Wernersson, ErikGelali, EleniGirelli, GabrieleWang, SuCastillo, DavidMattsson Langseth, ChristofferVerron, QuentinNguyen, Huy Q.Chattoraj, ShyamtanuMartinez Casals, AnnaBlom, HansLundberg, EmmaNilsson, MatsMartí Renom, Marc A.Wu, Chao-TingCrosetto, NicolaBienko, Magda2024-09-132024-09-132024Wernersson E, Gelali E, Girelli G, Wang S, Castillo D, Mattsson Langseth C, et al. Deconwolf enables high-performance deconvolution of widefield fluorescence microscopy images. Nat Methods. 2024 Jul;21(7):1245-56. DOI: 10.1038/s41592-024-02294-71548-7091http://hdl.handle.net/10230/61081Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.application/pdfeng© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41592-024-02294-7SoftwareWide-field fluorescence microscopyinfo:eu-repo/semantics/openAccess