Lsm980 multiphoton microscope

RBC membrane microviscosity was studied by fluorescence lifetime imaging (FLIM) technique using a molecular rotor (BODIPY-C10), whose fluorescence lifetime is dependent on the microviscosity of the environment. RBCs were labelled in suspension for 60 min at 37°C with 1 µM of BODIPY-C10 dissolved in 1 mg/mL DMEM-BSA. After incubation, RBCs were washed in fresh DMEM, dropped off in plastic chambers (ibidi) for 8 min at RT. Cells were observed with the Zeiss LSM980 multiphoton microscope, which was equipped with a time-correlated single-photon counting (TCSPC) FLIM module (PicoQuant) for high-resolution microscopy. BODIPY-C10 was excited by a coherent (Chameleon Discovery) pulsed laser (80 MHz) at 800 nm. The emission was captured with a 505–545 nm bandpass filter at a resolution of 512 × 512 pixels. The fluorescence lifetimes for each pixel of the image corresponding to the cells were recorded to create the FLIM images. A minimum of 1,000 photons in the brightest pixel were acquired before stopping the FLIM acquisition. The FLIM images were analyzed using the SymPhoTime64 software (PicoQuant, Germany).

RBC membrane microviscosity was studied by fluorescence lifetime imaging (FLIM) technique using a molecular rotor (BODIPY-C10), whose fluorescence lifetime is dependent on the microviscosity of the environment. RBCs were labelled in suspension for 60 min at 37°C with 1 µM of BODIPY-C10 dissolved in 1 mg/mL DMEM-BSA. After incubation, RBCs were washed in fresh DMEM, dropped off in plastic chambers (ibidi) for 8 min at RT. Cells were observed with the Zeiss LSM980 multiphoton microscope, which was equipped with a time-correlated single-photon counting (TCSPC) FLIM module (PicoQuant) for high-resolution microscopy. BODIPY-C10 was excited by a coherent (Chameleon Discovery) pulsed laser (80 MHz) at 800 nm. The emission was captured with a 505–545 nm bandpass filter at a resolution of 512 × 512 pixels. The fluorescence lifetimes for each pixel of the image corresponding to the cells were recorded to create the FLIM images. A minimum of 1,000 photons in the brightest pixel were acquired before stopping the FLIM acquisition. The FLIM images were analyzed using the SymPhoTime64 software (PicoQuant, Germany).