Förster resonance energy transfer efficiency measurements on vinculin tension sensors at focal adhesions using a simple and cost-effective setup.

Significance: Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use. Aim: We prove that absolute FRET efficiency can be measured on a simple setup, an order of magnitude more cost-effective than a standard FRET microscopy setup, by applying it to vinculin tension sensors (VinTS) at the focal adhesions of live CHO-K1 cells. Approach: Our setup located at Université Paris-Saclay acquires donor and acceptor fluorescence in parallel on two low-cost CMOS cameras and uses two LEDs for rapid switching of the excitation wavelength at a reduced cost. The calibration required to extract FRET efficiency was achieved using a single construct (TSMod). FRET efficiencies were measured for VinTS and the tail-less control VinTL, lacking the actin-binding domain of vinculin. Measurements were confirmed on the same cell type using a more standard intensity-based setup located at Rutgers University. Results: The average FRET efficiency of VinTS ( Conclusions: Force measurements, resolved spatially inside a cell, can be achieved using FRET-based tension sensors with a cost effective intensity-based setup. This will facilitate combining FRET with techniques for applying controlled forces such as optical tweezers.