Molecular brightness ($b$)

Molecular brightness is the detected fluorescence signal contributed by one labelled molecule or fluorescent particle under specified illumination and collection conditions. It is often expressed as detected photons per second per particle, or counts per particle per acquisition interval. It depends on the label, its environment and the apparatus, so it is not an intrinsic universal constant of the protein carrying the label.

For one uniformly observed species in a simple additive model, background-subtracted count rate $F$ and mean particle number $N$ satisfy $F=bN$, where $b$ is the molecular brightness. Ten particles with $b=1000$ counts per second and five particles with $b=2000$ counts per second both yield $F=10,000$ counts per second. Mean intensity alone cannot distinguish the two samples. A particle carrying two independent identical labels can approach twice the molecular brightness of one label, but quenching or incomplete labelling can invalidate that comparison.

Yan Chen and colleagues used the distribution of photon counts as particles diffuse through a tiny illuminated observation volume to estimate both molecular brightness and mean occupancy. This uses fluctuations beyond the average, with the illumination profile included in the measurement model.^[ref]