How to measure instrument-dependent sensitivity in flowcytometer?

The sensitivity of a flowcytometry system is ability of an instrument to resolve dim populations from each other and from background.

Resolution sensitivity is a function of three independent instrument factors:

1. Qr (Fluorescence detection efficiency): Qr is photoelectrons per fluorescence unit and indicates how bright a reagent will appear on the sample when measured in a specific detector.

2. Br (Background “noise”): Br is a measure of true optical background in the fluorescence detector, which helps indicate how easily (dim) signals may be resolved from unstained cells in that detector

3. Electronic noise (SDen): SDen is the background signal due to electronics

Qr and Br are independent variables, but both affect sensitivity. Increases in Br or decreases in Qr can reduce sensitivity and the ability to resolve dim populations.

What Factors Affect Qr , Br and Electronic Noise (SDen)?

1. For Qr: Laser power, Optical efficiency, PMT sensitivity (red spectrum), Poor PMT performance, Dirty flow cell, Dirty or degraded filter.

2. For Br: dirty flow cell, damaged optical component

3. For SDen: PMT connections / PMT noise, Cables too near power sources, Digital error

Courtesy: BD Biosciences