FLIM Strategies for Intracellular Sensing

Abstract

Since the very early years of microscopy development, scientists have pursued the ability to observe live cellular activity in order to probe the processes occurring inside cells in real time. Fluorescence microscopy has become an extraordinary tool for unraveling the myriad cellular processes and interactions that are relevant to understanding cell physiology. The intracellular sensing of certain analytes is of crucial importance to understanding some of these processes, such as the relation between cellular pH and metabolic states or the roles of specific ions in signaling pathways. However, the acquisition of quantitative information from the interiors of cells is not a trivial challenge. Ratiometric, intensity-based fluorescence microscopy approaches are commonly used, but they suffer from many systematic difficulties that make them unsuitable for quantitative sensing. Fluorescence lifetime imaging microscopy (FLIM) detects the time duration of fluorescence emission, taking advantage of the multidimensional nature of photon emission. FLIM-based intracellular sensing approaches, especially in the time domain in single-photon timing (SPT) mode, overcome many of the limitations of fluorescence-intensity methods. Herein, we review strategies for the FLIM-based intracellular sensing of local pH, ion concentration, and biomolecular interactions. In the first section, we demonstrate how in-depth knowledge of the photophysics of dyes can be useful in the development of FLIM sensors. Then, we review the growing field of nanoparticle-based FLIM sensors. Finally, the expanding detection capabilities of FRET and the use of FLIM for the larger-scale analysis of tissues are discussed.