3D imaging of moving cells within droplets with a staggered bifocal microlens array

Three-dimensional (3D) observation of moving cells offers critical insights into fundamental biological processes. However, the limited temporal resolution of traditional scanning-based 3D imaging techniques constrains their use in live-cell imaging. Light-field microscopy (LFM) has emerged as a scanning-free alternative, enabling rapid 3D volumetric data acquisition. Despite this advantage, LFM's spatial resolution and reconstruction artifacts remain challenges that hinder its broader application in biological imaging. This study proposes a custom-designed staggered bifocal microlens array (MLA), fabricated through a cost-effective, high-throughput method, to overcome these limitations. This approach enhances spatial resolution while minimizing reconstruction artifacts compared to conventional uniform MLAs. We demonstrate the technique through 3D imaging of microbeads at varying depths and live cells suspended in droplets. This bifocal MLA presents a promising advancement for extending LFM's applications in biological research.