Dual-feature validated depth reconstruction enabling accurate and fast two-photon volumetric imaging

Abstract

Biological systems exhibit complex three-dimensional distributions and rapid dynamics, posing significant challenges for in vivo volumetric imaging with high spatiotemporal resolution. Two-photon microscopy (TPM) employing elongated point spread functions (PSFs) enables fast volumetric imaging and depth decoding; however, acquiring depth information becomes unreliable when signals from different axial positions overlap or exhibit similar spatial profiles. To address this limitation, we develop a dual-view gradient excitation TPM (Dvge-TPM) that introduces an engineered intensity imbalance between the left and right PSF arms. This design encodes the axial information simultaneously into two independent features: the spatial separation distance and the fluorescence intensity ratio of the projection pair. Through dual-feature cross-validation, Dvge-TPM enables simultaneous depth reconstruction across a 60-μm-thick volume with a depth localization error of only 1.37 μm. We further demonstrate the capability of Dvge-TPM by tracking C. elegans nuclear division, monocyte/macrophage migration, and neuronal calcium dynamics, highlighting its potential for high-speed, accurate three-dimensional imaging of complex, dynamic biological systems.