Spc 150n

Fluorescence lifetime images of NAD(P)H and FAD in cells were captured by a customized multiphoton fluorescence lifetime microscope (Marianas, 3i) equipped with a time-correlated single photon counting (TCSPC) electronics module (SPC-150N, Becker & Hickl). A stage-top incubator (okolab) was set to 37°C, 5% CO₂, and 85% relative humidity to maintain a physiological environment while imaging. NAD(P)H and FAD in macrophages were excited by a tunable Ti: sapphire femtosecond laser (COHERENT, Chameleon Ultra II) at 750 nm (∼27 mW) and 890 nm (∼35 mW) respectively. The fluorescence lifetime images of NAD(P)H and FAD were obtained sequentially by photomultiplier tube (PMT) detectors (HAMAMATSU, H7422PA-40) and isolated by a 447/60 nm bandpass filter and a 560/88 nm bandpass filter, respectively. For each imaging dish, both NAD(P)H and FAD fluorescence lifetime images were captured in at least five random positions, and each fluorescence lifetime image (256 × 256 pixels) was acquired with a pixel dwell time of 50 μs and 5 frame repeats. The NAD(P)H and FAD fluorescence lifetime images in the cancer cell metabolic inhibitor experiment were previously collected by AJ Walsh and L. Hu with the same imaging system, and the details of autofluorescence imaging are covered in Hu et al. (Hu et al., 2022 ).

Slices were maintained in a continuous perfusion of modified artificial CSF (ACSF) containing the following (in mM): 119 NaCl, 2.5 KCl, 3 CaCl₂, 26.2 NaHCO₃, 1 NaH₂PO₄, and 11 glucose, bubbled and equilibrated with 5% CO₂/95% O₂. Regarding Fig. 2, the ACSF solution was replaced to ACSF solution including 50 mM KCl, and then this solution was replaced to original ACSF solution again upon washout process. For Fig. 3, the ACSF solution was replaced to ACSF solution including 20 µM glutamate (0 min) and 1 mM glutamate (16 min). Time-lapse imaging was carried out using a two-photon microscope (Fluoview 1000, Olympus) equipped with a Tsunami laser (Sprectra-Physics) at 910 nm. Epi-fluorescence was detected with a PMT (HPM-100–40; Hamamatsu) that was placed after the wavelength filters (Chroma, HQ510/70-2p for GFP and Brightline multiphoton filter 680SP). Fluorescence lifetime images were produced on a PCI board (SPC-150 N; Becker-Hickl). SPC imaging (Becker-Hickl) was used to generate fluorescence lifetime images. To analyze the fluorescence lifetime value of dendritic spines, a whole region including a neck and body of a dendritic spine was averaged. The fluorescence lifetime value of dendritic shafts was averaged from the base regions (white squares in Figs. 2, 3, 4) of dendritic spines regardless of the edges and centerline of dendrites.