Multimodal Multiphoton Microscope Setup

This platform consists of a Leica TCS SP8 multimodal multiphoton fluorescence benchtop microscope (Leica Microsystems, Wetzlar, Germany) controlled via Leica’s acquisition software, where its technical details were published elsewhere (15 (link)). Briefly, this platform includes a Ti : Sapphire tunable laser source from 690 nm to 1,040 nm (Mai Tai DeepSee, Spectra-Physics). The objective used is a 25× water-immersion (HCX IRAPO L 25X NA 0.95, Leica). A hybrid internal detector (HyD, Leica, Germany) was used for spectral measurements, while two other external non-descanned hybrid detectors (HyD-RLD, Leica Microsystems, Wetzler, Germany) were used for two-photon fluorescence, SHG, and FLIM imaging. The first one is dedicated to select the nicotinamide adenine dinucleotide (NADH) fluorescence signal when using 800 nm as excitation wavelength and SHG signal when using 890 nm, using a 448 ± 20 nm band-pass filter (Semrock, FF01-448/20-25). The second one is dedicated to select the flavins (FAD) fluorescence signal using a 520 ± 30 nm band-pass filter (Semrock FF01-520/35-25). For fluorescence lifetime imaging acquisition, a time-correlated single-photon counting module (PicoQuant TCSPC module, Berlin, Germany) was coupled with the two external hybrid detectors, which permit performing NADH and FAD lifetime imaging at 800 nm.