Original AIS quantification
This is the custom-written Matlab script we’ve used to quantitatively assess axon initial segment (AIS) location, length, and localisation in dissociated neuronal cultures (see Grubb & Burrone 2010 Nature 465: 1070-74). It takes in 2D z-projected or single z-slice tiff files, so won’t work if you want to analyse AISs in 3D, and it asks you to draw a profile along the axon before outputting various AIS measures, so you’ve still got a bit of work to do! Hopefully the annotations and instructions make things self-explanatory, but if you discover any bugs or need some help, please contact us. And, of course, please feel free to make any alterations you like – if they’re interesting, or make the code more efficient (not hard…) we’d be grateful if you’d send them back to us.
Update April 2011
Below is an updated version of our code for AIS analysis, with instructions for use. The basic principles are all the same, but there are a few important changes: the script now takes an RGB tiff file as input, allows analysis of AIS parameters in up to 3 channels, gives measures of fluorescent intensity, and gives more accurate measures of axonal distances.
Update September 2014
We now have a semi-automated programme for measuring AIS length, written by Dennis Kruijssen. It’s the ‘ais_auto.m’ file below, and comes with its own set of instructions as well as a ‘batch_ais_auto’ script that lets you work through multiple files rapidly. You’ll need to download the ‘lsmread.m’ and ‘tiffread.m’ files to get it to work, and you’ll need the Matlab Image Processing toolbox too. Finally, and most importantly, you’ll need some very clean AIS label – this just does not work with even moderate levels of background fluorescence.
We’d be happy to send out any of our published cDNA constructs to interested researchers in the public domain – just get in touch. But to make this process even easier, we’ve started depositing our plasmids with Addgene who archive, document and distribute constructs on a not-for-profit basis. If you go here you should see all the plasmids available from us.