Three-dimensional tracking of dynamic structures in living cells using single-plane imaging with focus feedback

Tracking dynamic subcellular processes in live cells presents a major challenge in biological research, as it often requires high precision in three-dimensional imaging. Here this protocol introduces a focus-feedback microscopy algorithm that enables the accurate tracking of structures within a single optical plane over time, thereby overcoming the limitations of traditional z-stack imaging and reducing imaging intervals and light exposure. This results in prolonged imaging sessions with minimal phototoxicity, making it ideal for studying dynamic molecular processes in living cells. The protocol provides step-by-step guidance for integrating the focus-feedback algorithm within Zeiss Zen or custom microscope software, incorporating cylindrical lenses for z-position detection, performing bead-based calibration and analyzing time-lapse data using both standard and custom tools. It is designed to be accessible to researchers with varying levels of experience and, depending on research question, can be completed within a single day. Focus-feedback microscopy has been successfully applied to track single gene loci within the nucleus and has potential extensions to cytoplasmic structures such as organelles or vesicles. Its compatibility with multichannel and single-molecule imaging makes it a powerful tool for studying dynamic cellular processes with precise spatial and temporal resolution.