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Axon Initial Segment characterization/alignment tools
Matlab routines for analyzing fluorescence intensity profile along axon's initial segment AIS (in 1D).
Written similar to Grubb MS, Burrone J. Nature 2010 paper, check out authors own version.
Includes three modules:
- AISquantPlot is for characterizing and inspecting individual profiles
- AISquantBatch is for analyzing many profiles in batch
- AISalign is for averaging and aligning multiple staining of two different proteins with respect to each other
You will need MatLab to use it. Download all .m files (including padadd.m) to separate folder. Navigate inside it using MatLab file browser. If you want to work outside of it, just select the folder in MatLab browser panel and right-click "Add to Path"->"Selected folder".
Also you would need to install ImageJ to open your images and create intensity profiles.
Both programs analyze profile/find reference points exactly in the same way. The only difference is that AISquantPlot takes as an input a single text file (profile) and plots final results. While AISquantBatch takes folder, analyzes every text file inside in the same fashion and doesn't show any plot. Both programs generate Excel file with results in the current folder.
Launch AISquantPlot and choose trace_example_ImageJ.txt to see how it works.
Here is detailed step-by-step manual for AISquantBatch, including ImageJ part (written by Sam van Beuningen).
AISquantPlot is intended for initial estimation of analysis parameters (smoothing window, threshold), since it can visualize intensity profile and reference points. It is recommended to play with it and your data before performing "large-scale" analysis of many files with AISquantBatch.
This routine aligns stainings of two proteins in different cells with respect to some reference point in one control protein's intensity distribution along AIS. This approach allows to average multiple stainings with respect to some feature point: maximum of intensity, 30% of max from cell body, etc.
The script works with file output from AISquantBatch. So first you need to run it on a folder with specific order of files/filenames. It should be following:
cell01_trace_of_protein1.log cell01_trace_of_control_protein.log cell02_trace_of_protein1.log cell02_trace_of_control_protein.log ...etc.
Where cell01_trace_of_protein1.log and cell01_trace_of_control_protein.log are line traces along the same profile line in two different channels.
Then the generated Excel file must be loaded to the AISalign script and you will get averaged aligned normalized result.
Developed in Cell Biology group of Utrecht University.
E-mail for any questions.