Near-infrared fluorescence lifetime imaging: fluorophores, methods, and applications

Abstract

Abstract. Significance Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides molecular and functional contrast that encodes local biochemical and biophysical environments beyond what fluorescence intensity alone can reveal. Realizing this potential across biological imaging and emerging clinical applications requires rigorous fluorophore characterization and a critical assessment of the photophysics, measurement capabilities, and conditions governing reliable lifetime interpretation. Aim We comprehensively survey the contemporary NIR FLI landscape, spanning fluorophore photophysics, imaging instrumentation, lifetime analysis, and biomedical applications, to identify application-specific criteria for fluorophore selection, quantitative measurement, and translational validation. Approach Within the NIR range, we critically compare organic dyes, fluorescent proteins, inorganic fluorophores, and endogenous emitters, integrating photophysical evidence with acquisition and analysis methods for multiplexing, Förster resonance energy transfer (FRET), environmental sensing, and clinical imaging. Results Reported NIR fluorophore lifetimes span tens of picoseconds to over 100 ns and depend on molecular structure, solvent, binding state, and aggregation. Multiplexing requires reproducible, distinguishable decay signatures; sensing requires selective, calibrated lifetime responses; and FRET requires reliable donor-only references. Uncorrected instrumental effects, tissue photon migration, and inappropriate analysis assumptions can distort lifetime estimates. Incomplete characterization and inconsistent reporting further limit reproducibility and comparison across studies. Conclusions Quantitative NIR FLI requires precise probe characterization, standardized measurement validation, and application-specific performance assessment. We provide practical recommendations for probe selection, lifetime contrast interpretation, and clinical utility evaluation, establishing a framework for reproducible NIR FLI from mechanistic cell studies to image-guided interventions.