To clarify the exchange between Gerhard and me: He had written >Just to clarify a point that might sound a bit confusing here (at least it >caught me out). Howard says that in the slit scanning the pulse peak is >proportional to the fluorescence in the cell, not the area which goes for the >distribution within the cell. I don't think so; I said that the area (integral) is *always* proportional to the total fluorescence, and the peak is proportional to the total fluorescence when the beam height is (at least a few times) bigger than cell diameter and not proportional when it is smaller. When the beam is smaller than the cell (which is the case in slit-scanning), the peak reflects fluorescence density or the "brightness" of the cell. Where do you think I said the opposite? Not, as far as I can see, in my answer to Bob Zucker's question. Gerhard acknowledges he misread me due to caffeine deficiency; I can sympathize. >Using a small beam diameter suitable for >peak/area measurement for doublet discrimination gives already away that area >remains the more accurate measurement for the DNA content of the measured >event >as the peak signal would not detect the presence of twice as much DNA in the >doublet. Correct. > The manual of the EPICS Elite actually has a nice table to highlight >which beam shapers produce what beam dimensions and for what applications they >can be used. > >Now what happens if you use an arc lamp and how much alteration comes from the >operation mode of the PMT...? What happens with arc lamps depends on the illumination geometry. In the old Phywe (and, I assume, some of the newer Partec) instruments, the cell travels along the axis of the illumination/collection optics, so the signal is an integral by nature. In the Skatron/Bio-Rad and other instruments following the Lindmo/Steen design, the illuminated region is an image of a slit; it's probably big enough so that peak is proportional to integral for bacteria, but you'd have to ask Tore or Harald what the situation would be for larger cells. PMT operating mode shouldn't make much difference; the tubes can get nonlinear at low voltage (or at high current if there is a dynode chain), but that shouldn't have different effects on peak and integral of pulses. And the pun to which Gerhard is referring is: >P.S. >I would prefer to be characterised height - I think height makes me look >slimmer. It could also make you look brighter... I didn't copy the mailing list first time around; that's what happens when you have to give up caffeine. -Howard
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