Andrew Beernink wrote- >Can anybody fill me in on the use of 543 nm HeNe lasers for flow? >Specifically, is 1-3 mW enough power for most applications, or does >someone make one in the 10 to 50 mW range? Aside from PE and its >conjugates, what will this excite? As far as I know, 3 mW is about as much as can be gotten out of a 543 nm HeNe laser. For PE excitation, this is at least as good as 15 mW at 488 nm, and there is much less excitation of autofluorescence from mammalian cells at 543 nm than at 488 nm, so the stained cells and the unstained ones are further apart. The green He-Ne works well in instruments with very efficient light collection; Beckman Coulter offers one as an option in the Elite and Altra cell sorters, and, while B-D has not offered the option in the FACSCAlibur, the FACSCount, a relatively inexpensive dedicated flow cytometer for CD4/CD8 counting, does use a green He-Ne. The green He-Ne laser would be less useful in a typical stream-in-air sorter, such as a B-D FACSVantage or Cytomation MoFlo, because their light collection is somewhat less efficient. Green frequency-doubled YAG lasers, emitting at 532 nm, would be a good choice here and in other instances where higher power is needed; they are small and energy efficient, and units with up to several watts of power output plug into a standard 110V outlet, draw under 100 watts, and are air-cooled. In fact, the availability of Green YAG lasers is probably keeping manufacturers from trying to build more powerful green He-Ne's, which would probably be at least as expensive and substantially larger. Also in their favor, single-mode green YAG lasers have very low noise, and are better for scatter measurements than the noisier green He-Ne lasers. Both 532 and 543 nm are good for excitation of PE and its tandems, also for Cy3, rhodamine dyes, pyronin Y, some of the Molecular Probes POPO, BOPRO, etc. cyanine nucleic acid dyes and Alexa protein labels, ethidium, propidium, 7-aminoactinomycin D, LDS751, the DiICn(3) cyanine dyes (membrane potential sensitive for n 6 or less, tracking dyes for n 14 or more), PKH26, and resorufin-based fluorogenic substrates. For many of these, 532 if preferable to 543 because the Stokes shifts are relatively small, as well as because more excitation power is available from Green YAG lasers than from green He-Ne lasers. On the negative side, neither 532 nor 543 nm lasers will excite fluorescein or its derivatives, GFP and its genetically modified variants, or acridine orange, and both wavelengths would interfere (532 more than 543) with measurement of these materials using a second 488 nm beam, although some games might be played with emission filters to minimize the interference. -Howard
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