Building Fluorescence Lifetime Maps Photon-by-Photon by Leveraging Spatial Correlations.

Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative characterization of subcellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution. Second, existing methods deduce photon ratios instead of absolute lifetime maps. Next, the number of fluorophore species contributing to the signal is unknown, while excited state lifetimes with <1 ns difference are difficult to discriminate. Finally, existing analyses require high photon budgets and often cannot rigorously propagate experimental uncertainty into values over lifetime maps and number of species involved. To overcome all of these challenges simultaneously and self-consistently at once, we propose the first doubly nonparametric framework. That is, we learn the number of species (using Beta-Bernoulli process priors) and absolute maps of these fluorophore species (using Gaussian process priors) by leveraging information from pulses not leading to observed photon. We benchmark our framework using a broad range of synthetic and experimental data and demonstrate its robustness across a number of scenarios including cases where we recover lifetime differences between species as small as 0.3 ns with merely 1000 photons.

Déclaration de conflit d'intérêts

The authors declare no competing financial interest.