RT Journal Article SR Electronic T1 Building Fluorescence Lifetime Maps Photon-by-photon by Leveraging Spatial Correlations JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.11.29.518311 DO 10.1101/2022.11.29.518311 A1 Mohamadreza Fazel A1 Sina Jazani A1 Lorenzo Scipioni A1 Alexander Vallmitjana A1 Songning Zhu A1 Enrico Gratton A1 Michelle A. Digman A1 Steve Pressé YR 2022 UL http://biorxiv.org/content/early/2022/11/30/2022.11.29.518311.abstract AB Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative analysis of sub-cellular 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 lifetime components 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 components 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 fluorescent species (through beta-Bernoulli process priors) and absolute lifetime maps of these species (through Gaussian process priors) by leveraging information from pulses not leading to observed photon. We benchmark our algorithm 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 components as small as 0.3 ns with merely 1000 photons.Competing Interest StatementThe authors have declared no competing interest.