Three-dimensional near-isotropic super-resolution imaging via axial interference speckle illumination

Optical super-resolution microscopy has significantly advanced nanoscale biological imaging. However, its limited axial resolution still poses challenges for investigating complex three-dimensional structures in biological specimens. In this study, we present a three-dimensional near-isotropic super-resolution imaging technique based on axial interference speckle illumination. A reflective silver mirror integrated into a single-objective microscope generates axially confined speckle patterns, enhancing axial resolution. Combined with fluctuation-based reconstruction methods, our approach achieves more than fourfold improvement in axial resolution compared to a diffraction-limited system. We demonstrate multi-color imaging and tracking of lysosomal dynamics in live U2OS cells. This simple, cost-effective system enables near-isotropic super-resolution imaging without requiring dual-objective alignment, providing a practical and versatile tool for investigating subcellular processes in living biological systems.