Hpm 100 40 c

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).

Fluorescence lifetime measurements were carried out using a confocal fluorescence microscope with a super continuum laser (Fianium SC-400–4, frequency of 40 MHz). The output of the laser was filtered by a bandpass filter (Chroma Technology D525/30 m) and focused onto the sample solution using a 60 × objective lens (Nikon Plan Apo IR) with a numerical aperture (NA) of 1.27. The excitation power was set at 0.3 μW at the entrance port of the microscope. Fluorescence photons were collected by the same objective lens and guided through a confocal pinhole as well as a bandpass filter (Chroma Technology D585/40 m), and then detected by a hybrid detector (Becker & Hickl HPM-100–40-C). For each photon signal detected, the routing information was appended by a router (Becker & Hickl HRT-82). The arrival time of the photon was measured by a TCSPC module (Becker & Hickl SPC-140) with the time-tagging mode.^{37 (link)} The time resolution was evaluated by detecting the scattering of the incident laser light at a cover glass, and it was typically 180 ps at FWHM.