Digital defocus aberration interference for automated optical microscopy

Automation in optical microscopy is critical for enabling high-throughput imaging across a wide range of biomedical applications. Among the essential components of automated systems, robust autofocusing plays a pivotal role in maintaining image quality for both single-plane and volumetric imaging. However, conventional autofocusing methods often struggle with implementation complexity, limited generalizability across sample types, incompatibility with thick specimens, and slow feedback. We observed that the digitally summed Fourier spectrum of two images acquired from two-angle illumination exhibits interference-like fringe modulation when the sample is defocused. These digital fringes correlate directly with defocus through a physics-based relation. Based on this principle, we developed an automatic, efficient, and generalizable defocus detection method termed digital defocus aberration interference (DAbI). Implemented with a simple two-LED setup, DAbI can quantify the defocus distance over a range of 443 times the depth-of-field for thin samples and 296 times for thick specimens. It can additionally extend the natural depth-of-field of the imaging system by 20-fold when integrated with complex-field imaging. We demonstrated the versatile applications of DAbI on brightfield, complex-field, refractive index, confocal, and widefield fluorescence imaging, establishing it as a promising solution for automated, high-throughput optical microscopy.