Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

You can use negative contrast imaging and confocal microscopy. Put a dye
like calcein in the extracellular medium and then do your through-focus
imaging. The dark voids represent the cells. Any photobleaching will be
negated by diffusion of new dye into the field. But I am surprised you
are having so much photobleaching. We can image whole volumes of cells
without a problem using confocal microscopy and suitable attenuation of
the excitation laser.

See:
Nishimura, Y., and J.J. Lemasters (2001) Glycine blocks opening of a
death channel in cul-tured hepatic sinusoidal endothelial cells during
chemical hypoxia. Cell Death Differ. 8, 850-858.

Also:
Nishimura, Y., and J.J. Lemasters (2001) Glycine blocks opening of a
death channel in cul-tured hepatic sinusoidal endothelial cells during
chemical hypoxia. Cell Death Differ. 8, 850-858.

Chacon, E., J.M. Reece, A.-L. Nieminen, G. Zahrebelski, B. Herman, and
J.J. Lemasters (1994) Distribution of electrical potential, pH, free
Ca2+, and volume inside cultured adult rabbit cardiac myocytes during
chemical hypoxia: a multiparameter digitized confocal mi-croscopic
study.  Biophys. J. 66, 942-952.

Hope this helps.

John

Aryeh Weiss wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> On Monday 10 February 2003 12:49 pm, you wrote:
>
>>We are trying to image human fibroblasts live in
>>collagen gels. While we have no problem with other
>>types of cells, these fibroblasts are so huge that our
>>standard marker (GFP) bleaches so fast that by the
>>time we have gone through the whole cell volume, there
>>is almost no fluorescence left (let alone for doing a
>>time-lapse...). We have used both confocal and
>>standard fluo/CCD imaging but even after all tricks
>>tried (reducing the number of slices, of time points,
>>minimal light exposure...), we are still limited to 2
>>or 3  times the cell volume before the fluo dies. We
>>have tried using a standard cell dye CFDA-SE, but with
>>no better results. Would any one have any ideas on how
>>to label these big  cells (whole volume or at least
>>membrane) with a more "unbleachable" approach?
>>
>
>
> Are you imaging specific cellular components, or are you
> just trying to image the cell volume and/or its boundaries.
>
> It it is the latter, then you can use nM concentrations of
> dyes such as sulforhodmaine B or sulforhodamine 101 to
> define the cell location and volume. Fluorescein, Oregon
> Green and  alexa dyes also work. Fluorescein
> and its analogs may damage the cells more than the the
> rhodamines and alexas. The common denominator is that
> they all have net negative charge.
>
> I have done this, and then observed the cells while continuously
> scanning for 5-10 minutes at a time. The cells wiggle, send out
> various processes to neighboring cells (which can be seen in
> the scans), and otherwise appear  happy.
>
> --aryeh
>
>
>
>


--
John J. Lemasters, MD, PhD
Professor of Cell & Developmental Biology and Surgery, and
Director of Cell and Molecular Imaging
Department of Cell and Developmental Biology
University of North Carolina at Chapel Hill
CB# 7090, 236 Taylor Hall
Chapel Hill, NC 27599-7090 USA
Tel: 919-966-5507
FAX: 919-966-7197
E-mail: [log in to unmask]