Fluorescence: Poisson statistics and algorithms for spectral decomposition

english

2026

volume 13, issue 1

fluorescence   fluorescence microscopy   Poisson statistics   Richardson–Lucy algorithm   spectral decomposition

Univerza v Ljubljani, Fakulteta za matematiko in fiziko
(Obvezni izvod spletne publikacije)

Fluorescence microscopy is a key technique in biophysics, yet its quantitative interpretation is often limited by spectral crosstalk and noise. In this paper, the photon counting statistics of fluorescence emission are examined, and it is experimentally confirmed that, under typical imaging conditions, detected photons follow Poisson statistics. Small deviations are observed only at high fluorophore concentrations, where fluorescence quenching becomes significant. This result is essential for spectral decomposition, as the Richardson--Lucy unmixing algorithm relies on a Poisson noise model. A comparison between the standard linear unmixing algorithm and the Richardson--Lucy algorithm is performed, showing that the latter provides superior performance, especially in the case of low signal-to-noise ratio data. Together, these findings demonstrate that improved multicolor fluorescence imaging and more reliable biological interpretation are enabled by accurate unmixing algorithms.

23.09.2026