Development of fluorescence lifetime imaging-based biosensors for quantitative visualization

Fluorescent biosensors have become essential tools in life sciences, enabling the visualization of the spatiotemporal dynamics of signaling molecules at the cellular level. In particular, intensity-based sensors-where changes in the concentrations of signaling molecules are detected as changes in fluorescence intensity-are widely used due to their versatility. However, such sensors are often affected by several factors, including variations in biosensor concentration, photobleaching, optical path settings, and focus drift, which hamper quantitative analysis. To overcome these challenges, we have been developing fluorescence lifetime imaging microscopy (FLIM)-based biosensors that utilize fluorescence lifetime-a parameter independent of probe concentration and imaging conditions-as a robust and reliable readout. Our research has focused on the quantitative visualization of physiological parameters, particularly those relevant to skeletal muscle homeostasis and ion channel activity. One example is a small-molecule fluorescent temperature sensor designed to quantify temperature changes in subcellular compartments. This sensor, based on an organic dye, enables targeting to organelle membranes and provides high spatial resolution, allowing precise detection of local heat production, such as that occurring in the mitochondria of brown adipocytes. In parallel, we have developed genetically encoded fluorescent protein-based sensors that correlate fluorescence lifetime values with the concentrations of signaling molecules such as ATP. These sensors have enabled the quantitative imaging of ATP dynamics in various cell types and multicellular systems. Furthermore, we are constructing a flexible sensor development platform, paving the way for the creation of diverse biosensors that can contribute to comprehensive studies in muscle physiology.