3D refractive index imaging via vortex beam dynamic speckle interference microscopy

We propose a label-free, depth-resolved quantitative phase and refractive index imaging method that extends our previously reported two-dimensional Vortex beam Speckle Interference Microscopy (VSIM) into the third dimension by integrating axial scanning with a layer-wise recursive reconstruction algorithm. By exploiting the unique angular properties of perfect optical vortex (POV) beams, the method generates dynamic high-spatial-frequency speckle illumination, simultaneously enhancing lateral resolution and axial sectioning capability. The axial sectioning arises from spatial coherence engineering—the ring-shaped POV beam, after passing through a rotating diffuser, produces a speckle field whose axial correlation length is controlled by the illumination pupil geometry, enabling depth-resolved imaging without requiring a broadband light source or confocal pinhole. The system employs a common-path interferometric design with a four-step phase-shifting algorithm for quantitative phase retrieval, providing intrinsic robustness against environmental disturbances. The method was validated using polystyrene microspheres, red blood cells (RBCs), and CT26 cancer cells. Under the projection approximation, the layer-wise recursive algorithm converts the measured phase stack into an estimate of the three-dimensional refractive index distribution, enabling the visualization of depth-dependent subcellular structures without exogenous labeling. We discuss the relationship of this approach to optical diffraction tomography, spatial-coherence phase microscopy, and programmable spatial coherence tomography. These findings underscore the potential of this technique for broad applications in cellular physiology, biopharmaceutical development, and nanomaterial characterization.