Re: Photobleaching

Posted by jross34 on Sep 09, 2015; 5:09pm
URL: http://imagej.273.s1.nabble.com/Photobleaching-tp5014227p5014291.html

If I completely understand your suggestion, I'm not sure it would work.

I'm using GCaMPs to assess action potential generation in an area of brain. I begin imaging sometime before I expect action potentials to occur and then continue to image for several seconds. At some point in response to a stimulus, cells will begin to generate action potentials and will have dramatic changes in fluorescence above background. However, because I'm imaging over time, I'm subject to photobleaching meaning that I will have decreasing background fluorescence overtime and the amplitude of each sequential action potential generation will look smaller, which may or may not be true.

So what I'm looking to do is generate an equation to fit the bleaching of the frames before any action potentials generate and then fit it to my entire stack. So far, all I've been able to do is determine an averaged constant for simple subtraction, which doesn't really work, and make an equation off the entire stack, which doesn't work because the higher fluorescence values associated with action potentials pulls my line equation up falsely.

Hopefully the additional information is useful.