A quick response to some of the below:
 
Natalie James wrote:
> Hello all,
> I have a flow chamber to study calcium signalling in endothelial cells under
> exposure to fluid flow (cell culture medium).  The indicator used is Fluo-3.
> Dye loading of 1.5 um Fluo-3 for 20 min RT is used to give low levels of
> intracellular dye, in order to minimise buffering of the calcium responses.
> I have heard that there are potential fixation effects due to the aldehyde
> degradation products of fluorescein-derived probes which are produced during
> photobleaching, which occurs during scanning.  The original papers on the
> development of Fluo-3 and its application in living cells (JBC 1989 (264)
> 8171-8 & 8179-84), don't discuss the possibility of this complication (& the
> illumination was with a xenon lamp rather than a laser).
>
I have not personally encountered "fixation" effects in scanned cells
after loading with Fluo-3. We use 2,5 - 10uM Fluo in cardiac
myocytes, and we would clearly see if contractility was affected.
However, I remember Steve Cody showing  some examples of contractile
dysfunction in parts of a cell excessively scanned. SO maybe the
scanning mode is the critical factor here. We use line scan mode mostly
(for XT plots) and the minimal dye excitation and positive effects of
dye diffusion could account for insignificant photobleaching and lack
of photodamage.
 
Perhaps Steve or others could fill us in about the aldehyde/fixation
effects ?
 
Ian.
 
> 1.  Does anyone know whether aldehyde-related fixation is likely at the
> concentrations of fluo-3 used for calcium measurements?
> What intracellular concentrations of a fluorescein-derived compounds could
> yield aldehydes that would give this problem?
> Does anyone have references discussing this problem?
>
> 2.In addition this factor was suggested as a possible reason for loss of
> contractility in experiments involving scanning of smooth muscle cells.  Any
> comments?
>
> I would very much appreciate any information on this question.
> Thanks in advance.
>
> Natalie James
> -----------------------------------------------------------------------
> Natalie James, PhD
> CSIRO Division of Biomolecular Engineering
> Riverside Corporate Park
> PO Box 184
 
 
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Dr Ian Harper
Experimental Biology Programme
Medical Research Council
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Microscopy Society of Southern Africa
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