Re: Fluoview 1000 and FCS

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fluoview-1000-and-FCS-tp4594644p4605241.html

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:

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