Fluorescence lifetime imaging microscopy

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique offering profound insights into a broad spectrum of biological processes such as metabolic imaging, protein–protein interactions and live-cell intracellular dynamics. The future of FLIM appears promising, with continuous technological advancements for time-resolved measurements pushing the boundaries for spatiotemporal information. However, the growth of the FLIM community has been slower, owing to the requirement for specialized training and technology. This Primer aims to address this gap by providing a comprehensive overview of FLIM principles, methods and analysis. We discuss various methods for measuring fluorescence lifetimes, including time-tagging and phase-modulation shift methods, along with their implementations and setup variations. Additionally, we explore different avenues for data analysis, with a specific focus on the phasor approach and its crucial considerations. Furthermore, we present a range of applications demonstrating versatility and usability of FLIM. Limitations and optimization strategies are also discussed, covering methodological constraints, equipment limitations and potential errors, along with their solutions. By sharing our expertise, we aim to expand FLIM to broader audiences while reinforcing concepts within the FLIM community. This Primer seeks to inspire bioimaging researchers to fully embrace FLIM, thereby advancing our understanding of complex biological systems.