Re: Fluoview 1000 and FCS

Posted by Jean-Pierre CLAMME-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fluoview-1000-and-FCS-tp4594644p4662918.html


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: [phone]
> >> >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: [phone]
> >> >> >>
> >> >> >
> >> >> >
> >> >> >
> >> >> >--
> >> >> >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: [phone]
> >> >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: [phone]
> >Email: [hidden email]