Oops. I see that in my recent posting on this topic I didn't answer Alexander Shvalov's last question: "Question: what reflects the fluorescence amplitude? Integral of the signal from Photoamplifier? Amplitude? or some classic combination of those parameters with width of the signal." The integral (area) of the pulse most closely represents the total amount of fluorescent material present in or on the cell or particle, subject to two fundamental assumptions: 1) That the cells or particles are passing through the system at the same velocity. This will not be the case if there are problems with the fluidics, and most of us have noted that turbulence resulting from partial obstruction to flow results in a larger coefficient of variation of measurements from supposedly identical particles such as calibration beads. 2) That the intensity of illumination is uniform within the experimenter's tolerance over the width of the sample stream. In systems with high laser power (typically over 100 mW) this requirement may be relaxed because photon saturation results in maximum emission from cells or particles even in regions where illumination intensity is substantially below the maximum. If the height of the beam (the size of the focal spot along the axis of flow) is several times the particle diameter, the entire particle will be illuminated for some time during its transit of the illuminating beam; in this case, the amplitude (height) of the pulse will be proportional to the integral, and also to the total amount of fluorescent material present in or on the cell. Pulse width will be approximately the same for all particles substantially smaller than the beam height. If the height of the beam is close to or smaller than the particle diameter, particles of different sizes will produce pulses of different widths, and pulse height will not be proportional to the total amount of fluorescent material, but will instead reflect the amount of fluorescent material per unit size, that is, the fluorescence density or brightness of the particle. Logarithmic amplification can introduce a complication. Log amps are typically placed between the output of a detector preamplifier and the input of a peak detector or integrator. The desired measurement is the log of the integral. However, an integrator operating on the output of the log amp will produce the integral of the log, which is not the same thing. The usual solution is to pass the pulse from the preamplifier through a low pass filter, which acts as an integrator, before it goes into the log amp. The height of the filtered pulse is then proportional to the integral of the original pulse. The height of the log amplified pulse will then be proportional to the desired log of the integral, and this value can be captured accurately by a peak detector (but not an integrator). Digital pulse processing will eventually make all of this simpler, and will facilitate further investigations of the relationships of pulse integral, height, width and other aspects of pulse shape to the properties of cells and other particles. -Howard
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