Confocal and lifetime microscopy were performed on a Nikon A1 laser scanning microscope with an integrated Picoquant time-correlated single photon counting (TC-SPC) system (Berlin, Germany). Generalized polarization (GP)-imaging was done as previously described52 (link). Briefly, emission was collected at I1=595 and I2=700 nm and GP was calculated as GP=(I1-G*I2)/(I1+G*I2) where G, the instrumental response factor, was determined according to protocol30 (link).
For FLIM, Di4 emission was collected at >560 nm and 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 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 104 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 τDi4.
For FLIM, Di4 emission was collected at >560 nm and 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 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 104 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 τDi4.
