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

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

18. Extensive branching of an axon from a dendrite

20. 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.

21. 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

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

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

24. 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

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

26. 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.

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

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

29. 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

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

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

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

33. 3D STICHING OF IMAGES

34. 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

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

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

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

38. 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