Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes

Measuring forces within living cells remains a technical challenge. We developed hydrophobic mechanosensing fluorescent probes called Flippers, which fluorescence lifetime depends on lipid packing and can report membrane tension. We describe here technical optimization of the probe imaging, and diverse characterizations in various biological and in vitro systems. We propose to the cell biology community a guideline to measure biophysical parameters of cellular membranes by FLIM microscopy with Flipper probes.i