Fluorescence Lifetime Imaging Microscopy

The FLIM system (LSM kit, Picoquant, Berlin, Germany) was incorporated into the Olympus IX70 and consisted of a pulsed diode laser (485 nm, FWHM = 83 ps, 20 MHz) and was equipped with a time-correlated single-photon counting module from PicoQuant (Berlin, Germany). The emission signal was collected through a band-pass emission filter centered at 510 nm (FWHM = 20 nm) using single-photon avalanche diode detectors (SPADs, PicoQuant, Berlin, Germany). Fluorescence lifetime measurements were acquired using software (SymPhoTime 64 version 2.0, PicoQuant) that controlled both the TCSPC system and the scanner system. Acquisition times were adjusted to achieve 1000 photons per pixel (with a typical acquisition time of 3 min). FLIM data were analyzed with a binning of 2 using the FLIMfit v5.1.1 software (https://flimfit.org/, accessed on 15 January 2021), which uses a separable nonlinear least square algorithm to recover the lifetimes from the fluorescence decays [42 (link)]. The whole-cell lifetime intensity decays were fitted with the mono-exponential decay function deconvoluted with the instrument response function (IRF) to generate FLIM images showing the apparent lifetime of each pixel. The reduced chi-squared (χ2) value from the mono-exponential function is sufficient to describe the lifetime data in the present study.