Confocal FLIM of Genetically Encoded FRET Sensors for Quantitative Ca²⁺ Imaging

Abstract

Fluorescence lifetime imaging (FLIM) is a powerful imaging mode that can be combined with confocal imaging. Changes in the fluorescence decay time of a donor in an intramolecular Förster resonance energy transfer (FRET)-based biosensor provide intrinsic quantitative data. Here, we describe a protocol using both the Ca²⁺ sensor TN-XL, which uses troponin C, as the Ca²⁺-sensing unit, and the FLIM technology based on time-correlated single-photon counting.