Re: Zeiss FCS Analysis

Posted by Sudipta Maiti on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-FCS-Analysis-tp7582564p7582570.html

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Dear Eric,
There could be a hardware reason that makes the time resolution of the count
rate trace much lower. Most autocorelator cards use a "multiple Tau"
algorithm, which lets them calculates an autocorrelation trace very fast
(usually with a few ns resolution). However, the raw count rate data cannot be
saved at that resolution due to memory transfer rate (and memory size) issues.
The count rate data is therefore binned at a much lower time resolution, and
the details are lost forever. There are some modern cards, usually much
pricier, which will give you the full thing.
I do not know if this is the issue with Zeiss, as I don't use their FCS, but I
suspect it is.
LabView, and even some spreadsheet softwares (e.g. Origin) these days have
autocorrelation functions built in (no commercial interest). But I suspect
Jay's algorithm will trump these (+ the best things in the world are free
;-)). Cheers.
Sudipta
 On Wed, 27 Aug 2014 16:11:18 +0000, Unruh, Jay wrote

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> Hi Eric,
>
> The .fcs output file should contain information at whatever
> resolution you calculate the correlation at.  There is probably an
> internal setting for that.
>
> I have free software for fcs analysis in ImageJ here:  
> http://research.stowers.org/imagejplugins/fcs_plugins.html  That
> software requires you to save the .raw files.  There should be a
> setting in the zeiss software for that as well.  It recalculates the
> autocorrelation functions using a slightly different method than the
> Zeiss software, so you can select the appropriate temporal sampling
> frequency for your analysis.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Eric Griffis
> Sent: Wednesday, August 27, 2014 10:43 AM To:

[hidden email]

> Subject: Zeiss FCS Analysis
>
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> Hi All,
>
> We are using a Zeiss 710 with a GaAsP detector to perform FCS. We
> are using a dilute (500nM) solution of Rhodamine B and the free on-
> board FCS software. We were hoping to use the known diffusion rate
> and concentration of this molecule to calculate the structural
> parameter of our FCS volume. When we look at our sample, we can see
> a reasonable count rate, a correlation of around 1, and a CPM value
> greater than 1. When we run the experiment, we see a nice
> autocorrelation curve. However, when we output the data, the best
> time resolution we can obtain is 1ms. There is clearly higher
> frequency data that is being used to generate the autocorrelation
> curves, but we cannot get it. Does anyone have any experience with
> using the data from a 710 to generate your own autocorrelation
> curves and do your own analysis? Additionally, if anyone could
> recommend any free software (Matlab or R) for performing FCS
> analysis, we would greatly appreciate it. Thanks for your help.
>
> Best regards,
>
> Eric
>
> Centre for Gene Regulation and Expression College of Life Sciences
> University of Dundee MSI/WTB/JBC Complex Dow Street Dundee DD1 5EH
> United KIngdom
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> [phone]
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Prof. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Road, Colaba
Mumbai 400005, India
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