Fluorescence Lifetime Imaging for Tension Sensing

The fluorescence lifetime images were acquired by a custom-made fluorescence lifetime imaging microscope built on a laser scanning confocal microscope (FluoView 1000, Olympus). A picosecond pulsed laser with the wavelength of 450 nm (LDH-D-C-450, Picoquant) was coupled with the laser scanning module, and the excited fluorescent signal was filtered by a band-pass filter 490-40 (ET490/40 × , Chroma) before entering a photon counting detector (PD-100-CTC, MPD). All signals were recorded in the Time-Correlated Single Photon Counting (TCSPC) mode with a data acquisition board (TimeHarp 260, Picoquant). The FRET efficiency of the TS module, μ, was calculated based on the lifetime of the donor molecule: $$\mu =1-\frac{{\tau }_{AD}}{{\tau }_D}$$ where τ_AD is the fluorescence lifetime of the donor molecule in the presence of the acceptor, and τ_D is the fluorescence lifetime of the donor molecule without the acceptor. The value of τ_AD/τ_D is obtained by fitting the decay curve in the software Symphotime (Picoquant)^{31 (link),32 (link)}. The tension force is calculated based on the calibration curve of the TSmod reported in ref.^{10 (link)}.