Photoactivated localization microscopy (PALM)

[gkiverm]

I have been reading up on PALM works and am still confused about a few things. In particular, (1) how can you only excite a few molecules at a time and then only photobleach those molecules? Won't other molecules also automatically be excited and eventually photobleached? (2) When you fit the point-spread function to a Gaussian function, what exactly are you doing? Isn't it basically just taking the weighted average of all the intensities at each pixel? Thanks!

 

[UltrafastPED]

The probe is limited to a very precise, very small area; the scanning process allows an image to be built up for larger regions.

See http://zeiss-campus.magnet.fsu.edu/articles/superresolution/palm/introduction.html