p Analysis tab of the main NCTracer window Radius Stiffness Optimization Step Size The Eliminate Small Trees button allows the user to automatically delete trees the total length of which is less than a user defined length threshold Loops in the trace if present can be displayed by pressing the Show Hide Loops button 10 7 Manage Plugins tab adding and removing MatLab and Image plugins Fie Image Processing Automated Tracing Trace Editing amp Analysis Manage Plugins view _ Macros Settings _ Stack 0 06 E aA a e o p NCTracer allows the user to import plugins designed for ImageJ To import an Image plugin check the ImageJ Plugins radio button and click on Add Plugins The user will need to find and open the main file jar java or class associated with the plugin The plugin will be copied into the My Documents NCTracer plugins folder and will automatically appear in the list of plugins in the Image Processing tab Functions written in MatLab can also be converted into NCTracer plugins In this case the user must first deploy MatLab Builder JA http www mathworks com products javabuilder to create a jar file associated with the MatLab function The version of MatLab Builder JA must match the version of the MatLab Run
20. ving this log will create 16 62 a text file in the My 17782 18 62 Documents NCTracer logs folder je 21 62 22 62 23 62 24 62 25 62 4 i Neural Circuit Trace eS eon Image Processing Automated Tracing _ Trace Editing amp Analysis Manage Plugins View Macros wy A J The user can create save and run sequences of operations macros to be performed on image stacks To create a macro click on the Macros tab in the main NCTracer window and assemble the desired macro components in the Macro Selection panel image on the next page You will be prompted to provide parameter values for every function included in the macro Click on Save to create a text file containing the macro sequence The file will be saved in the My Documents NCTracer macros folder To run the macro click on the Run Macro button and select the macro file ImageJ plugins cannot be included in a macro in this version of the software 14 Macro Selection Panel ast Marching Initial Trace Calculate Costs Eliminate Small Trees 10 Settings tab properties user manual and website Fie Image Processing _ Automated Tracing _ Trace Editing amp Analysis Manage Plugins View Macros cove wei a ay a This tab allows the user to modify the default parameters used by the Fast Marching Voxel Coding trace optimization
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