READ ME



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BEFORE BEGINNING MACRO
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The DEFiNE macro was designed to run in FIJI 1.51w using Java 1.8.0_66. 

There is no guarantee that DEFiNE will work on any other version.



How to check which version of FIJI is running:	
      
Help > About ImageJ 



How to change to FIJI 1.51w:	
      
Help > Update ImageJ > Upgrade to: v1.51



To install the macro, go to Plugins > Macros > Install and select the DEFiNE file.




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DEFiNE
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When running either function in DEFiNE, all images to run should be saved in one folder.

For the Clean Images function, all images should have the same channel order.



Clean Images Function

Purpose: To remove autofluorescence and fluorescent artifacts from stacked images of axonal fibers.
 

Requirements: 
-Images cannot contain more than six channels. 

-Images must all have the same channel order.

-Images must be stacked. Maximum intensity projections cannot be processed. 

Recommendation:

-Image acquisition should include an 'autofluorescence' channel for optimal cleanup. 

Quantify Fibers Function

Purpose: To quantify the axonal fibers present in an image, calculated as the area of the image occupied by pixels at or above a set intensity.


Requirements: 

-Images generated using the 'Clean Images' function are always in the proper format. 

-Images must be maximum intensity projections. 

-Images must contain only one channel. 




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TEST IMAGES
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To test DEFiNE, begin the macro and select the Test_Images when prompted for an input folder. 
-The images must be processed using the Clean Images function before the Quantify Fibers function.



Test Images Channel Information:

Total channels: 5

Channels to clean: 3

Autofluorescence channel number: 3

First channel to clean: 2

First channel color: green

Second channel to clean: 4

Second channel color: red

Third channel to clean: 5

Third channel color: magenta