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Morphometric or 3D reconstruction

Morphometric or 3D reconstruction. Chinmaya Sadangi. Outline. Define Morphometric Image J Simple Neurite tracer plugin Guide to reconstruction using image J. Morphometry. Studies on brain shape has given immense data

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Morphometric or 3D reconstruction

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  1. Morphometric or 3D reconstruction Chinmaya Sadangi

  2. Outline • Define Morphometric • Image J • Simple Neurite tracer plugin • Guide to reconstruction using image J

  3. Morphometry • Studies on brain shape has given immense data • Morphometric measurements have been obtained from brain regions that can be clearly defined and results in a lot of findings related to these measurements. • Used for localizing significant structural differences, or overall brain structure. Also used for characterizing essential differences or for classification.

  4. 3D Reconstruction • To understand the neuronal morphology • To investigate neuronal function • Understand the complicated relation between physiology (function) & morphology (form) of neurons. • Characterization of cell types & investigation of neuronal development.

  5. Open imageJ 64 bits If image is in .oib format convert it to .tiff format as shown in the dig.

  6. Simple Neurite tracer • Semi-automatic tracing of neurons. • Efficiently find the best path between points in neurons. • Measurement of neuron length & volume, 3D visualization. • Best suited for fast tracing of higher order neural branches. Bioinformatics (2011):Mark H. Longair. Simple neurite tracer: open source software for reconstruction, visualization, & analysis of neuronal processes.

  7. Go to plugins – segmentation – simple neurite tracer

  8. New dialog box – convert image to 8 bits. This dialog box won’t appear if image is already in 8-bits Another dialog box – No 3D view

  9. 2 new window opens You can chose different color

  10. Use Parts in nearby slices If you have a saved trace file you can load it using FILE – LOAD TRACES/SWC FILE

  11. You can select a different background. Go to LUT – dropdown menu ---- select your choice. For eg: selecting FIRE (LUT) would give something like this.

  12. Click on the cell body --- first start point Click at an end point to complete a path

  13. Click on YES to confirm path. Alternatively, PRESS “Y” To complete path click on complete path. Alternatively, PRESS “F”

  14. Traced path shows up in “All path” window You can designate it as soma, axon, dendrite

  15. To save the trace file --- Go to FILE --- SAVE TRACE FILE

  16. How to know if you are doing it correctly? All path should be connected to the soma. If there is something not connected, check for the floating branch.

  17. How to identify an axon and a dendrite? An axon would be thinner as compared to a dendrite An axon usually branches from a dendrite or the soma An axon branches extensively.

  18. Extensive branching of an axon from a dendrite

  19. JOINING PATHS Sometimes it may happen that you have to join paths as you missed them out before or you have to delete a path and rejoin it to some other path.

  20. Select the path by pressing G. Mark the start point Then move the cursor to the path where you want to join and select by pressing G Click on that path New dialog box would appear. Press Y

  21. Paths are now connected a new path would appear like this

  22. Fill outs • Filling out paths to volume • Gives an idea about the shape of the neuron

  23. Select all paths except soma. Delete all paths (but don’t save it) Now select the soma Click on Fill out Repeat the same for axons & dendrites

  24. New dialog box would appear. Click on create on mask and transparent fill

  25. The soma starts to fill-out. Click on pause and click on the overfilled area to remove it. Avoid overfilling the soma After filling, click on create image stack from fill. Save the stack file.

  26. Right click and click on Z project. Save the Z projection. Repeat the same for dendrites & axons

  27. MPI Overlays On simple neurite tracer --- click view ---- click on Show MIP overlays

  28. Z projection with 20% opacity is created and you can trace on this Z projection. Can be turned off by going to view & clicking on MPI Overlays

  29. Z Projection • Creates a 2-D image of the 3-D image

  30. Go to Image --- stacks --- Z Project New dialog box --- (default) Max Intensity – If not change to Max Intensity --- Click OK

  31. You will see Z projection of the image you are tracing

  32. 3D STICHING OF IMAGES

  33. Place both images side by side Select areas in both of them Crop them using control+shift+x Convert both of them to 8 bits

  34. Go to plugins --- stiching --- Depreciated--- 3D stiching

  35. Once the stichingstarts,a log appears and shows STARTING and when it ends it appears FUSION COMPLETED A new dialog box would appear. Check first stack and second stack Go to fused image --- select max intensity

  36. Keyboard shortcuts • Y --- confirms current path • F – Finishes current path • N – cancels temporary path • G – Select nearby path • + -- Zooms in • - (--) Zooms out

  37. Why use TIFF images? (TAGGED IMAGE FILE FORMAT • TIFF images format are LOSSLESS. • Lossless – file is uncompressed & of higher image quality. • JPEGS & other file format are LOSSY. • Compressing image takes away quality of image. • Image turned to JPEG can’t be reverted back

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