Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine

SIGNIFICANCE: Fluorescence lifetime imaging microscopy (FLIM) measures the decay rate of fluorophores, thus providing insights into molecular interactions. FLIM is a powerful molecular imaging technique that is widely used in biology and medicine. AIM: This perspective highlights some of the major advances in FLIM instrumentation, analysis, and biological and clinical applications that we have found impactful over the last year. APPROACH: Innovations in FLIM instrumentation resulted in faster acquisition speeds, rapid imaging over large fields of view, and integration with complementary modalities such as single-molecule microscopy or light-sheet microscopy. There were significant developments in FLIM analysis with machine learning approaches to enhance processing speeds, fit-free techniques to analyze images without a priori knowledge, and open-source analysis resources. The advantages and limitations of these recent instrumentation and analysis techniques are summarized. Finally, applications of FLIM in the last year include label-free imaging in biology, ophthalmology, and intraoperative imaging, FLIM of new fluorescent probes, and lifetime-based Förster resonance energy transfer measurements. CONCLUSIONS: A large number of high-quality publications over the last year signifies the growing interest in FLIM and ensures continued technological improvements and expanding applications in biomedical research.