mzylka at fas.harvard.edu
Sun Apr 7 23:23:51 EST 1996
Since the cells are fixed (dead), you might try UV irradiating the fixed
cells for 2 minutes BEFORE you perform antibody incubations (esp abs conj
to your fluorochrome). There are numerous sources of autofluorescence in
cells. For example, nicotinamide adenine dinucleotide derivatives
fluoresce blue when excited with light around 380 nm (UV). Perhaps the
biggest source of tissue autofluorescence comes from flavenoids (ex
riboflavin). Flavins have an excitation maxima around 450 nm and an
emission maxima around 525 nm. Obviously, a standard fluorescein filter
set with an excitation filter that passes/excites with light from 450-490
nm and passes/emits light between 515-565 nm does an excellent job of
exciting flavin autofluorescence (it looks punctate yellow-green).
Flavins are very photolabile and can be bleached with UV light (thus the
UV light treatment). Confocal microscopes that excite with the 488 nm
line will also excite flavin autofluorescence, although to a much lesser
extent than a standard fluorescein filter set.
I have found UV treating works, at least for living cells. I was
interested in observing live cells over a long period of time in standard
growth medium but was unable to see the cells very well due to lots of
yellow-green autofluorescence. I noticed that the "haze" would dissapear
after a few seconds of illumination with the fluorescein filter set.
This observation coupled with a little library research on
autofluorescence made me decide to UV treat the medium prior to viewing.
When I did this, I could clearly see the cells immediately. Thus, I
predict you should be able to eliminate a lot of autofluorescence in
cells merely by pretreating them with UV-light.
If it helps, let me know.
Program in Neuroscience
Harvard Medical School
mzylka at student.med.harvard.edu
More information about the Cellbiol