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| Accurately Measuring Cytometer Sensitivity |
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Last Updated
6th of January, 2009
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“Minding your B’s and Q’s”
Many cytometer manufacturers today rate the sensitivity of their flow cytometers using a fluorescence threshold. To perform this a set of beads with known Mean Equivalent Soluble Fluorescence (MESF) is run, and MESF vs. channel number is plotted. Then a blank bead is run, and the MESF of the blank bead is determined by extrapolating down from the known MESF beads. This number is preferred by manufacturers, because it results in a very low MESF figure.
A related method measures the separation between blank and stained beads with a known brightness. The separation is closely related to the fluorescence threshold value.
However, most users are more interested in the number of MESFs a particle must have to be resolved from noise. Most manufacturers do not cite this number because it is considerably higher. A study analyzed the resolution limit of several instruments in terms of Q (photoelectrons/MESF) and b (background MESF) (Cytometry 33:267), and concluded there was a good relationship between the Q of an instrument and the resolution limit.
As discussed in the Cytometry paper, the resolution limit and fluorescence threshold are not necessarily correlated. The reason for this is the functional dependence of the two measurements on the Q and b of the cytometer are different. The fluorescence threshold is given by sqrt(b/Q), and the closely related channel separation is given by 64*log((sqrt(Q/b)*f), where f is the MESF of the Calibrite bead. The resolution limit is determined by solving for f in the equation:
3√(Qb)=Qf-2√(Q(f+b))
The left side is proportional to the noise mean plus 2 Standard deviations (SDs), and the right side is proportional to the stained mean minus 2 SDs of the stained population. Because the resolution limit takes into account the width and means of the noise and stained distributions and the fluorescence threshold (and channel separation) only takes into account means, one would expect different answers for the two measurements. A typical Q value for FL1 is .013, and a typical b value for FL1 is 1,000. This gives 277 FITCs for the fluorescence threshold, 121 for the channel separation (assuming 22,000 FITCs for the Calibrite bead), and 1,420 FITCs for the resolution limit.
Cytek measures the Q and B of its Multi-colored upgrades as part of its QA procedure to assure optimal performance.
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