Edge pass optical filters

The fluorescence lifetime imaging measurements of the live cultured cells were performed at room temperature in air using an inverted Eclipse Ti-U microscope equipped with a DCS-120 confocal scanning FLIM system and HPM-100-40C hybrid single-photon detector (Becker & Hickl GmbH, Berlin, Germany). An oil immersion objective (PLAN APO 60X/1.4 NA, Nikon, Minato, Japan) was used for the image acquisition. The two-photon fluorescence was excited by pulses at 750 nm obtained using the femtosecond optical parametric oscillator TOPOL (Avesta Ltd., Moscow, Russia) pumped by a Yb femtosecond laser TEMA-150 (Avesta Ltd., Moscow, Russia) and driven by an 80 MHz repetition rate (pulse duration 140 fs). The fluorescence was registered in the range of 500 to 650 nm, limited by edge pass optical filters (Thorlabs, Newton, MA, USA). The image recording time in all cases did not exceed 3 min. The SPCImage software (Becker & Hickl, Berlin, Germany) was used to extract the lifetime distributions from the collected images. The post-processing of the data was performed using Origin Pro 2015 (OriginLab Corporation, Northampton, MA, USA).

The fluorescence decay data of EGFP-Y145L/S205V were recorded using a single-photon counting (SPC) detector (HPM-100-40C, Becker & Hickl, Berlin, Germany) in the spectral range from 500 to 650 nm, limited by edge pass optical filters (Thorlabs, Newton, MA, USA). An oil immersion objective (PLAN APO 60X/1.4 NA, Nikon, Minato, Japan) was used for the fluorescence excitation. The two-photon fluorescence was excited at 750 nm (repetition rate 80 MHz, pulse width 150 fs, optical power 20 mW) using a femtosecond optical parametric oscillator (TOPOL-1050-C, Avesta Project Ltd., Moscow, Russia) pumped by a Yb femtosecond laser (TEMA-150, Avesta Project Ltd., Moscow, Russia). The post-processing and visualization of the calculated data were performed using Origin Pro 2015 (OriginLab Corporation, Northampton, MA, USA).