Luminescence Lifetime Imaging: Key Concepts and Advances in the Image Acquisition, Processing, and Analysis Pipeline

There is rapidly growing interest in applying luminescence lifetime imaging, i.e., FLIM and PLIM (fluorescence and phosphorescence lifetime imaging microscopy, respectively), across natural and life sciences. This includes developing new chemistries for luminescence lifetime read out, new instrumentation and data analysis approaches, as well as diverse applications in chemical, environmental and life sciences. The methodology captures the luminescence lifetime, an intrinsic fingerprint that is sensitive to the local physicochemical environment and specific molecular interactions, making it a valuable quantitative imaging technique for tissue characterization, live-cell metabolic studies, materials science studies, and more. Luminescence lifetime imaging is both robust and reproducible when properly executed but demands careful experimental design and substantial user expertise in terms of choice of imaging modality, instrumentation parameters, image acquisition settings and subsequent image processing and analysis. Therefore, this review addresses the principal determinants of imaging data and reviews practical methods and state-of-the-art advances to address them. Concurrently, this provides a foundation to discuss and highlight the current frontiers and future directions of this important imaging modality.