Fluoview 1000 and FCS
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Hi all, I'm thinking about modifying our fluoview 1000 to do FCS. The fluoview has the possibility to do single point measurement. However as we can still choose the pixel time in this mode, I was wondering if the laser beam always stays at the same point of interest and continuously illuminates the point or if it actually "blinks". If it doesn't "blink", I would think it could hinder the FCS measurement et thus I'm wondering if there is an option to change this in the soft. ? Thanks, JP - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jean-Pierre CLAMME, PhD Senior Scientist Nitto Denko Technical 501 Via Del Monte Oceanside, CA 92058 E-mail: [hidden email] Phone: +760.435.7065 |
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Hi Jean,
You might want to have a look at Enrico Gratton's work and specially SimFCS wherein one can calculate diffusion coefficients from an image collected in a point-scan mode. I do not know if a blinking laser gives you the possibility of doing FCS (Do let me know if such a possibility exists. I would love to try it at my place.) We rather use a hardware correlator and a simple laser (diode/HeNe) Aseem On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME <[hidden email]> wrote: > > Hi all, > > I'm thinking about modifying our fluoview 1000 to do FCS. The fluoview has > the possibility to do single point measurement. However as we can still > choose the pixel time in this mode, I was wondering if the laser beam always > stays at the same point of interest and continuously illuminates the point > or if it actually "blinks". > > If it doesn't "blink", I would think it could hinder the FCS measurement et > thus I'm wondering if there is an option to change this in the soft. ? > > Thanks, > > JP > > > > > - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - > Jean-Pierre CLAMME, PhD > Senior Scientist > Nitto Denko Technical > 501 Via Del Monte > Oceanside, CA 92058 > E-mail: [hidden email] > Phone: +760.435.7065 > -- Aseem Mishra Senior Research Assistant, Malaria Group, International Centre for Genetic Engineering and Biotechnology, New Delhi-110067 INDIA |
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Jean, if you're present at this year's Biophysical Society meeting, make sure you check out this poster by Dr. Gratton's lab as well:
Iyrri L Salvemini, Enrico Gratton, and Pierre D J Moens. Single point FCS on a commercial confocal laser scanning microscope with analog detectors. 54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California. Biophys J. 2010; 98(3, Suppl 1): 180a, 935-Pos. Abstract: Fluorescence Correlation Spectroscopy is a technique invented in the early 1970s to measure diffusion coefficient, chemical reaction rates and photo physical processes. It is a common belief that in order to obtain single point FCS data, one needs either a sophisticated FCS instrument with photon counting detectors or avalanche photon detectors or an instrument custom made for this type of experiments. Here we show that we can obtain single point FCS data on a commercial confocal laser scanning microscope without any modifications (Nikon C1). We successfully measured the diffusion coefficient and the concentration of Rhodamine B in solution for concentrations ranging from 5 nM to 280 nM. We also determined the diffusion coefficient of two different labeled lipid analogs (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and BODIPY TMR phosphatidylinositol (4,5) bisphosphate) incorporated in the membrane of giant unilamellar vesicles. The results obtained for these lipid analogs are in good agreement with previously published data. Finally, we highlighted the fact that the actual proportion of labeled lipid analogs incorporated in the membrane of the giant unilamellar vesicle (formed by the electroformation method) is significantly different than the proportion of these lipids in the organic solvent stock solution. I'm sure there will also be a journal publication to follow up on this poster at some point. You could try emailing them for more details. Maybe other confocal brands can be setup for the same thing. John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging On 20-Feb-10, at 11:39 AM, aseem mishra wrote:
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In reply to this post by Jean-Pierre CLAMME-2
Hi,
I second Aseem's suggestion regarding SImFCS. A user of our core facility is using FV1000 and the SimFCS software from the Gratton lab, it appears to work quite well, as far as I know. The SimFCS software can be installed as a fully-functional 1-month demo, so you can test it yourself before making the final decision. One big advantage of the RICS approach that I see is the ability to subtract slow movements, such as sample or stage drift. A while ago I was told by Olympus that point scanning was not going to work for FCS on the FV1000 system, but I do not quite remember their technical explanation. From my tests, I remember there was an issue with the first or last datapoint being of incorrect value, due to the timing of the AOTF "shutter", or synchronization of the AOTF with data acquisition. You could try point-scanning of the fluorescent plastic slide from Chroma, the fluctuations that you see should indicate the instrument noise/instability, including the laser. The number of datapoints in point-scanning mode on FV1000 was limited to 4096, i.e., the max. permitted line width. I was told that before a scan a piece of code is sent to the control board(s), essentially reprogramming the firmware, and this somehow imposes a limit on the number of datapoints. You could do time-lapse point scan to acquire multiples of 4096 points,, but then you have an unknown amount of time between the time frames. Anyway, I think SimFCS is worth trying. Stan Vitha Microscopy and Imaging Center Texas A&M University On Sat, 20 Feb 2010 22:09:52 +0530, aseem mishra <[hidden email]> wrote: >Hi Jean, > >You might want to have a look at Enrico Gratton's work and specially >SimFCS wherein one can calculate diffusion coefficients from an image >collected in a point-scan mode. I do not know if a blinking laser >gives you the possibility of doing FCS (Do let me know if such a >possibility exists. I would love to try it at my place.) We rather use >a hardware correlator and a simple laser (diode/HeNe) > >Aseem > > >On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME ><[hidden email]> wrote: >> >> Hi all, >> >> I'm thinking about modifying our fluoview 1000 to do FCS. The fluoview has >> the possibility to do single point measurement. However as we can still >> choose the pixel time in this mode, I was wondering if the laser beam always >> stays at the same point of interest and continuously illuminates the point >> or if it actually "blinks". >> >> If it doesn't "blink", I would think it could hinder the FCS measurement et >> thus I'm wondering if there is an option to change this in the soft. ? >> >> Thanks, >> >> JP >> >> >> >> >> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - >> Jean-Pierre CLAMME, PhD >> Senior Scientist >> Nitto Denko Technical >> 501 Via Del Monte >> Oceanside, CA 92058 >> E-mail: [hidden email] >> Phone: +760.435.7065 >> > > > >-- >Aseem Mishra >Senior Research Assistant, >Malaria Group, >International Centre for Genetic Engineering and Biotechnology, >New Delhi-110067 >INDIA |
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Dear all
I can confirm that the FV1000 can be used for FCS measurements, we also used it with SimFCS software and it works very well. - the laser does not blink, it just scans continually at the designated point (use crosshair icon and select desired point on image for measurement) - the max number of points on FV1000 in point scanning mode is 32766. This number of points is sufficient for an accurate measurement. Acquisition in this way is that it is equivalent to capturing FCS data in "time mode" in ISS Vista software (the other system we use, where we can choose either "time mode" which counts photons/time-block, or "photon mode" which counts each photon with its respective time). In ISS Vista, we always exclude the first data point in the analysis, because it has no preceeding data points to be correlated with for the correlation analysis. I cant recall excluding the first point when we have analysed FV1000 point scan data in SimFCS, I'm not sure if there is an option to do so. If the first data point can be excluded in the analysis I would do so. Do not simply delete the first data point, as this would simply mean that now the second data point has no first point to be correlated to so the problem still remains. If the first point can not be excluded from the analysis, it is only one point whose correlation will be of reduced accuracy, out of the 32766 points, so I dont think this matters very much, and perhaps (? - maybe a mathematician can answer) it is not as important in time mode as in photon mode. SimFCS is definately worth trying, yes 30day demo licence is free, and the full licence is very economical anyway. Go to the website for the Laboratory for Fluorescence Dynamics; http://www.lfd.uci.edu/ For point-scanning data collection on the FV1000, configure for optimal resoluion, ie use a high NA objective etc, select photon counting mode, ie "photon cnt", chose appropriate pixel time eg 8-12.5us/pixel for solution or 12.5-40us/pixel for measurement on cells, select crosshair icon and set desired point in image, set number of frames to 32766 (if you type in an overly high number eg 1000000 it will automatically reset to the maximum available) with interval set to "freerun". Make sure you export the data as the "raw" 16-bit files. If you need a hand working through the analysis in the SimFCS software contact me offline. Note that the Wo (PSF waist) in SimFCS is defined using 1/e2 max intensity not half max intensity. Its always a good idea to include a reference dye for which you already have a good idea of expected or theoretical diffusion coefficient. Hope this helps Kind regards Jennifer -- Jennifer Clarke BSc (Hons) PhD Research Associate, Anatomy and Histology Centre for Neuroscience, School of Medicine & Facility Manager, Optical Microscopy Suite, Flinders Microscopy Flinders University GPO Box 2100, Adelaide 5001 Phone: 61 8 8204 6637 / 61 8 8204 6454 Email: [hidden email] -------------------------------------------------------- Quoting Stanislav Vitha <[hidden email]>: > Hi, > I second Aseem's suggestion regarding SImFCS. > > A user of our core facility is using FV1000 and the SimFCS software from the > Gratton lab, it appears to work quite well, as far as I know. The SimFCS > software can be installed as a fully-functional 1-month demo, so you can > test it yourself before making the final decision. One big advantage of the > RICS approach that I see is the ability to subtract slow movements, such as > sample or stage drift. > A while ago I was told by Olympus that point scanning was not going to work > for FCS on the FV1000 system, but I do not quite remember their technical > explanation. From my tests, I remember there was an issue with the first or > last datapoint being of incorrect value, due to the timing of the AOTF > "shutter", or synchronization of the AOTF with data acquisition. You could > try point-scanning of the fluorescent plastic slide from Chroma, the > fluctuations that you see should indicate the instrument noise/instability, > including the laser. > The number of datapoints in point-scanning mode on FV1000 was limited to > 4096, i.e., the max. permitted line width. I was told that before a scan a > piece of code is sent to the control board(s), essentially reprogramming the > firmware, and this somehow imposes a limit on the number of datapoints. > You could do time-lapse point scan to acquire multiples of 4096 points,, but > then you have an unknown amount of time between the time frames. > > Anyway, I think SimFCS is worth trying. > > Stan Vitha > Microscopy and Imaging Center > Texas A&M University > > On Sat, 20 Feb 2010 22:09:52 +0530, aseem mishra <[hidden email]> > wrote: > > >Hi Jean, > > > >You might want to have a look at Enrico Gratton's work and specially > >SimFCS wherein one can calculate diffusion coefficients from an image > >collected in a point-scan mode. I do not know if a blinking laser > >gives you the possibility of doing FCS (Do let me know if such a > >possibility exists. I would love to try it at my place.) We rather use > >a hardware correlator and a simple laser (diode/HeNe) > > > >Aseem > > > > > >On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME > ><[hidden email]> wrote: > >> > >> Hi all, > >> > >> I'm thinking about modifying our fluoview 1000 to do FCS. The fluoview > has > >> the possibility to do single point measurement. However as we can still > >> choose the pixel time in this mode, I was wondering if the laser beam > always > >> stays at the same point of interest and continuously illuminates the > point > >> or if it actually "blinks". > >> > >> If it doesn't "blink", I would think it could hinder the FCS measurement > et > >> thus I'm wondering if there is an option to change this in the soft. ? > >> > >> Thanks, > >> > >> JP > >> > >> > >> > >> > >> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - > >> Jean-Pierre CLAMME, PhD > >> Senior Scientist > >> Nitto Denko Technical > >> 501 Via Del Monte > >> Oceanside, CA 92058 > >> E-mail: [hidden email] > >> Phone: +760.435.7065 > >> > > > > > > > >-- > >Aseem Mishra > >Senior Research Assistant, > >Malaria Group, > >International Centre for Genetic Engineering and Biotechnology, > >New Delhi-110067 > >INDIA > -- Jennifer Clarke BSc (Hons) PhD Research Associate, Anatomy and Histology Centre for Neuroscience, School of Medicine & Facility Manager, Optical Microscopy Suite, Flinders Microscopy (available for training and assistance on Mondays only) Flinders University GPO Box 2100, Adelaide 5001 Phone: 61 8 8204 6637 / 61 8 8204 6454 Email: [hidden email] |
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In reply to this post by Stanislav Vitha
Hi,
we always recorded a large number of files with a limited number of data points each. Each file was autocorrelated and then we took the average of all the files which gives a nice autocorrelation curve. This was done with APDs and multi channel scalar (MCS) cards for data aquisition on a home built setup but should also work for the standard confocal setups. Just the detector sensitivity is lower and noise is higher for the PMTs. Andreas -----Original Message-----
From: Stanislav Vitha <[hidden email]> To: [hidden email] Sent: Mon, 22 Feb 2010 22:41 Subject: Re: Fluoview 1000 and FCS Hi, |
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In reply to this post by Jean-Pierre CLAMME-2
Dear Jennifer,
when you do point scanning FCS on the Fluoview 1000, what scan size do you typically have prior to selecting the point scan tool? That is, how many pixels per line was in the live view image? I am just curious about the total number of datapoints that one would need e.g., for solutions or cells (pixels per line x timepoints). Thanks! Stan Vitha. Microscopy and Imaging Center, Texas A&M University On Tue, 23 Feb 2010 11:19:43 +1030, Jennifer Clarke <[hidden email]> wrote: >Dear all > >I can confirm that the FV1000 can be used for FCS measurements, we also used it >with SimFCS software and it works very well. > > - the laser does not blink, it just scans continually at the designated point >(use crosshair icon and select desired point on image for measurement) > > - the max number of points on FV1000 in point scanning mode is 32766. This >number of points is sufficient for an accurate measurement. > >Acquisition in this way is that it is equivalent to capturing FCS data in "time >mode" in ISS Vista software (the other system we use, where we can choose >"time mode" which counts photons/time-block, or "photon mode" which counts each >photon with its respective time). > >In ISS Vista, we always exclude the first data point in the analysis, because >it >has no preceeding data points to be correlated with for the correlation >analysis. >I cant recall excluding the first point when we have analysed FV1000 point scan >data in SimFCS, I'm not sure if there is an option to do so. If the first data >point can be excluded in the analysis I would do so. Do not simply delete the >first data point, as this would simply mean that now the second data point has >no first point to be correlated to so the problem still remains. >If the first point can not be excluded from the analysis, it is only one point >whose correlation will be of reduced accuracy, out of the 32766 points, so I >dont think this matters very much, and perhaps (? - maybe a mathematician can >answer) it is not as important in time mode as in photon mode. > >SimFCS is definately worth trying, yes 30day demo licence is free, and the full >licence is very economical anyway. Go to the website for the Laboratory for >Fluorescence Dynamics; http://www.lfd.uci.edu/ > >For point-scanning data collection on the FV1000, configure for optimal >resoluion, ie use a high NA objective etc, select photon counting mode, ie >"photon cnt", chose appropriate pixel time eg 8-12.5us/pixel for solution or >12.5-40us/pixel for measurement on cells, select crosshair icon and set desired >point in image, set number of frames to 32766 (if you type in an overly high >number eg 1000000 it will automatically reset to the maximum available) with >interval set to "freerun". Make sure you export the data as the "raw" 16-bit >files. > >If you need a hand working through the analysis in the SimFCS software contact >me offline. >Note that the Wo (PSF waist) in SimFCS is defined using 1/e2 max intensity not >half max intensity. > >Its always a good idea to include a reference dye for which you already have a >good idea of expected or theoretical diffusion coefficient. > >Hope this helps >Kind regards >Jennifer > >-- >Jennifer Clarke BSc (Hons) PhD >Research Associate, Anatomy and Histology >Centre for Neuroscience, School of Medicine >& >Facility Manager, Optical Microscopy Suite, Flinders Microscopy > >Flinders University >GPO Box 2100, Adelaide 5001 >Phone: 61 8 8204 6637 / 61 8 8204 6454 >Email: [hidden email] > >-------------------------------------------------------- > >Quoting Stanislav Vitha <[hidden email]>: > >> Hi, >> I second Aseem's suggestion regarding SImFCS. >> >> A user of our core facility is using FV1000 and the SimFCS software from the >> Gratton lab, it appears to work quite well, as far as I know. The SimFCS >> software can be installed as a fully-functional 1-month demo, so you can >> test it yourself before making the final decision. One big advantage of the >> RICS approach that I see is the ability to subtract slow movements, such as >> sample or stage drift. >> A while ago I was told by Olympus that point scanning was not going to work >> for FCS on the FV1000 system, but I do not quite remember their technical >> explanation. From my tests, I remember there was an issue with the first or >> last datapoint being of incorrect value, due to the timing of the AOTF >> "shutter", or synchronization of the AOTF with data acquisition. You could >> try point-scanning of the fluorescent plastic slide from Chroma, the >> fluctuations that you see should indicate the instrument noise/instability, >> including the laser. >> The number of datapoints in point-scanning mode on FV1000 was limited to >> 4096, i.e., the max. permitted line width. I was told that before a scan a >> piece of code is sent to the control board(s), essentially reprogramming the >> firmware, and this somehow imposes a limit on the number of datapoints. >> You could do time-lapse point scan to acquire multiples of 4096 points,, but >> then you have an unknown amount of time between the time frames. >> >> Anyway, I think SimFCS is worth trying. >> >> Stan Vitha >> Microscopy and Imaging Center >> Texas A&M University >> >> On Sat, 20 Feb 2010 22:09:52 +0530, aseem mishra <[hidden email]> >> wrote: >> >> >Hi Jean, >> > >> >You might want to have a look at Enrico Gratton's work and specially >> >SimFCS wherein one can calculate diffusion coefficients from an image >> >collected in a point-scan mode. I do not know if a blinking laser >> >gives you the possibility of doing FCS (Do let me know if such a >> >possibility exists. I would love to try it at my place.) We rather use >> >a hardware correlator and a simple laser (diode/HeNe) >> > >> >Aseem >> > >> > >> >On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME >> ><[hidden email]> wrote: >> >> >> >> Hi all, >> >> >> >> I'm thinking about modifying our fluoview 1000 to do FCS. The fluoview >> has >> >> the possibility to do single point measurement. However as we can still >> >> choose the pixel time in this mode, I was wondering if the laser beam >> always >> >> stays at the same point of interest and continuously illuminates the >> point >> >> or if it actually "blinks". >> >> >> >> If it doesn't "blink", I would think it could hinder the FCS measurement >> et >> >> thus I'm wondering if there is an option to change this in the soft. ? >> >> >> >> Thanks, >> >> >> >> JP >> >> >> >> >> >> >> >> >> >> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - >> >> Jean-Pierre CLAMME, PhD >> >> Senior Scientist >> >> Nitto Denko Technical >> >> 501 Via Del Monte >> >> Oceanside, CA 92058 >> >> E-mail: [hidden email] >> >> Phone: +760.435.7065 >> >> >> > >> > >> > >> >-- >> >Aseem Mishra >> >Senior Research Assistant, >> >Malaria Group, >> >International Centre for Genetic Engineering and Biotechnology, >> >New Delhi-110067 >> >INDIA >> > > >-- >Jennifer Clarke BSc (Hons) PhD >Research Associate, Anatomy and Histology >Centre for Neuroscience, School of Medicine >& >Facility Manager, Optical Microscopy Suite, Flinders Microscopy >(available for training and assistance on Mondays only) > >Flinders University >GPO Box 2100, Adelaide 5001 >Phone: 61 8 8204 6637 / 61 8 8204 6454 >Email: [hidden email] |
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Dear Stan
The preceeding scan size doesnt matter for point scanning FCS measurement, and the measurement volume is determined by the confocal volume. However yes the spatial resolution of the preceeding image would, in part, affect your accuracy for positioning the xyz coordinate of the point FCS measurement. For us the preceeding image is usually 256x256 (ie 256 pixels per line) or 512x512 with 60 or 63x lens and between 1 and 6x zoom for visualisation of details of the cell (again zoom doesnt matter for the FCS measurement, only for visualisation). For live cells, a much bigger problem here however is movement of the cellular components during the course of the FCS point measurement. This is where RICS and scanning FCS and the SimFCS software become invaluable as the bulk cell movement during the measurement can be taken into account in the analysis. For solution measurements we would typically take at least 3 measurements and average them. For measurements on cells, we are trying to measure the movement of receptors on the membrane and the movement of the respective ligands, which we add to the media. This is very difficult as the cell membrane is constantly in motion. We try to position several measurement points along the membrane according to an image. Due to movement of the cell since the image was taken and during the course of the measurements not all the measurement points end up actually including the membrane. Usually it is clear in the analysis which measurement points were entirely intracellular and which were extracellular. Our main problem now is how to accomodate enough components into the FCS analysis (eg for the ligand, diffusion components include 1 triplet state, 2 free diffusion in extracellular component of measurement volume, 3 restricted movement in vicinity of membrane, 4 receptor binding events, and potentially any internalised ligand as well) and the fact that different membrane measurement points will include different proportions of extracellular and intracellular space). So, on live cells it gets very complicated vey quickly! Here we have a Leica SP5 with Avalance Photo Diode detectors and an FCS System with ISS Vista software. This is fine for FCS. Unfortunately there are problems using the APD image data aquired in the Leica software (LASAF) for analysis with RICS and N&B as the LASAF data is somehow modified and no longer exists as true raw data. True raw data is required for analysis with RICS and N&B as these techniques analyse the intensity fluctuations, so the data cannot be in any way already smoothed or averaged. With Enrico Grattons help, we are trying to find a way around this problem via feeding the APD image data straight into ISS Vista, bypassing LASAF. Unfortunately we do not regularly have access to an Olympus FV1000, we just used one when we were fortunate to visit the Gratton lab last year. Hope this helps I am not an expert in this, so I would be interested in any other listers comments too Kind regards Jennifer Quoting Stanislav Vitha <[hidden email]>: > Dear Jennifer, > when you do point scanning FCS on the Fluoview 1000, what scan size do you > typically have prior to selecting the point scan tool? That is, how many > pixels per line was in the live view image? I am just curious about the > total number of datapoints that one would need e.g., for solutions or cells > (pixels per line x timepoints). > > Thanks! > > Stan Vitha. > Microscopy and Imaging Center, > Texas A&M University > > On Tue, 23 Feb 2010 11:19:43 +1030, Jennifer Clarke > <[hidden email]> wrote: > > >Dear all > > > >I can confirm that the FV1000 can be used for FCS measurements, we also used > it > >with SimFCS software and it works very well. > > > > - the laser does not blink, it just scans continually at the designated > point > >(use crosshair icon and select desired point on image for measurement) > > > > - the max number of points on FV1000 in point scanning mode is 32766. > This > >number of points is sufficient for an accurate measurement. > > > >Acquisition in this way is that it is equivalent to capturing FCS data in > "time > >mode" in ISS Vista software (the other system we use, where we can choose > either > >"time mode" which counts photons/time-block, or "photon mode" which counts > each > >photon with its respective time). > > > >In ISS Vista, we always exclude the first data point in the analysis, > because > >it > >has no preceeding data points to be correlated with for the correlation > >analysis. > >I cant recall excluding the first point when we have analysed FV1000 point > scan > >data in SimFCS, I'm not sure if there is an option to do so. If the first > data > >point can be excluded in the analysis I would do so. Do not simply delete > the > >first data point, as this would simply mean that now the second data point > has > >no first point to be correlated to so the problem still remains. > >If the first point can not be excluded from the analysis, it is only one > point > >whose correlation will be of reduced accuracy, out of the 32766 points, so > I > >dont think this matters very much, and perhaps (? - maybe a mathematician > can > >answer) it is not as important in time mode as in photon mode. > > > >SimFCS is definately worth trying, yes 30day demo licence is free, and the > full > >licence is very economical anyway. Go to the website for the Laboratory > for > >Fluorescence Dynamics; http://www.lfd.uci.edu/ > > > >For point-scanning data collection on the FV1000, configure for optimal > >resoluion, ie use a high NA objective etc, select photon counting mode, ie > >"photon cnt", chose appropriate pixel time eg 8-12.5us/pixel for solution > or > >12.5-40us/pixel for measurement on cells, select crosshair icon and set > desired > >point in image, set number of frames to 32766 (if you type in an overly > high > >number eg 1000000 it will automatically reset to the maximum available) > with > >interval set to "freerun". Make sure you export the data as the "raw" > 16-bit > >files. > > > >If you need a hand working through the analysis in the SimFCS software > contact > >me offline. > >Note that the Wo (PSF waist) in SimFCS is defined using 1/e2 max intensity > not > >half max intensity. > > > >Its always a good idea to include a reference dye for which you already have > a > >good idea of expected or theoretical diffusion coefficient. > > > >Hope this helps > >Kind regards > >Jennifer > > > >-- > >Jennifer Clarke BSc (Hons) PhD > >Research Associate, Anatomy and Histology > >Centre for Neuroscience, School of Medicine > >& > >Facility Manager, Optical Microscopy Suite, Flinders Microscopy > > > >Flinders University > >GPO Box 2100, Adelaide 5001 > >Phone: 61 8 8204 6637 / 61 8 8204 6454 > >Email: [hidden email] > > > >-------------------------------------------------------- > > > >Quoting Stanislav Vitha <[hidden email]>: > > > >> Hi, > >> I second Aseem's suggestion regarding SImFCS. > >> > >> A user of our core facility is using FV1000 and the SimFCS software from > the > >> Gratton lab, it appears to work quite well, as far as I know. The SimFCS > >> software can be installed as a fully-functional 1-month demo, so you can > >> test it yourself before making the final decision. One big advantage of > the > >> RICS approach that I see is the ability to subtract slow movements, such > as > >> sample or stage drift. > >> A while ago I was told by Olympus that point scanning was not going to > work > >> for FCS on the FV1000 system, but I do not quite remember their technical > >> explanation. From my tests, I remember there was an issue with the first > or > >> last datapoint being of incorrect value, due to the timing of the AOTF > >> "shutter", or synchronization of the AOTF with data acquisition. You > could > >> try point-scanning of the fluorescent plastic slide from Chroma, the > >> fluctuations that you see should indicate the instrument > noise/instability, > >> including the laser. > >> The number of datapoints in point-scanning mode on FV1000 was limited to > >> 4096, i.e., the max. permitted line width. I was told that before a scan > a > >> piece of code is sent to the control board(s), essentially reprogramming > the > >> firmware, and this somehow imposes a limit on the number of datapoints. > >> You could do time-lapse point scan to acquire multiples of 4096 points,, > but > >> then you have an unknown amount of time between the time frames. > >> > >> Anyway, I think SimFCS is worth trying. > >> > >> Stan Vitha > >> Microscopy and Imaging Center > >> Texas A&M University > >> > >> On Sat, 20 Feb 2010 22:09:52 +0530, aseem mishra <[hidden email]> > >> wrote: > >> > >> >Hi Jean, > >> > > >> >You might want to have a look at Enrico Gratton's work and specially > >> >SimFCS wherein one can calculate diffusion coefficients from an image > >> >collected in a point-scan mode. I do not know if a blinking laser > >> >gives you the possibility of doing FCS (Do let me know if such a > >> >possibility exists. I would love to try it at my place.) We rather use > >> >a hardware correlator and a simple laser (diode/HeNe) > >> > > >> >Aseem > >> > > >> > > >> >On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME > >> ><[hidden email]> wrote: > >> >> > >> >> Hi all, > >> >> > >> >> I'm thinking about modifying our fluoview 1000 to do FCS. The fluoview > >> has > >> >> the possibility to do single point measurement. However as we can > still > >> >> choose the pixel time in this mode, I was wondering if the laser beam > >> always > >> >> stays at the same point of interest and continuously illuminates the > >> point > >> >> or if it actually "blinks". > >> >> > >> >> If it doesn't "blink", I would think it could hinder the FCS > measurement > >> et > >> >> thus I'm wondering if there is an option to change this in the soft. ? > >> >> > >> >> Thanks, > >> >> > >> >> JP > >> >> > >> >> > >> >> > >> >> > >> >> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - > >> >> Jean-Pierre CLAMME, PhD > >> >> Senior Scientist > >> >> Nitto Denko Technical > >> >> 501 Via Del Monte > >> >> Oceanside, CA 92058 > >> >> E-mail: [hidden email] > >> >> Phone: +760.435.7065 > >> >> > >> > > >> > > >> > > >> >-- > >> >Aseem Mishra > >> >Senior Research Assistant, > >> >Malaria Group, > >> >International Centre for Genetic Engineering and Biotechnology, > >> >New Delhi-110067 > >> >INDIA > >> > > > > > >-- > >Jennifer Clarke BSc (Hons) PhD > >Research Associate, Anatomy and Histology > >Centre for Neuroscience, School of Medicine > >& > >Facility Manager, Optical Microscopy Suite, Flinders Microscopy > >(available for training and assistance on Mondays only) > > > >Flinders University > >GPO Box 2100, Adelaide 5001 > >Phone: 61 8 8204 6637 / 61 8 8204 6454 > >Email: [hidden email] > -- Jennifer Clarke BSc (Hons) PhD Research Associate, Anatomy and Histology Centre for Neuroscience, School of Medicine & Facility Manager, Optical Microscopy Suite, Flinders Microscopy (available for training and assistance on Mondays only) Flinders University GPO Box 2100, Adelaide 5001 Phone: 61 8 8204 6637 / 61 8 8204 6454 Email: [hidden email] |
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In reply to this post by Jean-Pierre CLAMME-2
Dear Jennifer,
thank you for the clarification. I understand what determines the measurement volume for FCS. It appears that our FV1000 behaves differently than your system (we have software version 1.7c). When I run a live view with certain scan size, e.g., 640 x 640 pixels, and then select the crosshair tool and do spot scanning, the software acquires 640 spot scans. If I set up a time lapse with e.g., 1000 "frames" (or timepoints), the software acquires 1000x 640 spot scans. Similarly, if in my live view the X had 2048 pixels, the point scan acquires multiples of 2048 points. So in our software, the scan size in live view prior to selecting the spot scan determines the minimum number of points in spot scanning. The timelapse point scan is displayed and saved as a 2-D image, where each line represents one 'timepoint". I believe that between the "lines", the laser gets blanked by the AOTF on the FV1000 system. Maybe somebody from Olympus could clarify or correct if I am wrong. I am always happy to learn something new from them, but sometimes it is difficult - Olympus would not even tell me what is the pin-out on the connector for the external shutters on our IX-81. It must be a really important design secret (luckily, probing the connector with a $1.99 multimeter from Harbor Freight Tools revealed which pins send the TTL pulse and what polarity it is). Stan On Fri, 26 Feb 2010 15:29:39 +1030, Jennifer Clarke <[hidden email]> wrote: >Dear Stan > >The preceeding scan size doesnt matter for point scanning FCS measurement, and >the measurement volume is determined by the confocal volume. > >However yes the spatial resolution of the preceeding image would, in part, >affect your accuracy for positioning the xyz coordinate of the point FCS >measurement. For us the preceeding image is usually 256x256 (ie 256 pixels >per line) or 512x512 with 60 or 63x lens and between 1 and 6x zoom for >visualisation of details of the cell (again zoom doesnt matter for the FCS >measurement, only for visualisation). > >For live cells, a much bigger problem here however is movement of the cellular >components during the course of the FCS point measurement. >This is where RICS and scanning FCS and the SimFCS software become >the bulk cell movement during the measurement can be taken into account in the >analysis. > >For solution measurements we would typically take at least 3 measurements and >average them. > >For measurements on cells, we are trying to measure the movement of receptors on >the membrane and the movement of the respective ligands, which we add to the >media. This is very difficult as the cell membrane is constantly in motion. >We try to position several measurement points along the membrane according to >an image. Due to movement of the cell since the image was taken and during the >course of the measurements not all the measurement points end up actually >including the membrane. Usually it is clear in the analysis which measurement >points were entirely intracellular and which were extracellular. Our main >problem now is how to accomodate enough components into the FCS analysis (eg >for the ligand, diffusion components include 1 triplet state, 2 free diffusion >in extracellular component of measurement volume, 3 restricted movement in >vicinity of membrane, 4 receptor binding events, and potentially any >internalised ligand as well) and the fact that different membrane measurement >points will include different proportions of extracellular and intracellular >space). >So, on live cells it gets very complicated vey quickly! > >Here we have a Leica SP5 with Avalance Photo Diode detectors and an FCS System >with ISS Vista software. This is fine for FCS. >Unfortunately there are problems using the APD image data aquired in the Leica >software (LASAF) for analysis with RICS and N&B as the LASAF data is somehow >modified and no longer exists as true raw data. True raw data is required for >analysis with RICS and N&B as these techniques analyse the intensity >fluctuations, so the data cannot be in any way already smoothed or averaged. >With Enrico Grattons help, we are trying to find a way around this problem via >feeding the APD image data straight into ISS Vista, bypassing LASAF. > >Unfortunately we do not regularly have access to an Olympus FV1000, we just >one when we were fortunate to visit the Gratton lab last year. > >Hope this helps > >I am not an expert in this, so I would be interested in any other listers >comments too > >Kind regards >Jennifer > > > >Quoting Stanislav Vitha <[hidden email]>: > >> Dear Jennifer, >> when you do point scanning FCS on the Fluoview 1000, what scan size do you >> typically have prior to selecting the point scan tool? That is, how many >> pixels per line was in the live view image? I am just curious about the >> total number of datapoints that one would need e.g., for solutions or cells >> (pixels per line x timepoints). >> >> Thanks! >> >> Stan Vitha. >> Microscopy and Imaging Center, >> Texas A&M University >> >> On Tue, 23 Feb 2010 11:19:43 +1030, Jennifer Clarke >> <[hidden email]> wrote: >> >> >Dear all >> > >> >I can confirm that the FV1000 can be used for FCS measurements, we also used >> it >> >with SimFCS software and it works very well. >> > >> > - the laser does not blink, it just scans continually at the designated >> point >> >(use crosshair icon and select desired point on image for measurement) >> > >> > - the max number of points on FV1000 in point scanning mode is 32766. >> This >> >number of points is sufficient for an accurate measurement. >> > >> >Acquisition in this way is that it is equivalent to capturing FCS data in >> "time >> >mode" in ISS Vista software (the other system we use, where we can choose >> either >> >"time mode" which counts photons/time-block, or "photon mode" which counts >> each >> >photon with its respective time). >> > >> >In ISS Vista, we always exclude the first data point in the analysis, >> because >> >it >> >has no preceeding data points to be correlated with for the correlation >> >analysis. >> >I cant recall excluding the first point when we have analysed FV1000 point >> scan >> >data in SimFCS, I'm not sure if there is an option to do so. If the first >> data >> >point can be excluded in the analysis I would do so. Do not simply delete >> the >> >first data point, as this would simply mean that now the second data point >> has >> >no first point to be correlated to so the problem still remains. >> >If the first point can not be excluded from the analysis, it is only one >> point >> >whose correlation will be of reduced accuracy, out of the 32766 points, so >> I >> >dont think this matters very much, and perhaps (? - maybe a mathematician >> can >> >answer) it is not as important in time mode as in photon mode. >> > >> >SimFCS is definately worth trying, yes 30day demo licence is free, and the >> full >> >licence is very economical anyway. Go to the website for the Laboratory >> for >> >Fluorescence Dynamics; http://www.lfd.uci.edu/ >> > >> >For point-scanning data collection on the FV1000, configure for optimal >> >resoluion, ie use a high NA objective etc, select photon counting mode, ie >> >"photon cnt", chose appropriate pixel time eg 8-12.5us/pixel for solution >> or >> >12.5-40us/pixel for measurement on cells, select crosshair icon and set >> desired >> >point in image, set number of frames to 32766 (if you type in an overly >> high >> >number eg 1000000 it will automatically reset to the maximum available) >> with >> >interval set to "freerun". Make sure you export the data as the "raw" >> 16-bit >> >files. >> > >> >If you need a hand working through the analysis in the SimFCS software >> contact >> >me offline. >> >Note that the Wo (PSF waist) in SimFCS is defined using 1/e2 max intensity >> not >> >half max intensity. >> > >> >Its always a good idea to include a reference dye for which you already have >> a >> >good idea of expected or theoretical diffusion coefficient. >> > >> >Hope this helps >> >Kind regards >> >Jennifer >> > >> >-- >> >Jennifer Clarke BSc (Hons) PhD >> >Research Associate, Anatomy and Histology >> >Centre for Neuroscience, School of Medicine >> >& >> >Facility Manager, Optical Microscopy Suite, Flinders Microscopy >> > >> >Flinders University >> >GPO Box 2100, Adelaide 5001 >> >Phone: 61 8 8204 6637 / 61 8 8204 6454 >> >Email: [hidden email] >> > >> >-------------------------------------------------------- >> > >> >Quoting Stanislav Vitha <[hidden email]>: >> > >> >> Hi, >> >> I second Aseem's suggestion regarding SImFCS. >> >> >> >> A user of our core facility is using FV1000 and the SimFCS software from >> the >> >> Gratton lab, it appears to work quite well, as far as I know. The SimFCS >> >> software can be installed as a fully-functional 1-month demo, so you can >> >> test it yourself before making the final decision. One big advantage of >> the >> >> RICS approach that I see is the ability to subtract slow movements, such >> as >> >> sample or stage drift. >> >> A while ago I was told by Olympus that point scanning was not going to >> work >> >> for FCS on the FV1000 system, but I do not quite remember their technical >> >> explanation. From my tests, I remember there was an issue with the first >> or >> >> last datapoint being of incorrect value, due to the timing of the AOTF >> >> "shutter", or synchronization of the AOTF with data acquisition. You >> could >> >> try point-scanning of the fluorescent plastic slide from Chroma, the >> >> fluctuations that you see should indicate the instrument >> noise/instability, >> >> including the laser. >> >> The number of datapoints in point-scanning mode on FV1000 was limited to >> >> 4096, i.e., the max. permitted line width. I was told that before a scan >> a >> >> piece of code is sent to the control board(s), essentially reprogramming >> the >> >> firmware, and this somehow imposes a limit on the number of datapoints. >> >> You could do time-lapse point scan to acquire multiples of 4096 points,, >> but >> >> then you have an unknown amount of time between the time frames. >> >> >> >> Anyway, I think SimFCS is worth trying. >> >> >> >> Stan Vitha >> >> Microscopy and Imaging Center >> >> Texas A&M University >> >> >> >> On Sat, 20 Feb 2010 22:09:52 +0530, aseem mishra <[hidden email]> >> >> wrote: >> >> >> >> >Hi Jean, >> >> > >> >> >You might want to have a look at Enrico Gratton's work and specially >> >> >SimFCS wherein one can calculate diffusion coefficients from an image >> >> >collected in a point-scan mode. I do not know if a blinking laser >> >> >gives you the possibility of doing FCS (Do let me know if such a >> >> >possibility exists. I would love to try it at my place.) We rather use >> >> >a hardware correlator and a simple laser (diode/HeNe) >> >> > >> >> >Aseem >> >> > >> >> > >> >> >On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME >> >> ><[hidden email]> wrote: >> >> >> >> >> >> Hi all, >> >> >> >> >> >> I'm thinking about modifying our fluoview 1000 to do FCS. The fluoview >> >> has >> >> >> the possibility to do single point measurement. However as we can >> still >> >> >> choose the pixel time in this mode, I was wondering if the laser beam >> >> always >> >> >> stays at the same point of interest and continuously illuminates the >> >> point >> >> >> or if it actually "blinks". >> >> >> >> >> >> If it doesn't "blink", I would think it could hinder the FCS >> measurement >> >> et >> >> >> thus I'm wondering if there is an option to change this in the soft. ? >> >> >> >> >> >> Thanks, >> >> >> >> >> >> JP >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - >> >> >> Jean-Pierre CLAMME, PhD >> >> >> Senior Scientist >> >> >> Nitto Denko Technical >> >> >> 501 Via Del Monte >> >> >> Oceanside, CA 92058 >> >> >> E-mail: [hidden email] >> >> >> Phone: +760.435.7065 >> >> >> >> >> > >> >> > >> >> > >> >> >-- >> >> >Aseem Mishra >> >> >Senior Research Assistant, >> >> >Malaria Group, >> >> >International Centre for Genetic Engineering and Biotechnology, >> >> >New Delhi-110067 >> >> >INDIA >> >> >> > >> > >> >-- >> >Jennifer Clarke BSc (Hons) PhD >> >Research Associate, Anatomy and Histology >> >Centre for Neuroscience, School of Medicine >> >& >> >Facility Manager, Optical Microscopy Suite, Flinders Microscopy >> >(available for training and assistance on Mondays only) >> > >> >Flinders University >> >GPO Box 2100, Adelaide 5001 >> >Phone: 61 8 8204 6637 / 61 8 8204 6454 >> >Email: [hidden email] >> > > >-- >Jennifer Clarke BSc (Hons) PhD >Research Associate, Anatomy and Histology >Centre for Neuroscience, School of Medicine >& >Facility Manager, Optical Microscopy Suite, Flinders Microscopy >(available for training and assistance on Mondays only) > >Flinders University >GPO Box 2100, Adelaide 5001 >Phone: 61 8 8204 6637 / 61 8 8204 6454 >Email: [hidden email] |
 
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Jean-Pierre CLAMME-2 |
Re: Fluoview 1000 and FCS
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Hi, Thank you all for your answers. I actually already have the Global software and I'm doing point FCS using the Soft and Michelle Digman's protocol. I was just wondering if, in the case I wanted to add my own detection system/card and a correlator, it would be possible. I did build a FCS system before but we always controled the shutter manually and the laser was parked continuously on the same point. Therfore I wasn't sure what the olympus soft was doing with the laser (does it get blanked by the AOTF between time points, etc.) Thanks, JP Confocal Microscopy List <[hidden email]> wrote on 02/26/2010 08:54:35 AM: > Dear Jennifer, > thank you for the clarification. I understand what determines the > measurement volume for FCS. It appears that our FV1000 behaves differently > than your system (we have software version 1.7c). > When I run a live view with certain scan size, e.g., 640 x 640 pixels, and > then select the crosshair tool and do spot scanning, the software acquires > 640 spot scans. If I set up a time lapse with e.g., 1000 "frames" (or > timepoints), the software acquires 1000x 640 spot scans. Similarly, if in my > live view the X had 2048 pixels, the point scan acquires multiples of 2048 > points. So in our software, the scan size in live view prior to selecting > the spot scan determines the minimum number of points in spot scanning. The > timelapse point scan is displayed and saved as a 2-D image, where each line > represents one 'timepoint". I believe that between the "lines", the laser > gets blanked by the AOTF on the FV1000 system. Maybe somebody from Olympus > could clarify or correct if I am wrong. > I am always happy to learn something new from them, but sometimes it is > difficult - Olympus would not even tell me what is the pin-out on the > connector for the external shutters on our IX-81. It must be a really > important design secret (luckily, probing the connector with a $1.99 > multimeter from Harbor Freight Tools revealed which pins send the TTL pulse > and what polarity it is). > Stan > On Fri, 26 Feb 2010 15:29:39 +1030, Jennifer Clarke > <[hidden email]> wrote: > >Dear Stan > > > >The preceeding scan size doesnt matter for point scanning FCS > measurement, and > >the measurement volume is determined by the confocal volume. > > > >However yes the spatial resolution of the preceeding image would, in part, > >affect your accuracy for positioning the xyz coordinate of the point FCS > >measurement. For us the preceeding image is usually 256x256 (ie 256 pixels > >per line) or 512x512 with 60 or 63x lens and between 1 and 6x zoom for > >visualisation of details of the cell (again zoom doesnt matter for the FCS > >measurement, only for visualisation). > > > >For live cells, a much bigger problem here however is movement of > the cellular > >components during the course of the FCS point measurement. > >This is where RICS and scanning FCS and the SimFCS software become > invaluable as > >the bulk cell movement during the measurement can be taken into > account in the > >analysis. > > > >For solution measurements we would typically take at least 3 measurements and > >average them. > > > >For measurements on cells, we are trying to measure the movement of > receptors on > >the membrane and the movement of the respective ligands, which we add to the > >media. This is very difficult as the cell membrane is constantly in motion. > >We try to position several measurement points along the membrane according to > >an image. Due to movement of the cell since the image was taken > and during the > >course of the measurements not all the measurement points end up actually > >including the membrane. Usually it is clear in the analysis which > measurement > >points were entirely intracellular and which were extracellular. Our main > >problem now is how to accomodate enough components into the FCS analysis (eg > >for the ligand, diffusion components include 1 triplet state, 2 > free diffusion > >in extracellular component of measurement volume, 3 restricted movement in > >vicinity of membrane, 4 receptor binding events, and potentially any > >internalised ligand as well) and the fact that different membrane measurement > >points will include different proportions of extracellular and intracellular > >space). > >So, on live cells it gets very complicated vey quickly! > > > >Here we have a Leica SP5 with Avalance Photo Diode detectors and anFCS System > >with ISS Vista software. This is fine for FCS. > >Unfortunately there are problems using the APD image data aquired > in the Leica > >software (LASAF) for analysis with RICS and N&B as the LASAF data is somehow > >modified and no longer exists as true raw data. True raw data is > required for > >analysis with RICS and N&B as these techniques analyse the intensity > >fluctuations, so the data cannot be in any way already smoothed or averaged. > >With Enrico Grattons help, we are trying to find a way around this > problem via > >feeding the APD image data straight into ISS Vista, bypassing LASAF. > > > >Unfortunately we do not regularly have access to an Olympus FV1000, we just > used > >one when we were fortunate to visit the Gratton lab last year. > > > >Hope this helps > > > >I am not an expert in this, so I would be interested in any other listers > >comments too > > > >Kind regards > >Jennifer > > > > > > > >Quoting Stanislav Vitha <[hidden email]>: > > > >> Dear Jennifer, > >> when you do point scanning FCS on the Fluoview 1000, what scan size do you > >> typically have prior to selecting the point scan tool? That is, how many > >> pixels per line was in the live view image? I am just curious about the > >> total number of datapoints that one would need e.g., for solutions or cells > >> (pixels per line x timepoints). > >> > >> Thanks! > >> > >> Stan Vitha. > >> Microscopy and Imaging Center, > >> Texas A&M University > >> > >> On Tue, 23 Feb 2010 11:19:43 +1030, Jennifer Clarke > >> <[hidden email]> wrote: > >> > >> >Dear all > >> > > >> >I can confirm that the FV1000 can be used for FCS measurements, > we also used > >> it > >> >with SimFCS software and it works very well. > >> > > >> > - the laser does not blink, it just scans continually at the designated > >> point > >> >(use crosshair icon and select desired point on image for measurement) > >> > > >> > - the max number of points on FV1000 in point scanning mode is 32766. > >> This > >> >number of points is sufficient for an accurate measurement. > >> > > >> >Acquisition in this way is that it is equivalent to capturing FCS data in > >> "time > >> >mode" in ISS Vista software (the other system we use, where we can choose > >> either > >> >"time mode" which counts photons/time-block, or "photon mode" which counts > >> each > >> >photon with its respective time). > >> > > >> >In ISS Vista, we always exclude the first data point in the analysis, > >> because > >> >it > >> >has no preceeding data points to be correlated with for the correlation > >> >analysis. > >> >I cant recall excluding the first point when we have analysed FV1000 point > >> scan > >> >data in SimFCS, I'm not sure if there is an option to do so. Ifthe first > >> data > >> >point can be excluded in the analysis I would do so. Do not simply delete > >> the > >> >first data point, as this would simply mean that now the second data point > >> has > >> >no first point to be correlated to so the problem still remains. > >> >If the first point can not be excluded from the analysis, it is only one > >> point > >> >whose correlation will be of reduced accuracy, out of the 32766 points, so > >> I > >> >dont think this matters very much, and perhaps (? - maybe a mathematician > >> can > >> >answer) it is not as important in time mode as in photon mode. > >> > > >> >SimFCS is definately worth trying, yes 30day demo licence is free, and the > >> full > >> >licence is very economical anyway. Go to the website for the Laboratory > >> for > >> >Fluorescence Dynamics; http://www.lfd.uci.edu/ > >> > > >> >For point-scanning data collection on the FV1000, configure for optimal > >> >resoluion, ie use a high NA objective etc, select photon counting mode, ie > >> >"photon cnt", chose appropriate pixel time eg 8-12.5us/pixel for solution > >> or > >> >12.5-40us/pixel for measurement on cells, select crosshair icon and set > >> desired > >> >point in image, set number of frames to 32766 (if you type in an overly > >> high > >> >number eg 1000000 it will automatically reset to the maximum available) > >> with > >> >interval set to "freerun". Make sure you export the data as the "raw" > >> 16-bit > >> >files. > >> > > >> >If you need a hand working through the analysis in the SimFCS software > >> contact > >> >me offline. > >> >Note that the Wo (PSF waist) in SimFCS is defined using 1/e2 maxintensity > >> not > >> >half max intensity. > >> > > >> >Its always a good idea to include a reference dye for which you > already have > >> a > >> >good idea of expected or theoretical diffusion coefficient. > >> > > >> >Hope this helps > >> >Kind regards > >> >Jennifer > >> > > >> >-- > >> >Jennifer Clarke BSc (Hons) PhD > >> >Research Associate, Anatomy and Histology > >> >Centre for Neuroscience, School of Medicine > >> >& > >> >Facility Manager, Optical Microscopy Suite, Flinders Microscopy > >> > > >> >Flinders University > >> >GPO Box 2100, Adelaide 5001 > >> >Phone: 61 8 8204 6637 / 61 8 8204 6454 > >> >Email: [hidden email] > >> > > >> >-------------------------------------------------------- > >> > > >> >Quoting Stanislav Vitha <[hidden email]>: > >> > > >> >> Hi, > >> >> I second Aseem's suggestion regarding SImFCS. > >> >> > >> >> A user of our core facility is using FV1000 and the SimFCS software from > >> the > >> >> Gratton lab, it appears to work quite well, as far as I know. The SimFCS > >> >> software can be installed as a fully-functional 1-month demo, so you can > >> >> test it yourself before making the final decision. One big advantage of > >> the > >> >> RICS approach that I see is the ability to subtract slow movements, such > >> as > >> >> sample or stage drift. > >> >> A while ago I was told by Olympus that point scanning was not going to > >> work > >> >> for FCS on the FV1000 system, but I do not quite remember > their technical > >> >> explanation. From my tests, I remember there was an issue withthe first > >> or > >> >> last datapoint being of incorrect value, due to the timing of the AOTF > >> >> "shutter", or synchronization of the AOTF with data acquisition. You > >> could > >> >> try point-scanning of the fluorescent plastic slide from Chroma, the > >> >> fluctuations that you see should indicate the instrument > >> noise/instability, > >> >> including the laser. > >> >> The number of datapoints in point-scanning mode on FV1000 was limited to > >> >> 4096, i.e., the max. permitted line width. I was told that before a scan > >> a > >> >> piece of code is sent to the control board(s), essentially reprogramming > >> the > >> >> firmware, and this somehow imposes a limit on the number of datapoints. > >> >> You could do time-lapse point scan to acquire multiples of 4096 points,, > >> but > >> >> then you have an unknown amount of time between the time frames. > >> >> > >> >> Anyway, I think SimFCS is worth trying. > >> >> > >> >> Stan Vitha > >> >> Microscopy and Imaging Center > >> >> Texas A&M University > >> >> > >> >> On Sat, 20 Feb 2010 22:09:52 +0530, aseem mishra <[hidden email]> > >> >> wrote: > >> >> > >> >> >Hi Jean, > >> >> > > >> >> >You might want to have a look at Enrico Gratton's work and specially > >> >> >SimFCS wherein one can calculate diffusion coefficients from an image > >> >> >collected in a point-scan mode. I do not know if a blinking laser > >> >> >gives you the possibility of doing FCS (Do let me know if such a > >> >> >possibility exists. I would love to try it at my place.) We rather use > >> >> >a hardware correlator and a simple laser (diode/HeNe) > >> >> > > >> >> >Aseem > >> >> > > >> >> > > >> >> >On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME > >> >> ><[hidden email]> wrote: > >> >> >> > >> >> >> Hi all, > >> >> >> > >> >> >> I'm thinking about modifying our fluoview 1000 to do FCS. > The fluoview > >> >> has > >> >> >> the possibility to do single point measurement. However as we can > >> still > >> >> >> choose the pixel time in this mode, I was wondering if the laser beam > >> >> always > >> >> >> stays at the same point of interest and continuously illuminates the > >> >> point > >> >> >> or if it actually "blinks". > >> >> >> > >> >> >> If it doesn't "blink", I would think it could hinder the FCS > >> measurement > >> >> et > >> >> >> thus I'm wondering if there is an option to change this in > the soft. ? > >> >> >> > >> >> >> Thanks, > >> >> >> > >> >> >> JP > >> >> >> > >> >> >> > >> >> >> > >> >> >> > >> >> >> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - > >> >> >> Jean-Pierre CLAMME, PhD > >> >> >> Senior Scientist > >> >> >> Nitto Denko Technical > >> >> >> 501 Via Del Monte > >> >> >> Oceanside, CA 92058 > >> >> >> E-mail: [hidden email] > >> >> >> Phone: +760.435.7065 > >> >> >> > >> >> > > >> >> > > >> >> > > >> >> >-- > >> >> >Aseem Mishra > >> >> >Senior Research Assistant, > >> >> >Malaria Group, > >> >> >International Centre for Genetic Engineering and Biotechnology, > >> >> >New Delhi-110067 > >> >> >INDIA > >> >> > >> > > >> > > >> >-- > >> >Jennifer Clarke BSc (Hons) PhD > >> >Research Associate, Anatomy and Histology > >> >Centre for Neuroscience, School of Medicine > >> >& > >> >Facility Manager, Optical Microscopy Suite, Flinders Microscopy > >> >(available for training and assistance on Mondays only) > >> > > >> >Flinders University > >> >GPO Box 2100, Adelaide 5001 > >> >Phone: 61 8 8204 6637 / 61 8 8204 6454 > >> >Email: [hidden email] > >> > > > > > >-- > >Jennifer Clarke BSc (Hons) PhD > >Research Associate, Anatomy and Histology > >Centre for Neuroscience, School of Medicine > >& > >Facility Manager, Optical Microscopy Suite, Flinders Microscopy > >(available for training and assistance on Mondays only) > > > >Flinders University > >GPO Box 2100, Adelaide 5001 > >Phone: 61 8 8204 6637 / 61 8 8204 6454 > >Email: [hidden email] |
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Hi all,
This happens to be a great discussion. Lots of suggestions from everybody. I am infact trying to build a FCS for in-cell measurements using hardware correlator cards. I would like to suggest that experienced users like Jennifer could take some time out a list a flow-chart (or a schematic/protocol) which she might be using for taking these measurements on live cells. I am sure it would be helpful for many of us. Also, it would allow us to find a way to do such measuremnts using other confocal systems. Would someone back me one this!!!!!!!!!!!! Jennifer...we look forward to hear from you. Aseem On Wed, Mar 3, 2010 at 12:23 AM, Jean-Pierre CLAMME <[hidden email]> wrote: > > Hi, > > Thank you all for your answers. > I actually already have the Global software and I'm doing point FCS using > the Soft and Michelle Digman's protocol. > I was just wondering if, in the case I wanted to add my own detection > system/card and a correlator, it would be possible. > I did build a FCS system before but we always controled the shutter manually > and the laser was parked continuously on the same point. > Therfore I wasn't sure what the olympus soft was doing with the laser (does > it get blanked by the AOTF between time points, etc.) > > Thanks, > > JP > > > > > Confocal Microscopy List <[hidden email]> wrote on > 02/26/2010 08:54:35 AM: > >> Dear Jennifer, >> thank you for the clarification. I understand what determines the >> measurement volume for FCS. It appears that our FV1000 behaves differently >> than your system (we have software version 1.7c). >> When I run a live view with certain scan size, e.g., 640 x 640 pixels, and >> then select the crosshair tool and do spot scanning, the software acquires >> 640 spot scans. If I set up a time lapse with e.g., 1000 "frames" (or >> timepoints), the software acquires 1000x 640 spot scans. Similarly, if in >> my >> live view the X had 2048 pixels, the point scan acquires multiples of >> 2048 >> points. So in our software, the scan size in live view prior to >> selecting >> the spot scan determines the minimum number of points in spot scanning. >> The >> timelapse point scan is displayed and saved as a 2-D image, where each >> line >> represents one 'timepoint". I believe that between the "lines", the laser >> gets blanked by the AOTF on the FV1000 system. Maybe somebody from Olympus >> could clarify or correct if I am wrong. >> I am always happy to learn something new from them, but sometimes it is >> difficult - Olympus would not even tell me what is the pin-out on the >> connector for the external shutters on our IX-81. It must be a really >> important design secret (luckily, probing the connector with a $1.99 >> multimeter from Harbor Freight Tools revealed which pins send the TTL >> pulse >> and what polarity it is). > >> Stan > >> On Fri, 26 Feb 2010 15:29:39 +1030, Jennifer Clarke >> <[hidden email]> wrote: > >> >Dear Stan >> > >> >The preceeding scan size doesnt matter for point scanning FCS >> measurement, and >> >the measurement volume is determined by the confocal volume. >> > >> >However yes the spatial resolution of the preceeding image would, in >> > part, >> >affect your accuracy for positioning the xyz coordinate of the point FCS >> >measurement. For us the preceeding image is usually 256x256 (ie 256 >> > pixels >> >per line) or 512x512 with 60 or 63x lens and between 1 and 6x zoom for >> >visualisation of details of the cell (again zoom doesnt matter for the >> > FCS >> >measurement, only for visualisation). >> > >> >For live cells, a much bigger problem here however is movement of >> the cellular >> >components during the course of the FCS point measurement. >> >This is where RICS and scanning FCS and the SimFCS software become >> invaluable as >> >the bulk cell movement during the measurement can be taken into >> account in the >> >analysis. >> > >> >For solution measurements we would typically take at least 3 measurements >> > and >> >average them. >> > >> >For measurements on cells, we are trying to measure the movement of >> receptors on >> >the membrane and the movement of the respective ligands, which we add to >> > the >> >media. This is very difficult as the cell membrane is constantly in >> > motion. >> >We try to position several measurement points along the membrane >> > according to >> >an image. Due to movement of the cell since the image was taken >> and during the >> >course of the measurements not all the measurement points end up actually >> >including the membrane. Usually it is clear in the analysis which >> measurement >> >points were entirely intracellular and which were extracellular. Our >> > main >> >problem now is how to accomodate enough components into the FCS analysis >> > (eg >> >for the ligand, diffusion components include 1 triplet state, 2 >> free diffusion >> >in extracellular component of measurement volume, 3 restricted movement >> > in >> >vicinity of membrane, 4 receptor binding events, and potentially any >> >internalised ligand as well) and the fact that different membrane >> > measurement >> >points will include different proportions of extracellular and >> > intracellular >> >space). >> >So, on live cells it gets very complicated vey quickly! >> > >> >Here we have a Leica SP5 with Avalance Photo Diode detectors and anFCS >> > System >> >with ISS Vista software. This is fine for FCS. >> >Unfortunately there are problems using the APD image data aquired >> in the Leica >> >software (LASAF) for analysis with RICS and N&B as the LASAF data is >> > somehow >> >modified and no longer exists as true raw data. True raw data is >> required for >> >analysis with RICS and N&B as these techniques analyse the intensity >> >fluctuations, so the data cannot be in any way already smoothed or >> > averaged. >> >With Enrico Grattons help, we are trying to find a way around this >> problem via >> >feeding the APD image data straight into ISS Vista, bypassing LASAF. >> > >> >Unfortunately we do not regularly have access to an Olympus FV1000, we >> > just >> used >> >one when we were fortunate to visit the Gratton lab last year. >> > >> >Hope this helps >> > >> >I am not an expert in this, so I would be interested in any other listers >> >comments too >> > >> >Kind regards >> >Jennifer >> > >> > >> > >> >Quoting Stanislav Vitha <[hidden email]>: >> > >> >> Dear Jennifer, >> >> when you do point scanning FCS on the Fluoview 1000, what scan size do >> >> you >> >> typically have prior to selecting the point scan tool? That is, how >> >> many >> >> pixels per line was in the live view image? I am just curious about the >> >> total number of datapoints that one would need e.g., for solutions or >> >> cells >> >> (pixels per line x timepoints). >> >> >> >> Thanks! >> >> >> >> Stan Vitha. >> >> Microscopy and Imaging Center, >> >> Texas A&M University >> >> >> >> On Tue, 23 Feb 2010 11:19:43 +1030, Jennifer Clarke >> >> <[hidden email]> wrote: >> >> >> >> >Dear all >> >> > >> >> >I can confirm that the FV1000 can be used for FCS measurements, >> we also used >> >> it >> >> >with SimFCS software and it works very well. >> >> > >> >> > - the laser does not blink, it just scans continually at the >> >> > designated >> >> point >> >> >(use crosshair icon and select desired point on image for measurement) >> >> > >> >> > - the max number of points on FV1000 in point scanning mode is 32766. >> >> This >> >> >number of points is sufficient for an accurate measurement. >> >> > >> >> >Acquisition in this way is that it is equivalent to capturing FCS data >> >> > in >> >> "time >> >> >mode" in ISS Vista software (the other system we use, where we can >> >> > choose >> >> either >> >> >"time mode" which counts photons/time-block, or "photon mode" which >> >> > counts >> >> each >> >> >photon with its respective time). >> >> > >> >> >In ISS Vista, we always exclude the first data point in the analysis, >> >> because >> >> >it >> >> >has no preceeding data points to be correlated with for the >> >> > correlation >> >> >analysis. >> >> >I cant recall excluding the first point when we have analysed FV1000 >> >> > point >> >> scan >> >> >data in SimFCS, I'm not sure if there is an option to do so. Ifthe >> >> > first >> >> data >> >> >point can be excluded in the analysis I would do so. Do not simply >> >> > delete >> >> the >> >> >first data point, as this would simply mean that now the second data >> >> > point >> >> has >> >> >no first point to be correlated to so the problem still remains. >> >> >If the first point can not be excluded from the analysis, it is only >> >> > one >> >> point >> >> >whose correlation will be of reduced accuracy, out of the 32766 >> >> > points, so >> >> I >> >> >dont think this matters very much, and perhaps (? - maybe a >> >> > mathematician >> >> can >> >> >answer) it is not as important in time mode as in photon mode. >> >> > >> >> >SimFCS is definately worth trying, yes 30day demo licence is free, and >> >> > the >> >> full >> >> >licence is very economical anyway. Go to the website for the >> >> > Laboratory >> >> for >> >> >Fluorescence Dynamics; http://www.lfd.uci.edu/ >> >> > >> >> >For point-scanning data collection on the FV1000, configure for >> >> > optimal >> >> >resoluion, ie use a high NA objective etc, select photon counting >> >> > mode, ie >> >> >"photon cnt", chose appropriate pixel time eg 8-12.5us/pixel for >> >> > solution >> >> or >> >> >12.5-40us/pixel for measurement on cells, select crosshair icon and >> >> > set >> >> desired >> >> >point in image, set number of frames to 32766 (if you type in an >> >> > overly >> >> high >> >> >number eg 1000000 it will automatically reset to the maximum >> >> > available) >> >> with >> >> >interval set to "freerun". Make sure you export the data as the "raw" >> >> 16-bit >> >> >files. >> >> > >> >> >If you need a hand working through the analysis in the SimFCS software >> >> contact >> >> >me offline. >> >> >Note that the Wo (PSF waist) in SimFCS is defined using 1/e2 >> >> > maxintensity >> >> not >> >> >half max intensity. >> >> > >> >> >Its always a good idea to include a reference dye for which you >> already have >> >> a >> >> >good idea of expected or theoretical diffusion coefficient. >> >> > >> >> >Hope this helps >> >> >Kind regards >> >> >Jennifer >> >> > >> >> >-- >> >> >Jennifer Clarke BSc (Hons) PhD >> >> >Research Associate, Anatomy and Histology >> >> >Centre for Neuroscience, School of Medicine >> >> >& >> >> >Facility Manager, Optical Microscopy Suite, Flinders Microscopy >> >> > >> >> >Flinders University >> >> >GPO Box 2100, Adelaide 5001 >> >> >Phone: 61 8 8204 6637 / 61 8 8204 6454 >> >> >Email: [hidden email] >> >> > >> >> >-------------------------------------------------------- >> >> > >> >> >Quoting Stanislav Vitha <[hidden email]>: >> >> > >> >> >> Hi, >> >> >> I second Aseem's suggestion regarding SImFCS. >> >> >> >> >> >> A user of our core facility is using FV1000 and the SimFCS software >> >> >> from >> >> the >> >> >> Gratton lab, it appears to work quite well, as far as I know. The >> >> >> SimFCS >> >> >> software can be installed as a fully-functional 1-month demo, so you >> >> >> can >> >> >> test it yourself before making the final decision. One big advantage >> >> >> of >> >> the >> >> >> RICS approach that I see is the ability to subtract slow movements, >> >> >> such >> >> as >> >> >> sample or stage drift. >> >> >> A while ago I was told by Olympus that point scanning was not going >> >> >> to >> >> work >> >> >> for FCS on the FV1000 system, but I do not quite remember >> their technical >> >> >> explanation. From my tests, I remember there was an issue withthe >> >> >> first >> >> or >> >> >> last datapoint being of incorrect value, due to the timing of the >> >> >> AOTF >> >> >> "shutter", or synchronization of the AOTF with data acquisition. You >> >> could >> >> >> try point-scanning of the fluorescent plastic slide from Chroma, the >> >> >> fluctuations that you see should indicate the instrument >> >> noise/instability, >> >> >> including the laser. >> >> >> The number of datapoints in point-scanning mode on FV1000 was >> >> >> limited to >> >> >> 4096, i.e., the max. permitted line width. I was told that before a >> >> >> scan >> >> a >> >> >> piece of code is sent to the control board(s), essentially >> >> >> reprogramming >> >> the >> >> >> firmware, and this somehow imposes a limit on the number of >> >> >> datapoints. >> >> >> You could do time-lapse point scan to acquire multiples of 4096 >> >> >> points,, >> >> but >> >> >> then you have an unknown amount of time between the time frames. >> >> >> >> >> >> Anyway, I think SimFCS is worth trying. >> >> >> >> >> >> Stan Vitha >> >> >> Microscopy and Imaging Center >> >> >> Texas A&M University >> >> >> >> >> >> On Sat, 20 Feb 2010 22:09:52 +0530, aseem mishra >> >> >> <[hidden email]> >> >> >> wrote: >> >> >> >> >> >> >Hi Jean, >> >> >> > >> >> >> >You might want to have a look at Enrico Gratton's work and >> >> >> > specially >> >> >> >SimFCS wherein one can calculate diffusion coefficients from an >> >> >> > image >> >> >> >collected in a point-scan mode. I do not know if a blinking laser >> >> >> >gives you the possibility of doing FCS (Do let me know if such a >> >> >> >possibility exists. I would love to try it at my place.) We rather >> >> >> > use >> >> >> >a hardware correlator and a simple laser (diode/HeNe) >> >> >> > >> >> >> >Aseem >> >> >> > >> >> >> > >> >> >> >On Fri, Feb 19, 2010 at 3:27 AM, Jean-Pierre CLAMME >> >> >> ><[hidden email]> wrote: >> >> >> >> >> >> >> >> Hi all, >> >> >> >> >> >> >> >> I'm thinking about modifying our fluoview 1000 to do FCS. >> The fluoview >> >> >> has >> >> >> >> the possibility to do single point measurement. However as we >> >> >> >> can >> >> still >> >> >> >> choose the pixel time in this mode, I was wondering if the laser >> >> >> >> beam >> >> >> always >> >> >> >> stays at the same point of interest and continuously illuminates >> >> >> >> the >> >> >> point >> >> >> >> or if it actually "blinks". >> >> >> >> >> >> >> >> If it doesn't "blink", I would think it could hinder the FCS >> >> measurement >> >> >> et >> >> >> >> thus I'm wondering if there is an option to change this in >> the soft. ? >> >> >> >> >> >> >> >> Thanks, >> >> >> >> >> >> >> >> JP >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - >> >> >> >> - >> >> >> >> Jean-Pierre CLAMME, PhD >> >> >> >> Senior Scientist >> >> >> >> Nitto Denko Technical >> >> >> >> 501 Via Del Monte >> >> >> >> Oceanside, CA 92058 >> >> >> >> E-mail: [hidden email] >> >> >> >> Phone: +760.435.7065 >> >> >> >> >> >> >> > >> >> >> > >> >> >> > >> >> >> >-- >> >> >> >Aseem Mishra >> >> >> >Senior Research Assistant, >> >> >> >Malaria Group, >> >> >> >International Centre for Genetic Engineering and Biotechnology, >> >> >> >New Delhi-110067 >> >> >> >INDIA >> >> >> >> >> > >> >> > >> >> >-- >> >> >Jennifer Clarke BSc (Hons) PhD >> >> >Research Associate, Anatomy and Histology >> >> >Centre for Neuroscience, School of Medicine >> >> >& >> >> >Facility Manager, Optical Microscopy Suite, Flinders Microscopy >> >> >(available for training and assistance on Mondays only) >> >> > >> >> >Flinders University >> >> >GPO Box 2100, Adelaide 5001 >> >> >Phone: 61 8 8204 6637 / 61 8 8204 6454 >> >> >Email: [hidden email] >> >> >> > >> > >> >-- >> >Jennifer Clarke BSc (Hons) PhD >> >Research Associate, Anatomy and Histology >> >Centre for Neuroscience, School of Medicine >> >& >> >Facility Manager, Optical Microscopy Suite, Flinders Microscopy >> >(available for training and assistance on Mondays only) >> > >> >Flinders University >> >GPO Box 2100, Adelaide 5001 >> >Phone: 61 8 8204 6637 / 61 8 8204 6454 >> >Email: [hidden email] -- Aseem Mishra Senior Research Assistant, Malaria Group, International Centre for Genetic Engineering and Biotechnology, New Delhi-110067 INDIA |
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