Rick Meister wrote- >I'm running an EPICS Elite with a gated amplifier and 5 PMTs. We have 3 >lasers, one of which is an old Coherent Innova 90 which we have been using >for its UV wavelengths. Unfortunately, it has been running "over pressure" >for some time, and now gives me an "under voltage" error message. It now >will not operate in "light regulation" mode, so I think that it's >usefullness as a UV source is at an end. So, what to do now (with a >limited budget)? > >I've heard that there are air-cooled UV lasers now available. Do any of >you have experience using one of these for flow? Are they adequate for >doing calcium studies with indo-1 or DNA with Hoechst 33342? What >manufacture/model do you recommend? > >Or, are water-cooled lasers still preferred for flow work? If so, please >give me your recommendation for getting the plasma tube replaced in our old >Innova 90. (Or is there some reason not to try to repair the Innova 90?) You've already got the plumbing and wiring for the Innova 90, and, if you don't have to pay your electric and water bills, you may find that retubing that laser will cost you less than getting anything air-cooled. I'm not sure because I haven't used water-cooled lasers for many years, and I'm not up to date on prices (or on 3rd party repairs for ion lasers). But fixing the UV laser should give you a UV source that will run for years; the Elite has more efficient light collection optics than other droplet sorters, and doesn't usually need very high power in the UV. He-Cd lasers, which are air-cooled, have been around for a long time. Melles Griot has absorbed the two U.S. manufacturers of He-Cd lasers, Omnichrome and Liconix; they seem to be selling what were the Omnichrome He-Cd lasers but possibly not the Liconix ones. He-Cd lasers are also made by Kimmon in Japan; B-D's LSR uses one. I have had long experience with both Omnichrome and Liconix UV (325 nm) He-Cd lasers; they can be used for both indo-1 and Hoechst or DAPI, but they tend to become noisy over a period of a year or two. The noise is not that much of a problem for calcium studies, because most of it is removed by ratioing, but it will result in CV's for DNA measurement creeping up. My guess is that a He-Cd laser from either manufacturer would cost more than fixing the Innova 90. There are solid state UV lasers, but they haven't yet made it into commercial cytometers. Light Age, a New Jersey company, has an experimental CW frequency-doubled diode-pumped Alexandrite laser that emits around 370 nm; I've done some good Hoechst stains using a borrowed prototype, but that wavelength is too long for indo-1. The laser would probably cost way too much in its current state, anyway. Lightwave electronics (www.lwecorp.com)recently introduced a line of pulsed, frequency-tripled, diode-pumped YAG lasers that emit at 355 nm, a good wavelength for both indo-1 and the Hoechst dyes or DAPI. Their web site quotes a maximum pulse repetition rate of 100 kHz (a 60 ns pulse every 10 usec); this would be a little slow for most flow cytometers. If they could get the rate up to 1 MHz, the laser could probably be used in a standard cytometer approximately as if it were a CW laser; with the present repetition rates, it would probably be necessary to run the laser pulsed, triggering it (with an appropriate delay) when a cell hit another beam upstream. This is also a pretty pricey laser. UV diode lasers are now up to 25 mW in power and down as low as 385 nm in wavelength; I've gotten good CV's from DAPI with them, but the wavelength is a bit long for most of the Hoechst dyes and way too long for indo-1. So, while there are a few new UV sources for flow cytometers, each has at least as many problems as the older ones. But the pace of progress has quickened somewhat in the past couple of years. -Howard
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