Large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter and quantitative brain function imaging

Abstract

Significance Two-photon microscopy (TPM) is ideally suited for in vivo brain function imaging because of its high resolution and deep tissue penetration. However, conventional TPM is limited by a restricted field-of-view (FOV), an inherent trade-off between the imaging area and temporal resolution, and an insufficient amount of information obtained using only intensity recording. Here, we propose large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) with 9-mm2 FOV, 0.7-μm lateral resolution, and high FLIM throughput (512 × 512 pixels, 30 Hz, two regions). Benefitting from these advantages, LD-2P-FLIM enables neurovascular coupling studies under both physiological and pathological conditions with simultaneous spatiotemporal recording, and quantitative imaging of calcium concentrations across a large neuronal population.