3D Image processing with MorphoGraphX User manual
Contents
1. Process Mesh Segmentation Watershed Segmentation Probably you will have segmentation errors They are easy to see by turning the Mesh visualization on and selecting Cells in the dropdown beside To fix them first clear the current label in the top left by clicking on the icon see chapter 1b Miscellaneous toolbar Then use the Fill label tool R from the Mesh toolbar not the Volume toolbar to clear the incorrect cells You can then re seed the empty cells using Add new seed and re run the watershed If you first select a label with the Pick label tool N from the Mesh toolbar you can then color cells with that label by using Fill label Q At this point it is very important to save your data Automatic seeding and segmentation The automatic seeding and segmentation consist of several sub processes which are described below for better understanding of the parameters which the user is supposed to set Please note you can perform it as one process or as a series of separate processes We suggest to try the latter option at the beginning because it will allow you to optimize the parameters for your sample and therefore make the procedure more effective e Blur the cells Run Process Mesh Signal Gaussian Blur Estimate the radius of the smallest cells in your sample in micrometers and put it in the radius window Try several values at the first attempt as over or under estimation can resul2. The stack Main store must be selected and the Work store deselected so that the original data is projected not the processed stack At this point you should see the outlines of the cells Notice the parameters Minimum and Maximum Distance These distances are in the negative direction of the surface normal and tell MorphoGraphX which part of the signal to project onto the surface For better visualization of the mesh you can turn down the opacity of the stack with the slider in the Main tab Save your work There is no Undo operation in MorphoGraphX so it is important to save your work frequently In the Mesh menu choose Mesh1 and Save Give your mesh a meaningful name such as Coarse Mesh TQ for example Visualize projected data If you would like to see exactly what data was projected onto the mesh run Process Stack Mesh Interaction Annihilate Make sure the distances are the same as those you used for the projection If you want to keep this stack you can use the stack menu option Stack1 Save You have now created a 22D image of the surface layer of cells in the meristem Have a look to see how it compares with the original sample data You can adjust the mesh and stack opacity to see both the labels and the signal at the same time If you turn on the mesh visualization to Cells you will see the wireframe outlines of the cells 7 Segment the cells It is now time to segment the image into cells The segmentation
3. Principal directions of growth PDGs describe the growth anisotropy at the level of individual cells The output of this process are vectors that for each cell give the direction of maximal and minimal growth and can be visualized as crosses PDGs are computed based on cell junctions 1 e the vertices that are at the intersection between 3 different labels Before we can compute the PDGs we have to identify which junctions in the second time point correspond to junctions in the first one Fix corners Run Process Mesh Cell Mesh Fix Corners for both meshes This will make sure that all the junctions between cells are correctly segmented and that there is no triangle in the mesh that is left without a label Fix Corners should report 0 vertices after running Otherwise run the process again Save each mesh once Fix Corners is complete Find the correspondence between junctions Since the labels are different between the two meshes load the parents in the second time point Run Process Mesh Cell Axis PDG Check Correspondence The output should be a color map with cells which are correctly identified in blue and cells causing problems in red The vertices from the first time point Stack1 that could not be correctly identified in the second time point Stack2 are selected and should appear in red Make sure that Mesh Lines and Points are selected for Stack1 At this point both meshes are simplified so that only the v
4. checkbox and the Points checkbox are selected and View option is set to Al This will enable the visualization of the mesh Click the Select points in mesh tool Elon the left and hold the Alt key to select the bottom vertices of the apex They should turn red Hit the delete key to remove them To make this easier it 1s nice to have the apex ina horizontal position You can do this by left double clicking on it Try to delete the bottom cleanly Smooth the mesh Run Process Mesh Structure Smooth Mesh several times You should now have a mesh following the global shape of the sample Check how accurate it is with the clipping planes Be sure to turn off the work store and turn the main store back on when you do this As the clipping planes are made thinner the image will get darker you can increase the opacity to counteract this You want your mesh to resemble the shape of the original data If the mesh is far away from the surface does not align tightly with the shape of your stack increase the threshold for the edge detect For the sample data try 25 000 Troubleshooting Symptom The Edge Detect results in a very strange shape If the stack is loaded upside down the Edge Detect will extract the shape of the sample s bottom Check if the stack is loaded in the correct orientation by pressing the A key and the Reset view button This will show you the orientation of the X Y and Z axis If
5. 1842 66 1393 64 5359 14 7436 pa SOE map ceh area 78 292 40 6395 0 896271 18 35 7367 35 7367 82 4887 34 1991 0 561473 19 48 8837 48 8837 82 2042 16 8996 7 12617 22 44 182 44 182 83 6806 7 03876 8 70603 23 25 3023 25 3023 82 2381 2 04306 9 36707 24 37 9115 37 9115 85 3274 4 73887 7 77237 Example of spreadsheet output after running Heat Map of type Area Geometry This file is contained in the sample data folder under meristem_TO_cell_area csv 10 Quantify Fluorescence When imagining it is possible to collect fluorescence signal for gene expression on another channel We can now analyze this data based on your segmentation e Load the Data Use the Stack menu to load the fluorescence channel you want to quantify into the Main or Work store of the stack corresponding to your mesh In this example you can load meristem TO DR5 mgxs into Stack1 Work e Project the data onto the mesh Run Process Mesh Signal Project Signal At this point be sure to have the surface visualization set to Normal the Mesh visualization set to Cells and the correct stack active e g Work Stack1 You should now see which epidermal cells are expressing DRS at a given depth The default parameters Min Dist and Max Dist are set to 1 and 5 which means that the signal is taken only between 1 and 5 um away from the curved surface This depth corresponds to the epidermal layer in the exampl
6. 8 292 82 4887 82 2042 83 6806 82 2381 85 3274 11 8224 73 0392 14 2471 70O 16249 Center_X Center _Y 50 4784 75 0226 56 0198 71 792 60 9629 69 9763 66 1393 64 5359 Cell center coordinates x y z 40 6395 34 1991 16 8996 T 03876 2 04306 4 73887 18 3676 38 0995 44 6245 AQ NIAT Center Z 15 8784 16 3212 17 7 705 14 7436 0 896271 0 561473 7 12617 8 70603 9 36707 T 1237 11 0794 9 84716 7 51381 C ANNO Example of spreadsheet output after running a heatmap with the parameters as shown above on Mesh1 This file is contained in the sample data folder under meristem_TO_T1_growth csv Fix problems If everything is perfect you will see a heat map like in the picture at the beginning of this section More than likely you will see obvious outliers individual cells which are much bluer than their neighbors or much redder Go to these cells and fix any segmentation errors Note that the error might be in either Stack or Stack2 or it might have been a problem with the parent labeling transfer Do not forget to save the parents after correction Create a cell proliferation map Run Process Mesh Lineage Tracking Note that this process will work only for Stack2 where you have Parents active This will display the heat map showing how many cells originate from one parent label 13 Principal directions of growth PDGs ae S ia Ze Rei RRR fS ESGSTERFEETES
7. The process also demonstrates how to use drop down pick boxes in the GUI and how to call a dialog for file selection ITKMedianImageFilter This plugin demonstrates how to cell filters from the Insight Toolkit ITK image processing library MorphoGraphX provides an image source for input and a sink that writes the data back to the work store To compile a plugin extract the archive into a directory You can then type qmake make sudo make install By default the processes are set to install into the system area In cases where you do not have sudo rights or you wish to install only for yourself you can copy the so file to your local process directory This will be in your home directory under local share data MorphoGraphX MorphoGraphX processes See above to list the process directories To create a new process it is easiest to copy an exisiting one For the StackGammaFilter process there are 5 files StackGammaFilter hpp C process headerfile StackGammafFilter cpp C process file StackGammaFilter png Icon file StackGammaFilter qrc Qt resource file points to icon file StackGammaFilter pro Qmake project definition file In order to compile processes you will need to have g and the Qt4 development tools installed 18 Compiling MorphoGraphX from source Linux The source code for MorphoGraphxX is released alongside the binary distributions CMake is used for the
8. bar if you try to use them You can drag and drop the toolbars around to change their position in the MorphoGraphxX window Since two different stacks Stack1 and Stack2 and associated meshes can be displayed simultaneously you have to specify which one is being processed Active stack Select the active stack and mesh by switching between the Stack1 and Stack2 tabs The input for stack operations 3D filters and the like is chosen by checking either the Main or Work store The output modified image is always stored in the Work store View tab File Stack Mesh Labeli amp Selection Help ePweBONcig k View Tab oo F Main VIEW Process View Quality Stack Editing Pinel Beit Ped Clip T Clipz Clip 3 Cuthing surface Dinu Main Stack 1 Active Global brightness and contrast can be tuned in the View tab as opposed to the brightness and contrast of individual stacks and meshes that are controlled from the Main tab Depending on the speed of your machine 3D rendering can run fast or slow You can alter the quality of 3D images display using the Slices and Screen sampling slides to make 3D rendering faster when the stacks are rotated Process tab File Stack Mesh Labels amp Selection Heip pe ai ee i z Zz h F 50 um Process Tab Main View Process i Fubers Apply Separable Kernel E apply Transfer Function MY Autoscale Stack Binanize Bng hten Darken
9. build system In order to compile MorphoGraphxX you will need to install the following packages g libqt4 dev libgt4 opengl dev libglew dev cimg dev libgslO dev libtiff5 dev cmake gui python dev doxygen Windows Currently only Linux is supported although previously versions have been compiled for Windows To compile on Windows you will need g for example with MinGW however Cuda requires MSVC To handle this the Cuda code is compiled to a separate dll It is pretty straightforward to set up MinGW and MS Visual Studio Express free for 32bit however this gets much more complicated if you would like to compile a 64 bit version Mac The OpenGL support on Mac too poor always changing means that it is unlikely to be possible to compile on Mac Neither Windows nor Mac have been tested so there are likely to be many bugs 19 File formats MorphoGraphX can read and write different formats for both mesh and stack files A summary of the formats available is a follows Stack Mgxs read write nr read write tiff read write Stack Import ITK read image series read Mesh Mgxv read write ply read write vtk read write mesh read write Stl write ob read write txt read write MorphoGraphX custom stack file format See the StackSave process in the Doxygen for a detailed description of the format Format developed at INRIA Montpellier Compatible with multipage tiff files written by
10. by the area of their parents on Stack1 so that a value of 1 means no growth and a value of 2 means 100 growth Difference will give the area of daughter cells minus the area of their parents The Growth option can be useful for comparing deformations occurring in series with different time intervals The resulting values are growth ratio of areas 1 time interval If you want to export the results in a spreadsheet check on the Report to Spreadsheet box NB You can also quantify the change in signal between two time points Run the Heat map process with the same parameters as for a growth map except for Heat Map Visualization Total signal Use the Signal average option if you want to normalize the signal by cell size Heat Map Type Heat Map Visualization Area Options Geometry _ Signal average i Report to Spreadsheet meristem TO T1_growth es Signal Geometry Border Interior _ Use manual range 0 000000 1 38335 1 32957 1 22034 1 18182 Value associated to color map ratio of cell area on both meshes Cell label 25 1 0921 27 1 00876 ae 1 05497 141e 773A Area um 48 8824 63 5403 55 2761 17 1842 94 1815 35 7367 48 8837 44 182 25 3023 37 9115 76 1304 56 1409 50 1476 AT 690 Global coordinates ii Change map Increasing Ratio ja gt Growth time 0 00001 Cancel
11. is done by propagating label seeds on the mesh surface using the watershed algorithm Label seeds can be placed either manually or automatically While the automatic seeding saves a lot of time we recommend to get familiar with the manual seeding first as it helps understand how to use the automatic procedure to its full capacity Manual corrections are also often needed in areas where the signal is less clear and leads to errors in the automatic segmentation Manual seeding and segmentation MorphoGraphxX uses manual seeding and the watershed algorithm for this process Blur the cell outlines Run Process Mesh Signal Gaussian Blur or Smooth Mesh Signal This will close potential gaps in the signal at the cell borders For the Gaussian blur use a radius close to the width of the cell border here 1 um or below Note that too much blurring can result in faulty segmentation Seed the cells Select the Add new seed tool x Ensure that the Surface and Labels checkboxes are selected With Alt pressed click on each cell to add a seed If you click and hold you can draw on the surface with the seed This can help to direct the segmentation or to label two cells with the same color When finished draw a line around the entire area with a single seed You can hold shift to continue with the current seed or select the Add current seed tool rd Propagate the labels with watershed Once all the cells of interest are seeded run
12. label color display Note that labeled stacks use a different color mapping from normal ones The numbers representing the voxels are mapped to a color table which is typically 16 colors and repeats If the number of labels exceed 16 cells with different labels can be displayed with the same colors Symptom The software is slow to run after mesh extraction Adjust the cube size for the 3D marching cubes to see the effect on the shape extraction of the cells Do not go too small or it will create too many vertices and be very slow to run If the cube size is too large then the shapes of the cells will not be accurately captured 15 Create your own workflow tasks While processing data you will often use the same processes in a particular order It is then convenient to organize the processes into tasks To create and edit tasks right click below the Tasks tab Click on New task in the Edit User Task window Select the processes and drag them in the right panel To save the tasks you can export them into a text file Saving the MorphoGraphX session file mgxv format will also save the parameters used in the tasks Reference 16 Installation MorphoGraphX is straightforward to install however it can be tricky to get the nVidia graphics drivers and Cuda installed correctly Install the newest driver for your graphics card and the newest version of Cuda that is available in the repository for your distribution If you have p
13. rotate the view to look from the top Looking from the top try to align a few center cells as precisely as possible If the growth was relatively large you can also scale Mesh to increase its size e Transfer labels from Mesh1 to parents of Mesh2 Make sure the Surface visualization and Parents is checked for Stack2 and that Stack is active 1 e Stack2 tab is selected All editing operations work only on the active stack Select the Grab Label E from the Mesh toolbar on the left Now when you hold the Alt key the tool will transfer the label of the cell you are looking through from Mesh1 onto parents of Mesh2 Try a few and verify that you are getting the correct labels Transfer all of the labels in this way In the second time point it may happen that some of the cells have divided Both daughter cells will get the same label but will remain separated e Save the parent labels Run Process Mesh Lineage Tracking Save parents NB make sure that the stack of the second time point in this case Stack2 is active when you save the parents Otherwise you will save an empty file Troubleshooting Symptom It is not possible to superimpose all cells at the same time This is normal Start parent labeling with a few cells in the center of your sample and then move Stack along Stack2 while gradually adding new parent labels Symptom Samples in corresponding time points have very different sizes In eith
14. stretchRatioMin stretchRatioNormal 0 808674 0 152521 0 568141 0 417878 0 530822 0 737296 1 36584 1 12149 0 0 0459534 0 429039 0 902116 0 91003 0 390428 0 139328 1 18375 1 09646 0 0 701228 0 0755756 0 70892 0 609485 0 579429 0 541101 1 21568 1 06645 0 0 157844 0 498626 0 852325 0 92973 0 365856 0 0418541 1 11782 1 01572 0 0 669186 0 549817 0 499891 0 726385 0 34214 0 596075 1 08283 0 934686 0 0 175587 0 718707 0 672778 0 945426 0 067433 0 318781 1 17112 1 02809 0 0 80365 0 57271 0 161708 0 521343 0 54652 0 655376 1 13657 1 0241 0 0 517941 0 795346 0 314901 0 82012 0 357042 0 447129 1 14812 0 947165 0 0 732752 0 280129 0 620163 0 474081 0 863932 0 169908 1 19901 1 05209 0 0 222034 0 95443 0 199412 0 916084 0 274236 0 292547 1 05828 0 948829 0 0 314793 0 94877 0 0272171 0 839277 0 291628 0 458877 1 11244 0 996109 0 0 0356987 0 960362 0 27646 0 91145 0 144742 0 385109 1 10891 0 971614 0 0 151915 0 974467 0 165334 0 879981 0 209515 0 426305 1 07984 0 990876 0 0 357347 0 922172 0 147993 0 823839 0 385875 0 415198 1 10429 0 949434 0 0 344349 0 93732 0 0534352 0 885577 0 343185 0 313015 1 101 0 991371 0 0 363329 0 919665 0 149023 0 929467 0 368778 0 00973245 1 14697 0 930028 0 0 0769949 0 942366 0 325604 0 993182 0 0438233 0 108022 1 23288 1 03614 0 0 068294 0 86515 0 49684 0 989862 0 120925 0 074504 1 25662 1 01234 0 0 555252 0 567086 0 60836 0 793557 0 580179 0 18346 1 15226 972300 oe staats L ereere TOTTESE 1 1597
15. the arrow of the Z axis is pointing downward run Process Stack gt Canvas gt Reverse Axes with parameters X No Y No Z Yes You can then over write your stack for further use Press the A key again to make the axis arrows disappear Symptom The mesh is full of holes Try first to run the whole procedure again with a lower threshold for the Edge Detect If this does not help use Process Stack Morphology Fill Holes just after running the Edge Detect e Symptom There are spikes on the sample shape after Edge Detect The threshold for edge detection is probably too low 5 Experiment with Processes All data sets present different features and there is no universal recipe to process them in the best way We recommend to experiment with processes in order to adapt them to your data The parameters we recommend for the sample data will not necessarily be optimal for your data NB remember to check which stack is currently active so that you know if the process runs on the original data Main store or on an image already modified by the last process Work store e in Process Stack Filters o Gaussian Blur Stack Try different values for the Sigma parameter i e the radius of Gaussian blur Compare the result in Work store and the original data in Main store using the clipping planes to get only a cross section Larger radius should be more efficient at reducing the i
16. the right click translates the point of view You can check the camera movements relative to the coordinate system X Y Z by pressing the A key Double left click to rotate the camera to the nearest view aligned with one of the axes X Y or Z To move each stack independently press the Ctrl key Specify which stack should be displaced using the check boxes in Control Key interaction within the Main tab Reset all the rotations and translations clicking on the lt icon on the miscellaneous toolbar Press the A key again to hide the coordinate system 2 Transfer functions In the Main Tab click on the m icon to display the color scale or transfer function of each stack A pop up window will appear Use the Predefined color maps menu to change the type of transfer function Double click on the histogram to add a marker black triangle and displace the markers to modify the color map Right click on the histogram to reverse the transfer function 3 Clipping planes In the View tab select one of the clipping planes in X Y or Z in the Clip1 Clip2 and Clip3 tabs To display the boundaries of the clipping plane check the Grid checkbox on Modify the depth of clipping plane with the slider To display only the part of the stack inside the clipping plane check Enabled on You can move the clipping planes around the stack by holding the Crtl key Note that the correct radio button for the Control Key Interaction m
17. which do not exist in Mesh1 An inspection of the signal projection in Mesh1 c and Mesh2 d reveals that the segmentation is wrong on Mesh1 due to fuzzy signal in this region The neighbor exchange is clearly made visible after running Check correspondence cells involved are colored in red and the junctions of Mesh that could not be identified in Mesh2 are selected arrows e The faulty cells in red are easy to spot on the whole meshes f Correction of segmentation error The neighbor exchange in Mesh a and b can be fixed c and d using the Pick label to select label A and D and Add current seed 4 to re label some of the mesh triangles Remember always save the mesh after correction Re run Check correspondence after correction The cells should now appear in blue e If the entire meshes are free of mistakes f you can now proceed to the PDG computation e Compute the PDGs Once the correspondence is complete run Process Mesh Cell Axis PDG Compute Growth Directions with Stack active e Save the PDGs with Process Mesh Cell Axis Cell Axis Save The maximal and minimal direction of growths 3D vectors will be saved together with the values of deformation stretch ratio associated to them as a csv file see image below a c l D E F G l 4 i j yMax zMax xMin yMin zMin stretchRatioMax
18. 12617 8 70603 9 36707 1 77237 11 0794 9 84716 Example of spreadsheet output after running Heat Map of type Area Total signal with Signal average This file is contained in the sample data folder under meristem_TO_signal csv 11 Parent labeling In order to make a growth map we need to first segment a second time point of the time lapse in the same way at the first one In this example we assume that data for the first time point is stored in Stack and for the second one in Stack2 Of course labels on the first time point will not correspond to the second time point In order to compute growth map we first need to associate the labels of the daughter cells in Stack2 with the labels of their parents in Stack1 MorphoGraphxX provides a very simple way to do that e Go to Stackl In the Main tab turn Stack off Turn Surface off with Labels active Turn Mesh on with View Cells option selected e Go to Stack2 Turn Stack off Turn Surface on with Parents active Turn Mesh on with View Cells option selected To easily distinguish the cell mesh of the second time point from the first one change its color using the Colors Editor z e Now set the Control Key Interaction to Stack1 on the Main tab Hold the Control key and move the wireframe cell out line above Stack2 Move it to be about 2x the height of the sample above Stack2 Now
19. 3D Image processing with MorphoGraphxX User manual www MorphoGraphxX org This manual will introduce you step by step to 3D image processing and analysis software in MorphoGraphxX Example confocal stacks from the tutorial can be downloaded from www MorphoGraphxX org The manual covers the software basics e MorphoGraphX Installation e How to get good quality confocal data for segmentation e Loading and viewing confocal image data e Extracting the cell outline on a curved surface 22D segmentation e Analysis of growth from time lapse data and quantification of fluorescence e Exporting results into other formats e 3D segmentation If you are only interested in using the 3D segmentation we recommend nevertheless to go through the entire tutorial since the procedures are very similar for curved surface 22D and 3D with the exception of chapter 13 Principal Directions of Growth New functions are constantly added to the software For a more detailed description of new or advanced function please refer to the internal documentation or visit www MorphoGraphX org Table of Contents Og CUI ts fo ee eer eee er ee ee ee ee ee ee ee 4 1 Introduction to WIT O Gi AON xy to ceirai orsactesecetaioevaantstoncdoaitecaceese see mnisevaanteteuduelesuacieieqrtoniee 4 EEEE E E E EEE E EE TE EES AE E 4 User interface m MorphoGraph A 2 sersiereimniriesa en nineT eea e EE EESE ETERNE 4 Main menu toolbars and Main TaD sisiavesecssavisnciav
20. 9 Stretch 0 248723 0 753985 0 848309 512431 0 133365 1 13804 0 484808 0 389915 0 717225 100729 0 689523 1 13436 ratio 9149 Growth direction Max Growth direction Min 288 gs in x y 2 in x y z cee direction 99 66 the first time point which is the same as the parent label on the second time point The columns xMax yMax and zMax give the x y Z coordinates of the direction unitary vector of maximal growth while the column stretchRatioMax contains the magnitude of stretch in this direction Similarly xMin yMin and zMin define the direction of minimal growth stretchRatioMin the stretch ratio stretchRatioNormal gives the value corresponding to the axis perpendicular to both minimal and maximal growth directions Since we compute only the 2D deformation of the cell in its average plane this third value is zero e Display the PDGs You can now re load the original meshes and display the PDGs on them Make sure that parent labels are also loaded on the second time point before loading the PDGs Load the PDGs with Process Mesh Cell Axis Cell Axis Load Type PDG In Process Mesh Cell Axis PDG Display Growth Directions you can choose what type of heat map to display StretchMax resp StretchMin is the value of deformation stretch ratio in the maximal resp minimal direction A stretch ratio of 1 means no deformation 2 means an elongation by 100 0 8 a shrinka
21. ImageJ Stack formats readable by ITK Insight Toolkit Series of 2D images Any format readable by CImg If ImageMagik is installed Cimg can convert from almost any format MorphoGraphX custom mesh format See the MeshSave process in the Doxygen for a detailed description of the format Stanford polygon file format VTK Visualization Toolkit format MeshEdit format StereoLithography CAD file format Wavefront OBJ format Custom simple text format See the MeshExport process in the Doxygen for a detailed description of the format 20 Command line options Configuration information is available by launching MorphoGraphX from the command line To view all the available options MorphoGraphx help Usage MorphoGraphx debug dir process all process help h FILE mgxv debug Launch MorphoGraphxX in debug mode dir Print the application directory and exit process Print the process directory and exit user process Print the user process directory and exit all process Print all the directories searched for processes and exit include Print the include directory and exit version Display the version and revision and exit help h Print this help
22. Y color Gradient yf Alter Gaussian Blur Stack fa inari Normalize Stack Parameter Value x Radius 1 Y Radius 1 Z Radius l Sheps 1 Active stack Main Stack 1 Active Processes are bundled in groups that you can unfold by clicking on the icon next to the group name To run a process press the Go button or double click on the process name While running a process make sure that the right stack number 1 or 2 and store Main or Work is active 2 Data collection MorphoGraphxX has mostly been used to process 3D confocal image stacks Data collection for use in MorphoGraphX can be a little different than for other uses In most cases people optimize data collection so that individual slices look good to the human eye This 1s not always the best for 3D data analysis and visualization The following are some tips to help get the most out of the images e 16 bits images If possible collect 16 bits per channel instead of 8 bits Although the pictures may look no different the 16 bits images will have higher dynamic range and it will be easier to extract features in darker areas of the image If the microscope only supports 12 bits that will still be better than 8 bits Cubic voxels Use a small z step Try to make the voxels close to cubic for best results For example if the XY resolution is 0 5 um then use 0 5 or um for the z step 0 5 is better Averaging There is always a trade betw
23. at the mesh is segmented into cells you can do some analysis on it To make a heat map of cell area Run Process Mesh Heat Map Heat Map A dialog will pop up asking for parameters Dialog Heat Map Type Area gt Options Heat Map Visualization Geometry A VEEGA Global dinat l amp i Report to Spreadsheet aia a meristem_TO_cell_area Change map Bl Geometry Signal Border Interior _ Use manual range Cancel OK Be sure the heat map type is set to Area and that the Report to Spreadsheet box is checked Type in a filename for the spreadsheet Hit OK and the cells will be colored by area In case one of the labels covers a much greater area than the other the automatic color map scaling will result in most of the cells being blue and the large cell e g border of the mesh colored in red Run Process Mesh Heat Map Rescale Heat Map with proper parameters in this example Min 20 and Max 250 to re adjust the color scaling The cell labels are listed along with their areas If you click the Map checkbox inside MorphoGraphX it will show the cell label numbers You can also use the select the Pick label tool N in the Mesh toolbar to find out which label belongs to which cell Cc D E F G jLabel fValue Area um Center X Center_Y Center Z g 11 48 8824 48 8824 90 4784 75 0226 15 8784 12 63 5403 63 5403 56 0198 1 792 16 3212 13 55 2761 55 2761 60 9629 69 9763 17 7705 14 77 1842 77
24. can be compiled into shared object so files and are loaded when MorphoGraphX starts They can be installed in a system area for all users or in the user s home directory Run the command MorphoGraphx all process to print the plug in directories An overview for the process documentation can be found in the process namespace in the Doxygen programmer documentation which is available from the help menu in MorphoGraphxX After installing MorphoGraphxX this documentation can also be found here usr local share doc MorphoGraphX html index html The best way to start developing processes is to start from a sample available from the MorphoGraphX website www MorphoGraphxX org In the Samples plug in pack there are the following simple processes StackGammaFilter This process implements a simple gamma filter on the stack It demonstrates how to read and write information from the stack and how to collect parameters from the user interface Note that this process inherits from the StackProcess class MeshGammafFilter This process implements a simple gamma filter on the mesh signal In order to use this process you will need to have extracted a mesh and projected the signal on in see Section 6 Note that this process inherits from the MeshProcess class ExportMeshToFile This process demonstrates how to write mesh data to a text file It sames the mesh in simple OBJ Wavefront and PLY Stanford Polygon formats
25. croscopes can capture different light frequencies to different channels simultaneously you can collect PI wall stain signal and GFP fluorescence marker signal in the same amount of time exposure MorphoGraphxX will use the PI wall stain for surface extraction and segmentation and then the GFP channel for gene expression analysis You will have to experiment a bit to get the best results for your samples Start in the right place Be sure to start data collection a few microns above the sample Many people are tempted to start collection when they see the first cells This will leave a flat spot at the top of the sample Also be careful not to miss the bottoms of cells if you are doing 3D segmentation as you can only quantify cells which are complete Time lapse imaging If you want to quantify growth or changes in signal intensity over time the same sample may have to be imaged multiple times up to several days Repeated exposure to a light source e g confocal laser results in considerable stress for biological samples and might interfere with the results The time intervals between image acquisitions should be long enough to allow the sample to recover On the other hand the sample should not grow too much in between images otherwise it can become too difficult to identify the cells between successive time points As a rule of thumbs the cells should not divide more than twice during the time intervals Use Fiji to get TIF files Most mic
26. e tomato meristem data Try to change the depth parameter e g 6 and 12 corresponding to the sub epidermal layer to see the effect on the projection and compare this with the whole stack e Make a heat map Run the same heat map as before only this time set the heat map visualization to Total Signal in the dialog and check Signal for the spreadsheet You now have a heat map of epidermal DRS expression Value associated to color map average signal total signal per cell area 24 J3 25 439 462 2T 295 324 Cell area sollte per cell 54 1815 35 7367 48 8837 44 182 29 3023 37 9115 76 1304 96 1409 20618 41918 A7944 7 30911 2 39 54 4 33456 4 16579 7 18 292 82 4887 82 2042 83 6806 82 2381 85 3274 17 8224 73 0392 Dialog Heat Map Type Area 3 Options Heat Map Visualization Total signal 3 Signal average Report to Spreadsheet Global coordinates meristem TO signal m Change map l amp i Geometry l Signal Growth time 12 _ Border Interior Use manual range 0 900000 0 000000 __ Cancel OK Area um Total Signal Center X Center _Y Center Z 649 427 48 8824 31745 6 50 4784 75 0226 15 8784 367 873 63 5403 223374 7 56 0198 71 792 16 3212 252 289 55 2761 13945 5 60 9629 69 9763 17 7705 YU 406 54 J sle 64 3 14 436 Cell center coordinates x y z 40 6395 0 896271 34 1991 16 8996 7 03876 2 04306 4 73887 18 3676 38 0995 0 561473 7
27. edcecshacidscaaeazerderdascseigaeiiciieuiaradeaiedeee 5 PNM a E E E E E EE 6 POCOS e A E E E E E 7 Do NACA COMICON cinta sucncstscactienei E A E E E E 7 3 Loadimne samples stack or MICS jenssarsirannr i E used sdiemanmseie 9 TOE OO a E A E E E cee 10 A EXtract the biological object SDAP vs ccssvscexeiess sates vontoeedseudvasepsasetiond E R EEE 11 TOME Te BOON aer E E E E E E E 12 Di Eee E ING Yu LES E o T EET EENT ET A ET T TT 13 AE B ogre ga cee E E E E EA A S AE E E E T 14 T T E O ee E E AEE E A EEA aetecsnatenenstaetacanesetene 14 Manual seeding and segmentation essssssssoeersssssssseetrressssssecteesssssssseeerrsssssssseeresssssssees 15 Automatic seeding and segmentation eesssssssseeeresssssssstteressssssseterrsssssssceresssssssseeeeeesssss 16 TOTOE ROO a E A E E E E E E E 17 ies Pe A a EEE E E E A EE E EA E 17 JAn Ee U AT a e E E E E E E E E 18 W Onna OTC SCC NCS ee E E EA ese dqutiasannesn needs 19 EED e A e e E E E A A A E S A A A E E swecgueese eeneisess 20 TOU FCS OO ANS e E EE EE E EE E E S 22 12 Heat map of growth and cell PrOMPCrAlOMN ca c2cieccstecsiecacensessdeoed dendeciduscaberssuaanentendienel deaaetseendaians 22 13 Principal directions of growth PDGs sssseeenesssssssseeersssssssssceersssssssscereessssssseeeeesssssssees 24 E MY Se S Ea o E E E A A E A E 28 TOU Fe BOON e E E EE E E E 29 15 Create your own workflow 1aSKS seein cccrescstucoraccdt viasesteasesncceubesestsusveneto
28. een resolution and how much exposure the sample can tolerate as well as how much time you have Frame averaging 1s used to get better individual slices but requires multiple scans per slice For 3D image processing it is generally better to have more real slices For example if you have 0 5 um for XY pixel size and are using a z step of 2 um with 4x frame averaging it would be better to turn the frame averaging to 1 and set the z step to 0 5 um Both should have the same exposure time but of course without frame averaging you get a 4x larger data file Image saturation It is best to have the images just below the point where they are saturated The controlling software of confocal microscopes usually has a mode to show saturated pixels Turn the gain up until you just start to see some saturated pixels Of course if you want to compare fluorescence data between samples to make a quantification of fluorescence levels it is necessary to not saturate the channels you want to quantify For wall stains where you do not need to quantify it can help to over saturate in order to get a good contrast between the inside and the outline of the cell Wall Stain To get a nice segmentation you need a method to stain the cell walls or the plasma membrane This can be done with propidium iodide PI or a marker line With the correct detection range PI can be reliably separated from both GFP and YFP using the same excitation wavelength Since most confocal mi
29. er Stack or Stack2 check the Scale box on the bottom of the stack tab Now you can scale your stack along all three axes which can make parent labeling easier 12 Heat map of growth and cell proliferation Now we are ready to make a map of cell growth and proliferation This will correspond to the change in area of cells and number of cells over the time point In order for this to work it is important that the parent labeling lineage tracking is perfect Mistakes will appear as outliers in the growth map and are very easy to spot It is very unusual for lineage tracking to be perfect the first time To create the growth map Run the heat map process In Mesh select Labels and in Mesh2 select Parents You should see corresponding cells on both meshes painted with the same color Run Process Mesh Heat Map Heat Map Select Area for the heat map type and Geometry for the visualization Also select the Change map box on This tells MorphoGraphxX to make a heat map comparing Stack and Stack2 The heat map can be visualized on either the first typically Stack1 or second Stack2 time point For the growth sample here select Increasing if Stack 1 is active or Decreasing if Stack 2 is active There are 3 options for the type of change map Ratio Difference or Growth Ratio combined with Increasing will display the area of daughter cells on stack2 divided
30. ertices at the junctions between cells are present Do not over write the meshes after running this process Correct the errors If you compare the red cells on both meshes you can identify the sources of errors in the correspondence between junctions Most of the time they will be caused either by wrong parent labeling which can be fixed easily by modifying the parent labels or by an exchange in neighborhood see illustrations below In this case the original meshes will have to be modified Open a separate MorphoGraphX session with the original meshes Keeping the first session open will help identifying which k junctions should be corrected Use the Pick label and Add current seed tools bd to modify the meshes Save the meshes after correction and re run Check Correspondence until all the cells are blue Mesh 1 labels Mesh 1 surface P ae eo as A Mesh 1 surface Check correspondance i hres _ e2 dl Typical segmentation error found by Check Correspondence Junctions between cells are recognized in both meshes based on the identity of cells in contact If 2 junctions are very close to each other it can happen that a small segmentation error lead to an exchange in neighbors For example in Mesh a the cells B and C are in contact forming 2 junctions C B A and C B D In Mesh2 b the cells A and D touch each other forming the junctions A D C and A D B
31. ge of 20 The product of StretchMax and StretchMin is equivalent to the areal growth of the cell polygon The color and size of the PDG vectors can also be modified By default vectors corresponding to expansion stretch ratio gt 1 are displayed in white while red is used to draw the direction of shrinkage stretch ratio lt 1 The Threshold parameter is used to display PDGs axis only in cells for which the anisotropy is above a given value 14 3D segmentation Rie Stack Mosh Labels amp Saecion Hap i ee a SF g a ce It is also possible to fully segment cells in 3D with sufficient quality confocal stacks Best results can be achieved with very dense stacks fine Z step and a small pinhole diameter Since it is particularly difficult to seed in 3D MorphoGraphX uses an auto seeded watershed from the C library called the Insight Toolkit ATK To do the 3D segmentation Load the stack You can use the example data radicle of a mature Arabidopsis embryo from the Bassel_2014 folder provided on www MorphoGraphX org Blur to reduce noise Run Process Stack Filters Gaussian Blur Use a radius slightly larger than the width of the cell walls in the sample In the example try 1 um Segment Run Process Stack ITK Segmentation ITK Watershed Auto Seeded This can take some time but you do not have to do any seeding Adjust the threshold according to the results of the segmentation here try 3000 If a sa
32. l which selects all vertices that have the same label Make a heat map You can now make a heat map to visualize the cell volumes Run Process Mesh Heat Map Heat Map Select Volume for the Heat Map Type to color the cells by volume It is also possible to make a heat map of area or to count fluorescence inside volumes if you have that collected on a separate channel Like the Area heat maps it is also possible to quantify cell expansion Note To get an optimal 3D segmentation experiment with different values for the blurring and the threshold parameter for the ITK segmentation to see if you can nicely segment all the cells with as little manual correction as possible You cannot split cells so 1f the stack is under segmented then you must re run the segmentation with a lower threshold If a stack is highly over segmented then extensive manual correction is needed Optimizing the segmentation in the first instance is the quickest way forward Troubleshooting Symptom Error message after running ITK Watershed Number of objects greater than maximum of output pixel type Increase the threshold to get less cells Symptom A lot of cells in the segmented stack have the same color Make sure that the cells are not fused have different labels using the Pick label from a volume tool If you have a lot of cells your samples more than 30 check on the 16bit option on the Work stack This will change the
33. l correctly The next task is to enable the ALT key In Linux Mint and Ubuntu the ALT key is used to move windows around MorphoGraphX uses this key for user interaction so you will need to change the key used by Linux to the Super or Window key In Mint cinnamon this is done by right clicking on tasks bar and going into All Settings Windows There you change the mouse modifier key from Alt to the Super key Laptop use MorphoGraphX will run on a laptop that has a dedicated nVidia graphics card Most newer laptops will use nVidia Optimus technology In this case the laptop will use the graphics card built into the processor for simple tasks and will only turn on the dedicated card when required This will be handled automatically by the MorphGraphX startup script but it is necessary to get the Bumblebee and or Primus software working on your machine Troubleshooting e Symptom The viewer window is not black you get shader errors in the terminal window or when you load a stack it is a single solid color This means that the nVidia graphics driver is not installed properly e Symptom Libraries so files not found Sometimes libraries are not found immediately after installation At the command line type sudo ldconfig e Symptom You see the following error message in the terminal window when MorphoGraphX starts Cuda holdMem cannot allocate 8 Meg giving up This means that the Cuda driver is not insta
34. lled properly If you can get any application that uses Cuda working then MorphoGraphX should work as well Note There appears to be a problem with the Cuda driver in the Ubuntu 14 04 repository To see if this problem affects you try starting MorphoGraphX as root at the command line sudo mgx If this solves the problem MorphoGraphX will work without root privilege until the system is rebooted The issue is a problem with the Cuda library not creating the device files properly it has been reported here httos devtalk nvidia com default topic 699610 linux 334 21 driver ret urns 999 on cuinit cuda This can be fixed by editing the file etc rc local and add the following lines to it modprobe nvidia uvm mknod m 666 dev nvidia uvm c grep nvidia uvm proc devices cut d f 1 0 chgrp video dev nvidia uvm This will create the necessary device files at startup Cuda 6 5 An alternative option is to install Cuda 6 5 which is the nVidia officially supported version for Ubuntu 14 04 If you go this route you will need to download a version of MorphoGraphX with Cuda6 5 in the package name Also you will need to put the following lines in your bashrc file export CUDA_HOME usr local cuda 6 5 export LD_ LIBRARY _PATH LD_LIBRARY_PATH CUDA_HOME lib64 export PATH CUDA_HOME bin PATH export 17 Writing custom processes plugins It is possible to extend MorphoGraphxX by writing your own processes Processes
35. loaded into the main store Once the original stack has been modified by a function the result e g the 3D image after blurring is stored separately in the work store without over writing the original image in the main store Each store has a separate color brightness transparency etc e Mesh A mesh is a triangulated curved surface Typically the mesh is extracted from a stack after some image processing operations are performed on it Currently MorphoGraphxX can handle 2 stacks each with a main and work store and 2 associated meshes in a single session e Process Most operations in MorphoGraphX are performed by processes There are 3 kinds of processes Mesh Stack and Global They are named after what they can modify Processes can be organized by users into custom pipelines called tasks User interface in MorphoGraphX Start a MorphoGraphX session with the example data loaded by clicking on the example_session mgxv file in the Kierzkowski_2012 folder Main menu toolbars and Main tab ue lt Menu bar save session load stacks amp meshes A rn j di lt Main Tab Main View Process Conmtrol Key Interaction Piek Unightne vr m Voxel size Active stack Main Stack 1 Active Tools from the toolbars will work only if the Alt key is pressed on Some of the tools are enabled under specific conditions which will be described in the information
36. mage noise but also results in a more blurry image o Normalize Stack This will increase the contrast in the image enhancing both the speckle noise and low signal areas As above use the clipping planes to observe the effect of normalization on the stack You can perform a Gaussian blur before normalization to get the best results o Invert Transforms low signal into high signal and vice versa This can be useful if you want to segment features which are dark in the original image e in Process Stack Morphology perform all the following operations with a clipping plane enabled to observe the effect on a cross section o Dilate o Erode o Close This is just Dilation followed by Erosion e in Process Mesh Signal o Project Mesh Curvature Project the Gaussian curvature For best results use change the transfer function to Jet Experiment with different neighborhood and Autoscale values 6 Create a 2 2D image Many samples have too much curvature to simply segment them in 2D This is one of the features of MorphoGraphX distinguishing it from other software It was designed to work on curved 22D images To create this image perform the following Subdivide the mesh Run Process Mesh Structure Subdivide then run Smooth Mesh process Project the signal onto the mesh Ensure that the Surface checkbox is selected and deselect the Mesh checkbox Then run the Process Mesh Signal Project Signal
37. mple is highly under segmented cells fused then decreasing the threshold is needed If the sample is over segmented single cells segmented into multiple pieces then the threshold needs to be increased Delete the outside The segmentation fills the entire volume with labels and the sample will be buried inside somewhere To remove the outside label select the Delete Picked Label in Volume x from the Volume tool bar Be careful what you delete as you cannot go back You can however save the segmented volume in the Work store under the Stack menu Correct the segmentation Auto seeded segmentation almost always requires correction for over segmented cells These multiple segments can be fused together into a single segment by using the color picker and bucket Q from the Volume tool bar to merge cells Use the clipping planes under View tab see section 3a to correct for errors inside the sample Extract the volumes When you are happy with the segmentation extract the mesh using Process Mesh Creation Marching Cubes 3D The Cube spacing parameter defines how far apart vertices are in the mesh For a fine and detailed mesh cube spacing should be smaller try 3 um and above The tradeoff to this is a larger file size for the mesh Cells are automatically labeled with the same values as the stack labels Once the mesh is extracted you can edit each cell individually using the Select Connected Area amp too
38. n m in the main store of the stack you just loaded In the Transfer function editor window click on Auto Adjust If the data is 8 or 12 bits only part of the histogram will be filled 1 16 or the full range in case of 12 bits data 1 256 in case of 8 bits Although it is possible to run all the stack processes on non 16 bits images we recommend you to re scale the data to 16 bits processing it Use Process Stack Filters Brighten Darken with an amount of 16 to convert 12 bits images or 256 for 8 bits images Next run Process Stack Multi stack Copy work to Main stack Reset the transfer function of the main stack in the Transfer function editor Now the stack should look fine and you can use it for further processing Save it using the Stack menu you can over write the original stack or save separately the 16 bits version e The stack voxel size might be wrong In the Main tab check the voxel size see chapter 1b If it does not correspond to the pixel size and the z steps used during acquisition resize the voxels using Process Stack Canvas Change Voxel Size If the size is already correct in some of the directions X Y or Z enter a value of 0 for these and their size will not be changed e The store into which you loaded your data might not be active Check the main and work checkbox in Main tab 4 Extract the biological object shape emEPT LHOY wR A B The first step in the segmentation p
39. our cells in the meristem sample data 5 um not the radius of the smallest ones e Merge the over segmented cells Run Process Mesh Segmentation Combine Labels Estimate the width of the cell border for your sample in micrometers for example try 1 um and put it as the value for the Border distance parameter The Threshold parameter indicates the ratio of cell wall brightness to the brightness of cell interior Therefore it should be higher than 1 The exact value depends on the quality of signal in your sample For the sample data try a value between 1 5 and 1 8 Two cells will be merged if their ratio of common border signal vs average signal is lower than the threshold value If you want to perform all the above processes as one run Process Mesh Segmentation Auto Segmentation You will be able to set all the necessary parameters and decide whether or not you want to perform signal normalization e Correct errors Examine the surface carefully and search for segmentation errors especially in the regions where the signal is relatively low Correct them in the same way as for manual segmentation Troubleshooting e Symptom Cells are under segmented not all cell walls are recognized Try lowering the cell radius during signal blurring and seeding e Symptom The mesh combine regions process creates a lot of errors separate cells get merged for no reason Try increasing the border di
40. reneseteasesesemaeeseteessece 29 PR E E E E A EO eae AE E A A E O N E 30 W O E E E E E E 30 After installation enabling the ALT k y ssesssseeensssssssseeeressssssseccresssssssesceeessssssseereessssss 30 E DLEE Ba E AT P O EA A AEE E E E AEE T 30 e e a E E A A EA AA AN A TE A 31 O Te E E ee ee eee ee 31 17 Writing custom processes plugins tac tapaccattuSacecstnaccenbusicaasiaasecaney teauvadnaecactustsaasasaaccaneysteates 32 18 Compiling MorphoGraphX from Source seeeeenssesssseeeeessssssseeerrsssssssserresssssssseeerrsssssssees 33 DAMN EOE ace AE A E A E ET A E T T 33 YY TIO 5 ae eco a eae nectar E E E E E E E 33 IVA S E E A TEE TEE E AE E E EE A A ETT 34 RETS Ena E E cass A E A AAA A E E T A A E E 34 20 Command ETC IN sase EEE dtepocatdaiasnetseescianse 34 User Guide 1 Introduction to MorphoGraphX MorphoGraphX is an open source platform for the visualization and processing of 3D image data MorphoGraphxX can manipulate 3D image stacks and use them to extract curved surfaces 22D from them or segment data in full 3D Some definitions e Stack A stack corresponds to a 3D image of a biological sample There can be 2 stores associated with each stack corresponding for example to the original 3D image and the same data after image processing The size of a stack is defined in voxels which are the equivalent of pixels for 3D images e Store There are 2 stores in each stack Usually the original sample is
41. roblems it may be necessary to install a newer version from the nVidia web site MorphoGraphX is available as a Debian package which can be downloaded from www MorphoGraphX org Binary versions are available for Linux Mint and Ubuntu It is important to get the correct release as different versions of Linux ship with different versions of the libraries that MorphoGraphX depends on This is particularly true for the Cuda GPU tools which are evolving rapidly Use your favorite software manager to install the MorphoGraphX package or just double click on it In order to use the 3D segmentation and other tools from the Insight Toolkit you will need to install the ITK package as well A compiled version of the ITK package is available on the MorphoGraphxX web site for convenience but does not differ from the version available from www itk org If you would prefer to compile ITK yourself be sure to enable the Module_ITKReview in CMake to use the Morphological Watershed filter After installation enabling the ALT key After MorphoGraphX has been installed it should show up in the menu system or you can start it by typing mgx at the command line or double clicking on a MorphoGraphX project mgxv mesh mgxm or stack mgxs file When MorphoGraphX first starts double check that there are no errors in the terminal window If the graphics card is identified properly and the is memory allocated to Cuda it is most likely everything instal
42. rocess is to extract the global shape of your sample This is performed by first blurring the sample slightly to reduce noise and then using filters to convert the object to a solid shape The surface of the object is then extracted from this shape as a triangular mesh To create a mesh perform the following Load the stack Make sure that the PI stack is loaded into Stack1 Main and Main Stack 1 is active Blur the stack Run Process Stack Filters Gaussian Blur Stack with values of 0 3 or Process Stack Filters A verage The blurred image is stored in Work stack and the active stack is automatically changed to Work Stack 1 Edge detect Use Process Stack Morphology Edge detect This will create a solid shape representing the global shape of your object Tip for optimal visualization of this solid shape turn the opacity of the work stack to the maximum At this point it is also possible to use the Pixel Edit tool Va with Alt key to erase parts of the stack that you do not need Note that this operation only works on the work stack Extract the surface Run Process Mesh Creation Marching Cubes Surface with a cube size of 5 um As a general rule the cube size should be roughly as large as the cells if you want to extract only the general organ shape and several times smaller if you want to capture the curvature of individual cells Trim off the bottom In the Main tab ensure that the Mesh checkbox the Lines
43. rom processing operations will be displayed in the terminal Load a stack Drag and drop a TIF image stack onto the main window This will load your sample into the main store of Stack1 Drag and drop the file with the Alt key pressed to load the sample into Stack2 You can also load samples with the Stack menu for example under Stack Stack1 Main Open Importing file series It is also possible to import image series with the Import series option in Stack menu In this case you will need to specify the Z step yourself Loading a pre existing mesh Meshes are usually extracted from stacks but they can also be loaded separately from the stacks Load meshes the same way as stacks either by dragging and dropping the files into the main MorphoGraphX window or by using the Mesh menu e g Mesh Mesh1 Load Experiment with the visualization tools MorphoGraphX uses QGL Viewer to handle the display of samples The left mouse button allows you to rotate the sample the right button or left button Shift key translates and the wheel is used to zoom Mouse and keyboard codes are listed under About QGLViewer in the Help menu There are many visualization tools in MorphoGraphX We recommend to load two different samples in Stack and Stack2 and take some time to experiment with 1 Rotation translation Left click on the display area and moving the mouse rotates both stacks that is changes the camera view angle while
44. roscope formats are proprietary The Loci bio formats group has reverse engineered the formats of the most popular microscopes and made plugins for ImageJ We recommend Fiji which is a distribution of ImageJ that contains these along with many other useful plugins After opening a stack in Fiji split it into separate windows for each channel as MorphoGraphX currently requires there be only one channel per file From Fiji save each channel as a multilayer TIF file If voxel sizes are wrong The TIF format has no standard way to specify the voxel size in Z ImageJ writes this its own way and MorphoGraphX is programmed to read it If this doesn t work for some reason then MorphoGraphX allows you to change the voxel size after you load the image see chapter 3b If you need to do this you can get the voxel sizes using the microscope software or from the metadata text file in ImageJ 3 Loading samples stack or mesh ereOePEs 19 47 The first step is to load your sample into MorphoGraphxX Start MorphoGraphx If there is a green MGX icon on your desktop double click the icon Another way to start MorphoGraphX is to open an existing session by double clicking on a mgxv file You can even create an empty mgxv file and open it After MorphoGraphX starts you will see the main window and a text window called terminal underneath Do not close the terminal if you do MorphoGraphX will shut down Informational messages f
45. stance or slightly lowering the threshold remember not to go below 1 If the cells have signal inside white spots inside the cells try to normalize the mesh signal 8 Refine the subdivision In order to minimize the number of vertices in the mesh we will first do a coarse segmentation and then refine it by subdividing only near the cell boundaries This is important especially if your computer is not very fast Proceed as follows e Subdivide near the walls Run Process Mesh Structure Subdivide Adaptive Near Border This will clear the labels within a given distance from the border and subdivide triangles larger than the max area specified in the parameters e Re project and re segment Re project the signal smooth it and then re run the segmentation This plus the previous operation can be repeated several times until the image is sharp Sometimes it is necessary to change the parameters if the distance is too small or the area too large The idea is to get to the resolution of the stack You will be able to tell when it is fine enough when you can start to see the voxels in the projected image when you zoom in Now the image projected onto your mesh should look nice and sharp and the cell borders should be much less jagged If your computer is fast enough or your stack small you can subdivide the entire mesh Process Mesh Structure Subdivide before segmentation and omit this point 9 Quantify Cell Area Now th
46. t in incorrect segmentation For the sample data try 2 um e Seed the cells automatically Run Process Mesh Segmentation Auto Seeding A seed will be put at local minima of signal dark regions within a given radius Set the same cell radius as in the previous process 1 e the radius of the smallest cells here 2 um If you use a larger radius the small cells might be under segmented fused e Re project the signal onto the mesh Run Process Mesh Signal Project Signal with the same Min and Max distance parameters as originally used see chapter 6 e Blur the cell outlines Run Mesh Signal Gaussian Blur with a radius roughly equal to half the cell outline width try 1 um or below e Segment the cells Run the watershed Process Mesh Segmentation Watershed Segmentation as in manual segmentation Probably you will observe that in some cases one cell has been seeded several times 1 e is over segmented Merge the over segmented cells based on signal using Process Mesh Segmentation Combine Labels described below e Normalize mesh signal optional This process can enhance the color contrast between the inside of the cell and its borders which is important for the next step merging cells However if the signal is strong and clear it is not necessary to run this process To try it run Process Mesh Signal Normalize Signal This time indicate a radius slightly higher than the average radius of y
47. ust be set in the View tab Reset the clipping planes rotation with the button To get a cross section of the sample align the camera angle with one of the axes e g Z and enable the clipping plane perpendicular to that axis e g Clip3 to obtain a cross section Select the Control key interaction for this plane Slice throughout the sample using the middle mouse button while pressing the Crtl key You can visualize more than one clipping plane at the same time 4 Slices Under View Quality in the View tab use the Slices slider to modify the number of stack slices displayed Try to rotate the stack while the slice number is either minimal or maximal and notice if it makes a difference in the rendering speed Save the MorphoGraphxX session You can save the session with all the parameters used and the path of the data loaded In the File menu select save or save as This will create a MorphoGraphX session mgxv file that you can start next time The data stacks and meshes will then be automatically loaded Troubleshooting Symptom The stack is loaded but not visible Try the following solutions in order e The stack or mesh can be out of the field of view Use the lt to reset the view e The stack color depth might be 8 or 12 bits instead of 16 bits By default the stacks are supposed to be 16 bits and the color mapping is optimized for this color depth Check the transfer function by clicking o
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