Monday, March 3, 2008

LSRII #2: What can I do with a Violet Laser?

The violet laser (typically a 405nm solid state) has become pretty much a standard laser on today's flow cytometers. The violet is typically cheaper and possibly longer lasting than a true UV laser. However, you may be asking yourself, what can I do with this laser line? The answer, Lots! Some fluorochromes have been specifically designed around the 405nm laser line, others, just happen to work well enough with it. Some common ones in the former group include Alexa 405, Pacific Blue, Pacific Orange, and Violet DyeCycle, while those in the latter group include DAPI, and Quantum Dots. For many years, people used UV sources on their flow cytometers simply to do "specialty" assays like Hoechst efflux (Side Population) or Calcium Flux (Indo-1) or just plain old cell cycle analysis (DAPI). However, no one really used the UV for immunophenotyping since the UV-excitable fluorchromes coupled to antibodies weren't bright enough. Now, with the necessity for doing more and more colors, we've run out of room on our Blue, Green, and Red lasers so we need to start using the lower wavelength lasers for more than these few specialty assays. The 405nm laser therefore allows us to open up the possibilities of more and more colors. Simultaneously, we could conceivable look at Pac Blue, Pac Orange, and a Q-dot 705 conjugate. This gives us 3 more usable channels for our multicolor experiments. Or, once Q-dots becomes readily available in direct conjugates, then you could use a few Q-dots in these channels. Also, the we've found the violet laser to work just fine for DAPI, even for cell cycle analysis. You don't get as good of CV's as you might with a true UV, but it's pretty decent (G1 CV<5.0).

So, what do you lose with a UV? Side Population with HO 33342 is not good at all. Maybe it's ok on bone marrow, but that's about it. But, there are alternatives. You could do side population with the violet dyecycle dyes from Invitrogen, or you could use the other LSRII with the UV laser on it. The other thing you lose is Indo-1. There's no way you're gonna be able to do any indo-1 on a violet laser. But, you can use other calcium sensitive dyes like Fura-Red and Fluo-3. These are blue excitable, and when used together, you can get similar ratiometric measurements as you would with indo-1. If you're a BFP user, switch to CFP or cerulean.

Other than the few things mentioned above, the absence of a UV laser may not be that bad depending on what type of user you are. Please note however, we will have a UV on our other LSRII for at least the near future, so if you need to use UV, you're still in luck!