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 recently discovered a phenomenon that the digitally summed Fourier spectrum of two images acquired from two-angle illumination exhibits interference-like fringe modulation when the sample is out-of-focus. 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 212 times the depth-of-field (DoF) for thin samples and 300 times for thick specimens. It can additionally extend the natural DoF of the imaging system by 20 folds when integrated with complex-field imaging.
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
A qualitative comparison of autofocusing methods
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}
}