Re: Fluorophore bleaching by excitation light sources
Posted by
Mario-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fluorophore-bleaching-by-excitation-light-sources-tp592521p592525.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Re: Fluorophore bleaching by excitation light
sources
Dave,
Depends on what you mean by easy. To narrow the problem a bit,
the important issue is how much UV reaches the sample. Any other
filter is more or less irrelevant with respects to cell viability,
unless there is so much back scattered UV that extra background noise
must be averaged out with extra exposures.
Ignoring the latter, one might deal with the former by using a
test object that is excited by UV. There are quite a few options
including UV sensitive inorganic phosphors that can be purchased as
fine powders or disks. The latter compounds can have luminescent
lifetimes that range from as short as 40 nsec. to milliseconds. The
latter often use a lanthanide such as europium (red) or terbium (blue,
green, red) with sharp emission lines. Some have broad emissions such
as P31, a ZnS based phosphor. Another possibility would be some
organic fluorophores including AMCA or well saturated nuclei stained
with DAPI or Hoechst (say 3 ug dye /ml of cells).
A slide made with a phosphorescent disk will last for years. As I
recall, some EM suppliers sell phosphorescent disks and related
materials for use in cathodoluminescent detectors. I have used or made
all of the above options in one form or another. A further option is
to use a fiber optic couple micro spectrofluorometer. I like the USB
ported version from Ocean Optics, Fl. (no financial interest). They
can provide a cable that is capped with a collection lens that carries
the excitation light from the microscope objective to the spectrometer
which uses a grating and linear CCD. As I recall, there are a few
grating options one of which provides sensitivity down near 220 nm on
up to 700 nm.
Anyway, some of these approaches are crude but they will tell you
if there is significant UV bleed through.
Mario
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I have used
the CoolLED system, and it is not operating in strobe mode. It
is continuous illumination. The UV question is interesting.
Is there an easy way to test if there is UV leaking through the
epi filter set with the mercury burner? Dave
On Mar 12, 2008, at 4:27 PM, Stanislav
Vitha wrote:
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Regarding the difference between LED illumination and Hg lamp -
Perhaps if the illumination is not continuous but is set up for
strobe
operation of the LED, this could allow dark state relaxation an
prolong
the life of the fluorophore (I am not sure the same would apply to
the
life of the cell). - The time between light pulses should be more than
one
microscecond.
reference:
Gerald Donnert, Christian Eggeling & Stefan W Hell: Major signal
increase
in fluorescence microscopy through dark-state relaxation. Nature
Methods -
4, 81 - 86 (2007)
Stan Vitha
Dr. David Knecht
Department of Molecular and Cell
Biology
Co-head Flow Cytometry and Confocal
Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
--
________________________________________________________________________________
Mario M. Moronne, Ph.D.