Gene Pizzo wrote- >With the remark I received concerning UV laser reliability in the LSR (see >below) I was >wondering if anyone could comment on Cytomations claim that their UV laser >in their new Cyan machine has less noise and whether the LSR could be >retrofitted >if need be with a less noisy HeCd. > >Gene/UCONN Health > > >If you used an LSR be >aware that those lasers won't allowed you to performed cell cycle >measurements >with reasonnable CV for all there life time. In our hands, usually you can >perform DNA content and cell cycle for a period of about 8 to 12 months(wich >is >in accordance with HM Shapiro, Practical flow cytometry).Those lasers became >noisy after only a few months and getting worst as time goes by.For calcium >flux they are perfect because with Indo-1 you will do a ratio measurement >wich >eliminated the noise problem. The progressive increase in noise in He-Cd lasers as they age is generally accepted to be due to an increase in helium pressure, and apparently occurs in all He-Cd lasers. It happens more slowly if the laser is run for at least a few hours a week. I have had the bad experience of having a He-Cd become unacceptably noisy when it was unused for a few months following a plasma tube replacement, and I don't think I've ever had one stay quiet for more than a year. I do consult for Cytomation, and have consulted for B-D, but neither company always takes the advice I offer. Cytomation apparently elected to put an argon (Coherent Enterprise) laser in their CyAn analyzer because it was less noisy than a He-Cd. Whether or not this is more economical than retubing a He-Cd every year, I'm not sure. It is fairly likely that violet diodes usable for cell cycle analysis in fixed or intact cells with DAPI and some Hoechst dyes will be usable in commercial apparatus within a year or two; the He-Cd, even if noisy, could then be retained for work with indo-1. If B-D adds the digital processing electronics now available for their sorters to the LSR, it should be feasible to monitor and compensate for He-Cd laser noise, allowing reasonably high resolution DNA/cell cycle analysis. This could also be accomplished with analog circuitry no more complex than they use for cross-beam compensation in the FACSCalibur (and, I believe, the LSR). I use such analog circuitry with my He-Cd lasers, and it has let me operate for a couple of years between retubings with good CV's on DNA analyses. The bottom line, though, is that you can do very good DNA analysis without noise compensation with a new, relatively quiet He-Cd; I've gotten CV's of 1.5%, which is as well as or better than my Cytomutts can do with an arc lamp, an argon or krypton laser, a violet diode, or an experimental frequency doubled diode pumped alexandrite UV laser. So the potential buyers should get the manufacturers to provide a warranty for a certain period within which they will replace a He-Cd if it gets noisy, or offer one of the alternatives mentioned above. -Howard
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