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> We have had extensive experience with PerCP and the Vantage using
> an Enterprise laser. We find it is very useful at all outputs of the
> Enterprise, although we usually run ours at 50-100mW 488. I appreciate
> your concern as I have recently had a discussion with Carl Stewart on
> this issue. He believes that the only reason we can run PerCP at higher
> laser power is that our instrument is not properly aligned. I disagree,
> as the CVs and intensities of our other fluorochromes indicate that all
> is well and our power outputs are in the ballpark. I certainly am not a
> big believer of the "high laser power bleaches PerCP" theory. At any
> rate, I recommend you do what works best for you.
>
>
> Phil McCoy
Let me advance an alternate theory: the signal-to-noise ratio of
PerCP peaks at a lower laser power than, say, PE, because the fluorescence
lifetime of PerCP is relatively long. When I was working at BD, we built
a flow cytometer that was capable of measuring fluorescence lifetimes
(Cytometry 14:123 [1993]). I remember measuring lifetimes of about 2.5
nanoseconds for PE and FITC, and just under 2 nanoseconds for PE-CY5. By
contrast, PerCP had about a 7 nanosecond lifetime.
An article published a few years ago discussed attempts to detect
single molecules of PE in a flow system (Mathies RA, Peck K, and Stryer L,
Analytical Chemistry 62:1786 [1990]). There is a nice discussion of
signal-to-noise statistics in that article. In a nutshell, one you reach
a certain rate of excitatory photon flux, your fluorochrome of interest
becomes saturated. However, most particles exhibit an intrinsic, though
weak, fluorescent capacity (e.g., cellular autofluorescence). This does
not saturate as readily. Photobleaching is probably not an issue in the
few microseconds that a cell spends in the viewing volume. However,
increasing the laser power brings up the autofluorescence.
-- Unique ID : Ladasky, John Joseph Jr. Title : BA Biochemistry, U.C. Berkeley, 1989 Location : Stanford University, Dept. of Structural Biology, Fairchild D-105 Keywords : immunology, music, running, Green
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