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 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.
Intro Video
This video introduces the principle of DAbI in microscopy autofocusing and digital refocusing.
System Overview
Principle of DAbI
DAbI for autofocusing
Autofocusing is a process that when the sample is out-of-focus, the system can automatically move the sample back to focal plane through mechanical actuation.
Graphic illustration of autofocusing
Example of DAbI for confocal microscopy autofocusing
DAbI for digital refocusning
Digital refocusing numerically computes an in-focus image from out-of-focus measurements without any mechanical movements. DAbI can serve as a defocus prior in image reconstructions, thus, it can significantly extend the digital refocusing capability in complex-field imaging.
Graphic illustration of digital refocusing
Digital refocusing in complex-field imaging
Comparison among brightfield microscopy, APIC (the state-of-the-art method), and DAbI-assisted APIC.
BibTeX
@misc{zhou2025DAbI,
title={Digital defocus aberration interference for automated optical microscopy},
author={Haowen Zhou and Shi Zhao and Yujie Fan and Zhenyu Dong and Oumeng Zhang and Viviana Gradinaru and Changhuei Yang},
year={2025},
eprint={2507.10867},
archivePrefix={arXiv},
primaryClass={physics.optics},
url={https://arxiv.org/abs/2507.10867}
}