Alexander Shvalov asks- > Could someone help with literature on FC electronics > (block - scheme or/and electronic scheme, though, > I'm afraid the recent is the subject to copyright) of > how signal from Fluorescence is processed. > > How it works? : > Pulse board, Pulse processor, Status board, > FSC Amplifier, SSC/FL2 .. FL1/FL2 ? > > or send electronic version > > We plan to add signal processing system to > fluorescent channel Scanning Flow Cytometer > unit, (principally new approach to the light > scattering)... > and would like to understand basic principles > of signal processing from FC fluorescent > Photoamplifier data processing. > > 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. I have tried to provide good qualitative descriptions of signal processing in flow cytometers in the various editions of "Practical Flow Cytometry", in a more recent paper (Shapiro HM, Perlmutter NG, Stein PG: A flow cytometer designed for fluorescence calibration. Cytometry. 1998; 33:280-287), and in the new version of the Flow Cytometry volumes in Methods in Cell Biology", now in press from Academic Press. Most of that stuff is subject to copyright, but the current 3rd Edition of "Practical Flow Cytometry" (Wiley-Liss, New York, 1995), which I wish were cheaper, still won't cost you as much as a photomultiplier tube. Complete schematics for linear signal processing electronics were contained in the 2nd Edition of "Practical Flow Cytometry"; Wiley allows me to reproduce and distribute these since they decided to omit construction details from the 3rd and subsequent editions and add more on applications. I will send you a copy if you want it; however, these schematics date back to the mid-1980's, and you would almost certainly want to do things in a different fashion now. A 1980's vintage flow cytometer used the following components: Detectors - photodiode for forward scatter (PMT if higher gain needed); PMT's for orthogonal scatter and fluorescence. Preamplifiers - current-to-voltage conversion using an op-amp, with baseline restoration provided through a feedback circuit integrating the DC-coupled signal, with the peaks clipped off by diodes, and subtracting this from the input signal. Additional stages of voltage gain may be included, and are required when diode detectors are used; when PMT's are used, it is advisable to control gain using PMT voltage adjustments, because this introduces less noise than voltage amplification. "Front End" Electronics - incorporate a comparator to sense when a cell is present, i.e., when a trigger signal level rises above an adjustable threshold, and timing electronics to deal with coincidences and generate control signals for peak detectors, integrators, and/or pulse width measurement circuits, and signals to initiate analog-to-digital conversion of the held peak, integral, and width values. Peak detectors - these hybrid analog circuits are well known if not always well behaved; peak signals are stored in capacitors, and a reset pulse closes a switch which grounds the capacitor. Integrators - these can be switched-capacitor integrators, or, in some cases, peak detectors, the input signals to which are passed through a low-pass filter, such that the peak height of the filtered signal becomes proportional to the integral of the raw input signal. Pulse width measurement circuits - basically integrators with a linear ramp input. To deal with large dynamic range signals, logarithmic amplifiers were interspersed between the preamplifier and the peak detector or integrator. Fluorescence overlap compensation was done by placing the circuits for linear combination of signals between the preamplifier output and the input to the logarithmic amplifier. Most commercial flow cytometers still work this way. However, by the mid-1990's, high-resolution (16 or more bits) analog-to-digital converters had become available, allowing logarithmic amplifiers and compensation circuits to be eliminated; compensation and log conversion could then be accomplished by digital computation. This was done in the Beckman Coulter EPICS XL and in instruments built in my lab and others'. This approach, while improving the precision and accuracy of compensation and log transformation, still requires the use of hybrid analog peak detectors, integrators, etc., and there may be problems with dynamic range because these circuits do not perform well at very low input signal levels. Most recently, there has been a trend toward digital processing of the signal pulses, using 14-bit converters with digitization rates of 1.25 megahertz and higher. The Luminex 100 flow cytometer, designed for multiplexed biochemical assays on dyed beads, incorporates digital pulse processing, and Becton-Dickinson showed prototype digital pulse processing electronics for the FACSVantage sorter at the recent ISAC meeting in Montpellier. Schematics of these systems are proprietary. However, they basically include a simple preamplifier and a fast A-D converter for each channel (the same old diodes and PMT's are used as detectors), and one or more DSP chips to deal with the signals. Threshold determination, triggering and gating, pulse height and width determination and integration, compensation, and log transformation are all done by the DSP processor(s), which can also do baseline restoration and even compensate for laser noise by sampling the laser output. When multiple DSP's are used, one is set up to supervise and distil data from the others. The DSP data is transferred to the PC (or Mac) used for data analysis through a fast link such as Ethernet, USB, or IEEE 1394. If you are going to develop a new system, this is the way to do it. Unfortunately, there is very little - or at least I've found very little - in the DSP literature about pulse processing; it all seems to be oriented to periodic signals and fast Fourier transforms. You have to figure it out for yourself (or find a DSP expert and get him or her to help you figure it out). -Howard
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