Reconstruction using Image J

Morphometric or 3D reconstruction

Chinmaya SadangiAG I Vida & AG D Schmitz

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