Single-shot multiplexed digital holographic microscopy for dual-sample phase imaging

A grating-based digital holographic microscopy (DHM) configuration is presented for single-shot, spatially multiplexed quantitative phase imaging of multiple samples on a single sensor plane. By using a 1D transmission diffraction grating, the proposed system enables simultaneous acquisition of two spatially separated samples while maintaining full spatial resolution. The zeroth diffraction order is utilized as the reference wave, while the first diffraction orders serve as object waves corresponding to different samples. The experimental results of multiplexed quantitative phase and thickness reconstruction for optically similar and dissimilar samples, including red blood cells (RBCs), microspheres, and pollen grains, demonstrate the capability of the proposed dual-sample configuration. The quantitative phase accuracy of the multiplexed system was evaluated using polystyrene beads, yielding mean absolute errors of 0.124 ± 0.058 rad and 0.101 ± 0.089 rad for the two field of views (FOVs) corresponding to both samples, corresponding to relative errors of 1.37% and 1.11%, respectively, indicating minimal degradation due to multiplexing. The configuration's dynamic bio-imaging capability is further illustrated by simultaneously monitoring temperature-dependent membrane fluctuations of RBCs under heated and ambient conditions within a single acquisition. In addition, a numerical simulation study is presented to examine the scalability of the proposed multiplexing system for multiple-sample imaging.