Microsphere-assisted digital in-line holographic microscopy

Abstract Digital in-line holographic microscopy (DIHM) is known for its compact, easy-to-implement, and phase-stable configuration that enables full-field three-dimensional (3D) reconstruction and non-invasive motion tracking. However, like all forms of light microscopy, its spatial resolution is fundamentally limited by diffraction, and most solutions for resolution enhancement compromise DIHM’s key advantages by increasing system complexity and cost. Microsphere-assisted microscopy (MAM) has recently attracted significant interest for its ability to improve imaging resolution in a simple yet effective manner. In this study, we demonstrate the feasibility of integrating MAM with DIHM. Proof-of-concept experiments are conducted on standard diffraction gratings, resolving features below the diffraction limit of conventional optical systems. As an application, we apply the technique to dynamic 3D tracking and visualization of freely swimming Chlamydomonas cells, demonstrating its potential as a low-cost, label-free method for high-resolution 3D morphological and dynamic analysis in life and material sciences.