autofluorescence

[Mark Zylka]

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.

Mark Zylka
Program in Neuroscience
Harvard Medical School
mzylka at student.med.harvard.edu