Needle beam two-photon microscopy for simultaneous multiplane neural and vascular imaging in awake mice.

Abstract

Comprehensive understanding of brain functions necessitates high-speed imaging of neuronal and vascular dynamics across extensive volumes. Functional neuroimaging investigations with two-photon microscopy are commonly hindered by its limited depth of field which restricts imaging rates across multiple planes. We introduce needle-shaped beam two-photon microscopy (NB-2PM), a versatile platform for high-throughput neurovascular imaging at sub-cellular resolution across multiple depths. It employs customized diffractive optical elements to generate single- or multi-plane needle beams with up to 10 times elongated depth of field relative to Rayleigh lengths and engineered axial energy distribution to effectively offset light attenuation with depth. The proposed method was applied to snapshot volumetric vascular imaging and multi-plane neurovascular dynamic recordings of resting state and stimulus-evoked activity in mice. NB-2PM can seamlessly be integrated into existing microscopy systems, thus providing a scalable platform for gaining comprehensive insights into the functional architecture of murine brain.

Supplementary information: The online version contains supplementary material available at 10.1186/s43074-026-00237-3.