Depth-invariant 3D resolution in multiphoton microscopy enables inertia- and aberration-free axial imaging

Conventional inertia-free multiphoton microscopy (MPM) suffers from depth-dependent degradation of lateral and axial resolution during axial scanning due to defocus-induced aberrations. We introduce truncated non-diffracting beams (tNDBs), generated by segmenting a Bessel beam into multiple axial sections with equalized focal lengths, enabling depth-invariant 3D focusing in laser-scanning MPM. The tNDB maintains constant lateral (~540 nm) and axial (~6 µm) resolutions across a ~29-μm depth range. The method is validated using bead phantoms and mouse brain tissue, consistently revealing sharper structures across depth. tNDBs provide an inertia-free and aberration-robust solution for fast volumetric MPM, enabling constant-resolution 3D imaging without modifying the detection geometry.