A software-based focus system for wide-field optical microscopy

TL;DR

This paper introduces a software-based autofocus method for wide-field optical microscopy that corrects focus drift using only a motorized Z-drive and side-placed marks, eliminating hardware needs.

Contribution

The authors develop and evaluate a software autofocus technique that matches hardware systems in performance, simplifying long-term biological imaging without additional hardware.

Findings

Performance comparable to hardware autofocus systems

Effective in live bacterial colony imaging

Works across multiple objectives

Abstract

Long-term time-lapse imaging of biological samples requires correcting for focal drift, which would otherwise gradually push the sample out of focus. We present a software-based method that eliminates this time-dependent blur using only a motorized Z-drive, with no additional hardware. The method relies on imaging marks made on the side of the coverslip opposite to the sample. We provide a Beanshell script implementation, evaluate its performance across multiple objectives, and benchmark it against a hardware autofocus system, finding comparable results. Finally, we demonstrate its effectiveness in live imaging of growing bacterial colonies.