Lesson 4.1: Cytometer Subsystems – Electronic

Electronic Subsystem

Our examination of the electronic subsystem begins with the detector. High-voltage power supplies are used to control the voltage applied to the photomultiplier tube (PMT). As stated in the optics discussion, increasing this voltage, increases the sensitivity of the PMT while decreasing voltage decreases sensitivity. As photons strike the PMT a current pulse is formed which passes from the detector to the amplifiers. These amplifiers provide a linear or logarithmic (generally user-selectable) amplification of the current pulse in order to increase the signal for easier measurement. The amplified signal then passes through a thresholding circuit.

As you can imagine, there are always stray photons striking the detector (unless the cytometer is kept in an absolutely light-fast environment, of course). This would make the identification of individual cell fluorescence impossible if not for the threshold system. In simple terms, the operator chooses a parameter (generally forward scatter) or combination of parameters (forward scatter plus fluorescence detector one) and a minimum signal level. Once the parameter(s) and signal level are selected, the threshold blocks any signal below the chosen level and only passes those events with a higher signal level than the chosen threshold.

Peak and Hold

It may surprise you at this point to learn that while the system generates a current pulse, measurements are generally not made (on analog systems) of this current pulse. Rather the system performs a current to voltage conversion to produce a voltage peak from the original current signal. The reasons are myriad, but from a practical point it is easier to manipulate and measure voltage signals than current signals with analog, solid state systems.

Once a signal is converted to a peak and passes thresholding, the analog cytometer systems hold the state of the electronic system (ignoring further input) for a user-defined period of time. This period is known as the instrument dead time as the instrument will ignore any additional signals passing threshold (Note: the careful reader will realize that this dead time is another limiting factor on the maximum speed at which a sample should be injected into the flow cell). During this dead time the electronics will quantify the requested peak characteristics (maximum deviation (height), width, area) and pass the values on to the software system.


DON’T PANIC! You will not be required to perform a Weasel analysis here!

Though the trend is increasingly for modern cytometers to perform compensation in the software, many cytometers (including the FACSVantage SE) perform compensation in the hardware. As you may recalll, compensation is simply the act of subtracting a percentage of one signal from another. Hardware compensation works in much the same way. The voltage peak to be subtracted is reduced by the appropriate factor, and the peak is inverted (producing a voltage peak of the opposite sign). This signal is added to the voltage peak of the signal to be compensated, thereby reducing the measured signal level. These modified signals are passed on to the software system.

Moving On

Once you’re comfortable with this material, please move on to the next subsytem.

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