Posted by
Jeff Spector on
URL: http://confocal-microscopy-list.275.s1.nabble.com/flash-4-0v2-for-single-molecule-imaging-tp7582355p7582366.html
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Thanks for the replies so far.
We do have access to EMCCD cameras and that is what we use most of the
time for single molecule dynamics. Most of what I have looked at says that
sCMOs is better than EMCCD but the crossover point seem to be right around
the S/N you get when doing single molecule imaging. I guess we thought that
since you can do single molecule localization you could do dynamics but I
guess in the localization case you really are pumping a lot more photons
out of the fluorophore (which I guess means you image it for a shorter
time?).
thanks...
-Jeff
On Thu, Jul 17, 2014 at 4:59 AM, Colin Coates <
[hidden email]> wrote:
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>
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>
> **commercial response**
> Hi Jeff,
> Bottom line, if you happen to have access to an EMCCD that's generally
> what I'd recommend for dynamic single molecule TIRF. After a few years of
> our single molecule imaging users comparing both our EMCCD and sCMOS
> cameras, the feedback has been pretty convincing. Its generalising, but the
> light levels involved in single molecule tend to be below the practical
> cross
> over point in the SNR vs photon flux curve, whereby an EMCCD has the
> advantage. Localisation super-resolution microscopy can be the exception,
> since the approach requires you to drive up photon levels to be successful.
>
> Having said that, if you are trying to adapt an sCMOS to dynamic single
> molecule, one option is to at least bring the effective pixel size closer
> to
> what you are used to with a larger pixel EMCCD. The best way is to use
> between 1.5x to 2x demagnification before the camera (depends on the
> objective as to what you can get away with and still oversample).
> Alternatively, 2x2 binning of the 6.5 um pixel (yielding a 13um binned
> pixel) can also be effective. The photons per pixel will quadruple, but be
> aware the read noise will also double - however, there's still a net gain
> in
> SNR.
>
> Best,
> Colin
>
> Dr Colin Coates
> Product Manager - Imaging
> Andor Technology
>