Users Manual
Contents
1. in the above window the two output coexpression files are AP_EN_EVE coexpress apo and AP_EN_EVE coexpress txt The former can be viewed by dragging and dropping to the Vaa3D 3d viewer The candidate cells that coexpress are highlighted by white The second file AP_EN_EVE coexpress txt save the detail information of the coexpressed cells One you set up these parameters click Finish button the program will produce the predicted coexpressing cells in a second Your job is to check the AP_EN_EVE coexpress apo and AP_EN_EVE coexpress txt and generate a manually curated file text format listing the confirmed coexpressing cells The format of this new text file is final cell name cell name in marker A cell name in marker B For example aCC A1L aCC pMAD A1L aCC eve A1L aCC A1R aCC pMAD A1R aCC eve A1R BDL1 A1L BDL1 pMAD A1L BDL1 bar A1L BDL1 A1R BDL1 pMAD A1R BDL1 bar A1R Also see cellname_mapping txt in the input folder This file will be used in Merge coexpressing cells see below Merge coexpressing cells Once you select this function the following window will pop up Pointcioud atlas merge coe x pressed ce lic e ditor Select an atlas file apo _oregan data final_data atlas_20110926_with_folders output atlas_PS1 apo File Existing atlases are Users longf work fly_oregan data final_data atlas_20110926_with_folders output atlas_AP apo Users longf work2. histogram gt image_blending gt gt gt gt gt gt gt gt FlyWorkstation_utilities Z 48 X 151 image_edge_detection Y 127 is image_filters image_geometry M Focus cross Link out V Linked image_registration image_resolution Zoom Regular x1 4 x8 Looking glass x4 image_ROI Segment all image objects adaptive thresholding followed by watershed apane image_stitching Label_Objects gt about Zy plane image_thresholding lobeseger gt u Xz plane linker_file J marker_utilities Use looking glass Reset movies neuron_toolbox neuron_tracing gt gt gt gt gt gt Tri view zoom 1 Click to set gt neuron_utilities gt gt gt gt gt gt gt Channels 3 Intensity Misc pixel_intensity 0000 al 4 Max principal_skeleton_detection sync3D E 00ff00 2 vw J Sum M T Information of your selections amp O0000ff c3 T i cei Vaa3D_PluginInterface_Demos Voxel type UINT8 Tri view zoom 1 Index Focus 151 127 48 RGB 29 0 0 Channel min max C1 min 0 max 253 C2 min 0 max 253 C3 min 0 max 253 MR MG MB Reset 0 255 Defined marker location Landmark controls Copy Paste Load Save Landmark Atlas Color Manager See in 3D Help After that a window for parameter setting will pop up EOR S Define watershed parameters Channel B Median filter size 2 Smoothing Gaussian filter size 1 Gaussian filter sigma 1
3. 10D07_4c tif 10D07_1 tif ano gray tif 10D07_5Sc tif pz 10D07_1 t 0 mask raw 3 chl_eve 3 10D07_3 tif ano ch2_10D07 e 10D07_3 tif ano apo 10D07_3 tif ano gray tif 10D07_3 t 0 mask raw 10D07_4 tif ano 10D07_4 tif ano apo 10D07_4 tif ano gray tif 10D07_4 t 0 mask raw 10D07_5 tif ano 10D07_5 tif ano apo 10D07_5 tif ano gray tif 10D07_5 t 0 mask raw FJ Bees B me For each channel in the original image there is a 1 ano file It can be opened with a text editor It contains the name of three associated files GRAYIMG 10D07_1 tif ano gray tif MASKIMG 10D07_1 tif ano mask raw ANOFILE 10D07_1 tif ano apo 2 tif file This is the original image data it can be dragged into Vaa3d to be viewed 3 mask raw file This is the result of segmentation it can be dragged into Vaa3d to be viewed 4 apo file This can be opened with a text editor or dragged into Vaa3d It is a csv file with information about each segmented object n orderinfo name comment z x y pixmax intensity sdev volsize mass color _r color_g color_b 1 1 A 10D07 T3R 45 191 179 253 000 208 939 52 313 1983 000 414326 000 251 38 17 2 2 85 25 248 253 000 195 720 61 394 3525 000 689914 000 51 176 176 Annotation Annotation is done using Vano software update 1 9 A detailed users guide is available using the following link http vano cellexplorer org how to use This program opens the original tif file the segmente
4. 121 max 253 C3 min 0 max 253 MrR MaMe Reset 0 255 Defined marker location Reset Defined marker location Landmark controls Landmark controls Copy Paste Load Save Copy Paste Load Save Landmark Atlas Color Manager Landmark Atlas Color Manager See in 3D See in 3D Help Help If the results are satisfactory i e most nuclei are separated and there are few over segmented nuclei phantom objects you may save it Otherwise you may change the parameters and retry To save your result choose File from Vaa3D menu and then click export At this point you should have four files for each channel see below for example All of the files associated with Eve should be placed in a single folder 10D07_I1c tif 10D07_1 tif ano 10D07_3c tif 10D07_1 tif ano apo 10D07_4c tif 10D07_1 tif ano gray tif 10D07_5Sc tif es 10D07_1 tif ano mask raw C chl_eve e 3 10D07_3 tif ano J ch2_10D07 10D07_3 tif ano apo 10D07_3 tif ano gray tif es 10D07_3 tif ano mask raw 3 10D07_4 tif ano 10D07_4 tif ano apo 10D07_4 tif ano gray tif rs 10D07_4 tif ano mask raw 10D07_5 tif ano 10D07_5S tif ano apo 10D07_S tif ano gray tif es 10D07_5S tif ano mask raw In addition all of the files associated with additional markers should be placed in separate folders The file names in each folder should be identical for example 10D07_1c tif 10D07_1 tif ano 10D07_3c tif 10D07_1 tif ano apo
5. are displayed in gray You may click index radio button on the right to display them in color You may also use See in 3D function to visualize the cells in 3D vaa3d64 File Image Data Visualize Advanced Plug In Window Work Mode Help eoo Vaa3D pry YF Volumes LaCie laptop_20110331 fly_oregon_final_data atlas_data_complete_20110223 repo_hb9_eve ch3_ev eoo Segmented Image Image data Opti Image data Options a Views XY upper left ZY upper right XZ lower left Focus Coord Views XY upper left ZY upper right XZ lower left Focus Coord Z 65 ra 65 x 151 X 151 Y 113 ls Ve 113 ily M Focus cross Link out V Linked M Focus cross M Link out Linked Zoom Regular x1 4 x8 Looking glass x4 Tri view zoom 1 Click to set Zoom Regular x1 4 x8 Looking glass x4 XY plane XY plane ZY plane ZY plane XZ plane XZ plane Use looking glass Reset Use looking glass Reset Tri view zoom 1 Click to set Channels 3 Intensity Misc Channels 1 Intensity Misc E ff0000 c1 M Max affffff cl Max E 400ff00 c2 vw Sum Sum Mean 3 3 Mean Information of your selections HB oo00f 3 M j Information of your selections OIT OIT Voxel type UINT8 Tri view zoom 1 ndex Voxel type UINT16 Tri view zoom 1 Index Focus 151 113 65 RGB 11 157 2 Focus 151 113 65 RGB 0 0 0 Channel min max C1 min 0 max 253 C2 min 0 Channel min max C1 min 0 max
6. fly_oregan data final_data atlas_20110926_with_folders output atlas_EN apo Users longf work fly_oregan data final_data atlas_20110926_with_folders output atlas_REPO apo Users longf work fly_oregan data final_data atlas_20110926_with_folders output atlas_PSl apo Remove Select the coexpression file gata final_data atlas_20110926_with_folders input cellname_mapping txt File Output folder Users longf work fly_oregan data final_data atlas_20110926_with_folders output Prefix of the names of output merged files atlas AP_EN REPO PS1 final Cancel Finish This window is similar to the previous window except 1 Select the coexpression file You select the file semi automatically generated by the last step For example here we use cellname_mapping txt we generated This file lists all the coexpressing cells in the current atlas 2 Prefix of the names of output merged files If you use atlas AP_ EN REPO PS1 final then the final atlas will be name as atlas_AP_EN_REPO_PS1_final apo and the statistics of cells in this atlas will be atlas_AP_EN_REPO_PS1_final apo_cellStatistics txt Note that the suffix cellStatistics is defined in your configuration file i e atlas_config txt under plugins folder You may change it by simply editing the atlas_config txt or by using the Adjust preference function In addition each marker will have its own final atlas named by atlas_ marker name _merged a
7. no matter the annotated ratio You don t have to provide this file if you don t force any cell to be included You can find a list of forceAddCells in the input folder After setting up these parameters you click OK The program will run and generate the initial atlas shortly You may find your results under the output folder that you ve setup in the Adjust preference step 1 For each marker the atlas is named by atlas marker apo 2 The initial atlas that combines all the markers is named by atlas_all apo 3 The statistics of cells expressing each marker is saved in atlas maker apo_cellStatistics txt 4 The atlases of different cell types are named by atlas celltype apo Common errors 1 Build the atlas hangs Did you create cellnamelist_marker txt 2 atlas_marker apo is empty cellnamelist_marker txt may have hidden characters 3 Standard deviation is really large Possible mis annotation of a cell identity or the segment is off by one relative to the Eve channel Detect coexpressing cells The initial atlas you build using the above steps may contain duplicated cells that express different markers You may use Detect coexpressing cells function to detect the co expressing cells Once you select this function the following window will pop up 1 2 3 oexnress Select an atlas file apo jregan data final_data atlas_20110926_with_fol
8. the pointcloud atlas builder linker file editor see the following window Common reference e g gene name EVE EVE Signal name e g gene name 10D07 Signal files and reference files match Signal folder uilding_1 9 real_data 10D07 ch1_eve Folder Contains 4 point cloud apo files Reference folder ling_1 9 real_data 10D07 ch2_10D07 Folder Contains 4 point cloud apo files Previous existing 0 signal s are Tip If any texts above become red color you need to enter valid file folder info If the common target pointcloud ref file is left empty then define target automatically Cancel Finish Add a new signal This editor allows you to create a linker file that will be used by the software to build the atlas It lets you proceed by adding markers one by one Common reference The common reference marker is EVE Signal name The name of the marker of interest should be entered in Signal name e g gene name Here we use 10D07 as an example Note that signal name 10D07 corresponds to cellnamelist_10D07 txt created as described in the Annotation section Signal folder is where the marker channels of your annotated marker image stacks are saved apo generated by manual annotation using VANO software using the names deposited in the cellnamelist_eve_nocluster txt Reference folder is where the reference channels EVE of the dataset are saved As mentioned above file names i
9. 00 Thresholding Global 2D Segmentation Method Intensity based watershed Cancel OK Channel You need to specify which channel you want to segment Channel number starts from 1 yD Smoothing You need to adjust Median filter size Gaussian filter size and Gaussian filter sigma to reduce noises in your image before segmentation Thresholding You may choose Adaptive Global 3D and Global 2D to separate background from signal This information is used in later segmentation Adaptive thresholding detects the threshold values at uniformly sampled pixels based on the statistical information of the local context and then produces pixel resolution thresholds by interpolation Global 3D detects a unified intensity threshold value on all pixels assuming the background level does is more or less constant across pixels in an image stack Global 2D is similar but thresholds are computed for each 2D plane rather then a 3D volume and a smoothing step is applied to produce continuous thresholds between adjacent 2D planes Segmentation Method You may choose intensity based watershed or shape based watershed The former is more sensitive to noise Once you setup all the parameters you may click OK button The software will segment cells from the specified channel of the loaded stack The result image will be displayed in another image window see below The segmented cells
10. Image acquisition Co stain with anti Even skipped 2B8 1 50 often this needs to be pre absorbed several times for optimal staining or 3C10 1 50 available at DSHB Use stage 16 embryos see Pereanu et al 2007 for morphological staging criteria Images should be acquired at least 8 bit 512x512 resolution or better using a 60x or 63x objective and 0 3 to 0 5 microns steps Segments T2 T3 and A1 should be included Images can be cropped e g using ImageJ to eliminate regions without cells of interest Segmentation First start Vaa3d http Vaa3d org Peng et al 2010 To segment cells in an image stack drag and drop the image stack into Vaa3d The file will be opened and displayed Then you choose from Vaa3D Plug In menu the Image_segmentation plug in Then you choose Cell_Segmentation and then segment all image objects adaptive thresholding followed by watershed Note these are not currently present in the Windows release this is available for Mac only vaa3d64 File Image Data Visualize Advanced Window Work Mode_ Help eoo Plug in manager Vaa3D Re scan all plugins _Vaa3D_plugin_creator SP celegans Volumes LaCie laptop_20110331 fly_oregon_final_data atlas_data_complete_20110223 repo_hb9_eve ch3_ev Y color_channel Image data Options data_lO Views XY upper left ZY upper right XZ lower left Focus Coordinates Ca data_type gt gt gt gt gt gt A gt
11. RP2 eve AL lt Sivi gt 5 Color gt gt n 1 a E 000000 91 16 9 100 607 333 AP2 ewe AIR lt sivi gt s Current Atlas projects J 000000 6 14 16 100 797 RP2 eve T2 T T ET Name Comments ammm 1 2 Ea pa 000000 42 1 i 100 794 222 AP2 ewe TIR Sivi Cilie Ailes Current Atlas projects _ 1 3 rw E 00000 26 is 7 100 724 444 RP2 seve TIL lt Sivi gt s Current Atlas_project Oprens o 000000 27 15 10 100 687 33 RP2 eve TIR lt Sivi gt Oilie Ailes Current Atlas_project _ Attached to 30 view 133 46 9 100 698 889 Ul eve AlL lt Stvi gt Cilie Hhes Current Atias_project Valiente _ 1 4 Find Next Prewous Mighligh all hits Panel E Main window 1 Object manager button clicking this button opens the Object Manager window shown in panel F 2 Surface cut the default view for the 3D atlas data in the main window 3 X Y Z rotation controls spinning the analog dials rotates the 3D atlas around the indicated axis and using the digital window below can be used for the same purpose 4 Zero button this realigns the 3D dataset to 0 0 0 degrees Panel F Object manager This menu is used to select desired cells manipulate cell color add or read comments about the cells It can be set to two formats to manipulate individual cells by selecting the Point Cloud function 5 or to manipulate all the cells that express a single marker by selecting the Point Cloud Set function 6 Wit
12. _type_motoneuron txt cellname_type_secretory txt You can find them in your input folder To remove any of these files click the Remove button on the right Then you click the File button on the right of the cell type file It will pop up a window allowing you to select a file The selected file will be added into the Existing cell type file box You may add more files by repeating this process The files in the input folder do not need to be edited 6 Cell statistics file suffix is the suffix of the cell statistics file that will be generated by the program For example if you use cellStatistics txt then the Statistics of the AP maker is saved in atlas_AP apo_cellStatistics txt Note that currently the program always use atlas_ marker name apo as the initial atlas output file before coexpression detection and merging It uses atlas_all apo as the initial atlas combining all markers This does not need to be changed 7 Marker map file is the name of file indicating for each cell which marker it expresses 1 expresses 0 does not express It has the following format Markerl marker 2 marker n Cell 1 1 O O Cell 2 0 1 1 Cell 3 L 1y s 0 You may name it differently but this does not need to be changed 3 Create a new point cloud atlas linker file Once you Select Create a new point cloud atlas linker file it will pop up
13. ata final_data atlas_20110926_with_folders input cellname_type_secretory txt Cell statistics file surfix cellStatistics txt Marker map file genemap txt Cancel Finish When you select Adjust preference from the menu you will see the above window pop up The program reads the information in atlas_config txt and fills them in the screen 1 2 3 4 5 Input folder is where the user puts input files such as cellname lists that tells the computer what cell names to expect The user needs to create a file called input In the input folder place all of the files from input zip found in the Users Manual section of the supplemental methods Using the Folder navigate to the input folder Output folder is where the user puts files produced by the atlas builder such as linkers and new apos see below Create a file called output Using the Folder navigate to the output folder Cell name file prefix is the prefix of cell name lists For example the names of cells expressing AP markers are saved in cellnamelist_ap txt So cell name file prefix is cellnamelist This is shared by all the markers There is no need to change this field Cell name file suffix is the suffix of cell name lists In this example it is txt There is not need to change this field Cell type file is a list of cell type file names e g cellname_type_interneuron txt cellname
14. d mask raw file and the apo file with annotations for each object in a single interface It allows the user to navigate through the images and insert annotations Note that during annotation cell segmentation can be manually adjusted Depending on the quality of the starting image the user may need to extensively manually edit the segmentation It is important to have a single annotation for each cell of interest Any updates will change the mask raw file as well as the apo file Also note if an object is left unannotated by the user it will be ignored in subsequent steps For the Even skipped pattern a user must annotate the following cells aCC eve T2L aCC eve T2R aCC eve T3L aCC eve T3R aCC eve A1L aCC eve A1R pCC eve T2L pCC eve T2R pCC eve T3L pCC eve T3R pCC eve A1L pCC eve A1R RP2 eve T2L RP2 eve T2R RP2 eve T3L RP2 eve T3R RP2 eve A1L RP2 eve A1R U1 eve T2L U1 eve T2R U1 eve T3L U1 eve T3R U1 eve A1L U1 eve A1R U2 eve T2L U2 eve T2R U2 eve T3L U2 eve T3R U2 eve A1L U2 eve A1R U3 eve T2L U3 eve T2R U3 eve T3L U3 eve T3R U3 eve A1L U3 eve A1R U4 eve T2L U4 eve T2R U4 eve T3L U4 eve T3R U4 eve A1L U4 eve A1R U5 eve T2L US eve T2R U5 eve T3L U5 eve T3R U5 eve A1L U5 eve A1R The easiest way to do this is to cut and paste from the cellnamelist_eve_nocluster txt file found in the input folder see below The program does not tolerate typos but does tolerate extra spaces at the beginning or end o
15. ders output atlas_EVE apo File Existing atlases are Users longf work fly_oregan data final_data atlas_20110926_with_folders output atlas_AP apo Users longf work fly_oregan data final_data atias_20110926_with_folders output atlas_EN apo Users longf work fly_oregan data final_data atlas_20110926_with_folders output atlas_EVE apo Remove Coexpression rules v Symmetry rule Distance less than pixels 5 Y Bilateral rule output folder Users longf work fly_oregan data final_data atlas_20110926_with_folders output Prefix of the names of output coexpression files AP EN EVE Cancel Finish Select an atlas file apo You may select a set of atlas files to detect their coexpression Click File it opens a window allowing you to select an atlas file The selected file will be shown in the Existing atlases are box You may select multiple atlas files You may also remove any of them by first selecting the files in the box and then click the Remove button on the right hand side Coexpression rules Three rules are used by the program to detect coexpression You may define the distance between coexpressing cells You may also selecting symmetry if two cells in the left hemisegment coexpress their right hemisegment parterners should also coexpress and bilateral coexpressing cells must be in the same hemisegment or midline rules Prefix of the names of output coexpression files if you use AP_EN_EVE
16. f the annotation For other patterns a user must annotate any cell of interest with a unique cell name The convention we use is as follows cellname marker segment e g aCC eve T2L Each cellname should also be listed in a text file named cellnamelist_marker txt Note creating a cellname list with hidden characters when using Excel for example can produce an error so it is best to use a manual text editor to compile this file The cellname file should be saved in the input folder see below with the name cellnamelist marker txt Vaa3D interface The image is reproduced from Figure 1E F A list of the controls is given below Volume Sef Otect GREED Anes Save Mowe vo Boots box Acemmate gt gt Paralel Gcale dar n ceee C eme Vol Cotormap Object Messer a pT 2 vosme Cut T Surface Cut m Lo X man X man an o _ vom SS l ma l ean nal eee GEE 200m 4 sun x Y z 3 i o 90 Freeze Ge Zero ee 4 F Marker 0 Label Surface 0 Neuron line Structure 0 Bolnt Cloud 90 Poirt Clowd Set 1 ont co o y a y Select All O 7 o y a 000000 os 64 19 100 547 111 CLCPM eve AIR lt SEVE gt s Current Atlas projects Select None _ 8 a v Boo o 71 15 100 537 125 CLCPM eve TIL lt SEVED Single Color 9 E acoooo 6 s 13 100 683 333 ELCPM TIR Seve TS NAO ED 4 L leve lt gt Random Color Mapping On on r 1 0 a aooo e9 11 7 100 724 556
17. hin each of these functions the following controls can be used 7 Select all this selects all the cells or point clouds markers and once a cell marker is selected you can apply a color to it or manipulate it in other ways 8 Select none this de selects all selections 9 Select inverse this toggles selected to deselected and deselected to selected 10 On off on adds the selected cell marker to the atlas view display in panel A off takes the selected cell marker out of the display 11 Color clicking this button opens the color choice window red line to left with three choices Single Color allows you to choose a single color to apply to the selected cells markers Hanchuan s Color Mapping automatically assigns a pre ordered color selection uniquely mark each cell marker selected and Random Color Mapping gives every cell marker a random color 12 Name Comments this button brings up a window allowing the user to edit the name comments fields 13 Undo this button reverses the last action 14 Accumulate allows sequential search strings using field 19 to accumulate 15 On off column the boxes in this column show selected cells markers They can be toggled individually or using the controls 5 7 16 Color column shows the color the cell marker presents in the display 17 Name this column shows the cell marker name 18 Comment this column shows the markers exp
18. ill see the following functions 1 Adjust preference 2 Create a new point cloud atlas linker file 3 Edit an existing point cloud atlas linker file 4 Build the atlas 5 Detect coexpressing cells 6 Merge coexpressing cells 7 About this plugin 2 Adjust preference The basic parameters used for the atlas builder are saved in a configuration file atlas_config txt You can find this file under the atlas_builder folder that you put under v3d_external bin plugins or v3d_external v3d plugins depending on your version of Vaa3D Currently this file contains the information used for running atlas builder on an example machine You need to run Adjust preference function only once to setup your own Once the parameters are set you do not need to reset them unless you rename your files or move the data Input folder longf work fly_oregan data final_data atlas_20110926_with_folders input Folder Output folder jngf work fly_oregan data final_data atlas_20110926_with_folders output Folder Cell name file prefix celinamelist Cell name file surfix txt Cell type file File Existing cell type files rork fly_oregan data final_data atlas_20110926_with_folders input cellname_type_galia txt rork fly_oregan data final_data atlas_20110926_with_folders input cellname_type_interneuron txt rork fly_oregan data final_data atlas_20110926_with_folders input cellname_type_motoneuron txt Remove rork fly_oregan d
19. lthough you may choose any annotated file apo as your target file you can only use Eve apo if you want to map your atlases to our atlases Eve apo can be found in Supp File 2 Reference marker control point file for registration click File button to select the cellnamelist_eve_nocluster txt under your input folder Note that if you use a different target file you also need to provide a different control point file Save registration data if you select this radio button the 3D positions of each cell in each image stack before and after registration will be saved into before_registration_marker txt for markers after_registration_marker txt for markers before_registration_ref txt for reference marker i e EVE after_registration_ref txt for reference marker i e EVE Annotated stack must pass a ratio 0 1 of this is where you may select the ratio of annotated stacks in order to include a cell in the final atlas For example if you set 0 3 and you annotated 10 stacks then a cell can be included in the atlas if it appears in more than 0 3 10 3 stacks We have found that the more often a cell appears the more reliable its final position So building an atlas at a ratio of 0 5 will have fewer cells but the ones that are present will be at more reproducible positions 6 Select force added cell file select the file that list the cells that must be included into the atlas
20. n the signal ch2_10D07 and reference ch1_eve should be identical To select these folders you need to click the Folder buttons to the right of Signal folder and Reference folder The software will automatically test if the files in signal folder match those in the reference folder If any mismatch occurs for example a particular stack has only a marker channel but no reference channel the font in the two text area on the right of Folder will be changed to red You need to check the problem of your data before moving on to the next step If no error is found the marker added will be displayed in the text area below You can click Add a new signal to add another marker or click Finish to end adding new markers Once you click finish a window allowing you to select where the linker file should be saved will pop up You may put it under your output folder 4 Edit an existing point cloud atlas linker file Select a linker file silding_1 9 output_folder linkers 10D07_linker pc_atlas File Select a target file for registration to_update Supp File 2 eve apo EVE apo File Reference marker control point file for registration as_building Atlas_building_1 9 input_folder cellnamelist_eve_nocluster txt File e Save registration data Annotated stack must pass a ratio 0 1 of g Select force added cell file jlding_1 9 input_folder forceAddCells_final txt File Cancel OK After you c
21. po e g atlas_AP_merged apo and the statistics file is atlas_AP_merged apo_cellStatistics txt Once you click Finish button it will generate the merged atlas
22. reate the atlas linker file you may need to add new markers as your data accumulate This can be achieved by clicking Edit an existing point cloud atlas linker file and choosing the linker file that you want to edit It pops up a window similar to the one when you create a linked file but can display all the existing markers in the linker file You may follow the same steps you used in creating the linker file 5 Build the atlas Once you setup your parameters and create atlas linker file you may build the atlas For that you select Build the atlas in the atlas builder the following window will pop up Select a linker file ta atlas_20110926_with_folders output import pc_atlas File Select a target file for registration eve_7 ano ano_new_new_new adjust apo File 1 2 3 4 5 Reference marker control point file for registration al_data atlas_20110926_with_folders input cellnamelist_eve_nocluster txt File e Save registration data Annotated stack must pass a ratio 0 1 of 0 Select force added cell file _data atlas_20110926 forceAddCells_final txt File Cancel OK Select a linker file click File button to select the linker file you created through Create a new point cloud atlas linker file or Edit an existing point cloud atlas linker file steps Select a target file for registration click File button to select the target file Eve apo for registration A
23. ressed by each cell 19 21 Search function Type search query 19 and select either next to find the next occurrence of the search term or previous to find the previous occurrence 20 or Highlight all hits 21 which highlights all occurrences of the term After the search terms are highlighted they can be selected deselected by using buttons 7 8 Successive searches can be combined by toggling the Accumulate button 14 prior to starting the search Registration 1 Main menu Put the atlas_builder plug in into the plugins folder for Vaa3d Mac only The atlas builder plug in can be found in Supp File 5 Users Manual folder as a zip file Click Plug in from Vaa3D menu select atlas_builder You will see pointcloud_atlas_builder Plug in manager Re scan all plugins _Vaa3D_plugin_creator atlas_builder pointcloud_atlas_builder P Adjust preference Create a new point cloud atlas linker file Edit an existing point cloud atlas linker file Build the atlas Detect coexpressing cells Merge coexpressing cells About this plugin image_blending image_edge_detection image_filters image_geometry image_registration image_resolution image_ROI image_segmentation image_stitching image_thresholding linker_file movies neuron_utilities pixel_intensity principal_skeleton_detection refExtract Vaa3D_PluginInterface_Demos vvvvvvvvvvvvvvvvvyvy Wwasv Select pointcloud_atlas_builder you w
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