Posted by
Ian Dobbie-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Hardware-Questions-TIRF-Cameras-tp590221p590224.html
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalStephen Bunnell <
[hidden email]> writes:
[snip]
> I have two options, and a question associated with each:
>
> (1) Move to a TIRF system. What are your opinions about how much gain in
> sensitiviy this may provide? Has anyone worked with the Zeiss TIRF module
> for the Axiovert 200M?
This will lead to a massive gain in signal to noise. So long as your
proteins of interest are in the magic 100nm from coverslip range you
can get down to signal molecule sensitivity. Haven't a clue about the
Zeiss TIRF module I'm afraid.
> (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.
> Is there a better option, that offers high resolution, high sensitivity, and
> low background noise? Obviously, there will be compromises. What are your
> opinions of intensified CCD cameras?
The only real advantage of EMCCDs is in imaging FAST. An EMCCD should
allow you to image all the colour ranges you want in a fraction of a
second (of course depending on you filter changer speed) rather than
several seconds. The two drawbacks are increased pixel size, but this
shouldn't be a factor if you use an optavar, and effectively 1/2 the
QE. A back-thinned EMCCD shouldn't be too much worse than a front
illuminated conventional CCD such as the ORCA-ER.
As mentioned before in this thread a combination of both TIRF and an
EMCCD would be ideal but you may well not have the budget for that. As
you have tried some EMCCDs and not found them too useful and you don't
want to image really quickly, it is likely that TIRF would be the best
single option to try. I would try to get a loan of some equipment for
a week or two to see how it goes.
Ian