Hello Folks,

>In UltraView, effectively what is happening from the fluorochrome point of
>view is a repetitive (pulsed 360/s) excitation which is very week (~0.1%)
in
>comparison to a short blast of total laser exposure in a classical
confocal.

>Fedja Bobanovic
>PerkinElmer Life Sciences

     The comments on Nipkow disk's decreased photobleaching/phototoxicity
are interesting and to the point.  One curious thing is that in my lab we
are
having a difficult time in producing **enough** phototoxicity!

     We can easily image cells (on and off) for hours on a BioRad MRC 600
using
a 40x 0.75 water objective, while using laser attenuations of 95 or 99%.  We
are
imaging calcium dynamics in individual nerve cells in the brainstem of
intact
larval zebrafish.  We then (often) try to kill the same cells by turning the
laser
power up to maximum, focusing all of the laser power on the center of the
cell
and "cooking" it for up to 10 minutes.  While this will always photobleach
the
cell, surprisingly, it often does not kill the cell!  If one goes back and
looks at
the cells the next day, some are missing and presumed dead, while others
have
regained their fluorescence [presumbly from dye located in distal axons that
were not bleached] and look quite healthy.  Behavioral studies similar to
Liu and Fetcho's 1999 confirms both the cell kills and the cells that appear
to
survive.   It is interesting to me that cells could survive such treatment--
I think
the fact that the cells are in vivo, i.e. inside an animal with a
functioning
circulatory system that can carry toxic wastes away, may have something
to do with this, but this is pure speculation.  We are now looking into ways
to
make the Krypton-Argon laser more lethal to these cells [we can't go to UV
or
add a pulsed nitrogen laser onto the system].
        Suprisingly, Alexa Dextran, which labels these cells extremely
brightly, and
produces excellent retrograde labeling, is not a very useful dye for
photo-killing.
Oregon green BAPTA dextran and calcium green dextran seem to work better,
to the extent described above.  I agree that briefer, more intense
stimulation would
seem likely to be more harmful: trying to fry ants with a magnifying glass,
on a
cloudy day, might be futile no matter how long you tried!  The bottom line,
I think,
is that neither photobleaching nor phototoxicity is well understood.
     Anyone know any good references on these mechanisms?

Donald M. O'Malley, Ph.D.
Assistant Professor
Department of Biology
414 Mugar Hall
Northeastern University
Boston, MA 02115
[log in to unmask]
www.omalleylab.neu.edu