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It is based on localizing single fluorophores by rapid probing with a patterned beam that features a local intensity minimum. Current implementations, however, are complex and expensive and are limited in speed and robustness. Here, we show that a combination of an electro-optical modulator with a segmented birefringent element such as a spatial light modulator produces a variable phase plate for which the phase can be scanned on the MHz timescale. Bisected or top-hat phase patterns generate high-contrast compact excitation point-spread functions for MINFLUX localization in the x, y, and z-direction, respectively, which can be scanned across a fluorophore within a microsecond, switched within 60 microseconds and alternated among different excitation wavelengths. We discuss how to compensate for non-optimal performance of the components and present a robust 3D and multi-color MINFLUX excitation module, which we envision as an integral component of a high-performance and cost-effective open-source MINFLUX.<\/jats:p>","DOI":"10.1038\/s41377-024-01487-1","type":"journal-article","created":{"date-parts":[[2024,6,7]],"date-time":"2024-06-07T06:02:16Z","timestamp":1717740136000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Simple and robust 3D MINFLUX excitation with a variable phase plate"],"prefix":"10.1038","volume":"13","author":[{"given":"Takahiro","family":"Deguchi","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6640-9250","authenticated-orcid":false,"given":"Jonas","family":"Ries","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,6,7]]},"reference":[{"key":"1487_CR1","doi-asserted-by":"publisher","first-page":"606","DOI":"10.1126\/science.aak9913","volume":"355","author":"F Balzarotti","year":"2017","unstructured":"Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606\u2013612 (2017).","journal-title":"Science"},{"key":"1487_CR2","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1038\/s41592-019-0688-0","volume":"17","author":"KC Gwosch","year":"2020","unstructured":"Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217\u2013224 (2020).","journal-title":"Nat. Methods"},{"key":"1487_CR3","doi-asserted-by":"publisher","first-page":"20607","DOI":"10.1073\/pnas.2009364117","volume":"117","author":"JK Pape","year":"2020","unstructured":"Pape, J. K. et al. Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOS proteins. Proc. Natl Acad. Sci. USA 117, 20607\u201320614 (2020).","journal-title":"Proc. Natl Acad. Sci. 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Science 379, 1004\u20131010 (2023).","journal-title":"Science"},{"key":"1487_CR7","doi-asserted-by":"publisher","first-page":"1010","DOI":"10.1126\/science.ade2676","volume":"379","author":"T Deguchi","year":"2023","unstructured":"Deguchi, T. et al. Direct observation of motor protein stepping in living cells using MINFLUX. Science 379, 1010\u20131015 (2023).","journal-title":"Science"},{"key":"1487_CR8","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21652-z","volume":"12","author":"R Schmidt","year":"2021","unstructured":"Schmidt, R. et al. MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nat. Commun. 12, 1478 (2021).","journal-title":"Nat. Commun."},{"key":"1487_CR9","doi-asserted-by":"publisher","DOI":"10.1038\/s41377-022-00763-2","volume":"11","author":"LA Masullo","year":"2022","unstructured":"Masullo, L. A. & Stefani, F. D. Multiphoton single-molecule localization by sequential excitation with light minima. 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T.D. and J.R. are co-inventors.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}}],"article-number":"134"}}