There are a few suggestions to reduce photodynamic damage. Briefly, higher-NA
objective lenses collect more of the fluorescence emission light. For a given
lens there is also a theoretical setting of the zoom magnification that best
matches the resolution required to the allowable dose. When imaging planes are
more than 10 um below the coverslip, water-immersion lenses should be used to
avoid the signal loss caused by spherical abberation when oil lenses are used
to image at those depths. Another way to reduce light damage is to minimize
the number of scans used during experiments. If one is able to alter the
hardware of the confocal microscope, one can increase the time constant of the
integrator (i.e., decrease the bandwidth of the amplifier) in front of the
digitizer stage of the frame buffer. One can also reduce photodynamic damage
by adding antioxidants to the medium.
Oxyrase is an enzyme additive used to deplete oxygen. It has been used at 0.3
unit/ml to reduce photodynamic damage. Another approach is to include ascorbic
acid in the medium. This reducing agent is typically used at 0.1-1.0 mg/ml.
(Handbook of Biological Confocal Microscopy, James B. Pawley, 1995)

Further suggestions and more details can be found in the book mentioned above.

Robin Kamperman
Laboratory of Virology, Wageningen University
Wageningen, the Netherlands


- - - - - - - - - - - - - - Original Message - - - - - - - - - - - - - -
Can anyone please recommend a protocol to diminish live cells phototoxicity
during imaging?  I am thinking about antioxidants and free radical scavengers,
but any suggestions are welcome.

Thanks!

Anda Cornea, Ph.D.
Oregon Regional Primate Research Center
505 NW 185th Avenue
Beaverton, OR 97006
ph: (503) 690-5293
fax:(503) 690-5384

- - - - - - - - - - - - End of Original Message - - - - - - - - - - - -