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

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.