Zeiss FCS Analysis
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 [hidden email]<mailto:[hidden email]> http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html The University of Dundee is a registered Scottish Charity, No: SC015096 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Eric, As far as I know the Zeiss software does not allow you to export the raw data. My understanding is that the ACF is generated "online" and that the user can only export fluctuation traces which are binned in certain time intervals. This seems to be a limitation of the software if you want to generate your own ACF. However if you only want to use custom software (e.g. Matlab) to fit the ACF to a certain model you can of course work using the ACF exported from the Zeiss software. Best regards, Philipp Philipp A. Steffen, PhD Vienna (Previously at IMBA, Ringrose lab) > Am 27.08.2014 um 17:42 schrieb Eric Griffis <[hidden email]>: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > 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 > [hidden email]<mailto:[hidden email]> > http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html > > > > > The University of Dundee is a registered Scottish Charity, No: SC015096 |
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In reply to this post by Eric Griffis
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 [hidden email]<mailto:[hidden email]> http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html The University of Dundee is a registered Scottish Charity, No: SC015096 |
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In reply to this post by Philipp Steffen-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** There may be a bug of some sort in your Zeiss installation. There may also be a setting under the maintain menu to keep the raw trajectories. Our LSM 780 exports the raw photon mode trajectories just fine as .raw files. You have to parse the binary data, but it is certainly there for detailed analysis. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philipp Steffen Sent: Wednesday, August 27, 2014 11:06 AM To: [hidden email] Subject: Re: Zeiss FCS Analysis ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Eric, As far as I know the Zeiss software does not allow you to export the raw data. My understanding is that the ACF is generated "online" and that the user can only export fluctuation traces which are binned in certain time intervals. This seems to be a limitation of the software if you want to generate your own ACF. However if you only want to use custom software (e.g. Matlab) to fit the ACF to a certain model you can of course work using the ACF exported from the Zeiss software. Best regards, Philipp Philipp A. Steffen, PhD Vienna (Previously at IMBA, Ringrose lab) > Am 27.08.2014 um 17:42 schrieb Eric Griffis <[hidden email]>: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > 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 > [hidden email]<mailto:[hidden email]> > http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.h > tml > > > > > The University of Dundee is a registered Scottish Charity, No: > SC015096 |
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In reply to this post by Unruh, Jay
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > 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: > Subject: Zeiss FCS Analysis > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > 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 > [hidden email]<mailto:[hidden email]> > http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html > > The University of Dundee is a registered Scottish Charity, No: SC015096 Prof. Sudipta Maiti Dept. of Chemical Sciences Tata Institute of Fundamental Research Homi Bhabha Road, Colaba Mumbai 400005, India Ph. +91 222 278 2716 Alternate e-mail: [hidden email] webpage: biophotonics.co.in |
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In reply to this post by Unruh, Jay
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I think you need the FCS license module to export the data, the basic analysis works without FCS included in the license. You could record a long time series with the required time resolution and correlate yourself. Andreas -----Original Message----- From: "Unruh, Jay" <[hidden email]> Sent: 27/08/2014 17:31 To: "[hidden email]" <[hidden email]> Subject: Re: Zeiss FCS Analysis ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** There may be a bug of some sort in your Zeiss installation. There may also be a setting under the maintain menu to keep the raw trajectories. Our LSM 780 exports the raw photon mode trajectories just fine as .raw files. You have to parse the binary data, but it is certainly there for detailed analysis. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philipp Steffen Sent: Wednesday, August 27, 2014 11:06 AM To: [hidden email] Subject: Re: Zeiss FCS Analysis ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Eric, As far as I know the Zeiss software does not allow you to export the raw data. My understanding is that the ACF is generated "online" and that the user can only export fluctuation traces which are binned in certain time intervals. This seems to be a limitation of the software if you want to generate your own ACF. However if you only want to use custom software (e.g. Matlab) to fit the ACF to a certain model you can of course work using the ACF exported from the Zeiss software. Best regards, Philipp Philipp A. Steffen, PhD Vienna (Previously at IMBA, Ringrose lab) > Am 27.08.2014 um 17:42 schrieb Eric Griffis <[hidden email]>: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > 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 > [hidden email]<mailto:[hidden email]> > http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.h > tml > > > > > The University of Dundee is a registered Scottish Charity, No: > SC015096 |
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In reply to this post by Unruh, Jay
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Jay, I see, I have not looked into the binary data. But am I right, that there is no way to export the raw data in a simple format (e.g. csv)? BTW: Your web page is a great resource! Best, Philipp > Am 27.08.2014 um 18:30 schrieb "Unruh, Jay" <[hidden email]>: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > There may be a bug of some sort in your Zeiss installation. There may also be a setting under the maintain menu to keep the raw trajectories. Our LSM 780 exports the raw photon mode trajectories just fine as .raw files. You have to parse the binary data, but it is certainly there for detailed analysis. > > Jay > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philipp Steffen > Sent: Wednesday, August 27, 2014 11:06 AM > To: [hidden email] > Subject: Re: Zeiss FCS Analysis > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear Eric, > > As far as I know the Zeiss software does not allow you to export the raw data. My understanding is that the ACF is generated "online" and that the user can only export fluctuation traces which are binned in certain time intervals. This seems to be a limitation of the software if you want to generate your own ACF. > However if you only want to use custom software (e.g. Matlab) to fit the ACF to a certain model you can of course work using the ACF exported from the Zeiss software. > > Best regards, > > Philipp > > Philipp A. Steffen, PhD > Vienna > (Previously at IMBA, > Ringrose lab) > > >> Am 27.08.2014 um 17:42 schrieb Eric Griffis <[hidden email]>: >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> 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 >> [hidden email]<mailto:[hidden email]> >> http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.h >> tml >> >> >> >> >> The University of Dundee is a registered Scottish Charity, No: >> SC015096 |
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In reply to this post by Sudipta Maiti
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of maiti Sent: Wednesday, August 27, 2014 11:39 AM To: [hidden email] Subject: Re: Zeiss FCS Analysis ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > 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: > Subject: Zeiss FCS Analysis > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > 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 > [hidden email]<mailto:[hidden email]> > http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.h > tml > > The University of Dundee is a registered Scottish Charity, No: > SC015096 Prof. Sudipta Maiti Dept. of Chemical Sciences Tata Institute of Fundamental Research Homi Bhabha Road, Colaba Mumbai 400005, India Ph. +91 222 278 2716 Alternate e-mail: [hidden email] webpage: biophotonics.co.in |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 [hidden email]<mailto:[hidden email]> http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html On 28 Aug 2014, at 16:34, "Unruh, Jay" <[hidden email]<mailto:[hidden email]>> wrote: ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of maiti Sent: Wednesday, August 27, 2014 11:39 AM To: [hidden email] Subject: Re: Zeiss FCS Analysis ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** 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 [hidden email]<mailto:[hidden email]> http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.h tml The University of Dundee is a registered Scottish Charity, No: SC015096 Prof. Sudipta Maiti Dept. of Chemical Sciences Tata Institute of Fundamental Research Homi Bhabha Road, Colaba Mumbai 400005, India Ph. +91 222 278 2716 Alternate e-mail: [hidden email] webpage: biophotonics.co.in The University of Dundee is a registered Scottish Charity, No: SC015096 |
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