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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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>Drosophila<\/jats:italic>\n                      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\n                      <jats:italic>Amira<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      To facilitate simple application within\n                      <jats:italic>Amira<\/jats:italic>\n                      or other frameworks, we share the source code, which is available at\n                      <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"https:\/\/github.com\/zibamira\/filopodia-tool\" ext-link-type=\"uri\">https:\/\/github.com\/zibamira\/filopodia-tool<\/jats:ext-link>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12859-026-06385-4","type":"journal-article","created":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T13:07:54Z","timestamp":1771247274000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Semi-automatic geometrical reconstruction and analysis of filopodia dynamics in 4D two-photon microscopy images"],"prefix":"10.1186","volume":"27","author":[{"given":"Bla\u017e","family":"Brence","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Josephine","family":"Brummer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincent J.","family":"Dercksen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mehmet Neset","family":"\u00d6zel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abhishek","family":"Kulkarni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neele","family":"Wolterhoff","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steffen","family":"Prohaska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter Robin","family":"Hiesinger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Baum","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,2,16]]},"reference":[{"key":"6385_CR1","doi-asserted-by":"publisher","first-page":"10","DOI":"10.1016\/j.semcdb.2022.03.038","volume":"133","author":"CB Wit","year":"2023","unstructured":"Wit CB, Hiesinger PR. 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