Semi-automatic Geometrical Reconstruction and Analysis of Filopodia Dynamics in 4D Two-Photon Microscopy Images

Semi-automatic Geometrical Reconstruction and Analysis of Filopodia Dynamics in 4D Two-Photon Microscopy Images | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF software Semi-automatic Geometrical Reconstruction and Analysis of Filopodia Dynamics in 4D Two-Photon Microscopy Images Blaž Brence, Josephine Brummer, Vincent J. Dercksen, Mehmet Neset Özel, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4681083/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Feb, 2026 Read the published version in BMC Bioinformatics → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Filopodia are thin and dynamic membrane protrusions that play a crucial role in cell migration, axon guidance, and other processes where cells explore and interact with their surroundings. Historically, filopodial dynamics have been studied in great detail in 2D in cultured cells, and more recently in 3D culture as well as living brains.However, there is a lack of efficient tools to trace and track filopodia in 4D images of complex brain cells. Results: To address this issue, we have developed a semi-automatic workflow for tracing filopodia in 3D images and tracking the traced filopodia over time.The workflow was developed based on high-resolution data of photoreceptor axon terminals in the in vivo context of normal Drosophila brain development, but devised to be applicable to filopodia in any system, including at different temporal and spatial scales. In contrast to the pre-existing methods, our workflow relies solely on the original intensity images without the requirement for segmentation or complex preprocessing. The workflow was realized in C++ within the Amira software system and consists of two main parts, dataset pre-processing, and geometrical filopodia reconstruction, where each of the two parts comprises multiple steps.In this paper we provide an extensive workflow description and demonstrate its versatility for two different axo-dendritic morphologies, R7 and Dm8 cells.Finally, we provide an analysis of the time requirements for user input and data processing. Conclusion: To facilitate simple application within Amira or other frameworks, we share the source code, which is available at https://github.com/zibamira/filopodia-tool. 4D image analysis filopodia two-photon microscopy tracing tracking semi-automatic workflow Full Text Additional Declarations No competing interests reported. Supplementary Files Video1.mpg Cite Share Download PDF Status: Published Journal Publication published 16 Feb, 2026 Read the published version in BMC Bioinformatics → Version 1 posted Editorial decision: Revision requested 04 Dec, 2025 Reviews received at journal 23 Oct, 2025 Reviewers agreed at journal 22 Oct, 2025 Reviews received at journal 20 Oct, 2025 Reviewers agreed at journal 20 Oct, 2025 Reviewers invited by journal 20 Oct, 2025 Submission checks completed at journal 01 Oct, 2025 First submitted to journal 30 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4681083","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"software","associatedPublications":[],"authors":[{"id":534090012,"identity":"937506e1-2d14-4988-afb7-77837a88a61f","order_by":0,"name":"Blaž Brence","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYBACAwhlw8AmwcAgQYqWNNK1HAarJ06LufThYw9+VJy355NuYLzxgRgtln1p6YY9Z24zs8kcYLacQZTDzvCYSTO23WZjk0hgk+YhTgv/N2nGf+d4wFr+EGkLmzRjwwEJsBZidDBY9rCZSfYcSzZgkznYbNlDjBZzHuZnEj9q7OzlZzcfvPGDKGsQgLGBRA2jYBSMglEwCnACANbGKLlISiKrAAAAAElFTkSuQmCC","orcid":"","institution":"Zuse Institute Berlin","correspondingAuthor":true,"prefix":"","firstName":"Blaž","middleName":"","lastName":"Brence","suffix":""},{"id":534090013,"identity":"0db222b5-ced3-4e8e-ac88-d0b92c22ef1f","order_by":1,"name":"Josephine Brummer","email":"","orcid":"","institution":"Zuse Institute Berlin","correspondingAuthor":false,"prefix":"","firstName":"Josephine","middleName":"","lastName":"Brummer","suffix":""},{"id":534090014,"identity":"3ed921d0-386b-41ab-be5f-41da97a35ed6","order_by":2,"name":"Vincent J. 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