>
> Hi,
>
> Do any of you have experience wih doing confocal z-sections on small ( <
> 0.5uM)  organelles in cells labeled wih a ratio dye.
>
>
> Any comments will be very much appreciated.
>
> Thanks
>
> Nihal Altan
> Rockefeller University NYC NY 10021
> (212) 327 8946
> Nihal Altan
> Box 221
> Laboratory of Cellular Biophysics
> Rockefeller University
> 1230 York Avenue
> New York, N.Y. 10021 USA
> 212-327-8946 (voice)
> 212-327-7543 (fax)
> [log in to unmask] (internet)
>
>

Good afternoon, Nihal,

I am just doing the final editing procedure on a paper which covers
exactly the problem you are mentioning. I have already published one
paper on a CSLM for experiments on Fura-2 loaded specimens. It's

Helm PJ, Franksson O, Carlsson K (1995),
A confocal scanning laser microscope for quantitative ratiometric 3D
measurements
of [Ca2+] and Ca2+ diffusions in living cells stained with Fura-2,
Pfluegers Archiv - European Journal of Physiology, 429:672-681

As I am going to outline in the paper which I mentioned first, the main
problem of ratiometric measurements is to collect a sufficient number
of photons. The ratio-imaging algorithm is so much noise-enhancing
that a sufficiently large number of detected fluorescence photons is
a conditio  sine qua non for a complete quantitative ratiometric
analysis of the scanned image.

A way how to detect a sufficiently large number of photons (i.e.
something between 1,000 and 10,000) is, e.g.,  outlined in two other
papers:

Markram H, Helm PJ, Sakmann B (1995),
Dendritic calcium transients evoked by single back-propagating action
potentials in rat neocortical pyramidal neurons,
J.Physiol., 485(1):1-20

and

Helm PJ (1996),
A microscopic setup for combined, and time coordinated
electrophysiological and confocal fluorescence microscopic experiments on
neurons in living brain slices,
Rev.Sci.Instr., 67(2):530-534


Might be these articles are helpful. The essential information, however,
will, as I personally think, be a content of the article in preparation
which I mentioned above. It will be sent off to the magazine in January.


Yours sincerely

Paul Johannes Helm


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Paul Johannes Helm

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