FCS/FCCS advice

[Wolfgang Staroske]

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Dear list users,

we have a Zeiss LSM780 and attached a Confocor 3 (Zeiss FCS machine). So
I would like to comment on this. You can do with both the APDs in the
confocor module and the 32-channel GaAsP in the LSM 780, very nice FCS
measurements. Both detectors can be run in photon counting mode of
course and the improvement from standard PMT to GaAsP (PMT Mode) to
GaAsP (photon counting mode) is always visible.

We have both, because quantum efficiency behaves different for both
detector types. The APDs have there maximum in the orange/red, while the
GaAsP has it in the blue/green, with a constant decrease to the red. The
confocor 3 has only two channels and is still filter based, while on the
32-channel GaAsP you have full spectrum flexibility. Dark counts are
comparable between both for a the same spectral region below 200Hz. I
prefer still the confocor3 for FCCS measurements, because of the better
sensitivity in red. Because no fluorescent protein is as good as EGFP in
points of brightness and photo stability.

Coming back to the original question. I can only advice your user, to go
in a lab which has experience with FCS/FCCS and ask for a collaboration.
There are many pitfalls starting from the choice of fluorophores,
suitable expression levels, positive and negative controls, data
evaluation and so on. I know what I'm talking about, because I handle
the FCS/FCCS service here in Dresden, Germany.

Additionally, I can only endorse, what was written before, if you want
to do intracellular measurements, leave the hands from any home built
solution. You need a reliable, every day ready, turn key solution,
otherwise you will waste a lot of time for sample preparation, because
the machine is not working properly today for any reason. I can here
only comment on Confocor3 from Zeiss, which is such a solution and is
attachable to LSM510. The other systems mentioned here I know, but
haven't used them in all day work, so I can not comment on daily
reliability.

Of course you are welcome to ask any questions.

Bye Wolfgang


Am 20.04.2012 06:04, schrieb Stanislav Vitha:

> <pre wrap>
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>
> Hallo,
> what Michelle said.
> Our facility has a FV1000 confocal, one of our users was contemplating
> building a FCS hardware box, in the end went ahead and tried the Raster
> Image Correlation Spectroscopy on the FV1000 (and published a paper).
> You can download a fully functional 1-month evaluation copy of the
> RICS/FCS
> software from Laboratory for Fluorescence Dynamics, so I think it is
> definitely worth a try - no money, just time invested.
>
> Besides RICS, at least on the FV1000 you can park the beam and do spot
> scanning, so I think standard-type FCS should be possible (but I have not
> tried that myself.
>
> I agree that detector efficiency is crucial. Zeiss, Olympus and Leica  all
> have their new detectors (GaAsP PMTs or Hybrid detectors) as an upgrade
> option - I have not talked to Nikon lately, so I do not know if they have
> one or not.
> It is my understanding that RICS or FCS analyses are more
> straightforward if the
> detection is in photon counting mode.  Nikon does not have photon counting
> (yet?). During our recent Leica STED/confocal  demo I used the HyD in
> photon
> counting regime and compared to the
> standard PMT.  It is a huge improvement in sensitivity; I have not
> tried the
> Zeiss and Olympus GaAsP detectors yet, so I cannot comment on their
> suitability for photon counting -  sometimes increased quantum efficiency
> comes with increased dark noise. I would love to hear from users who
> tested
> them.
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&amp;M University
>
>
>
>
>
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--
Dr. Wolfgang Staroske

Single Molecule Specialist
Light Microscopy Facility

Technische Universität Dresden
Biotechnology Center
Tatzberg 47/49
01307 Dresden, Germany

Tel.: [phone]
Fax.: [phone]
E-Mail: [hidden email]
Webpage: www.biotec.tu-dresden.de