Re: Fluoview 1000 and FCS

Posted by Stanislav Vitha on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fluoview-1000-and-FCS-tp4594644p4621592.html

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: [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 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: [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]