Near IR Excitation

Howard Shapiro hms at shapirolab.com
Tue Jan 9 22:58:44 EST 2007


Carol Oxford wrote:

> I have an investigator who is interested in equipping our new  
> sorter with laser lines at 680 nm and 780 nm to excite dyes like  
> Cy7 and other near IR excited dyes.  I've searched the Purdue list,  
> and other than an interesting discussion from Howard in 1997, I  
> don't see any related messages.  My search of PubMed wasn't very  
> interesting either.  Is anyone looking at these excitation  
> wavelengths?

680 nm is too short for Cy7 and 785 nm is too long. Indocyanine  
green, which works as a protein stain (not covalently binding) is  
well-excited at 785 nm and emits around 805. There may be some long- 
wavelength styryl dyes that would work as nucleic acid stains with  
785 nm excitation, but most dyes that absorb in that region tend to  
be unstable. In my limited experience, dating back to the early  
1990s, it is nontrivial to set up a flow cytometer for 785 nm  
excitation, because the beam is pretty much invisible, and it's hard  
to be sure it's focused on (or even anywhere near) the sample stream.  
On the other hand, high-power 785 nm diode lasers are dirt cheap.

Linda Lee et al, then at Biometric Imaging, which has since  
disappeared into BD, described some dyes usable as labels for  
antibodies and other proteins that were excitable at 785 nm; I don't  
think the dyes were ever produced commercially. The reference and  
abstract appear below.

-Howard

Lee LG, Woo SL, Head DF, Dubrow RS, Baer TM: Near-IR dyes in three- 
color volumetric capillary cytometry: cell analysis with 633- and 785- 
nm laser excitation. Cytometry. 1995; 21:120-8.
Several fluorescent dyes that absorb in the near-infrared are  
described. The photostability and aggregation properties of the dyes  
were examined. Two of the dyes, BHMP and BHDMAP, emit at 805 nm and  
were useful dyes for protein labeling. A dual-laser, three-color  
scanning instrument was constructed. CD3+CD4+ and CD3+CD8+  
populations were enumerated in undiluted, whole blood based on the  
fluorescence of Cy5, Cy5.5 and BHMP.





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