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Three-dimensional tracking of dynamic structures in living cells using single-plane imaging with focus feedback
Simona V Antonova1,2, Wim Pomp1, Joseph V W Meeussen1
1Division of Gene Regulation, The Netherlands Cancer Institute, Oncode Institute, Amsterdam, The Netherlands.
Nature Protocols
|July 28, 2026
Summary
This study introduces a focus-feedback microscopy algorithm for precise tracking of subcellular structures in live cells. This method reduces phototoxicity and imaging time, enabling longer observation of dynamic molecular processes.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Biophysics
Background:
- Tracking dynamic subcellular processes in live cells is challenging due to high 3D imaging precision requirements.
- Traditional z-stack imaging limits temporal resolution and increases phototoxicity.
- There is a need for advanced microscopy methods to observe live cellular dynamics with high spatial and temporal accuracy.
Purpose of the Study:
- To introduce a focus-feedback microscopy algorithm for accurate tracking of structures within a single optical plane over time.
- To overcome the limitations of traditional z-stack imaging in live-cell studies.
- To enable prolonged imaging sessions with minimal phototoxicity for studying dynamic molecular processes.
Main Methods:
- Development and integration of a focus-feedback microscopy algorithm.
- Utilizing cylindrical lenses for z-position detection.
- Bead-based calibration and analysis of time-lapse data using standard and custom tools.
- Step-by-step guidance for implementation in Zeiss Zen or custom microscope software.
Main Results:
- Accurate tracking of subcellular structures within a single optical plane over time.
- Reduced imaging intervals and light exposure compared to z-stack imaging.
- Enabled prolonged imaging sessions with minimal phototoxicity.
- Successful application in tracking single gene loci within the nucleus.
Conclusions:
- Focus-feedback microscopy offers a powerful solution for tracking dynamic subcellular processes in live cells.
- The protocol facilitates precise spatial and temporal resolution, ideal for studying molecular dynamics.
- Potential extensions include tracking cytoplasmic structures like organelles and vesicles, compatible with multichannel and single-molecule imaging.
