ProtocolPublished: 28 July 2026Simona V. Antonova ORCID: orcid.org/0009-0006-2977-52741 nAff2,Wim Pomp1,Joseph V. W. Meeussen ORCID: orcid.org/0000-0002-6963-39601 &…Tineke L. Lenstra ORCID: orcid.org/0000-0002-4440-99621 Nature Protocols (2026) Cite this articleSave articleView saved researchSubjectsKineticsMolecular biologySingle-molecule biophysicsWide-field fluorescence microscopyAbstractTracking 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.Key pointsThe focus-feedback microscopy algorithm enables precise single-plane tracking of subcellular structures, reducing imaging intervals and phototoxicity for prolonged live-cell fluorescent imaging.The focus-feedback technique is highly sensitive, versatile, accessible and compatible with single-molecule applications, making it powerful for molecular and subcellular dynamics studies.This is a preview of subscription content, access via your institutionAccess optionsAccess Nature and 54 other Nature Portfolio journalsGet Nature+, our best-value online-access subscription27,99 € / 30 dayscancel any timeLearn moreSubscribe to this journalReceive 12 print issues and online access269,00 € per yearonly 22,42 € per issueLearn moreBuy this articlePurchase on SpringerLinkInstant access to the full article PDF.39,95 €Prices may be subject to local taxes which are calculated during checkoutFig. 1: Schematic of the cylindrical lens setup and the relationship between PSF shape and z-position.Fig. 2: Monitoring FocusFeedbackGUI performance in real-time.Fig. 3: Example of image re-alignment using the ‘Warp image’ option in FocusFeedbackGUI.Fig. 4: Example of fluorescent bead PSF elongation at different z positions.Fig. 5: Bead calibration of the focus-feedback system.Fig. 6: FocusFeedbackGUI Configuration tab.Fig. 7: Kymographic analysis of tracked spots.Fig. 8: Fluorescence intensity and position of tracked spots over time.Data availabilitySource data were derived from Pomp et al.3 and are available via Zenodo at https://doi.org/10.5281/zenodo.16994120 ref. 27.Code availabilityFocusFeedbackGUI and the Jupyter notebooks detailing the interval time and total imaging duration calculation, axial range determination and data analysis code are available at: https://github.com/Lenstralab/FocusFeedbackGUI and https://github.com/Lenstralab/FocusFeedbackAnalysis.ReferencesPark, H. 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AntonovaPresent address: Department of Human Genetics, Leiden University Medical Center, Oncode Institute, Leiden, The NetherlandsAuthors and AffiliationsDivision of Gene Regulation, The Netherlands Cancer Institute, Oncode Institute, Amsterdam, The NetherlandsSimona V. Antonova, Wim Pomp, Joseph V. W. Meeussen & Tineke L. LenstraAuthorsSimona V. AntonovaView author publicationsSearch author on:PubMed Google ScholarWim PompView author publicationsSearch author on:PubMed Google ScholarJoseph V. W. MeeussenView author publicationsSearch author on:PubMed Google ScholarTineke L. LenstraView author publicationsSearch author on:PubMed Google ScholarContributionsConceptualization: T.L.L. Methodology: W.P. and J.V.W.M. Software: W.P. Visualization: J.V.W.M. and W.P. Funding acquisition: T.L.L. Supervision: T.L.L. Writing—original draft: S.V.A. Writing—review and editing: W.P., J.V.W.M. and T.L.L.Corresponding authorCorrespondence to Tineke L. Lenstra.Ethics declarationsCompeting interestsThe authors disclose that the discussed tracking algorithm3 has been licensed to Zeiss, a microscope company, for commercial use.Peer reviewPeer review informationNature Protocols thanks Wulan Deng, who co-reviewed with Bo Wang; Carl Wu, who co-reviewed with Anand Ranjan; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Key referencePomp, W., Meeussen, J. V. W. & Lenstra, T. L. Transcription factor exchange enables prolonged transcriptional bursts. Mol. Cell 84, 1036–1048.e9 (2024): https://doi.org/10.1016/j.molcel.2024.01.020Supplementary informationRights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article