Picoharp300 dual channel spad system

FRET-FLIM was performed on a PicoHarp300-Dual Channel SPAD system (PicoQuant) in combination with Nikon A1Rsi microscope and on Leica TCS SP5 X scan head coupled to internal Hybrid (HyD) detector (Becker & Hickl). The excitation pulses were generated by a Picosecond Pulsed Diode Laser LDH-D-C-485 (PicoQuant) and supercontinuum tunable white light laser (488 nm), resulting in excitation pulses of 100 ps at a repetition of 40 MHz. 60×/1.20 water-immersion and 63×/1.20 water-immersion objectives were used. Images with a frame size of 128 × 128 pixels were acquired, and the average count rate was around 10⁴ photons per second for an acquisition time of ±60 s. From the time resolved fluorescence intensity images, the fluorescence decay curves were calculated per pixel and fitted with double-exponential decay model using the SymPhoTime 64 (PicoQuant) and the SPCImage 3.10 software (Becker &Hickl). The fitting procedure was performed without fixing any parameters. Several cells (n > 10) for each sample were analyzed and Student's t-test was used to determine statistical significance of differences between samples (*P < 0.001). Error bars represent standard error of the mean (SEM) (52 (link)).