Fluorescence lifetime: Beating the IRF and interpulse window

Mohamadreza Fazel, Alexander Vallmitjana, Lorenzo Scipioni, Enrico Gratton, Michelle A. Digman, Steve Pressé

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes—outside the intermediate, Goldilocks, zone—for data drawn from a single confocal spot leveraging the tools of Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP lifetime analysis, using a range of synthetic and experimental data. Moreover, we show that the BNP lifetime analysis method can distinguish and deduce lifetimes using photon counts as small as 500.

Original languageEnglish (US)
Pages (from-to)672-683
Number of pages12
JournalBiophysical journal
Issue number4
StatePublished - Feb 21 2023

ASJC Scopus subject areas

  • Biophysics


Dive into the research topics of 'Fluorescence lifetime: Beating the IRF and interpulse window'. Together they form a unique fingerprint.

Cite this