A1 laser scanning microscope

RBL cells were treated for 24 h prior to staining and imaging. Outer leaflet staining of RBL cell PMs was achieved by incubating cells at 4 °C for 8 min with Di4 at 1 μg/ml in phosphate-buffered saline. Cells were quickly washed prior to immediate imaging in phenol red-free MEM. Lifetime microscopy was performed on a Nikon A1 laser scanning microscope with an integrated Picoquant time-correlated single photon counting (TC-SPC) system (Berlin, Germany). The instrument response function (IRF) was determined with a saturated erythrosine B and KI solution at pH=10 according to the manufacturer’s (Picoquant) protocol. Di4 emission was collected at >560 nm, and images were acquired using 20 MHz pulse frequency. The photon count rate was kept under 10% of the pulse rate by adjusting a manual shutter, and enough frames were acquired to obtain at least 8 × 10³ photons cumulative signal intensity. The fluorescence decay curves were fitted to a bi-exponential re-convolution function adjusted to the IRF, and the average lifetime was calculated and represented in the FLIM images as τ_Di4. The experiment was repeated four times, imaging 15–30 cells per condition each time.