Re: localization precision in PALM/STORM

Posted by Alex Knight on
URL: http://confocal-microscopy-list.275.s1.nabble.com/localization-precision-in-PALM-STORM-tp7219699p7224449.html

James Pawley wrote

However, while it is certainly important to know noise levels of the various photodetectors, there may be other noise sources that may be even more important.

What about the statistical and other errors related to the very small numbers of fluorescent molecules actually localized?

Hi James, you make a lot of excellent points here, and ones that it is easy to forget about.

One could add to your list:
- what about the molecules that you never see because they don't blink during your acquisition, or photobleach first?
- what about overlapping molecules which are not rejected by the software, but give rise to mis-localisations?
- worst of all, what about mechanical drift occurring in the system during the acquisition? If this is not controlled, it can seriously degrade your images.

I could go on. Really the importance of all these various factors depends on the biological question you are trying to address and the type of sample you are using.

The fluorophore orientation issue you raise is interesting, but I think that in most situations the fluorophores rotate freely enough that polarisation is not a problem.

Cheers

Alex

PS For those of you who are on LinkedIn we have just set up a super-resolution/nanoscopy group which may be of interest.

Alex Knight, National Physical Laboratory