Posted by
Eric Griffis on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-FCS-Analysis-tp7582564p7582595.html
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Hi Everyone,
Thanks to everyone who helped us with this issue. Zeiss provided us with some off-list advice on how to select the correct options in the FCS window to allow us to export the raw data. However, these options were all greyed out (along with our ability to select the GaAsP detector we wanted to use) until we installed the FCS dongle. Without the dongle, we were unable to select a bin of smaller than 1ms for the data. Now that we have the dongle, we can calculate our own autocorrelation curves to compare to the ones generated by the Zen software, and we can now start to troubleshoot the rest of the experiment.
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
+44 (0)1382 385118
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http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.htmlOn 28 Aug 2014, at 16:34, "Unruh, Jay" <
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This is not true for the Zeiss hardware/software. They faithfully record the temporal spacing between photons at the max detection rate (this was 20 MHz for the confocor3 and I think 15 MHz for the LSM 780).
In response to Steffen's recent comment, I think it would in many cases be unwise to export the data in text format, simply given the large nature of fcs data and the slowness of text io and processing. In my experience, the majority of FCS acquisition and processing software uses some sort of binary data format.
I'm in the process of updating my plugins to include a direct importer for Zeiss raw trajectories in either time or photon mode formats. The resulting plot window will support csv or tab delimited export for those who prefer those formats. Just don't try to open it in excel:)
Jay
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Sent: Wednesday, August 27, 2014 11:39 AM
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Subject: Re: Zeiss FCS Analysis
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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
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Sent: Wednesday, August 27, 2014 10:43 AM To:
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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
+44 (0)1382 385118
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