Hi - 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. 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. 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. i hope this doesn't confuse the issues more. Marty Bigos Gladstone Institutes Flow Core >At a local flow cytometry users group ( RTCA) on Feb 22 2001, we heard a >about the BD DIVA digital electronics. It was impressive and like all new >developments in flow cytometry it is a welcomed addition. >BD is now measuring area on all pulses instead of height that is normally >used on previously versions of BD equipment. For you BD users, there is >one board in the machines currently that can yield area measurements which >are required for DNA measurements using Doublet discrimination. Now I >remember that our old Ortho 50H yielded area measurements on all signals >and this is the way we always ran our machine. We reluctantly gave up this >measurement to use the User friendly BD FacsCalibur with their CellQuest >program. > >My questions are the following: >How is our Flow data going to change by using area instead of peak height >measurements >It should be more accurate with area but how bad is the height measurement >that we have obtained for many many years.. >This Question was raised at the meeting but it was not adequately answered >by anyone in the audience. How bad is height measurements?. Is it only bad >( inaccurate) for linear measurements like DNA??? Let's discuss this. I >think it is important. >Bob > > > >Robert M. Zucker, PhD >U.S. Environmental Protection Agency >MD 72 >National Health and Environmental Effects Research Laboratory >Research Triangle Park, North Carolina, 27711 >Tel: 919-541-1585; fax 919-541-4017 >e-mail: zucker.robert@epa.gov
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