{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T15:15:09Z","timestamp":1774624509792,"version":"3.50.1"},"reference-count":29,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2019,10,8]],"date-time":"2019-10-08T00:00:00Z","timestamp":1570492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"funder":[{"name":"Spanish Ministry of Economy and Competitiveness","award":["DPI2015-64221-C2-2"],"award-info":[{"award-number":["DPI2015-64221-C2-2"]}]},{"name":"Spanish Ministry of Economy and Competitiveness","award":["RTI2018-094494-B-C22"],"award-info":[{"award-number":["RTI2018-094494-B-C22"]}]},{"name":"Spanish Ministry of Economy and Competitiveness","award":["MCIU\/AEI\/FEDER, UE"],"award-info":[{"award-number":["MCIU\/AEI\/FEDER, UE"]}]},{"name":"Spanish Ministry of Economy and Competitiveness","award":["RTC-2015-4167-1"],"award-info":[{"award-number":["RTC-2015-4167-1"]}]},{"name":"Spanish Ministry of Economy and Competitiveness","award":["RTC-2017-6218-1"],"award-info":[{"award-number":["RTC-2017-6218-1"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2020,3,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:sec>\n                  <jats:title>Motivation<\/jats:title>\n                  <jats:p>Recent advances in multiplex immunostaining and multispectral cytometry have opened the door to simultaneously visualizing an unprecedented number of biomarkers both in liquid and solid samples. Properly unmixing fluorescent emissions is a challenging task, which normally requires the characterization of the individual fluorochromes from control samples. As the number of fluorochromes increases, the cost in time and use of reagents becomes prohibitively high. Here, we present a fully unsupervised blind spectral unmixing method for the separation of fluorescent emissions in highly mixed spectral data, without the need for control samples. To this end, we extend an existing method based on non-negative Matrix Factorization, and introduce several critical improvements: initialization based on the theoretical spectra, automated selection of \u2018sparse\u2019 data and use of a re-initialized multilayer optimizer.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Our algorithm is exhaustively tested using synthetic data to study its robustness against different levels of colocalization, signal to noise ratio, spectral resolution and the effect of errors in the initialization of the algorithm. Then, we compare the performance of our method to that of traditional spectral unmixing algorithms using novel multispectral flow and image cytometry systems. In all cases, we show that our blind unmixing algorithm performs robust unmixing of highly spatially and spectrally mixed data with an unprecedently low computational cost. In summary, we present the first use of a blind unmixing method in multispectral flow and image cytometry, opening the door to the widespread use of our method to efficiently pre-process multiplex immunostaining samples without the need of experimental controls.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Availability and implementation<\/jats:title>\n                  <jats:p>https:\/\/github.com\/djimenezsanchez\/Blind_Unmixing_NMF_RI\/ contains the source code and all datasets used in this manuscript.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Supplementary information<\/jats:title>\n                  <jats:p>Supplementary data are available at Bioinformatics online.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1093\/bioinformatics\/btz751","type":"journal-article","created":{"date-parts":[[2019,10,1]],"date-time":"2019-10-01T19:27:59Z","timestamp":1569958079000},"page":"1590-1598","source":"Crossref","is-referenced-by-count":30,"title":["NMF-RI: blind spectral unmixing of highly mixed multispectral flow and image cytometry data"],"prefix":"10.1093","volume":"36","author":[{"given":"Daniel","family":"Jim\u00e9nez-S\u00e1nchez","sequence":"first","affiliation":[{"name":"IDISNA , Ciberonc and Solid Tumours and Biomarkers Program, Center for Applied Medical Research, University of Navarra, Pamplona 31008, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mikel","family":"Ariz","sequence":"additional","affiliation":[{"name":"IDISNA , Ciberonc and Solid Tumours and Biomarkers Program, Center for Applied Medical Research, University of Navarra, Pamplona 31008, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos\u00e9 M\u00e1rio","family":"Morgado","sequence":"additional","affiliation":[{"name":"Cytognos SL , Salamanca 37900, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iv\u00e1n","family":"Cort\u00e9s-Dom\u00ednguez","sequence":"additional","affiliation":[{"name":"IDISNA , Ciberonc and Solid Tumours and Biomarkers Program, Center for Applied Medical Research, University of Navarra, Pamplona 31008, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carlos","family":"Ortiz-de-Sol\u00f3rzano","sequence":"additional","affiliation":[{"name":"IDISNA , Ciberonc and Solid Tumours and Biomarkers Program, Center for Applied Medical Research, University of Navarra, Pamplona 31008, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2019,10,8]]},"reference":[{"key":"2023060910374902100_btz751-B1","first-page":"V-621","article-title":"New algorithms for non-negative matrix factorization in applications to blind source separation","volume":"5","author":"Cichocki","year":"2006","journal-title":"2006 IEEE International Conference on Acoustics Speed and Signal Processing Proceedings, Toulouse, France"},{"key":"2023060910374902100_btz751-B2","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1162\/neco.1994.6.4.559","article-title":"What is the goal of sensory coding?","volume":"6","author":"Field","year":"1994","journal-title":"Neural Comput"},{"key":"2023060910374902100_btz751-B3","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1002\/cyto.a.20311","article-title":"Spectral imaging: principles and applications","volume":"69A","author":"Garini","year":"2006","journal-title":"Cytometry Part A"},{"key":"2023060910374902100_btz751-B4","doi-asserted-by":"crossref","first-page":"e542","DOI":"10.1016\/S1470-2045(16)30406-5","article-title":"Predictive biomarkers for checkpoint inhibitor-based immunotherapy","volume":"17","author":"Gibney","year":"2016","journal-title":"Lancet Oncol"},{"key":"2023060910374902100_btz751-B5","first-page":"1457","article-title":"Non-negative matrix factorization with sparseness constraints","volume":"5","author":"Hoyer","year":"2004","journal-title":"J. Mach. Learn. Res"},{"key":"2023060910374902100_btz751-B6","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1186\/s12938-015-0107-4","article-title":"Two-hierarchical non-negative matrix factorization distinguishing the fluorescent targets from autofluorescence for fluorescence imaging","volume":"14","author":"Huang","year":"2015","journal-title":"BioMed. Eng. Online"},{"key":"2023060910374902100_btz751-B7","first-page":"192","article-title":"Blind Source Separation based on ICA algorithm applied to multispectral fluorescence imaging","volume":"11","author":"Lafi","year":"2016","journal-title":"Int. Rev. Comp. Software"},{"key":"2023060910374902100_btz751-B8","doi-asserted-by":"crossref","first-page":"882","DOI":"10.1002\/cyto.a.20774","article-title":"Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism","volume":"75A","author":"Le Roy","year":"2009","journal-title":"Cytometry Part A"},{"key":"2023060910374902100_btz751-B9","first-page":"556","article-title":"Algorithms for non-negative matrix factorization","volume":"13","author":"Lee","year":"2001","journal-title":"Adv. Neural Inform. Process. Syst"},{"key":"2023060910374902100_btz751-B10","doi-asserted-by":"crossref","first-page":"15792.","DOI":"10.1038\/s41598-017-15952-y","article-title":"Statistically strong label-free quantitative identification of native fluorophores in a biological sample","volume":"7","author":"Mahbub","year":"2017","journal-title":"Sci. Rep"},{"key":"2023060910374902100_btz751-B11","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1109\/TGRS.2006.888466","article-title":"Endmember extraction from highly mixed data using minimum volume constrained non-negative matrix factorization","volume":"45","author":"Miao","year":"2007","journal-title":"IEEE Trans. Geosci. Remote Sens"},{"key":"2023060910374902100_btz751-B12","doi-asserted-by":"crossref","first-page":"3791","DOI":"10.1016\/j.bpj.2008.10.068","article-title":"Blind source separation techniques for the decomposition of multiply labeled fluorescence images","volume":"96","author":"Neher","year":"2009","journal-title":"Biophys. J"},{"key":"2023060910374902100_btz751-B13","doi-asserted-by":"crossref","first-page":"508","DOI":"10.1002\/cyto.a.22272","article-title":"Generalized unmixing model for multispectral flow cytometry utilizing non-square compensation matrices","volume":"83A","author":"Novo","year":"2013","journal-title":"Cytometry Part A"},{"key":"2023060910374902100_btz751-B14","doi-asserted-by":"crossref","first-page":"13380.","DOI":"10.1038\/s41598-017-13942-8","article-title":"Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues","volume":"7","author":"Parra","year":"2017","journal-title":"Sci. Rep"},{"key":"2023060910374902100_btz751-B15","first-page":"43","article-title":"Novel platforms of multiplexed immunofluorescence for study of paraffin tumor tissues","volume-title":"J. Cancer Treat. Diagn.","author":"Parra","year":"2018"},{"key":"2023060910374902100_btz751-B16","first-page":"2079","volume-title":"Microscopy: Science, Technology, Applications and Education. Microscopy Book Series\u2013\u2013Number 4","author":"Pengo","year":"2010"},{"key":"2023060910374902100_btz751-B17","doi-asserted-by":"crossref","first-page":"e78504.","DOI":"10.1371\/journal.pone.0078504","article-title":"Efficient blind spectral unmixing of fluorescently labeled samples using multi-layer non-negative matrix factorization","volume":"8","author":"Pengo","year":"2013","journal-title":"PLoS One"},{"key":"2023060910374902100_btz751-B18","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1007\/s10851-011-0276-0","article-title":"On endmember identification in hyperspectral images without pure pixels: a comparison of algorithms","volume":"42","author":"Plaza","year":"2012","journal-title":"J. Math. Imaging Vis"},{"key":"2023060910374902100_btz751-B19","doi-asserted-by":"crossref","first-page":"1776","DOI":"10.1109\/TGRS.2016.2633279","article-title":"Matrix-vector non-negative tensor factorization for blind unmixing of hyperspectral imagery","volume":"55","author":"Qian","year":"2017","journal-title":"IEEE Trans. Geosci. Remote Sens"},{"key":"2023060910374902100_btz751-B20","doi-asserted-by":"crossref","first-page":"874","DOI":"10.1109\/TIM.2016.2516318","article-title":"Target\/background classification regularized non-negative matrix factorization for fluorescence unmixing","volume":"65","author":"Qin","year":"2016","journal-title":"IEEE Trans. Instrum Meas"},{"key":"2023060910374902100_btz751-B21","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1109\/LGRS.2014.2325874","article-title":"Spectral unmixing of hyperspectral imagery using multilayer NMF","volume":"12","author":"Rajabi","year":"2015","journal-title":"IEEE Geosci. Remote Sens. Lett"},{"key":"2023060910374902100_btz751-B22","doi-asserted-by":"crossref","first-page":"1706.","DOI":"10.3390\/rs10111706","article-title":"Inertia-constrained pixel-by-pixel non-negative matrix factorisation: a hyperspectral unmixing method dealing with intra-class variability","volume":"10","author":"Revel","year":"2018","journal-title":"Remote Sens"},{"key":"2023060910374902100_btz751-B23","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1002\/1097-0320(20011101)45:3<194::AID-CYTO1163>3.0.CO;2-C","article-title":"Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats","volume":"45","author":"Roederer","year":"2001","journal-title":"Cytometry"},{"key":"2023060910374902100_btz751-B24","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1038\/nri.2016.56","article-title":"Computational flow cytometry: helping to make sense of high-dimensional immunology data","volume":"16","author":"Saeys","year":"2016","journal-title":"Nat. Rev. Immunol"},{"key":"2023060910374902100_btz751-B25","volume-title":"Workshop on Hyperspectral Image and Signal Processing, Evolution in Remote Sensing, Tokyo, Japan","author":"Tong","year":"2017"},{"key":"2023060910374902100_btz751-B26","doi-asserted-by":"crossref","DOI":"10.1109\/JSTARS.2015.2508448","article-title":"Hypergraph-regularized sparse NMF for hyperspectral unmixing","volume":"9","author":"Wang","year":"2016","journal-title":"IEEE J. Sel. Top. Appl. Earth Obs. Remote Sens"},{"key":"2023060910374902100_btz751-B27","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1109\/TIP.2010.2079810","article-title":"Autofluorescence removal by non-negative matrix factorization","volume":"20","author":"Woolfe","year":"2011","journal-title":"IEEE Trans. Image Process"},{"key":"2023060910374902100_btz751-B28","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1109\/TGRS.2011.2161320","article-title":"Coupled non-negative matrix factorization unmixing for hyperspectral and multispectral data fusion","volume":"50","author":"Yokoya","year":"2012","journal-title":"IEEE Trans. Geosci. Remote Sens"},{"key":"2023060910374902100_btz751-B29","first-page":"245","article-title":"Spectral imaging and linear unmixing in light microscopy","volume":"95","author":"Zimmermann","year":"2005","journal-title":"Adv. Biochem. Eng.\/Biotechnol"}],"container-title":["Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/academic.oup.com\/bioinformatics\/advance-article-pdf\/doi\/10.1093\/bioinformatics\/btz751\/30267342\/btz751.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article-pdf\/36\/5\/1590\/50552849\/bioinformatics_36_5_1590.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article-pdf\/36\/5\/1590\/50552849\/bioinformatics_36_5_1590.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,6,9]],"date-time":"2023-06-09T10:38:30Z","timestamp":1686307110000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article\/36\/5\/1590\/5583734"}},"subtitle":[],"editor":[{"given":"Robert","family":"Murphy","sequence":"additional","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2019,10,8]]},"references-count":29,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2020,3,1]]}},"URL":"https:\/\/doi.org\/10.1093\/bioinformatics\/btz751","relation":{},"ISSN":["1367-4803","1367-4811"],"issn-type":[{"value":"1367-4803","type":"print"},{"value":"1367-4811","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2020,3]]},"published":{"date-parts":[[2019,10,8]]}}}