excitation and emission

Howard Shapiro hms at shapirolab.com
Mon Nov 7 22:42:27 EST 2005


Mark D'Amico wrote:

>I have a 532nm laser for excitation. I am using R-PE as my marker. 
>The excitation peak is at 488nm. Is my laser wavelength too long?

R-PE does have an excitation peak at 488 nm; it is a local maximum of 
the excitation (and absorption) spectrum, representing 
absorption/excitation by/of the urobilin chromophores in R-PE. 
However, the true excitation maximum is up around 560 nm, about 15 nm 
shorter than the emission maximum. Excitation at 532 nm is 
substantially better than at 488 nm, and 532 nm typically excites 
much less cellular autofluorescence (at least from mammalian cells) 
than is excited at 488 nm, so the signal to background from R-PE is 
much better with 532 nm excitation. One can get even better 
excitation at 543.5 nm from a green He-Ne laser or 546 nm from a 
mercury arc lamp, but these sources do not provide nearly as much 
power as can be obtained from a 532 nm diode-pumped frequency doubled 
YAG laser, which is why the YAG lasers are becoming more widely used 
for cytometry. So 532 nm is certainly not too long for R-PE, but it 
is too long for fluorescein, which has a true absorption/excitation 
maximum close to 488 nm.

When you do have questions of this kind, it is usually helpful to 
look at the full spectra of the probes involved, rather than relying 
on tables of absorption/excitation and emission maxima. Spectra for 
many commonly used dyes and labels are available online, e.g., at 
http://probes.invitrogen.com/resources/spectraviewer/

-Howard





More information about the Cytometry mailing list