Transcript Morphometric or 3D reconstruction

Morphometric or 3D
reconstruction
Chinmaya Sadangi
Outline
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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 point
Click 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
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.
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.
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
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
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 using
control+shift+x
Convert both of them
to 8 bits
Go to plugins --stiching --Depreciated--- 3D
stiching
Once the stiching starts,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
Keyboard shortcuts
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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