[Cytometry] 532 vs 568 laser

Howard Shapiro hms at shapirolab.com
Fri Feb 6 09:14:55 EST 2009

Bill Telford wrote-
> I agree with Marty's comments - just to provide our own experience:
> (1)  When we do a 532 nm versus 561 nm comparisons using power-matched
> lasers (50 mW) and the same optical path/PMTs/detection filters,
> excitation of PE is very similar for both.  You do need to use a
> slightly longer PE filter with the 561 nm (590/20 nm), which causes a
> small loss of PE sensitivity.  We have not looked specifically at the PE
> tandems, but I would expect that the 532 nm laser at 150 mW would work
> better than the 561 nm 50 mW as Marty sees in his comparisons.  The 561
> nm might also be exciting the Cy5 and Cy5.5 portion of the PE tandem to
> some extent, which could also be contributing to the signal spreading
> Marty sees.  532 nm lasers are available at higher power levels than 561
> - the current commercial max for 561 nm is 100 mW.
Note that PE itself will not tolerate excitation at high power; it tends 
to go into a nonfluorescent and persistent triplet state, with the 
result that the PE intensity vs excitation power curve will plateau or 
even dip. You'd probably want to keep PE excitation at 532 nm well below 
100 mW; higher powers would be tolerated at wavelengths at which the 
excitation cross-section is lower. Other fluorescent proteins also carry 
the liability; PerCP is notorious for being a very good label in 
analyzers that use 15-20 mW at 488 nm and next to useless in a 
stream-in-air sorter with a 200 mW 488 nm beam, and I believe APC also 
forms triplets. I wouldn't bet that the fluorescent proteins don't but 
don't know the literature well.

Luckily, the situation changes for PE, PerCP, and APC when they are in 
tandems, because the availability of the acceptor decreases the 
probability of transition to the triplet state. That makes PerCP-Cy5.5 a 
much more tractable label than PerCP itself.


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