Marty Bigos wrote- >I think Howard hit upon the major issues here, but I will take a stab >at obfuscation. > >In general THERE IS NOTHING WRONG WITH MEASURING PEAK HEIGHTS in an >analog system. There are limitations, and the primary one is dynamic >range. To measure the peak height one needs a way to detect the peak. >In analog circuitry, these are known as "peak detect circuits", "peak >sense and hold" circuits, etc. They do not have a four decade dynamic >range. So for linear measurements, e.g. DNA, or ratios that vary by >less than 20-fold or so, these circuits are fine. For >immunofluoresence, the dynamic range is too great, so range >compression is needed. This is where the log amps come in. The >outputs of the log amps can then be measured by peak detection >circuits, yielding a four decade range at a lower resolution than the >linear scaling. Well, not quite. There is something wrong with measuring peak heights in an analog system when the beam height is close to or smaller than the cell diameter, because, under those conditions, the peak height doesn't give you the answer you're looking for, i.e., how much fluorescent stuff is there in or on the cell, because the peak height isn't proportional to the area. >In analog circuitry, area measurements suffer similar design >limitations as peak detection circuits. So if your measurements are >scalable to a linear domain, analog area measurements of linearly >amplified signals will work fine. This is very useful, as pointed >out, for doublet discrimination. If the dynamic range is too great, >there is no range compression fix; as Howard pointed out the area of >the log is not meaningful. And, as I noted, one can get the log of the area by low pass filtering the preamp signal, in effect integrating it, and making the peak height proportional to the area or integral of the original signal, before putting the signal through the log amp and detecting the peak of the log signal. >I also want to point out that many preamplification systems used in >flow electronics are bandwith limited, which, in essence, means they >are doing some integration as well. In general, this has mostly >helped the accuracy of the measurements. It decreases the accuracy of peak measurements, but makes them closer to area measurements - two wrongs sort of make a right, in this instance. >i hope this doesn't confuse the issues more. Me too. -Howard
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