Digital defocus aberration interference for automated optical microscopy

TL;DR

This paper introduces DAbI, a novel autofocusing method for optical microscopy that uses digital interference fringes to accurately and efficiently determine defocus across various imaging modalities and sample types.

Contribution

The authors developed DAbI, a simple, physics-based autofocusing technique using two-LED illumination that improves speed, accuracy, and generalizability over existing methods.

Findings

DAbI quantifies defocus over 443 times the DoF for thin samples.

DAbI extends the natural DoF by 20 times with complex-field imaging.

DAbI is applicable to multiple microscopy modalities including brightfield and fluorescence.

Abstract

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 (DoF) for thin samples and 296 times for thick specimens. It can additionally extend the natural DoF of the imaging system by 20 folds 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.