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1000
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Abstract/Summary
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Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) is a powerful tool for studying conformational changes of biosensors, protein-protein interactions and the dynamics of molecular processes in living cells. This approach enables measurements of molecular distances at the nanometer scale, with high spatial and temporal resolution. FLIM-FRET relies only on donor and acceptor lifetime changes induced by energy transfer thus avoiding possible artifacts of pure intensity-based FRET measurements. This tutorial provides a step-by-step workflow of widefield FLIM measurements of immune competent cells, including instrument setup, sample preparation, data acquisition and analysis. Practical guidelines are offered to optimize experimental conditions and troubleshoot some common issues. A case study highlights the application of FLIM-FRET to investigate protein conformational changes during T-cell receptor activation, demonstrating the technique’s sensitivity and versatility in the context of cellular signaling.
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