Depth-Resolved Volumetric Two-Photon Fluorescence Microscopy Based on Power-Exponent-Phase Vortex Autofocusing Beam

There is an urgent need for volumetric microscopy techniques that can provide depth information while reducing scanning frequency. Since its proposal in 2010, the abruptly autofocusing beam (AAB) has attracted significant research interest owing to its distinctive autofocusing properties. However, efforts to generate AABs with both extended depth of field and depth-resolving capabilities continue to face major challenges. In this study, we present a volumetric two-photon confocal microscopy (TPCM) system based on power-exponent-phase vortex autofocusing beams (PVABs), which demonstrates depth-resolving functionality. The power-exponent-phase vortex (PV) enhances the axial imaging range, significantly increasing acquisition speed. PVABs serve as excitation sources to illuminate the specimen, with depth information extracted from variations in their side lobes. The depth-resolving range of this volumetric TPCM system reaches up to 100 μm, approximately 10 times greater than that of AABs. Compared with Bessel-beam-based TPCM, this approach offers superior depth-resolving performance and requires fewer scans than traditional Gaussian-beam-based TPCM. Experimental comparisons show that the imaging speed of the PVAB-based TPCM system is approximately 100 times faster than that of conventional Gaussian TPCM. This depth-resolved, high-speed volumetric imaging technique holds strong potential for investigating spatial distribution and dynamic processes in neural biology.