Practical guide to fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) has emerged as a powerful modality that offers sensitivity to molecular environments, fluorophore concentration independence, local environment sensing, characterization of molecular binding events with multiplexing capabilities, and monitoring metabolism. Recently, we adapted FLIM for quantitatively sensing RNAs in live cells, establishing the Riboglow-FLIM platform. More broadly, FLIM has the potential to complement and advance traditional intensity-based microscopy platforms. Here, we provide a practical guide to make use of FLIM for diverse fluorescence sensors. We discuss FLIM basics, the experimental setup, data-fitting principles, and real-world case studies of well-understood fluorescent proteins to demonstrate the applicability of these workflows. This perspective provides a basic "best practices" guide for designing and executing FLIM experiments, with the goal of introducing researchers to concepts that will help empower them to utilize this emerging technology.