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An affordable fiducial marker strategy for reliable autofocus in long‐term live microscopy

2026/06/11 by Ilyas Djafer‐Cherif, Ilyas Djafer-Cherif, Bartlomiej Waclaw +1
Engineering · Biochemistry, Genetics and Molecular Biology · #Image Processing Techniques and Applications #Cell Image Analysis Techniques #Advanced Fluorescence Microscopy Techniques

paper · doi:10.1111/jmi.70127

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 motorised 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.

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