Fluorescence-Based In Situ Quantitative Imaging for Cellular Lipids.

Membrane lipids are dynamic molecules and their local concentrations serve as regulatory signals for diverse biological processes. To achieve quantitative in situ imaging of various lipids, we developed a ratiometric analysis using fluorescence biosensors, each of which is composed of an engineered lipid-binding protein and a covalently attached solvatochromic fluorophore. To cover a wide range of lipid concentration, lipid-binding proteins are engineered to have variable dynamic ranges. These tunable sensors allow robust and sensitive in situ quantitative lipid imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of key signaling lipids. The sensor strategy is also applicable to in situ quantification of multiple cellular lipids or a single lipid in the opposing leaflets of cell membranes.