Posted by
Jennifer Clarke on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fluoview-1000-and-FCS-tp4594644p4616079.html
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]