OK, Howard. Perhaps I should have said that I wasn't sure that 440nm excitation was compared directly with 413nm by Tsien's group. Although the excitation of CFP may peak closer to the 440nm line, that line is getting closer to the excitation of flavins, perhaps generating more autofluorescence. I thinking particularly of experience with BFP (both our own data, and that of others, notably Ken Bauer); the 407nm line is better even though you are not at peak excitation of BFP, because of lower autofluorescence. You're right about the HeCad's. Most don't have the 440, actually 441.6nm line. I was thinking of a laser by Kimmon, that is switchable between 325 (10mW) and 442(50mW) and sells for about $17,000. (Although they have one that is higher power, but more expensive) Just a clarification! Kevin Kevin L. Holmes, Ph.D. Head, Flow Cytometry Section Research Technologies Branch NIAID, NIH Bldg. 7, Room 01 NIAID, NIH Phone: 301-496-9071 FAX: 301-402-4532 Email: kholmes@niaid.nih.gov -----Original Message----- From: Howard Shapiro [mailto:hms@shapirolab.com] Sent: Thursday, December 14, 2000 7:45 PM To: cyto-inbox Cc: Cytometry Mailing List Subject: RE: cyan fluorescent protein... Kevin Homes wrote, in response to Andrew Beernink's query about excitation for cyan fluorescent protein - >We've used the 413 line from our krypton in recent studies (Science Jun 30 >2000: 2354-2357 and (www.stke.org/cgi/content/full/OC_sigtrans;2000/38/pl1); >Roger Tsien suggested that the HeCad 440 line is optimal, but this was for >microscopy. I'd like to see if this is true for flow as well, but I don't >have a HeCad to try at the moment. The excitation spectrum is what it is; if 440 nm is optimal for microscopy, it should be for flow as well. Note that most of the He-Cd lasers used in flow cytometers emit at 325 nm and do *not* have interchangeable mirrors, so you'd need a new 440 (closer to 442, actually) nm He-Cd laser. But you can get 100 mW of 442 nm excitation out of a He-Cd laser which is air-cooled and runs on 110 VAC, although it will cost you $20K or so. -Howard
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