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