Morphometric or 3D reconstruction Chinmaya Sadangi.
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Transcript of Morphometric or 3D reconstruction Chinmaya Sadangi.
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
• 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.
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.
Open imageJ 64 bits
If image is in .oib format convert it to .tiff format as shown in the
dig.
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.
Go to plugins – segmentation – simple
neurite tracer
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
2 new window opens
You can chose different color
Use Parts in nearby slices
If you have a saved trace file you can load it using
FILE – LOAD TRACES/SWC FILE
You can select a different background. Go to LUT –
dropdown menu ---- select your choice.
For eg: selecting FIRE (LUT) would give something like this.
Click on the cell body --- first start pointClick at an end point to complete a path
Click on YES to confirm path. Alternatively,
PRESS “Y”
To complete path click on complete path.
Alternatively, PRESS “F”
Traced path shows up in “All path”
window
You can designate it as soma, axon,
dendrite
To save the trace file --- Go to FILE --- SAVE TRACE FILE
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?
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
Extensive branching of an axon from a dendrite
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.
Select the path by pressing G.Mark the start
pointThen 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
Paths are now connected a new path would appear like this
Fill outs
• Filling out paths to volume• Gives an idea about the shape of the neuron
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
New dialog box would appear. Click on create on mask and transparent fill
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.
Right click and click on Z project.Save the Z projection.
Repeat the same for dendrites & axons
MPI Overlays
On simple neurite tracer --- click view ---- click on Show MIP overlays
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
Z Projection
• Creates a 2-D image of the 3-D image
Go to Image --- stacks --- Z Project
New dialog box --- (default) Max Intensity – If not change to Max
Intensity --- Click OK
You will see Z projection of the image you are tracing
3D STICHING OF IMAGES
Place both images side by side
Select areas in both of them
Crop them usingcontrol+shift+x
Convert both of them to 8 bits
Go to plugins --- stiching ---
Depreciated--- 3D stiching
A new dialog box would appear. Check first stack and second stackGo to fused image --- select max
intensity
Once the stiching starts,a log appears and shows STARTING and when it ends it appears
FUSION COMPLETED
Keyboard shortcuts• Y --- confirms current path• F – Finishes current path• N – cancels temporary path• G – Select nearby path• + -- Zooms in• - (--) Zooms out
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