Laser Noise/Cooling; Cyanine Dyes
Howard Shapiro (HPANDA@HARVARDA.HARVARD.EDU)
Wed, 08 Feb 95 16:54:06 EST
Bruce Milthorpe asks whether 0.75% RMS (6% peak-to-peak) noise in the UV
from an Enterprise laser will cause practical problems with DNA measure-
ments. If everything else is perfect, the minimum CV will be 0.75%, due
to the noise alone. In my experience, UV ion lasers can be noisier than
that; I had an Innova with consistent 20% peak-to-peak noise, and could
not get CV's better than about 3% running 100 mW or so output. The folks
who sort chromosomes using 500 mW or more UV get good CV's because the
power level is sufficient to saturate the dye (see van den Engh and Farmer
in Cytometry a couple of years ago); at the lower power levels typical of
the Enterprise and He-Cd lasers, you don't have saturation to compensate
for power fluctuations and the CV's show it. Commercial cytometers don't
incorporate noise compensation circuitry, which could be done fairly cheaply,
so there isn't much most people can do about the laser noise (my Innova
never got fixed because the manufacturer never specified a UV noise level,
which, technically, meant the laser wasn't out of spec, warranty or no).
Jacek Witkowski cautions against using cyanine dyes for membrane
potential estimation in mammalian cells because of possible contributions
to fluorescence from dye in mitochondria. In many cell types, e.g.,
peripheral lymphocytes, cyanine dye measurements in the presence and absence
of mitochondrial inhibitors give pretty much the same results, indicating
that the mitochondrial contribution is not significant; in such cases, it's
OK to use cyanines. When in doubt, check your cells with and without
inhibitors. Also, be advised that cyanines are pumped out of cells with
an active glycoprotein efflux pump. This is a much bigger problem when
you wash cells than it is when you leave cells in equilibrium with dye, as
you should when measuring membrane potential.
If I were Peter Bercz I wouldn't use a nonrecirculating cooling system;
there are environmental activists and antienvironmental conservatives who
could unite to embarrass the EPA over that issue.
--Howard
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
