http://confocal-microscopy-list.275.s1.nabble.com/Fluoview-1000-and-FCS-tp4594644p4673138.html
This happens to be a great discussion. Lots of suggestions from
everybody. I am infact trying to build a FCS for in-cell measurements
I am sure it would be helpful for many of us. Also, it would allow us
>
> 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]