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Dear List,

I have compared the photo bleaching of virus particles carrying YFP label in
TIRF (514 nm Argon laser line) with 1% n-propyl gallate in PBS/5% Ethanol
(no oxyrase) and the same sample mounted with ProlongGold and analyzed by
conventional wide-field microscopy. The pixel intensity was set to be
similar in TIRF versus the wide-field microscopy.

In theory, evanescent wave should not cause significant photo bleaching of
the fluorophore (especially of YFP that is rather photo stable). However,
the difference in YFP bleaching between the TIRF and wide-field imaging was
more than 3 fold. 

Does anyone has any experience with other anti-fading agents for TIRF, or
how to increase the solubility of n-propyl gallate in PBS (according to
Sigma, the highest concentration of n-propyl gallate in water is 0.35%)
without adding large amounts of glycerol (high N) or ethanol.

Best,

Vitaly
NCI-FREDERICK,
301-846-6575