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Here is the manual for VAST Lite RC2 as PDF

1. VAST comes with a client side implementation in Matlab which is used by the VastTools toolbox and included in the vast_package zip file In Matlab the VAST API is implemented as a class VASTControlClass m Internally it uses the jtcp library for TCP IP communication in Matlab There are class methods available for the different API functions Using the VAST API is very simple With VASTControlClass m and its helper functions available in the path simply make an instance of the class and use its functions Here is a short example which connects to VAST reads out some basic information and disconnects vast VASTControlClass res vast connect 127 0 0 1 22081 1000 if res 0 warndlg Connecting to VAST at 127 0 0 1 port 22081 failed Error else vinfo vast getinfo disp vinfo vast disconnect end B 3 API FUNCTION REFERENCE 49 You can also use Vast Tools to handle the connection Simply run Vast Tools and connect then run your own script VastTools uses a global variable vdata To access the APT functions simply enable access to the global variable then use the VAST API like so global vdata vinfo vdata vast getinfo disp vinfo The following API functions are currently available Version 1 as returned by getapiversion res connect host port timeout Tries to connect to VAST via TCP IP Returns 1 if connected 0 if connection failed res disconnect Disconnects the TCP
2. Use getscreenshotimage to request a correctly reshaped two three or four dimensional image Screenshot images are always returned in RGB for mat three bytes per voxel If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code screenshotimage res getscreenshotimage miplevel minx maxx miny maxy minz maxz collapseseg Same as getscreenshotimageraw but reshapes the one dimensional array to a matrix of the requested dimensions The order of dimensions in the matrix is Y X Z C C for color R G B If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code 58 APPENDIX B TECHNICAL INFORMATION Bibliography 1 CARDONA A SAALFELD S SCHINDELIN J ARGANDA CARRERAS l PREIBISCH S ET AL TrakEM2 Software for Neural Circuit Reconstruc tion PLoS ONE 7 6 e38011 2012 doi 10 1371 journal pone 0038011 SAALFELD S FETTER R CARDONA A AND TOMANCAK P Elastic volume reconstruction from series of ultra thin microscopy sections Nature Methods 9 7 2012 717 720 SRUBEK TOMASSY G BERGER D CHEN H KASTHURI N HAY WORTH K VERCELLI A SEUNG S LICHTMAN J AND ARLOTTA P Distinct Profiles of Myelin Distribution Along Single Axons of Pyra midal Neurons in the Neocortex Science 344 6181 2014 319 324 doi 10 1126 science 1249766 59 60 BIBLIOGRAPHY
3. The grouping can also be applied when segmentations are exported Segment hierarchies are visualized and edited in the Segment Colors tool window This tool window uses a tree view control similar to the navigation pane of a windows explorer window which makes usage very intuitive Most 22 CHAPTER 3 WORKING WITH VAST advanced functions can be found in the tool window s menu which opens either by clicking the Menv button or right clicking into the tool window 3 4 3 Re ordering and moving segments in the tree To re order the segments simply drag and drop them with the mouse You can only select and drag one segment at a time but if the segment has children the whole branch will be moved including all children Please note that to make a segment the first child of another segment you have to drag it to the right side of the tool window right of the new parent segment The new parent segment will then be highlighted in blue instead of the black line indicating the target space between two segments You can move any segment with exception of the Background segment The Background segment can also not have any children Because it can be cumbersome to move hundreds of items from one folder to another one by one VAST currently supports two functions Make all siblings children and Make all children siblings which can help in certain situations see section 3 4 7 3 4 4 Collapsing and expanding tree b
4. dialog Decrease tooltip diameter Increase tooltip diameter N or Keypad Zoom out M or Keypad Zoom in F Flash selected segment I 0 P Set paint mode to Paint all Background Parent HOME or G Go to anchor point of selected segment lt gt Select previous next segment in recently selected list Table A 3 Other Controls A 6 TERMS OF USAGE AND PRIVACY STATEMENT 45 A 6 Terms of Usage and Privacy Statement This version of VAST the software is free of charge and may be distributed freely but not sold Commercial usage is allowed You are using this software at your own risk Even though it has been tested extensively it is not free of bugs Please keep backup copies of your data THE SOFTWARE IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTIC ULAR PURPOSE AND NONINFRINGEMENT IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM DAMAGES OR OTHER LIABILITY WHETHER IN AN ACTION OF CON TRACT TORT OR OTHERWISE ARISING FROM OUT OF OR IN CON NECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE VAST does not collect usage statistics or other data In particular it does not transmit any of your image or segmentation data anywhere This software uses easyzlib c which is based on the zlib library by Jean loup Gailly and Mark Adler VastTools uses jtcp m by Kevin
5. 8 if accurate boundary tracing is not needed Painting is also optimized to be highly responsive and the user interface provides quick access to functions which are used often navigating through the image stack changing the tooltip size color picking and switching between paint and delete mode To reduce the amount of required memory and to enable interactive painting speed with any tooltip size VAST implements multi scale painting Considering that alternative labeling methods are likely to be more error prone and will probably require more time until an acceptably low error rate is reached voxel painting might be the overall faster alternative for fully manual labeling Tf an object skeleton is needed for example to compute length of a dendrite or number of spines etc it can be computed from the voxelized segmentation whereas the inverse operation computing accurate volumes from skeletons is much harder In a preliminary test we found that using VAST a very experienced user can produce a dense segmentation of well stained and well aligned cortex neuropil data 6x6x30 nm voxel size at a speed of about 4 cubic microns per hour If cross sections are labeled with a color dot in the middle rather than accurately outlining and filling the cross sections the labeling speed can be increased by a factor of 3 at the expense of accuracy 12 cubic microns per hour VAST is also very portable and light weight It consists of a single Windows
6. Bartlett 2Except if you set that up explicitly using the Remote Control API Server of course You can make it transmit segmentation data through the API to Vast Tools for example but that is under your control 46 APPENDIX A FAQ AND TROUBLE SHOOTING Appendix B Technical Information B 1 Size limitations Maximal file size for EM images and segmentations Limited by maxi mal file size on disk theoretical maximum 2 bytes Largest EM stack that has been imported into VAST so far 5 Terabytes Maximal number of labels supported currently 216 1 but needs lots of RAM 600 bytes per label Largest image supported at full resolution 2 1 231 1 B 2 Supported file formats for importing ex porting Importing of EM images Currently 8 bit graylevel and 24 bit RGB image stacks are supported Stacks and tiled stacks JPG PNG TIF 3D Volume files NII NIfTD will be converted to 8 bits when imported Importing from TIF images under Windows 7 can result in an image stack that has only 16 gray levels instead of 256 because of a bug in the Windows GDI TIFF routines Consider converting the TIFF images to PNG before importing Exporting of EM images Stacks and tiled stacks PNG 8 bit indexed TIF uncompressed RAW Importing of segmentations Segmentations can be imported from RGB TIF and PNG image stacks The segment number for each pixel is encoded in the RGB value of the image as 47
7. Include Vast folder names in file names If enabled the VAST folder names of the segment hierarchy will be added to all OBJ file names and also the names of the exported objects This makes it easy to select and process subsets of the objects based on their names Invert Z axis In enabled the models will be mirrored so that they lie below the z 0 plane This reflects the actual shape of the objects in the tissue if slices are counted up as the are cut off the surface of a block Close surface sides If enabled meshes will be closed at the sides where they exit the extraction region Write 3dsMax bulk loader script to folder If enabled a small file called loadallobj_here ms will be saved to the target directory This is a 3dsMax script which if executed in 3dsMax will batch load all objects in the same directory This is very useful if you are working with many object files Save surface statistics to file If this is enabled the export script will also compute the surface area of each exported object by summing up the triangle surface areas and save the result to the text file with the provided name This file will always be stored in the same folder as the OBJ files the target folder so please give only a file name in the text field not a file name with target path Computing the surface area will take some time so disable it if you don t need it Skip model file generation Enable this option if you just wan
8. Please note that sequences of the same value defined by one pair can loop around at the end of a row and or extend into the next plane Run lengths longer than 21 1 65535 are encoded in several pairs segimage res getsegimageRLEdecoded miplevel minx maxx miny maxy minz maxz surfonlyflag Same as getsegimage above except that the data is transmitted run length encoded RLE from VAST to Matlab and then decoded on the Matlab side This can improve speed because it reduces the size of the transmitted data If surfonlyflag is 1 all interior voxels any nonzero voxels for which all six direct neighbors have the same ID are set to 0 See getsegimage above for information on volume coordinates If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code values numbers res getRLEcountunique miplevel minx maxx miny maxy minz maxz surfonlyflag This function returns the segment IDs values and number of voxels of each ID numbers in the requested subvolume See getsegimage above for informa 56 APPENDIX B TECHNICAL INFORMATION tion on volume coordinates If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code segimage values numbers res getsegimageRLEdecodedcountu nique miplevel minx maxx miny maxy minz maxz surfonlyflag Combines the functions getsegimageRLEdecoded and getRLEcountunique above and re
9. VAST will keep existing non zero voxels and ignore imported non zero voxels at the same place Similar to segmentation merging VAST will not save the segmentation af ter importing automatically You can do that yourself after importing using File Save Segmentation As from the main menu 28 CHAPTER 3 WORKING WITH VAST f Export Data ua Export as Single tle stack 8192 8192 EM data target Source Layer 0 E yAST Thousands_6nm_spec_lossless vsv Region to export Specify coordinates at full resolution X from 0 to 10746 1077 E Export EM Data as Y from 0 to 12894 12895 4 BBox WASTThousands_6 or Slice from o to 150 o y a E VAST Thousands_6r t Resolution viplevel 2 1 4 24 0 24 0 30 0 nm px y Every sice y Segmentation target 7 NP MATO aa Source Layer Image size 2686 3223 EM data size 59 7 GB Seg data size 119 4 GB Y Export Segmentation as PNG y 24bitjpx RGB encoded gt Screenshot target E Collapse labels as in current view E Don t save empty tiles E Export Screenshots as pri E Export currently selected label and child tree only Segmentation output file name template H Threecylindermerge_May08_db14_final3_screenshot Browse H Threecylindermerge_May08_db14_final3_export Browse Ceen ona Figure 3 4 Export dialog 3 8 Exporting Image Stacks Exporting EM volumes and or segmentations as ima
10. You can load several image stacks at once provided they are the aligned and the same size and each one can be RGB or graylevel In addition you can tint different image stacks in different colors to distinguish different channels Does VAST support 4 dimensional data sets for example time lapse data of a 3D structure No How do I open a VSS file without a matching VSV file in VAST You can not Actually you can with a work around If you load any VSV and then open the VSS in question and the VSS file has a different size VAST will tell you its dimensions size in pixels in the error message window Note down these numbers Now create a dummy VSVR with these dimensions VSVR files are just text txt files with a different file name extension Set the field ServerName to an empty string to make it a dummy layer Then restart VAST open the dummy VSVR as image stack and open your VSS which should have the same size on top Here is an empty dummy VSVR example Comment Empty Dummy Layer ServerType openconnectome ServerName 39 40 APPENDIX A FAQ AND TROUBLE SHOOTING ServerFolder SourceDataSizeX 49152 SourceDataSizeY 32768 SourceDataSizeZ 255 TargetDataSizeX 49152 TargetDataSizeY 32768 TargetDataSizeZ 255 OffsetX O QffsetY O OffsetZ 0 OffsetMip O TargetVoxelSizeXnm 6 TargetVoxelSizeYnm 6 TargetVoxelSizeZnm 30 TargetL
11. can click on the magnifying glass right of the text edit field to go to the next segment that matches your text The F3 key has the same function provided that the segment tree sub window of the Segment Colors tool window is active You can also search for a segment with a particular ID To do this type an opening bracket into the find edit field followed by the ID number you are looking for 3 4 15 The Collect tool The Collect tool Collect Segment Mode can be selected in the toolbar by clicking the icon with an arrow pointing at a folder When in Collect mode segments you click will be moved in the segment tree to become children of the currently selected segment This can be useful to quickly classify different objects into different types Simply make a folder segment for each type select it and click on the objects in the image that are of that type with the Collect tool Using this tool is a bit dangerous because there is no Undo If you click the wrong object it might be difficult to remember where it came from and there is currently no easy way to move it back Secondly when an segment is moved all its child segments are moved with it but not its parent s So if you are dealing with objects which consist of several parts make sure that you move the parent segment of the object and not only a side branch of its tree 3 5 Saving Segmentations Important VAST DOES NOT SAVE AUTOMATICALLY while
12. can export segmentations as image stacks for using them in other analysis programs or to render the segmented objects in a 3D animation program like 3D Studio MAX You can also import segmentation image stacks that were generated externally 3 1 Image Stack Importing Typically volumetric image data is stored as a series of 2D images or as a serial 3D block of data which is not suitable for fast interactive viewing When you import such a data set into VAST it puts the images into a single file containing a diced data structure and computes and includes mipmaps for the images Using diced data does not only speed up loading of parts of images but will in the future also enable fast loading of volumetric sub regions or 2D sections at other orientations through the image data 1 VSVOL can be used instead and may be preferable because Microsoft thinks VSV is a Microsoft Visio file 2You can use VSSEG instead of VSS as an alternative file name extension for VAST segmentation files 3A mipmap is a downsampled version of an image VAST uses power of two 2D XY mipmaps For example for an original image of 1024x1024 pixels it will compute mipmaps of 512x512 and 256x256 pixels It does this for every slice image in a stack 9 10 CHAPTER 3 WORKING WITH VAST VAST does currently not include image alignment and stitching functions If you are starting with an unaligned stack of images you will first have to align the images w
13. larger file size depending on your data Variable Bitdepth Compression should be fastest when reading from the compressed files Quantization specifies whether the compression should be lossy or lossless Lossyness is achieved by quantizing meaning throwing away bits For example if you set Quantization to 2 bits graylevel images will have only 6 bits resolution 64 different gray levels rather than 8 bits 256 different gray levels Throwing 3 1 IMAGE STACK IMPORTING 13 bits away reduces both file size and image quality Voxel Order defines in which order the pixels in the images will be stored This can have an effect on compressed file size 2D Swizzle stores pixels in 2D Z order I usually get best results using 2D Swizzle but 3D Swizzle might be superior for very well aligned data e g FIB SEM Next VAST will ask you to specify a target location and file name for the resulting vsv image volume file Use vsv as extension for the file name Choose a location where you have enough storage space for the file The file will not only contain the original image data but also the mip maps For example if you import 1024 images of 1024x1024 pixels each and store uncompressed the vsv file will be approximately 1024 1024 1024 512 512 1024 256 256 1024 1409286144 Bytes 1 3 Gigabytes large Lossless compression will reduce the file size and lossy compression even more but by how much depends strongl
14. means that if segments are removed from a segmentation the other segments will move up to keep the list continuous This happens when you use the functions Delete Segment Subtree Weld Segment Subtree or save a new vss file with a subset of the current segments using the Save Segmentation As Special function If you are using the internal IDs to identify particular segments and do not want them to change avoid those functions For example you can put deleted segments into a A 2 TYPICAL USE CASES 41 Deleted folder and or re use them If you use Merge Segmentations in or Import Segmentation with certain settings the internal segment IDs of the imported segmentation will also be changed so that they don t overlap with existing IDs Why is it called VAST Lite and not just VAST The Lite in the name emphasizes that this is not supposed to be the final version of the software It is currently a usable tool with a limited set of ca pabilities which is provided to the scientific community without restrictions Development of VAST continues and there may at some point be a released version with more features Also VAST Lite is a better name for Google searches than VAST A 2 Typical Use Cases Analyzing synaptic connectivity in a cortex EM stack It is possible to define the location of synapses and their synaptic partners by painting the synaptic membrane We do this in two st
15. retrieve the error code B 3 API FUNCTION REFERENCE 55 minx maxx miny maxy minz and maxz are given in voxels at the resolution defined by miplevel You can retrieve the size of the volume at full resolution mip level 0 using the function getinfo To compute the size of the volume in X and Y at a different mip level miplevel use the following code xmin bitshift xmin miplevel xmax bitshift xmax miplevel 1 ymin bitshift ymin miplevel ymax bitshift ymax miplevel 1 Note that the size of the volume in Z does not change with mip level this is the number of slices segimageRLE res getsegimageRLE miplevel minx maxx miny maxy minz maxz surfonlyflag Reads the requested volume region and returns it as an runlength encoded RLE string This function is useful if you want to directly process the RLE string otherwise use one of the functions below If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code See getsegimage above for information on volume coordinates The runlength encoded image is an array of pairs of unsigned 16 bit values The first value in a pair is the segment ID which follows and the second value is the number of voxels with that ID to follow The encoding order of voxels in the volume is row by row X from left to right concatenated for one plane Y from top to bottom and planes are concatenated from the first to the last slice of the volume
16. see section 4 2 You do not have to generate model files if you just want to measure surface area check Skip model file generation in the parameter setup dialog 4 4 4 Euclidian Distance Measurement Tool This will open a dialog in which you can make simple 3D distance measurements between points in VAST First go to a location in VAST use the center cross Then in VastTools click the Get button next to the first coordinate to read the current location from VAST Then go to the other location and click Get next to the second coordinate The distance between the two locations will show up as a distance in voxels and in nanometers You can easily jump back to the first and second coordinate in VAST by pressing the GO button Make sure you are using the right voxel size to get accurate nanometer measurements Click Update to read the current voxel size from VAST To change the voxel size you currently have to set the voxel size in the vsv file used 38 CHAPTER 4 THE VASTTOOLS MATLAB TOOLBOX in VAST To do this go to Info Volume Properties in VAST and change the voxel size see also section 3 1 4 Then click Update in the VastTools Euclidian Distance Measurement Tool You can copy the values in the edit fields in the Euclidian Distance Mea surement Tool to paste them into different programs but editing these values directly will currently not work 4 5 Target Lists Target lists can store the
17. when you pan the view You can also center any point by right clicking that point with the mouse in Move mode and selecting Center from the context menu The dropdown listbox in the Coordinates tool window lists the up to 64 most recent locations you visited A new entry is added every time you pan the view but currently not if you scroll through Z You can go back to previous locations by selecting the coordinates from this list 3 2 4 Layers VAST can open several image stacks at the same time provided that they have the same stack size Each image stack and also the segmentation stack are listed as a Layer in the Layers tool window For image stacks not the seg mentation the order in the list defines the order of the layers Layers BELOW in the list are in front The segmentation layer is always rendered on top of all image stack layers You can change the order of the layers by drag and drop in the list If you can not see all layers in the list increase the size of the tool window by dragging a corner Below the list of layers the Layers tool window shows a number of Layer Properties for the currently selected layer 6You can set the opacity of the center cross in the Preferences under Center Cross Opacity 3 3 PAINTING 17 e Solo If this function is enabled only the currently selected image layer will be displayed e Editable Disabled for image layers e Visible Trans
18. 3 4 5 Using anchor points 000 22 3 4 6 Adding new segments 2 20040 23 3 4 7 Helper functions for arranging segments 23 3 4 8 Select recently selected segments 23 111 lv CONTENTS 3 4 9 Global operations Deleting and welding segment subtrees 23 3 4 10 Segment tags o lt u ea s send da aten e d ee ee 24 3 4 11 Editing the color of a segment 24 3 4 12 Exporting segment metadata 24 3 4 13 Segment information 00 0 24 3 4 14 Searching for a segment with a given name orID 25 3 4 15 The Collect tool o o a 25 3 5 Saving Segmentations 0 000 000 25 3 5 1 Save Segmentation As Special 26 3 6 Segmentation Merging aoaaa 26 3 7 Importing Segmentations From Image Stacks 27 3 8 Exporting Image Stacks o o 28 The VastTools Matlab Toolbox 31 4 1 Getting started with the VastTools Matlab Toolbox 31 4 2 Exporting 3D Models o o 32 4 3 Exporting Projection Images o o 35 4 4 Measuring a dd A de o ae te 37 4 4 1 Measure Segment Volumes o 37 4 4 2 Measure Segment Lengths o 37 4 4 3 Measure Segment Surface Area 37 4 4 4 Euclidian Distance Measurement Tool 37 4 0 TargetLists i aa a a a de da deal 38 FAQ and Trouble Shooting 39 A 1 Frequ
19. 48 APPENDIX B TECHNICAL INFORMATION follows Bits 0 7 of the label number are expected in the blue channel bits 8 15 in the green channel and bits 16 23 in the red channel This is the same format used for exporting segmentations to image stacks see below Please be aware that VAST can currently only handle 16 bit segmentations Exporting of segmentations When exporting segmentations the available file formats depend on the range of segment numbers used For example if the highest segment number is greater than 255 8 bit indexed file formats will not be available In that case the label numbers will be encoded into the color channels for example for RGB bits 0 7 of the label number will be put into the blue channel bits 8 15 into the green channel and bits 16 23 into the red channel Stacks and tiled stacks PNG TIF uncompressed RAW 3D Volume files NII NIfTT 8 bit only currently unsupported Exporting of screen shots Stacks and tiled stacks PNG TIF uncompressed RAW all 24 bit RGB 3D Volume files NII Nifti 8 bit only currently unsupported B 3 API Function Reference VAST includes an API through which external programs can communicate with VAST The communication is done through a TCP IP connection This makes it possible to write client programs in any programming language which sup ports the TCP IP protocol It also allows client programs to run on separate computers and communicate through the network
20. AM the more the better e DirectX 11 compatible graphics card e 2 TB of disk space depends on the size of the data you work with e Wacom Cintiq 13HD or other pen touch screen with two button pen Minimal system configuration e Windows PC with 64 bit Windows Vista e 2 GB of RAM e DirectX 11 compatible on board graphics card e Standard screen and mouse 6 CHAPTER 2 GETTING STARTED 2 2 Program Setup To use VAST simply copy the executable program into a folder where you have read write access and set up links on your desktop start menu and or taskbar if desired It is important that VAST has read and write access to the folder where the executable is because it will write a configuration file vast_preferences dat into the same folder to store your settings Start the executable 2 2 1 Try It Out The quickest way to try out VAST is to use an online data set Several online data sets are included in the ZIP package of supplementary files as VSVR files You can save this package from the executable by clicking Yes in the First Start pop up window when you start VAST for the first time or by choosing Save Documentation ZIP To Disk from the Info menu Unzip the package Then in VAST go to Open EM and select one of the VSVR files in the VAST_package Online Datasets folder for example openconnectome_kasthuril1 vsvr This will load images of a big EM stack from the Johns Hopkins Openconnecto me s
21. IP connection to VAST Always returns 1 If the request fails a Java Error will stop the script so a return of 0 is currently not possible errornumber getlasterror Retrieves the error code of the last call of an API function Error codes have the following meaning 0 No error 1 Unknown error 2 Unexpected data received from VAST API mismatch 3 VAST received invalid data Command ignored 4 VAST internal data read failure 5 Internal VAST Error 6 Could not complete command because modifying the view in VAST is disabled 10 Coordinates out of bounds 11 Mip level out of bounds 20 Segment number out of bounds 21 No segmentation available 22 RLE overflow RLE encoding makes the data larger than raw please request as raw 30 Layer number out of bounds 31 Invalid layer type 50 sourcearray and targetarray must have the same length Table B 1 VAST API Error Codes as returned by getlasterror 50 APPENDIX B TECHNICAL INFORMATION apiversion res getapiversion Returns the version of the API provided by the currently connected VAST ex ecutable The API version described here is version 1 If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code info res getinfo Reads out general information from VAST If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code Returns a struct with the following fields if successful or an em
22. TTOOLS MATLAB TOOLBOX image should be stretched in Z for projections along the X or Y axis if the slice thickness is different from the pixel size The stretching will be determined by the voxel size of your image stack as set in VAST see section 3 1 4 File name Target folder Enter the target file name and location here The projection image will be saved after generation if Save to file is enabled Segmentation preprocessing Select here how you want to use the segmentation for the projection image This determines which parts of the segmentation are used and for the Screenshots image source whether the segments should be colored as displayed in VAST collapsed or in their individual native colors uncollapsed Expand segments by n pixels Makes your segmented regions larger Useful when using the segmentation as a mask Blur edges by n pixels Blurs the edges of the segmentation to smooth out the projection image Image source Select here what you want to use as source images for the projection image Select Screenshots if you want to use images as they are shown in VAST using all brightness and contrast settings layer blending and tinting segmentation transparency patterns etc Opacity source Determines whether you want to only use segmented areas only unsegmented areas or all Blending mode Select here whether you want to alpha blend layers with the selected trans parency map add all layers o
23. The area is defined by minimum and maximum values for the ranges in x y and z This function transmits the segmentation image as raw data a one dimensional array of bytes with either one or three bytes per voxel depending on whether the layer has one or three color channels B 3 API FUNCTION REFERENCE 97 Use getemimage to request a correctly reshaped two three or four dimensional image If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code emimage res getemimage layernr miplevel minx maxx miny maxy minz maxz Same as getemimageraw but reshapes the one dimensional array to a matrix of the requested dimensions The order of dimensions in the matrix is Y X Z C C for color R G B if the layer has three color channels If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code screenshotimage res getscreenshotimageraw miplevel minx maxx miny maxy minz maxz collapseseg Reads out the EM and segmentation stack as currently displayed in VAST as a voxel image for a given mip level resolution and area The area is defined by minimum and maximum values for the ranges in x y and z If collapseseg is 1 the color of the segmentation will appear as displayed in VAST if it is 0 each segment will be colored in its native uncollapsed color This func tion transmits the segmentation image as raw data a one dimensional array of bytes
24. VAST Lite Volume Annotation and Segmentation Tool User Manual VAST Lite RC2 Daniel R Berger March 23 2015 il Contents 1 Introduction 1 2 Getting Started 5 2 1 System Requirements e 5 2 2 PTOsTany Set p a a ia 6 221 Try Om AA Sh a 6 2 2 2 Preferences o ee ee 6 2 3 Additional Files Included With VAST 8 3 Working with VAST 9 3 1 Image Stack Importing o e 9 3 1 1 Importing image stacks Pattern based names 10 3 1 2 Lossless and lossy compression 12 3 1 3 Importing 3D volume files 13 3 1 4 Image scale and description o 13 3 1 5 The RAM usage indicator 14 3 2 Viewing and Navigating an Image Stack 14 3 2 1 Remote image stacks 0 o 14 32 2 The sidebar swe es aa 15 3 2 3 Getting and setting coordinates 15 322 Layers ds a a a al a a 16 39 Pantig do o a Ba ee Lhe A a a E anait 17 3 3 1 Multi scale painting 19 3 3 2 Automatic Z filling o oo 20 3 3 3 Using conditional painting 21 3 4 a oe HN Eg be Be es E E eer a 21 3 4 1 Picking segments 21 3 4 2 The segment hierarchy o o 21 3 4 3 Re ordering and moving segments in the tree 22 3 4 4 Collapsing and expanding tree branches 22
25. Voxel Order 20 Swizzle x Cancel px E E 4 Figure 3 2 Second dialog for importing EM image stacks Image compression options codes like 4d integer number or 404d integer number with zero padding to 4 digits VAST will then fill in those numbers for each image For more information about format strings refer to a C manual or ask the internet In the combo box below select which coordinates are used in the file names and in which order they appear The edit boxes below let you specify the range of integer numbers for the three coordinates After you entered all parameters press OK 3 1 2 Lossless and lossy compression Next VAST will show a dialog where you can specify the color mode and image compression options Figure 3 2 Multi file chunking and triple mip mapping are currently not fully implemented and are therefore disabled Under Color Mode please select if you want to import the images as 8 bit graylevel or 24 bit color images and for graylevel which source color channel to use When importing from graylevel images please select the first option from RED channel Under Compression you can specify compression options Under T ype you can select the compression method Uncompressed Variable Bitdepth Compres sion zlib Compression and Spectral Compression The three different compres sion algorithms are by themselves lossless but might produce slightly smaller or
26. abeled segments and export 3D surface models Current limitations e Only one segmentation layer can be opened at a time e Currently only 16 bit segmentations are supported e There is no Undo function 4 CHAPTER 1 INTRODUCTION e Exporting of 3D models of segmented objects is currently only supported externally using the VastTools Matlab script e No 3D model display e No multi user support e Not an open source project but the VAST Lite executable can be copied and used freely see section A 6 for details Please note that VAST is currently under development and is subject to change as new features are added and bugs are fixed For bug reports or helpful suggestions please contact me at danielberger fas harvard edu Chapter 2 Getting Started 2 1 System Requirements VAST currently only runs on 64 bit Windows computers that support DirectX 11 These are Windows Vista Windows 7 and Windows 8 all 64 bit with DirectX 11 or later installed Windows XP and older versions will not work Currently only 64 bit versions of these operating systems are supported because 32 bit programs are limited in the amount of RAM they can handle 4 GB max theoretically less realistically The computer also has to have a DirectX 11 compatible graphics card Luckily in most modern computers even the on board graphics chips support DirectX 11 Recommended system configuration e Windows PC with 64 bit Windows 7 or Windows 8 e 16 GB of R
27. at have not been labeled yet visual pop out It also allows for some additional functionality for example reliably determining neighbor regions using voxel overlap of several segmentations for merging regions or determining synaptic connectivity and conditional painting Such functions are also difficult to implement when using outline labeling drawing vectorized lines around process cross sections in 2D slices In addition outline labeling can introduce problems in identifying corresponding outlines in different slices and can cause region overlap Last but not least many machine learning algorithms for automatic segmentation rely on voxelized labelings for training and the output of many such algorithms are labeled voxelized regions VAST can be used to generate volumetric training data sets and can to some extent also be used for importing proof reading and correcting results of segmentation algorithms The disadvantages of voxel painting which are mentioned most often are that it is slower than for example skeletonization or bread crumbing and that it needs more storage space than alternative methods especially when working with very large data sets VAST tries to alleviate both disadvantages For voxel painting the time needed to label an object is largely determined by the number of outlines that have to be drawn VAST can do automatic convex Z filling to 2 CHAPTER 1 INTRODUCTION reduce the number of outlines by a factor of up to
28. ayerName Empty Dummy Layer How do I deal with self touching objects If you need to be able to recover the true shape of an object for example for correct skeletonization or computation of the surface area places where there are self touches for example a dendritic spine touching the dendritic shaft can be problematic One way to get around this is by using sub objects and glue Just like a plastic model which is constructed from parts you would make the spine a child of the parent and add glue a different segment which you treat specially in the analysis to the interface where parent and child object are actually connected How do I make shiny 3D pictures and animations from the segmen tations I painted in VAST VAST does currently not have 3D rendering capabilities I use Matlab scripts to extract OBJ Wavefront OBJ model files of objects segmented in VAST see section 4 2 These models can then be loaded into 3D rendering programs I use Autodesk 3dsMax I accidentally pressed Home How do I get back to where I was just painting The tool window Coordinates in the upper right corner of the VAST window keeps a temporally ordered list of recently visited places Select the second entry from the bottom in the drop down menu list to jump back Suddenly all internal segment IDs changed What happened VAST currently keeps a continuous list of segment IDs between 1 and n for n segments at all times This
29. current view coordinates zoom level and selected segment together with comments You can add the current view in VAST to the target list by clicking the Add current VAST location button To move the view in VAST back to a stored location click on the GO field of the row in the table You then have to make VAST the active window to see the change Functions to delete rearrange and add separator lines to the list are provided in the Edit menu You can save target lists to a file and load them back later You can have several target lists open at once and cut copy paste between them You can also select the list or part of the list and copy Ctrl C and paste Ctrl V the contents into other programs like a text editor or Excel Target lists are stored as simple mat Matlab files so you can generate them yourself if you fill a mat file with the appropriate variables Simply load a target list file into Matlab to see which variables it contains Appendix A FAQ and Trouble Shooting A 1 Frequently Asked Questions My image stack is not aligned How do I get it aligned into VAST VAST does not have any stack alignment not stitching functionality You ll have to use other programs to render an aligned image stack for example Adobe Photoshop or plugins in Fiji 1 2 and then import that aligned stack into VAST or you ll have to work on an unaligned image stack Can I analyze multi channel optical image stacks in VAST Yes
30. d in will be changed so that they do not overlap with existing segments VAST does not save the merged segmentation automatically nor does it change any of the segmentation files It will generate the merged segmentation in the segmentation cache and if it does not fit in RAM it will end up in the tempo rary segmentation file You will have to save the resulting merged segmentation if you want to keep it for example via File Save Segmentation As from the main menu 3 7 IMPORTING SEGMENTATIONS FROM IMAGE STACKS 27 3 7 Importing Segmentations From Image Stacks VAST can import segmentations from image stacks either generating a new segmentation or merging with a previously loaded segmentation Just like the image files generated during segmentation exporting see section 3 8 below the RGB values of the image pixels should encode the segment numbers the least significant 8 bits 0 7 are in the blue channel bits 8 15 in the green channel and bits 16 23 in the red channel Please remember that VAST can currently only handle 16 bit segmentations To import select File Import Segmentation from Images from the main menu VAST will ask you to select one or several image files of the image stack you want to import Then it will open a dialog where you can specify the parameters for segmentation importing File name s You have to specify a name template for all image files in the stack see section for more informat
31. der next to Contrast or Sel Alpha linfo blendmode The current blend mode see below linfo blendoradd 0 alpha blending 1 additive blending linfo tintcolor Tint color as 32 bit RGBA value Default white Oxffffffff linfo name Name of the layer Table B 4 getlayerinfo struct Blend modes are 0 no transparency Flat 1 the darker mean r g b the more transparent 2 the brighter mean r g b the more transparent 3 the darker max r g b the more transparent 4 the brighter max r g b the more transparent segdata res getallsegmentdata This function is similar to data getsegmentdata id above but retrieves the data for all segments at once and returns a cell array of structs instead of a single struct If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code segname res getallsegmentnames This function is similar to name getsegmentname id above but retrieves the names of all segments at once and returns them in a cell array If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code 54 APPENDIX B TECHNICAL INFORMATION res setselectedsegmentnr segmentnr Sets the selected segment in VAST to the segment with the given ID number If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code selectedsegmentnr res getselectedsegmentnr Returns the ID number of th
32. e on the other For this it is easiest to set one pen button to right click and the other one to middle click If that does not work for your system sometimes Windows uses the Right Click event in a special way for example you can configure the pen buttons to simulate equivalent key presses see Appendix A 5 If you see brief circular animations when you use the pen and drawing is delayed you should go to Pen and Touch in the Windows Control Panel and switch off Flicks On the tab Flicks uncheck Use flicks to perform common actions quickly and easily and click Apply Microsoft what were you thinking 44 APPENDIX A FAQ AND TROUBLE SHOOTING A 5 Keyboard Shortcuts in VAST You can open a window which lists all the keyboard shortcuts in VAST from the main menu under Window Keyboard Shortcuts Here is a summary SHIFT or ENTER Pick segment color by mouse click CTRL or INSERT Temporarily go to move mode TAB or Move mode Click left and move pen up down to zoom Paint mode Click left and move pen up down to change pen diameter or DELETE Paint mode Erase mode H or L Hides segmentation while held down Table A 1 Mode Modifiers hold down UP or A One slice up DOWN or Z One slice down PAGEUP or MAXPAINTDEPTH slices up PAGEDOWN or X MAXPAINTDEPTH slices down Table A 2 Slice Navigation MAXPAINTDEPTH is the value set under Max Paint Depth in the Drawing Properties
33. e which can slow down processing a lot To fix that problem tell VAST to A 4 SETTING UP VAST WITH A WACOM SCREEN 43 use less memory by REDUCING the maximal RAM cache memory sizes in the Preferences dialog This should not cause problems even if you are working with large image stacks since VAST does not need to load all data in RAM at once The only effect which reducing the cache memory size should have is that VAST may have to load parts of your data set from disk more often which is slower than reading from RAM A 4 Setting up VAST with a Wacom screen When it comes to fast and accurate manual painting on a computer tablets and in particular tablet screens can improve productivity significantly We are using various Wacom Cintiq screen tablets While the larger Cintiqs have a better screen they can be expensive and bulky 1 personally prefer the Cintiq 13HD which can be laid flat onto a desk and close to the user An alternative could be tablet laptops if they fulfill the system requirements for VAST We tried the Asus EEE Slate EP121 it works but it is a bit slow and pen button presses tend to be unreliable I can currently not recommend the Microsoft Surface Pro 3 because its pen buttons can apparently not be customized Wacom tablets come with driver software which lets you configure the pen buttons for each program For optimal workflow in VAST I find it most useful to have erase on one pen button and change tooltip siz
34. e Drawing Properties Figure 3 3 provides options for drawing whereas the lower Segment Colors lets you select and organize the segment labels and their colors in the segmentation When in paint mode you can paint on top of the currently displayed EM image Select a color label number from the Segment Colors window at the right by clicking on it Then click the left mouse button where you want to paint in the image You will see the outline of your current tooltip as a circle Even though you can use VAST with a mouse it is designed to be used with a pen tablet 18 CHAPTER 3 WORKING WITH VAST Lc Drawing Properties Pen Diam px Lock Y Fill Paint All 9 Background Parent Restrict No paint all mip levels x Z Scrolling During Paint Stroke Conditional Painting Enable Continuous only EM pixel between T 0 00 0 50 Figure 3 3 The Drawing Properties tool window By clicking and dragging the mouse you can paint larger regions All painting happens in an overlay plane which is blended over the EM image the EM image itself will not be changed You can use the Alpha checkbox in the toolbar to switch the painted overlay on and off and the slider right of it to set the opacity of the painted overlay Most colors are not solid colors but have patterns Use the Pattern checkbox to switch patterns on and off and use the slider right of it to manipulate the c
35. e currently selected segment or 1 if an error oc curred If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code res setselectedlayernr obj Sets the selected layer in VAST to the layer with the given ID number counting from 0 as the first layer in the list If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code selectedlayernr selectedemlayernr selectedsegmentlayernr res getselectedlayernr Returns the numbers of the currently selected layer EM layer and segment layer or 1 if there is no such layer The selected layer will always be either the selected EM layer or the selected segment layer whichever is actually highlighted in the Layers tool window in VAST The other number indicates the most recently selected layer of the other type If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code segimage res getsegimageraw miplevel minx maxx miny maxy minz maxz Reads out the segmentation as a voxel image for a given mip level resolution and area as defined by minimum and maximum values for the ranges in x y and z This function transmits the segmentation image as raw data a one dimensional array of bytes consider using getsegimageRLEdecoded below for a version with faster transmission If the call succeeded res will be 1 otherwise 0 Use getlasterror to
36. e folder the one you picked an image from and are named consistently with numbers for slices rows and columns It also assumes that the set of images is complete which means that there s an image for every slice row column combination in the range you give In this dialog you specify these ranges as well as a schema to derive the filename for a given slice row column coordinate Let s say for example you have a data set called reallybigstack which has 1000 slices numbered from 0 to 999 and each slice has 10x8 tiles numbered from 1 to 10 and 1 to 8 You use a naming scheme so that the first image in the upper left corner of the first slice is called reallybigstack_s0000_x01_y01 png the image tile right of it is called reallybigstack_s0000_x02_y01 png and so on The last image in the lower right corner of the last slice would be called reallybigstack_s0999_x10_y8 png First make sure that the file name in the edit box at the top contains the correct C format string as it is used by printf In general numbers which specify the slice column and row coordinates have to be replaced by 12 CHAPTER 3 WORKING WITH VAST r Sere gt Image Compression Options Th Enable Multi File Chunking with chunk size 1000 Enable Triple 2x1 Mip Mapping Color Mode 8 bit Graylevel from Red channel v Compression Type Variable Bitdepth Compression y Quantization None Lossless m
37. ently Asked Questions o a 39 A 2 Typical Use Cases 2 o ee 41 A 3 Some Performance Tips o 42 A 4 Setting up VAST with a Wacom screen 43 A 5 Keyboard Shortcuts in VAST o o 44 A 6 Terms of Usage and Privacy Statement 45 Technical Information 47 Bel Size limitations s ece e ee ee e Ad iS 47 B 2 Supported file formats for importing exporting 47 B 3 API Function Reference o e e 48 Chapter 1 Introduction VAST is a utility program for manual annotation and segmentation of large volumetric voxel data sets It enables users to work with voxel data sets in the Terabyte or even Petabyte range at interactive speeds to explore them visually and to label structures of interest by voxel painting Voxel painting has a number of advantages over other manual segmentation approaches like bread crumbing placing a number of labeled points inside each object and skeletonization placing labeled points and connecting them with edges to form a skeleton It reveals the true shape of objects and makes visualizations of the data more comprehensive It also allows measurement of volume and surface shape properties of the labeled objects When working on a dense segmentation the fact that voxel painting labels areas rather than single points or lines in the cross sections of objects makes it a lot easier to spot objects th
38. ents in the new segmentation file will be changed to maintain a gap free list of IDs from 0 to n for n segments The settings in the lower part of the dialog provide limited functionality to adjust the resolution of the saved segmentation and target canvas size Normally VSS files only open on top of an image stack of the same size in voxels You can use this function to save an existing segmentation so that you can open it on a stack which has slightly different size and or is scaled up or down by a power of two Currently the saved segmentation will stay aligned to the upper left top corner of the stack Also only powers of two scaling is possible If you want to translate the segmentation to a different location or scale in the target stack you ll have to export the segmentation as an image stack modify the images accordingly and re import 3 6 Segmentation Merging If you have two or more for example partial segmentations VSS files of the same image stack you can merge them into a single segmentation For this select File Merge Segmentations in from the main menu and choose the VSS file s you would like to merge in with the currently opened segmentation During merging VAST will add the selected VSS files to the current segmentation as if someone painted them on top in Paint All mode You can select several VSS files at once for merging which will then be added one by one Segment IDs of the files which are merge
39. eps First we paint the axons and dendrites with individual colors Then we save this data set and generate a second data set in which just the synapse membranes are labeled or in which the synapse membranes are over painted with a special synapse color with a pen with fixed size for example 16 pixels diameter Make sure that the 3D region painted for each synapse is a single connected component and that different synapses are separate connected components We then export both data sets as image stacks and use a Matlab script to find each synapse by doing connected component analysis For each synapse we then find the axon and dendrite which occupy the same voxels as the synapse in the other data set which gives us the connectivity information Counting and classifying objects by painting Just as for the synapses in the previous example you can use connected component analysis in Matlab to count other objects in the stack for example neuron cell bodies You can use paint all neurons of one type in the same color and use different colors for different types Connected component analysis can be used to separate the different cell bodies for each type given that they are separated in space The connected component analysis will also give you the number of objects of each type and their volume if you count the painted voxels for each connected component Segmenting out a single cell from a confocal light microscopy image stack VAST can als
40. erver Your computer has to have internet access for this to work Click and drag the EM slice to pan Use the mouse wheel to zoom Use UP DOWN arrow keys to scroll through the stack Click on the little pencil icon in the toolbar to switch to paint mode Choose Yes in the popup window Click and drag over the image to paint You can select different paint colors in the Segment Colors tool window To erase hold down the Delete key while painting or click and hold the right and left mouse buttons together Select Keyboard Shortcuts from the Window menu for a list of available keyboard functions 2 2 2 Preferences In the main menu of VAST go to File Preferences to open the Pref erences dialog window Here you can set the parameters for data cacheing and display VAST will set up the preferences for you when you run it for the first time on a computer whenever it cannot find the preferences file You can edit these preferences if you want to You should at least check once whether the folder in which VAST puts its temporary disk cache is on a hard drive with lots of free space Depending on what you do temporary disk cache files can get similarly large as the segmentation files you are working with in particular if you use global segmentation editing functions like merging segment deleting or segment welding Remote EM images are loaded using the openconnecto me cutout service over HTTP The VSVR fi
41. es olution Smaller Than value specified in the VAST Preferences A value of 8 zooms out by a factor 2 and shows the next lower mip level texture If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code nroflayers res getnroflayers Returns the number of layers currently loaded in VAST If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code B 3 API FUNCTION REFERENCE 53 linfo res getlayerinfo layernr Returns information about the layer with the given number layernr can be a number between 0 and getnroflayers 1 If the function succeeds it will return a struct layerinfo with fields as described below and res will be 1 If it fails it will return an empty struct and res will be 0 This returns the values which are also visible in the VAST Layers tool window If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code linfo type 0 EM layer VSV 1 Segmentation layer 3 remote EM layer linfo editable 1 if the layer is editable 0 else linfo visible 1 if the layer is visible 0 else Visible checkbox linfo brightness Bright brightness or Pattern checkbox linfo contrast Contrast or Sel Alpha checkbox linfo opacitylevel Slider next to Visible which defines layer opacity linfo brightnesslevel Slider next to Bright or Pattern linfo contrastlevel Sli
42. executable file which is independent of third party libraries It does not require to be installed and can be easily copied and for example run from a memory stick Many tools for labeling voxel data exist but such tools usually have draw backs when it comes to painting in large data sets Most tools do not support voxel painting but instead do bread crumbing skeletonizing or storing object outlines as vector graphics for example splines Also most of them require the data to be loaded in RAM completely This is not possible for the data sets in the Terabyte and soon Petabyte range which are currently produced by serial section electron microscopy of biological samples Many of these tools are also developed as cross platform applications so that they can be run on Windows Mac and Linux systems This usually means that a non native GUI system has to be used e g Qt which makes the program a lot more bulky and more difficult to install and maintain VAST is a Windows only program and uses native Windows GUI and graphics functions VAST is currently being used in several studies to label cells in electron microscopic and light microscopic image stacks One recently published study for which it was used is 3 The key concepts of VAST are e Image stacks are imported into VASTs vsv file format where they are stored as dices Pre computation of mipmaps allows for fast panning and zooming through the data when it is opened in VAST e v
43. fines the size of the exported region edit fields change each other to stay consistent If you click the Full button the values will be set back to the full extent of your stack 3 8 EXPORTING IMAGE STACKS 29 The BBox button sets the region to the bounding box of the currently selected segment You can use this function to define a cut out region from a painted segment you can also use a new segment and just paint the upper left top and lower right bottom corners of the cutout region select it and go to Export Data Then click the BBox button to use the painted extent as an export region Under Resolution you can select at which mip level you want to export VAST does not support arbitrary scaling but can export image stacks at its native mipmap scales which are powers of two You can also subsample the stack by slices every nth slice Below you can see the image size resulting from your settings and an estimate of the raw data size that will be exported Compressed image formats like PNG can however produce much smaller file sizes depending on the image content Screenshot target If you want to export a stack of screenshots how the images look in the main window of VAST enable the checkbox Export Screenshot as VAST will reproduce the pattern blending and tinting settings as they are currently set in the main window in the exported screenshots Select a target image format and f
44. ge stacks can be useful to generate slice animations to transfer data to other programs like the Fiji 3D viewer or to process your results further externally To export parts of the image stack and or segmentation stack as a stack of image files select File Export in the main menu The dialog shown in Figure 3 4 will pop up You can export EM image stacks segmentation image stacks and screenshot image stacks You can specify a region of the data set to be exported and a resolution mip level for the images You can also export a data region which is too large for storing the whole slice in a single image as a tiled set of images Export as Choose here if you want to export an image stack as a single tile stack one tile per slice or a multi tile stack a grid of image tiles per slice For the second option you can define the tile size to be used Region to export Specify coordinates at full resolution This defines the region of the stack that should be exported for all three targets Screenshot EM image data Segmentation By default it is set to the full stack You can restrict the export region here X is the horizontal axis in the slice pointing rightwards Y is the vertical axis in the slice pointing downwards on the screen and Slice Z defines the range of slices that should be exported The first and second columns of the edit fields let you define start and end of the region for each axis the third column de
45. gmentation you are working on this can take a lot of time RAM and disk space Also it will change the internal ID numbers of the segments so please think twice before using these functions in case you are relying on absolute segment IDs in your analysis These functions are also not well tested please report any bugs you might encounter 3 4 10 Segment tags Each segment can have a Tag which you can select in the Segment Colors context menu under Tags A tag is a number between 0 and 15 which can indicate the type of the segment The tag value is exported with the Segment Colors text file and can be used to help external analysis By default the tag of all segments is 0 VAST uses tag 1 to indicate that the segment is a Folder Seg ment which is not a labeled object by itself but rather a folder which contains other folders and objects This information can be used to collapse all objects but expand all folders select Expand Only Segments Tagged as Folder un der Expand Collapse Child Folders in the Segment Colors tool win dow context menu Segments which have a tag that is not 0 will have an icon with a colored frame in the Segment Colors treeview Folder segments have a gray frame 3 4 11 Editing the color of a segment The color and pattern of any segment can be changed Right click on a segment and go to the sub menu Colors of the context menu then choose the desired option Each segment has a
46. hat it can not be stored in a single image file but is stored in a set of tiles which form a 3 1 IMAGE STACK IMPORTING 11 gt Specify File Naming Scheme For Image Stack Mz eal Make Single Tile Stack Using File Names and Order as Selected Use Pattern Based Names Parameters for Pattern Based Names Basic Filename String C sprintf format reallybigstack_s 04d_y 02d_x d png Derived from reallybigstack_s0999_y08_x10 png Parameter Order in String Slice Row Column Z Y X No of first column X 4 No of last column X 10 No of first row Y 1 No of last row Y 8 No of first slice Z 0 No of last slice Z 999 S Figure 3 1 First dialog for importing EM image stacks Specification of pattern based names regular grid Please note that these tiles should not be unstitched image tiles as they come off a microscope but they have to fit seamlessly If you have a set of raw microscopic images which are not yet stitched and aligned please use an external program to generate a stitched and aligned image stack first and store each slice as a single image or a set of image tiles You can then import those images into VAST VAST will use the file s you selected in the previous dialog to determine the source directory where the image files are and to generate a basic template for the file name It assumes that all images of the stack are in the sam
47. hese functions because currently there is no Undo 3 4 8 Select recently selected segments Under Select Recently Selected in the Segment Colors tool window s context menu you can find a list of the segments you had recently selected You can click on one of the listed segments to select it again 3 4 9 Global operations Deleting and welding segment subtrees Deleting and welding segments is actually more difficult than it seems because it involves traversing the whole segmentation data set and inspecting every single painted voxel When you choose to delete the selected segment and its children VAST will actually have to not only set all voxels with those segment numbers to 0 but also renumber all the other voxels so that the used label numbers will have no gaps all label numbers between 0 and n are used Welding will make the selected segment and all its children the same label It will renumber all voxels with label numbers of children of the selected label to the same number as the selected label and also similar to when deleting renumber the other segment voxels so that the resulting segmentation is free of label number gaps 24 CHAPTER 3 WORKING WITH VAST Because these functions change almost the complete segmentation and VAST is designed so that the opened segmentation file is not changed it basically has to copy almost the complete segmentation data to the temporary cache file Depending on what se
48. ice images in the correct order because images will be stacked in the same order in which they appear in the system s list of selected files The order is usually correct if you select the last image first then shift click hold the SHIFT key down and click left with the mouse the first image to select the whole range You can also try Select All by pressing CTRL A if the folder only contains the image files you want to import If you are worried about the order of the images and want more precise control you can use pattern based names If you make use of pattern based names to import single or multi tile image stacks it is sufficient to select one file but even better to select the first and the last file in your set of images Then click Open After selecting one or more image files not nii VAST will display the dialog shown in Figure 3 1 To import without pattern based names select Make Single Tile Stack Using File Names and Order as Selected and press OK If you select the second option Use Pattern Based Names the parame ters in the lower part of the dialog window will be enabled With pattern based names you specify a template string for the file names which contains placehold ers for numbers and ranges for these numbers With this you can also import image stacks in which each slice is stored in several image files multi tile image stacks This is useful for data sets in which a single slice is so large t
49. ilename prefix location EM data target This saves a stack of EM image data from the selected layer You can specify the target format filename prefix and location Segmentation target This saves the segment IDs of the segmentation layer or part of it as an image stack The segment ID of each pixel a 16 bit number will be encoded in the color of the pixel in the exported image Bits 0 7 will end up in the blue channel and bits 8 15 will end up in the green channel The red channel will currently stay 0 When you are done setting up the parameters for the export press Export and VAST will start exporting the image stack or stacks VAST can export more than one target at the same time 3D models target This function appears in the newest version of the export dialog but is disabled because it is not fully implemented yet For now please use Vast Tools to export 3D models of segmentations see section 4 2 10Note that the bounding box is not always correct in particular if you delete parts of what was painted before the bounding box will not shrink 30 CHAPTER 3 WORKING WITH VAST Chapter 4 The VastTools Matlab Toolbox VAST now includes an API through which Matlab can directly communicate with VAST using a TCP IP connection The VastTools toolbox which is in cluded with VAST Lite uses this API to provide users with a number of addi tional tools for VAST including target lists 3D surface exporting and mea
50. ion on the format of the string You can also specify the order and the value limits for the parameters of your file name template to define the range of names of all the images or image tiles in your stack Image Parameters Here you can rotate and flip the images if necessary and tell VAST where to put them into the currently opened volume The Tile Size is extracted from the image file you selected but you can adjust it here too The Start coordinates are currently not allowed to be negative Please crop your images prior to importing if necessary Segment Label Parameters If you have a segment metadata file for the imported segmentation stack same format as the file written in section 3 4 12 you can provide it here The options below specify how VAST should deal with the segments in the imported stack If you are importing into a previously loaded segmentation you can either keep the imported segments separate by renumbering and appending them to the existing segments or merge the imported segments which have already existing segment IDs with the existing ones New segments will be appended If you use the option Merge with existing segments Renumber unknown segments together with a metadata file the hierarchy in the metadata file will be ignored Voxel Combination Parameters If Overwrite existing non zero voxels is checked imported non zero voxels will overwrite existing non zero voxels If it is not checked
51. ith a different program Fiji or Photoshop for example and then save a stack of aligned images which all have the same dimensions and are named and numbered in a consistent way for example img000 png img001 png Put all images into the same folder VAST can import single tile image stacks multi tile image stacks and 3D volume files In a single tile image stack each slice of the stack consists of a single image file In a multi tile image stack each slice is composed of several tiles in a XY grid and each tile is stored in a separate image file A 3D volume file stores all slice images in a single file Currently the only 3D volume file format that VAST supports is NIfTI nii VAST will convert image data to either 8 bit graylevel or 24 bit RGB when importing For importing and dicing VAST will use the RAM cache which is normally used for cacheing EM image data during viewing and painting Having lots of cache memory available will make importing somewhat faster because images have to be re loaded less often You can set the size of the EM image cache in the Preferences see section 2 2 2 3 1 1 Importing image stacks Pattern based names In the main menu of VAST go to File Import EM VAST will show a file browser dialog in which you can select one or several image files For importing 3D NIFTI files please select only one file If you import a single tile stack and do not want to use pattern based names select all sl
52. ith binary zipped data through HTTP see http www openconnectomeproject org services chru Before you can 4Essentially VAST requests 128x128x16 pixel blocks of the data set by reading from the Open Connectome server with URLs which specify the requested region like 3 2 VIEWING AND NAVIGATING AN IMAGE STACK 15 access the remote image stack you have to generate a VSVR file which specifies the parameters of the data set VSVR files are text files in a JSON like format here is the content of the openconnectome_kasthuril1 vsvr file Comment Source http openconnecto me ocp ca kasthuril1 info ServerType openconnectome ServerName openconnecto me ServerFolder ocp ca kasthuril1 SourceDataSizeX 21504 SourceDataSizeY 26624 SourceDataSizeZ 1850 TargetDataSizeX 10747 TargetDataSizeY 12895 TargetDataSizeZ 1850 OffsetX 0 OffsetY 0 OffsetZ 0 OffsetMip 1 TargetVoxelSizeXnm 6 TargetVoxelSizeYnm 6 TargetVoxelSizeZnm 30 TargetLayerName Kasthurii1C0penConnectome VAST comes with a few pre defined vsvr files which you can use to open and view some example data sets 3 2 2 The sidebar VAST provides a sidebar for zooming and moving through the stack The sidebar is a region close to the left and the right edge of the main window When you move the mouse cursor to the left or right edge of the window you will see it appear as a t
53. le just defines the web address of the data set to load and its dimensions 2 2 PROGRAM SETUP 7 Memory and Cacheing On the left side of the Preferences dialog you can set how much cache memory VAST will use maximally for voxel images and for segmentations VAST chooses initial values which are reasonable for your system The rule of thumb is If you can afford it leave 1 2 GB for the system and split the rest 1 3 each for image cache segmentation cache and general usage of VAST don t assign On a system with 8 GB RAM this means to give 2000 MB to the image RAM cache and 2000 MB to the segmentation RAM cache If you are only viewing images and not using segmentations you can increase the size of the image cache and reduce the size of the segmentation cache accordingly If you plan to use other programs at the same time or run two instances of VAST please reduce these values as needed VAST will not immediately use all of the allotted memory but it will stop reserving new memory for cache blocks and re use old blocks when it reaches the limit In general do not allow VAST to allocate more memory than the system has This can result in severe performance issues There is a memory usage indicator in the upper right corner of the VAST window which shows you how much memory is currently used The blue frame indicates the maximum amount of RAM which VAST uses for image and segmentation cacheing If the memory indicator becomes red and your
54. leting has to be done in every slice individually Z Scrolling During Paint Stroke is by default disabled to prevent paint ing errors when accidentally switching to the next slice before the paint stroke is finished However if you enable it you can very quickly coarsely label a long neurite running through your stack vertically by scrolling through the stack while following the neurite with your pen provided that loading of the image stack keeps up with the update rate of the screen 3 4 SEGMENTS 21 3 3 3 Using conditional painting The last section of the Drawing Properties tool window handles the settings for conditional painting If you switch on conditional painting by clicking the check box Enable only pixels will be painted for which the EM image fulfills certain criteria You can choose from three methods which determine the paintable pixels depending on whether the normalized brightness of the image pixel of the selected image layer is in a certain range which you can set The value range for minimum and maximum brightness is 0 1 If you are using several image layers and conditional painting does not seem to work please make sure that the correct image layer is selected in the Layers window Currently conditional painting does not work very well with z filling I rec ommend not using z filling set it to 0 when you use conditional painting 3 4 Segments 3 4 1 Picking segments You can select the segmen
55. m nanometers to micrometers Model output offset By default the models will be placed so that the upper left corner of the exported region is at the origin 0 0 0 You can provide a constant offset here if you want to move the models somewhere else Processing block size As mentioned above models are exported in smaller blocks and then glued together this specifies the block size A smaller block size will reduce memory consumption and may increase the speed of isosurface computation but will increase the processing time needed for glueing model parts together Changing the block size should only have an effect on memory consumption and processing speed not on the exported models Export what Here you can select whether you want to export models of all segments or of a selected branch and you can choose whether to export all segments individually or glue them together as they are currently displayed in VAST by selecting a 34 CHAPTER 4 THE VASTTOOLS MATLAB TOOLBOX parent segment and collapsing and expanding folders in VAST This informa tion will be read from VAST when you press OK so you can make adjustments in VAST while the VastTools export dialog is open File name prefix All OBJ file names and also the names of the exported objects will start with this prefix string Target folder All generated OBJ and MTL files will be stored to this target folder Use the Browse button to select a different folder
56. main VastTools window The following parameters for the export can be set Render at resolution Here you can select the mip level at which the surfaces should be generated 4 2 EXPORTING 3D MODELS 33 Lower mip levels will result in more detailed models but also generate larger OBJ files with more triangles and take longer to process Models and surface computations will be automatically scaled to compensate Use every nth slice If you want to reduce the resolution in Z you can do so by skipping slices If you set this value to 2 every second slice will be used if you set it to 3 every third and so on Models and surface computations will be automatically scaled to compensate Render from area This specifies the region of the current volume in which you want to generate surface models By default the region is set to cover the whole volume but you can change the values to cover a smaller region if needed These values are always specified at full resolution no matter which mip level is set in the Render at resolution selector Voxel size full res These fields will be filled in automatically by the values provided by VAST The voxel size is used to scale your models correctly You can override these values manually by entering different values into the edit fields Scale models by Additional scaling factors for your models By default these factors are set to 0 001 1 1000 in all directions to convert the units fro
57. ndary colors and pattern of the segment with the given ID Values for rl g1 b1 r2 g2 and b2 have to be between 0 and 255 pl defines the pattern and has to be between 0 and 15 p2 is currently unused If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code 52 APPENDIX B TECHNICAL INFORMATION res setsegmentcolor32 id coll col2 Same as setsegmentcolor8 but the two colors for the segment are given as two 32 bit values If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code x y Z res getviewcoordinates Returns the current view coordinates in VAST in voxels at full resolution If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code zoom res getviewzoom Returns the current zoom value in VAST If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code res setviewcoordinates x y z Sets the current x y z coordinates in voxels at full resolution of the view center of window in VAST If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code res setviewzoom zoom Sets the current zoom value Zoom values are currently integer values and can be negative The higher the value the more magnified the view A zoom value of O sets the pixel size of the full resolution image to exactly the Target R
58. o load image stacks acquired in light microscopy If a subset of cells with overlapping branches is labeled in such an image stack VAST can be used to generate a Z projection image which shows only one cell For this first paint over all parts of the cell you want to show Then use the VastTools function 42 APPENDIX A FAQ AND TROUBLE SHOOTING Export Export Projection Image to generate a Z projection image of only the segmented regions of the image stack For this use Selected EM layer as image source Segmented areas as opacity source and Additive as blending mode If you use Screenshots as image source you can optimize brightness contrast and blending of several layers in VAST to tune the Z projection image Tracking objects in a video If you translate a video into a sequence of images you can of course import this image sequence as a stack into VAST even in color In the same way as you can label three dimensional structures in VAST you can label objects or regions or fiducial points as they move through the video You can then export the labelings as an image stack and analyze locations in the image and movement Defining fiducial points in an unaligned image stack for manually aided alignment Some EM image stacks are difficult to align with automatic methods for exam ple if the image quality is bad there is high contrast background or the tissue slices have folds Manually defined fiducial points which sh
59. ogram you can re open a previously diced data set by using File Open EM from the main menu VSV files you open will be added to a list under File Open Recent EM from where you can quickly access them again The list contains the 16 most recent VSV files You can also open VSV and VSS files by drag and drop from a file browser Windows Explorer onto the VAST window VAST currently has a Move mode a Paint mode a Collect Mode and an Eyedropper Mode which you can set by clicking the tool buttons in the toolbar The cross of arrows icon selects Move mode and the little pencil selects Paint mode In this section we will explain how to use the Move mode For an explanation of the other modes please refer to section 3 3 The easiest way to navigate in the image stack is by using the mouse in Move mode You can pan move the image sideways by left clicking and dragging it You can use the mouse wheel to zoom in and out Alternatively you can zoom using the N and M buttons or the sidebar see below Use the UP and DOWN arrow keys or A and Z to scroll through the slices of the stack or the sidebar to scroll more quickly 3 2 1 Remote image stacks In addition to using an image stack in VAST which has been imported into a local VSV file you can also open and access image stacks which are hosted online VAST supports the Open Connectome Project Cutout Service from http www openconnecto me w
60. ontrast of the patterns If you enable the SelAlpha checkbox the opacity of the selected segment and its children will be controlled separately by the SelAlpha slider You can use this to highlight a particular segment or set of segments You can also switch the EM image layer on and off by clicking on the EM checkbox in the toolbar This is sometimes useful if you want to inspect just the segmentation You can change the size of the pen tooltip The easiest way is if you are using a pen tablet and VAST is properly configured to hold down one of the pen buttons and to move the pen up or down on the screen You can also use the and buttons The current pen diameter is displayed in the Drawing Properties tool window The third way of changing the tooltip size is to edit the Pen Diam text field in the tool window You can also lock the current tooltip size if you don t want it to be changed accidentally for example if the size of the tooltip is important for your data analysis by switching on Lock The checkbox Fill next to it switches automatic filling of closed contours on and off If enabled after each paint stroke VAST checks a rectangular area with the approximate extent of the stroke for empty closed contours of paint color and fills them with the paint color Below you can choose from Paint All Background and Parent This determines which voxels in the current segmentation are paintable If you
61. ould end up in the same place from slice to slice can help improve the alignment You can load an unaligned stack into VAST and use manual painting to define fiducial points Use a different color for each feature you are tracking through the stack so that in the analysis you know which points belong together A 3 Some Performance Tips e If file access is very slow when you move through the stack and it takes time until the images appear consider storing the data locally and or on SSD drives In particular especially when using non SSD drives put files which you use together on physically separate drives The problem is often that two files for example an EM layer and the segmentation layer are loaded at the same time from the same non SSD hard drive which causes the read write head to jump forth and back between two locations at high speed This slows down file access a lot e If you experience a low frame rate the mouse cursor is jumping rather than moving smoothly try to reduce the size of the Maximal Window Width in the Preferences to something like 1280 On very large screens you can set the Target resolution smaller than to 4 Default 2 to help e If VAST slows down considerably after using it for a while check if the RAM of your computer is full check the RAM usage indicator in the upper right corner of the VAST main window see section 3 1 5 Once memory is full Windows might swap parts of the data to the hard driv
62. ow and therefore also not change its coordinates Getting or setting coordinates will also use the coordinates of the center of the screen as do the anchor points of segments see section 3 3 While you drag the slice with the mouse VAST displays a transparent cross which indicates the location of the center Once you load an image stack a tool window labeled Coordinates will appear in the upper right corner of the main window If the tool window is not displayed you can open it using Window Coordinates from the main menu It shows you the current center coordinates and allows you to read and set these values The edit field in the tool window is updated as you navigate through the stack To save the current location simply copy the coordinates from that text field mark with the mouse and press CTRL C then paste it into the text editor of your choice You can also set the coordinates by entering or pasting numbers here and pressing Enter VAST will then jump to the new coordinates The exact format of the string does not matter VAST simply looks for the first three numbers in the string VAST does not mind whether there are commas or brackets or other non numerical characters This function is quite useful if you want to store coordinates of interesting points in an external text file or spread sheet Please keep in mind that the coordinate denotes the center of the current view The center is indicated by transparent crosshairs
63. parency value for this layer Switch off to hide layer e Bright Image Brightness switch on to enable brightness control e Contrast Image Contrast switch on to enable contrast control VAST can blend layers with different transparency modes Click on the button Menu to access more layer options The different settings for Blend Mode are e Flat All pixels in the image share the same transparency Default e Dark Transparent The darker a pixel R G B 3 the more trans parent it is e Bright Transparent The brighter a pixel R G B 3 the more transparent it is e Max RGB Dark Transparent The darker a pixel Max R G B the more transparent it is e Max RGB Bright Transparent The brighter a pixel Max R G B the more transparent it is Color Filter will open a color selection dialog where you can choose a color by which the layer images should be filtered during display To not filter the images choose white 255 255 255 Default 3 3 Painting The main function currently provided by VAST is painting of segmentations as a colored overlay of the image data When a stack of EM images is loaded you can enter Paint Mode by clicking the little pencil icon in the toolbar When you start a new segmentation like this VAST will ask you if you want to add 16 segments label colors to your segment list Also two floating tool windows will appear at the right side The upper on
64. perties Connectivity Surface VastTools Meshes obj mtl 3D Studio Max Figure 4 1 Exporting from VAST using VastTools using a different IP or port for example because VAST and VastTools run on separate computers you can set the IP address and port to use under Connect Connection Options in the VastTools main menu Once connected you can use the different functions of the VastTools toolbox by selecting from the main menu of the VastTools window in Matlab 4 2 Exporting 3D Models 3D model exporting generates surface meshes of the painted voxelized segmen tation in VAST and saves the resulting meshes in Wavefront OBJ files which are widely supported by 3D rendering and animation programs VastTools uses Matlab s isosurface function to generate the meshes Typically it will gener ate surface meshes in parts of the volume to limit memory usage and then glue together the mesh pieces to generate the final objects The exporter also saves a MTL file for each OBJ file which defines the material properties color as defined in VAST You can also measure the surface area of all exported objects and save the results to a text file If you select Export Export 3D Surfaces as OBJ Files from the main menu in VastTools a dialog window will pop up in which you can specify all the parameters for the export Once you click OK the exporting will start and run until finished or until you press Cancel in the
65. primary color a secondary color and a pattern that is used to blend between them You can also randomize the colors of all segments or set the primary or secondary color of all segments to the same color not recommended Please remember that by collapsing segment folders you can quickly and reversibly switch the displayed color of a segment to the color of the collapsed parent 3 4 12 Exporting segment metadata The entry Save Segment Colors in the context menu lets you export the metadata of the segments to a text file which you can then for example parse with MATLAB to extract colors hierarchies names anchor points etc of the segments for analysis 3 4 13 Segment information Segment Info in the context menu opens a window which shows you the in ternal information associated with the selected segment You can use this infor 3 5 SAVING SEGMENTATIONS 25 mation to count children of the segment get its internal ID or other parameters The text can be copy pasted if needed 3 4 14 Searching for a segment with a given name or ID At the top of the Segment Colors tool window is an edit field which can be used to find segments As you type or paste a text string into this field VAST will select the next segment after the selected segment in depth first search order the name of which contains the typed sub string The edit field is case sensitive If there are more than one segment which contain this sub string you
66. pty struct if failed info datasizex X horizontal size of the data volume in voxels at full resolution info datasizey Y vertical size of the data volume in voxels at full resolution info datasizez Z number of slices size of the data volume in voxels info voxelsizex X size of one voxel in nm info voxelsizey Y size of one voxel in nm info voxelsizez Z size of one voxel in nm info cubesizex X size of the internal cubes used in VAST in voxels always 16 info cubesizey Y size of the internal cubes used in VAST in voxels always 16 info cubesizez Z size of the internal cubes used in VAST in voxels always 16 info currentviewx Current view X coord in VAST in voxels at full res window center info currentviewy Current view Y coord in VAST in voxels at full res window center info currentviewz Current view Z coord in VAST in voxels slice number info nrofmiplevels Number of mip levels of the current data set Table B 2 getinfo struct nr res getnumberofsegments Returns the number of segments of the current segmentation or if failed If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code data res getsegmentdata id Reads out information of the segment with number id This is the same infor mation that gets written to a text file by Save Segment Colors in VAST see section 3 4 12 If the call succeeded res will be 1 otherwise 0 Use getlasterror to ret
67. r Info View Scale Bar and for scaling models and measurements in Vast Tools In the main menu under Info EM File Information you can enter and view text which will be stored in your VSV file as well This can contain a description of the data copyright information or other 14 CHAPTER 3 WORKING WITH VAST 3 1 5 The RAM usage indicator At the right side of the toolbar you can see a little field names RAM which shows the current RAM usage in your computer The blue frame indicates how much RAM VAST will use maximally for segmentation and image cache com bined Make sure that this frame is not dedicating more than 2 3 of your total RAM you can adjust these settings in the Preferences see section 2 2 2 The solid blue block shows how much RAM VAST has currently allocated including blocks allocated for segmentation and image cache The green area shows you how much RAM the Windows system and other programs are using The colors will change to yellow if the total memory usage goes above 90 and red if they go above 96 Running out of available RAM can slow down your system sig nificantly However in some cases Windows uses large amounts of the available RAM for disk caching and can free those instantly if more RAM is needed by programs without affecting the system performance 3 2 Viewing and Navigating an Image Stack After you imported a stack of images you can view them interactively After you closed the pr
68. r do a maximum value projection Object opacity 0 1 Areas selected in Opacity source will receive this opacity Values between 0 and 1 are allowed Use 1 for fully opaque Use shadows Shadow cone angle Enable the check box if you want to include fake shadows in the rendering The shadow cone angle determines how much the shadow spreads out by means of an image blur filter from layer to layer 4 4 MEASURING 37 Depth attenuation far brightness 0 1 If you set this to values below 1 slices which are further away in the stack will be darkened This can support the impression of depth in the resulting projection image Normalize projection image If enabled the brightness of the projection image will be adapted so that it uses the whole brightness range without changing color hue 4 4 Measuring VastTools provides several functions to measure objects labeled in VAST 4 4 1 Measure Segment Volumes This function can count the number of voxels of different segmented objects in a source area and save the results to a text file Parameters in the setup dialog of this function are similar to those of exporting 3D models and measuring surface sizes see section 4 2 above 4 4 2 Measure Segment Lengths This function is not yet implemented 4 4 3 Measure Segment Surface Area Since this function relies on a surface mesh to estimate the surface area it is part of the Export 3D Surfaces as OBJ Files function
69. ranches You can collapse and expand tree branches which are displayed in the same way as folders and subfolders are in the Windows explorer by clicking on the little or sign left of parent segments When you collapse a folder in the segmentation layer all its children will be displayed in the same color as the parent If you pick a segment color from the segmentation layer by shift clicking and the selected segment is in a collapsed folder the folder will be automatically expanded to show the native color of the segment you selected 3 4 5 Using anchor points Each segment has an Anchor Point stored with it This is an XYZ coor dinate vector which indicates the location of the segment in the stack Ini tially the anchor point is set to the point at which the segment is painted first You can jump to the anchor point by right clicking on a used segment in the Segment Colors tool window and selecting Go To Anchor Point from the context menu You can quickly jump to the anchor point of the selected segment by pressing the Home key You can also set the anchor point of the selected segment to the current view location as indicated by the center cross by se lecting Set Anchor Point from the context menu You will have to confirm this action in a pop up window to prevent accidental setting of anchor points 9If you pressed the Home key accidentally and want to go back to where you were you can select
70. ransparent white overlay strip Clicking into the sidebar and dragging the mouse up or down will scroll through the slices of the stack left mouse button or zoom right mouse button If you move the mouse cursor too far away from the side of the window the view will jump back to the previous view If you move the mouse cursor very close to the top or bottom of the window while scrolling not zooming VAST will start to scroll continuously with a speed depending on mouse cursor position You can use this function to quickly scroll through a very large image stack 3 2 3 Getting and setting coordinates VAST uses a coordinate system with a zero point in the upper left corner of the first slice with positive X to the right and positive Y down in the slice and Z marking the slice number Coordinates are given in pixels at full resolution http openconnecto me ocp ca kasthuril1 zip 6 1 129 1 129 1 17 The received file is then unzipped to extract the image data 5You can set the opacity of the sidebar in the Preferences under Side Bar Opacity 16 CHAPTER 3 WORKING WITH VAST the coordinates are independent of the mip map displayed The coordinates displayed in the upper left corner of the main window show the current location of the center of the main window You can switch the displayed coordinates on and off by using Info View Coordinates from the main menu Zooming in or out will not move the center point of the wind
71. reater of the largest window you will be using in pixels This value is used to determine how many textured tiles are needed to fill the entire window at all zoom levels Setting this value smaller reduces memory consumption and increases cacheing speed but if the value is too small the image texture might not reach all the way to the sides of the window at all zoom levels Target Resolution Smaller Than lets you specify the effective resolution of the displayed textures on the screen in screen pixels per texture pixel This affects at what zoom levels which mipmaps are used 2 is a good setting for this 1 makes it more detailed but slower and more memory consuming and 4 makes it faster but blurry Texture size m texture is m 2 defines how large the texture tiles will be which are used for displaying image and segmentation textures De pending on the graphics card some texture sizes might be faster than others I recommend to leave this setting at 128 Texture Smoothing You can set here whether you want to use texture interpolation This reduces aliasing effects but can result in a slightly blurred appearance of the textures The most natural setting of this is in my opinion A11 except Mip 0 which will show pixels with sharp boundaries only if you zoom in more than the native resolution of the image data The remaining options are self explanatory Opacity values have to be set between 0 fully tran
72. rieve the error code B 3 API FUNCTION REFERENCE 51 Returns a struct with the following fields if successful or an empty struct if failed data id ID of the requested segment data flags Flags field of the segment as 32 bit value data col1 Primary color as 32 bit value data col2 Secondary color as 32 bit value data anchorpoint XYZ coordinates of the segment s anchor point in voxels data hierarchy IDs of parent child previous and next segment 0 if none data collapsednr If the segment is collapsed into a folder this is the folder ID data boundingbox Segment bounding box may be incorrect if voxels were deleted Table B 3 getsegmentdata struct name res getsegmentname id Returns the name of the segment with the given ID Returns if the ID is out of range If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code res setanchorpoint id x y z Sets the anchor point of the segment with the given ID to the given coordinates in voxels at full resolution Only non negative values are allowed for x y and Z If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code res setsegmentname id name Sets the name of the segment with the given ID If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code res setsegmentcolor8 id r1 g1 b1 pl r2 g2 b2 p2 Sets the primary and seco
73. select 3 3 PAINTING 19 Paint All you will paint over or delete anything no matter if it was painted before or not If it is set to Background previous paints will not change but your paint will only be applied to voxels which have not yet been painted to If you erase only the current paint color will be erased to empty background This is the most useful painting mode Instead of only affecting background pixels Parent mode will affect only pixels which have the color of the immediate parent of the current paint color see below for a description of segment hierarchies When erasing voxels with the current paint color will be changed back to the parent color This mode is only useful in special cases in particular when re labeling a previously painted area to a new color 3 3 1 Multi scale painting A specialty of VAST is that it allows you to paint at different resolutions In fact VAST limits you to always paint at the currently displayed resolution The advantage of this is that the amount of data that has to be manipulated when you paint a stroke is limited by the window size and screen resolution Otherwise for very large volumes one could easily get into a situation in which the amount of data that has to be written for a paint stroke is much larger than what can be loaded in RAM at one time which would cause all sorts of problems including very slow painting Also it does not make sense to paint at a resolution
74. sparent and 255 fully opaque Press OK after you re done configuring the preferences 2 3 Additional Files Included With VAST Under Info Save Documentation ZIP To Disk in the main menu you can save out additional files which are packaged into the VAST executable as a ZIP file Select a target location and save then unzip the ZIP file Currently this includes a set of vsvr files to access some large EM data sets remotely see section 3 2 1 some Matlab scripts which can be useful for analyz ing VAST data in Matlab including the VastTools toolbox which can communi cate directly with VAST through the API and provides additional functionality see chapter 4 and this documentation as a PDF file Chapter 3 Working with VAST VAST uses its own file format VSV to store image data It can open VSV files immediately and navigate in them quickly If your data is a stack of for example PNG images you will have to import it into VAST before you can use it During importing the data will be saved into a VAST specific VSV data file which allows quick access to arbitrary parts of the data After opening a VSV image file you can create a segmentation by painting on top of the images or you can open an associated segmentation file VSS and view image and segmentation together VSS files tend to get big quickly but can be packed efficiently for example in a ZIP file You can view segmentations modify and save them You
75. sure ment functions It can be found in the vast_package zip file Since Vast Tools is a Matlab script users can extend the interface with their own functions Documentation of the VAST API is provided in Appendix B 3 4 1 Getting started with the VastTools Matlab Toolbox To start the toolbox open vasttools m in Matlab and run it If Matlab asks change the current directory A small window should pop up with a menu message area and cancel button Before you can use any of the functions of the toolbox you have to connect VastTools to an instance of VAST which is currently running First in VAST you must enable the Remote Control API Server Select Window Remote Control API Server in the main menu of VAST In the tool window which then opens enable a TCP IP port for communication by clicking the Enable check box in the upper left corner If you are running VAST and VastTools on the same computer you can use the standard settings IP 127 0 0 1 and port 22081 on both the VAST and Matlab side Then in VastTools connect to VAST by selecting Connect Connect to VAST in the main menu of the VastTools window If the menu item Connect Connect to VAST changes to Disconnect Disconnect from VAST you are connected The message log in the VAST Remote Control Server window will also show when a remote connection has been accepted In case you are 31 32 CHAPTER 4 THE VASTTOOLS MATLAB TOOLBOX Analysis Object pro
76. sv files support lossless and lossy compression to reduce the resulting file size e Image data and segmentations are stored in single files which makes it easy to copy them from one place to another e Support for loading EM stacks from a web server over HTTP opencon necto me format e Dynamic multi threaded cacheing in RAM with pre loading for low latency display update e Several image stacks can be opened and displayed together with a number of blending and tinting options e Multi scale painting in VAST painting always happens at the currently displayed resolution Mip level e Automatic convex Z filling during painting to speed up coarse labeling e Automatic 2D filling of closed contours e Label color patterns to create a larger number of distinguishable label colors e Label hierarchies for fast and reversible grouping of labeled segments e Anchor points to quickly find a given segment in the volume e Exporting of segmentations EM stacks and mixed image stacks screen shots in multiple formats e Importing of segmentations from image stacks e File modification free editing Image files are not changed except if you explicitly update information in them Segmentation files are only changed it you save the segmentation back to the same file e TCP IP based API through which external programs can directly com municate with VAST and supporting Matlab script Vast Tools which provides functions to measure l
77. system slows down try to REDUCE the cache limits to allow Windows and VAST to use more RAM for other data Some of the segmentation cache is used for holding the currently displayed part of the segmentation in memory When you exit the preferences dialog VAST will tell you how much of the segmentation cache it needs for the current display settings and whether the cache size is sufficient Disk Cache Directory Here you can specify the folder where VAST stores its temporary disk cache Click the button to browse Set this to a folder where you have lots of free space more than the size of the largest segmentation file you will be working with since for certain functions VAST has to duplicate the segmentation data Painting The segmentation bit depth is currently fixed at 16 bits This allows for a maximum of 65535 labels however since each segment is represented in the tree view of the Segment Colors window in VAST memory limitations in the Windows system might prevent VAST from using that many labels Tablet Mode Pen Paints Finger Moves On some pen enabled tablet computers VAST can distinguish finger and pen input If this mode is enabled the pen will paint and the finger will move the view when in Paint Mode 21t runs fine with more than 6700 labels in one of our data sets 8 CHAPTER 2 GETTING STARTED Display Properties Maximum Window Width specifies the width or height whichever is g
78. t in the Drawing Properties tool 8I have to do this because at the time of painting at a low resolution not all higher resolution images may be available in RAM they may even be too large to be loaded in RAM 20 CHAPTER 3 WORKING WITH VAST window VAST will then enable painting only when the image stack is zoomed to display the selected mipmap 3 3 2 Automatic Z filling The time that has to be spent to manually paint a segment in VAST depends largely on the number of 2D outlines that have to be drawn Especially if you follow a process that runs vertically through the volume you have to paint almost the same outline over and over again for every slice If you want to just get a rough outline of an object and you re not interested in a high precision of the boundary you could increase the painting speed by a factor of n if you paint the outline only in every n th slice or paint n slices at a time In the first case you get gaps of n 1 slices between the painted outlines in the second case it is hard to determine what you are actually painting because you can t see where your color goes in most of the slices VAST uses a third method It supports automatic Z filling of intermediate slices where the regions of the lower and the upper painted region overlap It turns out that in most cases neuronal objects are locally convex Exceptions are branches for example when a spine neck runs very close to the dendritic shaft Au
79. t color to paint with in the Segment Colors tool window by clicking on it in the tree view You can also pick any color you see in the segmentation layer by using the pipette tool To do this hold down the SHIFT key and click on the segment you wish to select This makes is very easy to switch between segment colors while painting Alternatively you can use the Pipette mode which you can select in the main toolbar If you hover over segment colors in the main window when in picking mode VAST will display the name of the segment as a tooltip 3 4 2 The segment hierarchy VAST can arrange segments in a tree like hierarchy This means that each segment can have other segments as children which can themselves have chil dren and so on VAST also allows you to collapse and expand parts of the tree dynamically so that you can quickly switch between a visualization which shows a whole branch of the tree in the same color or individual sub branches in individual colors For example if all spines of a spiny dendrite are labeled as sub objects children of the dendritic shaft one can instantly flip between a display in which the whole dendrite has the same color or each spine has a different color by opening and closing the dendritic shaft folder Segments can also be used as folders to group segments for example to classify labeled objects You can use tags to designate certain segments as folders to help external anal ysis see section 3 4 10
80. t to measure model surface area and not generate OBJ files 4 3 EXPORTING PROJECTION IMAGES 35 2015 by Daniel Berger Lichtman Lab Harvard _ Figure 4 2 Example stack projection images with simulated shadows generated with VastTools in Matlab 4 3 Exporting Projection Images The Export Projection Image tool allows you to generate 2D projection images in which your microscopic image stacks and or segmentations are projected along a cardinal axis This is for example useful to generate renderings of your segmentation or a Z projection of part of a confocal light microscopic image stack The following parameters can be set Render at resolution Sets the mip level at which the projection image is rendered This affects the resolution of the source images in X and Y but not Z Render from area This determines the XYZ block from which the image will be generated You can use the buttons Set to current voxel and Extend to current voxel together with moving the center cross in VAST to different locations to help you set up a source area Use every nth slice To reduce the resolution in Z and speed up rendering of very large stacks you can skip slices if you set this to an integer value larger than 1 Projection axis stretching Select here along which cardinal axis you would like to project and from what side which side should be in front in the image and whether the resulting 36 CHAPTER 4 THE VAS
81. the previous location from the drop down menu in the Coordinates tool window 3 4 SEGMENTS 23 3 4 6 Adding new segments There are several functions to add segments in the context menu of the Segment Colors tool window You can add a segment as next sibling or as a child of the selected segment Add 10 Segments will add 10 segments immediately after the selected segment Add Skeleton Segments adds a set of child segments to the selected segment which can be used for rudimentary skeletonization I have not found this function particularly useful though The most sophisticated way to add segments is Add Named Segments which lets you specify a naming scheme and add multiple named segments at the desired target location in the segment tree VAST will attempt to guess a naming scheme from the name of the currently selected segment You can change the name of any segment in the same way as file names are changed in the Windows Explorer click a selected segment name a second time then rename it 3 4 7 Helper functions for arranging segments Under Arrange in the context menu you can find two useful functions to move many segments at once Make All Siblings Children will move all siblings of the selected segment into its folder make them children of the selected seg ment Make All Children Siblings moves all children of the selected seg ment out of its folder and makes them siblings Be careful with t
82. tomatic z filling will only fill in the volumes of the overlap between the specified painted regions and in most cases for convex objects the filled regions will stay inside the segmented object Z filling makes sense across a few slices only because there will be no overlap if your object moves too much from slice to slice runs oblique Also VAST has to load multiple slices in RAM to be able to fill in those slices The maximal distance across which VAST lets you fill in depends on the size of the image cubes used Currently the cubes are set to be 16 voxels large and VAST allows you to fill in up to 8 slices because it loads two layers of cubes at a time In the data sets we are using this is approximately as far as z filling makes sense and it speeds up painting by a factor of 8 You can set how far the z filling will reach by setting Max Paint Depth in the Drawing Properties tool window This value controls both the distance at which Z filling occurs and the stepping distance for navigating with S X or PageUp PageDown keys to ensure gap free painting Automatic Z filling is only applied while painting not when erasing This makes it easier to correct what has been filled in in the case of non convex neighborhoods This also means that the best strategy to draw an object coarse to fine is to try to paint conservatively try to stay within the object boundaries and correct by adding paint rather than removing paint because de
83. turns both the decoded subvolume and a list of segment IDs in the volume and number of voxels for each ID See getsegimage above for information on volume coordinates If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code segimage values numbers bboxes res getsegimageRLEdecod edbboxes miplevel minx maxx miny maxy minz maxz surfon lyflag Same as getsegimageRLEdecodedcountunique above but also returns the bounding boxes for all IDs See getsegimage above for information on vol ume coordinates If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code res setsegtranslation sourcearray targetarray Tells VAST how to translate the segmentation volume before transmitting it through getsegimage and getRLE functions sourcearray and targetarray are arrays of unsigned positive 16 bit values and have to have the same length VAST will convert all voxels with IDs in sourcearray to the corresponding val ues in targetarray Voxels with other IDs will be removed set to 0 To disable the segmentation translation call setsegtranslation If the call succeeded res will be 1 otherwise 0 Use getlasterror to retrieve the error code emimage res getemimageraw layernr miplevel minx maxx miny maxy minz maxz Reads out the EM image of the specified layer as a voxel image for a given mip level resolution and area
84. which is much higher than the screen resolution because mouse or pen precision is also limited Finally allowing low resolution painting can save a lot of memory if large objects are painted coarsely In VAST images are stored as a pyramid of mipmaps with reduced reso lution using powers of two factors Painting always happens at the resolution of the currently displayed mipmap This means that you can change the res olution at which you are painting by zooming A single segmentation can be composed of parts at different resolutions For example it is possible to draw a rough outline of an object at a low resolution and then to zoom in and cor rect the object s shape at a high resolution VAST will automatically upscale and downscale the displayed segmentation as you zoom but zooming will not change the painted segmentation The segmentation is stored at the resolution at which it was painted If you paint at a low resolution first and then correct at a high resolution part of the low resolution segmentation will be replaced by a high resolution version If you paint at a high resolution first and then correct at a low resolution part of the high resolution segmentation will be replaced by a low resolution version including pixels in the vicinity Sometimes you might want to make sure that a painted segmentation has a certain resolution You can enforce painting at only one resolution by restricting painting to a particular mipmap Restric
85. y on your data and the compression method used Then the images will be read diced and put into the target file After that VAST will compute the mipmaps and put those in the target file too Depending on the size of the data this process can take several hours For example a big data set of 350 GB takes about 5 hours to import on a recent desktop machine The limiting factor is the speed of hard drive access You can cancel the importing but the target file will then be incomplete corrupted and can not be used with VAST 3 1 3 Importing 3D volume files Importing a 3D volume file is easier than importing an image stack The only format currently supported by VAST is Nifti VAST will ask you to specify the name of the source nii file and the name of the target vsv file Currently VAST requires the whole Nifti file to be loaded at once into RAM so this only works for smaller volumes Also the data in the Nifti file currently has to be 8 bit per pixel 3 1 4 Image scale and description After importing you should set the voxel size of your data in the file To do this go to Info Volume properties in the main menu Here you can set the X Y Z size of a voxel in your data set in nanometers Press Save to file to store the information you entered in the VSV file This dialog also displays how large your image stack is in voxels The voxel size entered here is used for the scale bar which you can enable in the main menu unde
86. you paint Your tracings will be held in RAM and or a cache file on disk until you explicitly save them If you open a segmentation from a VSS file and work on it the file will not be changed unless you explicitly tell VAST to save the changes you made to the opened file by selecting Save Segmentation from the main menu If you want to keep the previous version and save to a new file use Save Segmentation As instead VAST will then take all data from the opened file the RAM cache and the segmentation cache file and combine them into a new file on disk 26 CHAPTER 3 WORKING WITH VAST We have had cases in which people had VAST open for several days without saving and lost a lot of work when the computer crashed Please save your work once in a while 3 5 1 Save Segmentation As Special Save Segmentation As Special provides you with two functions to save your current segmentation to a new file in a modified way First you can choose to save only the selected segment or subtree of the current segmentation to a new file Alternatively you can change the resolution of your segmentation and adjust the canvas size on which the saved segmentation will open this is currently only supported when saving all segments To save only the selected segment or the selected segment and its child tree to a new segmentation select that option from the drop down list and press Save Please be aware that the internal IDs of the segm

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