A1 mp confocal microscope

FLIM data were acquired with a Nikon A1-MP confocal microscope equipped with a 2-photon Ti:Sapphire laser (Mai Tai, Spectra Physics, Newport Beach, CA) by producing 80-fs pulses at a repetition rate of 80 MHz. A PML-SPEC 16 GaAsP (B&H, Germany) multi-wavelength detector coupled to a SPC-830 TCSPC/FLIM device (B&H, Germany) was used to collect the decay data. A 60 × oil-immersion objective, 1.2 NA, was used for all experiments. Samples were excited at 750 nm. Signals were integrated into the wavelength region of 408–496 nm. For image acquisition, the pixel frame size was set to 512 × 512 and the pixel dwell time was 60 µs. The average laser power at the sample was maintained at the milliwatt level. In the FLIM images, mitochondrial NAD(P)H responses were separated by the rest of the autofluorescence (cytoplasm, nuclei) by means of fluorescence intensity analysis.^{19 (link)}

A Nikon A1-MP confocal microscope outfitted with a 2-photon Ti:Sapphire laser (Mai Tai, Spectra Physics, Newport Beach, CA, USA), emitting 80-fs pulses at a repetition rate of 80 MHz, was used to characterize the functional properties of ARPE-19 cells. An on-stage incubator (OKOLAB) kept a constant temperature of 37 °C and a 5% level of CO₂. For the quantification of intracellular non-polar aggregates, the lipophilic probe Laurdan was used. Cells were treated with 1 µM of Laurdan. Laurdan intensity images (excitation: 740 nm) were recorded in the two emission ranges 450/50 nm and 525/50 nm with a 1024 × 1024-pixel resolution, and a 60× oil-immersion objective was used to visualize lipid aggregates.