Extended depth-of-field volumetric microscopy based on bidirectionally separated nonparaxial self-bending beam

Abstract

We propose a detection-side volumetric imaging strategy based on a nonparaxial self-bending beam (SBB), achieving an extended depth-of-field (DOF) of 70 μm in a 100×, numerical-aperture (NA) 1.45 fluorescence microscope. By combining cubic phase, asymmetric chirp, and nonparaxial residual-phase compensation, we engineer a nonparaxial SBB with robust depth encoding under high-NA focusing. With bidirectional separation, the nonparaxial SBB enables high-resolution 3D imaging. A standard-sample experiment validates the 3D imaging fidelity and reconstruction accuracy over a DOF of ∼40 μm. A volumetric fluorescent beads experiment demonstrates the ∼70 μm DOF and the near-diffraction-limited resolution. Imaging of a 16-μm-thick mouse kidney section further evaluates the applicability of the method to continuous biological tissue. This framework provides a practical paradigm for deploying structured beams to enhance imaging performance in high-NA volumetric microscopes.