Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid‐binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems‐level investigation of lipid‐mediated cell signaling and regulation.