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However, aligning images acquired with different modalities, settings or instruments remains challenging. Here, we introduce NucleiSky, a microscopy image registration framework that utilises the spatial arrangement of nuclei or other landmarks as an intrinsic biological fingerprint. NucleiSky represents images as constellations of centroids and aligns them using geometric algorithms and spatial consensus scoring. In benchmark datasets, NucleiSky could localise query regions within larger reference images using as few as five nuclei. We show that NucleiSky can locate high-magnification fields of view within low-magnification overview scans, map these alignments to additional channels, support live brightfield-to-fixed registration using synthetic nuclear labels and guide microscope re-targeting. We further show that the same constellation-matching principle can be extended to 3D localisation and to non-nuclear landmarks. These findings establish local landmark geometry as an intrinsic spatial fingerprint that enables localisation and registration across imaging scales, modalities and microscopy platforms. NucleiSky is available as an open-source Python package and as notebook-based applications.<\/jats:p>","DOI":"10.1242\/jcs.265152","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T10:27:56Z","timestamp":1786703276000},"source":"Crossref","is-referenced-by-count":2,"title":["NucleiSky enables cross-scale multimodal registration of microscopy data using nuclei constellations"],"prefix":"10.1242","volume":"139","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-8923-568X","authenticated-orcid":false,"given":"Iv\u00e1n","family":"Hidalgo-Cenalmor","sequence":"first","affiliation":[{"name":"Turku Bioscience Centre, University of Turku and \u00c5bo Akademi University 1 , FI-20520 Turku ,","place":["Finland"]},{"name":"\u00c5bo Akademi University 2 Faculty of Science and Engineering, Cell Biology , , FI-20520 Turku ,","place":["Finland"]},{"name":"InFLAMES Research Flagship Centre, University of Turku 3 , FI-20014 Turku ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ad\u00e1n","family":"Olgu\u00edn-Olgu\u00edn","sequence":"additional","affiliation":[{"name":"Turku Bioscience Centre, University of Turku and \u00c5bo Akademi University 1 , FI-20520 Turku ,","place":["Finland"]},{"name":"\u00c5bo Akademi University 2 Faculty of Science and Engineering, Cell Biology , , FI-20520 Turku ,","place":["Finland"]},{"name":"InFLAMES Research Flagship Centre, University of Turku 3 , FI-20014 Turku ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7406-9797","authenticated-orcid":false,"given":"Carolina","family":"Prieto","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa 4 , 2780-157 Oeiras ,","place":["Portugal"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8136-8928","authenticated-orcid":false,"given":"Johannes","family":"Kumra Ahnlide","sequence":"additional","affiliation":[{"name":"Lund University 5 Department of Clinical Sciences Lund, Division of Infection Medicine, Faculty of Medicine , , SE-221 84 Lund ,","place":["Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9481-9951","authenticated-orcid":false,"given":"Pontus","family":"Nordenfelt","sequence":"additional","affiliation":[{"name":"Lund University 5 Department of Clinical Sciences Lund, Division of Infection Medicine, Faculty of Medicine , , SE-221 84 Lund ,","place":["Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2043-5234","authenticated-orcid":false,"given":"Ricardo","family":"Henriques","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa 4 , 2780-157 Oeiras ,","place":["Portugal"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0430-1463","authenticated-orcid":false,"given":"Mario","family":"Del Rosario","sequence":"additional","affiliation":[{"name":"\u00c5bo Akademi University 2 Faculty of Science and Engineering, Cell Biology , , FI-20520 Turku ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9286-920X","authenticated-orcid":false,"given":"Guillaume","family":"Jacquemet","sequence":"additional","affiliation":[{"name":"Turku Bioscience Centre, University of Turku and \u00c5bo Akademi University 1 , FI-20520 Turku ,","place":["Finland"]},{"name":"\u00c5bo Akademi University 2 Faculty of Science and Engineering, Cell Biology , , FI-20520 Turku ,","place":["Finland"]},{"name":"InFLAMES Research Flagship Centre, University of Turku 3 , FI-20014 Turku ,","place":["Finland"]},{"name":"Foundation for the Finnish Cancer Institute 6 , Tukholmankatu 8, FI-00290 Helsinki ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2026,10,14]]},"reference":[{"key":"2026101405534176600_JCS265152C1","doi-asserted-by":"publisher","first-page":"1223","DOI":"10.1038\/s41467-019-09231-9","article-title":"Automating multimodal microscopy with NanoJ-Fluidics","volume":"10","author":"Almada","year":"2019","journal-title":"Nat. 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