Posted by
Beat Ludin on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Hardware-Questions-TIRF-Cameras-tp590221p590223.html
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalAt 18:38 31-08-2007, you wrote:
> (2) Change cameras. I have tried a few back thinned EM-CCDs, but did not
>find that they offered much benefit once the pixel size was corrected for.
I suspect that this is because you have relatively high background
fluorescence so you need high photon counts to get an acceptable S/N.
Under these circumstances, EM-CCDs may be not better than or even
inferior to normal CCDs. So trying a back-illuminated conventional
CCD would be a first advice although I wouldn't expect a dramatic
effect (the QE of the Orca-ER is pretty good already). But you may be
able to gain a lot more by reducing the background:
- use riboflavin-free and serum-free medium for imaging (you can use
a balanced salt solution like Earl's or Hank's for a first test to
see the effect)
- I found that culturing cells in the presence of Trolox-C can reduce
intracellular background several-fold. But you need to do the proper
controls and find the right concentration to avoid side-effects.
- optimize your filters
- minimize stray light (reduce ambient light)
- check different immersion oils, to find the best compromise between
losses due to autofluorescence and due to mismatch in refractive OR
dispersive index.
Of course, TIRF has the potential of reducing the background a lot,
if it is compatible with your application.
Regards, Beat