{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T02:10:06Z","timestamp":1773108606979,"version":"3.50.1"},"update-to":[{"DOI":"10.1371\/journal.pcbi.1009242","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,4,14]],"date-time":"2022-04-14T00:00:00Z","timestamp":1649894400000}}],"reference-count":12,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2022,4,4]],"date-time":"2022-04-04T00:00:00Z","timestamp":1649030400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006447","name":"Universit\u00e4t Z\u00fcrich","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100006447","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012390","name":"SystemsX.ch","doi-asserted-by":"crossref","award":["MecanX RTD"],"award-info":[{"award-number":["MecanX RTD"]}],"id":[{"id":"10.13039\/501100012390","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001711","name":"Schweizerischer Nationalfonds zur F\u00f6rderung der Wissenschaftlichen Forschung","doi-asserted-by":"publisher","award":["CR22I2-166110"],"award-info":[{"award-number":["CR22I2-166110"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.ploscompbiol.org"],"crossmark-restriction":false},"short-container-title":["PLoS Comput Biol"],"abstract":"<jats:p>\n                    Ratiometric time-lapse FRET analysis requires a robust and accurate processing pipeline to eliminate bias in intensity measurements on fluorescent images before further quantitative analysis can be conducted. This level of robustness can only be achieved by supplementing automated tools with built-in flexibility for manual ad-hoc adjustments. FRET-IBRA is a modular and fully parallelized configuration file-based tool written in Python. It simplifies the FRET processing pipeline to achieve accurate, registered, and unified ratio image stacks. The flexibility of this tool to handle discontinuous image frame sequences with tailored configuration parameters further streamlines the processing of outliers and time-varying effects in the original microscopy images. FRET-IBRA offers cluster-based channel background subtraction, photobleaching correction, and ratio image construction in an all-in-one solution without the need for multiple applications, image format conversions, and\/or plug-ins. The package accepts a variety of input formats and outputs TIFF image stacks along with performance measures to detect both the quality and failure of the background subtraction algorithm on a per frame basis. Furthermore, FRET-IBRA outputs images with superior signal-to-noise ratio and accuracy in comparison to existing background subtraction solutions, whilst maintaining a fast runtime. We have used the FRET-IBRA package extensively to quantify the spatial distribution of calcium ions during pollen tube growth under mechanical constraints. Benchmarks against existing tools clearly demonstrate the need for FRET-IBRA in extracting reliable insights from FRET microscopy images of dynamic physiological processes at high spatial and temporal resolution. The source code for Linux and Mac operating systems is released under the BSD license and, along with installation instructions, test images, example configuration files, and a step-by-step tutorial, is freely available at\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/github.com\/gmunglani\/fret-ibra\" xlink:type=\"simple\">github.com\/gmunglani\/fret-ibra<\/jats:ext-link>\n                    .\n                  <\/jats:p>","DOI":"10.1371\/journal.pcbi.1009242","type":"journal-article","created":{"date-parts":[[2022,4,4]],"date-time":"2022-04-04T13:37:10Z","timestamp":1649079430000},"page":"e1009242","update-policy":"https:\/\/doi.org\/10.1371\/journal.pcbi.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Fast and flexible processing of large FRET image stacks using the FRET-IBRA toolkit"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0914-6834","authenticated-orcid":true,"given":"Gautam","family":"Munglani","sequence":"first","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6552-4708","authenticated-orcid":true,"given":"Hannes","family":"Vogler","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0522-8974","authenticated-orcid":true,"given":"Ueli","family":"Grossniklaus","sequence":"additional","affiliation":[]}],"member":"340","published-online":{"date-parts":[[2022,4,4]]},"reference":[{"issue":"1","key":"pcbi.1009242.ref001","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1111\/j.1365-2818.2008.03109.x","article-title":"Robust approaches to quantitative ratiometric FRET imaging of CFP\/YFP fluorophores under confocal microscopy","volume":"233","author":"M Tadross","year":"2009","journal-title":"J Microsc"},{"key":"pcbi.1009242.ref002","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1016\/B978-0-12-407761-4.00025-7","article-title":"Quantitative ratiometric imaging of FRET-biosensors in living cells","volume":"114","author":"D Spiering","year":"2013","journal-title":"Methods Cell Biol"},{"issue":"12","key":"pcbi.1009242.ref003","doi-asserted-by":"crossref","first-page":"3267","DOI":"10.1093\/jxb\/erx032","article-title":"Oscillatory signatures underlie growth regimes in Arabidopsis pollen tubes: computational methods to estimate tip location, periodicity, and synchronization in growing cells","volume":"68","author":"D Damineli","year":"2017","journal-title":"J Exp Bot"},{"key":"pcbi.1009242.ref004","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1186\/1471-2105-14-297","article-title":"An automatic method for robust and fast cell detection in bright field images from high-throughput microscopy","volume":"14","author":"F Buggenthin","year":"2013","journal-title":"BMC Bioinformatics"},{"issue":"7","key":"pcbi.1009242.ref005","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1038\/nmeth.1984","article-title":"An image analysis toolbox for high-throughput C. elegans assays","volume":"9","author":"C W\u00e4hlby","year":"2012","journal-title":"Nat Methods"},{"issue":"3","key":"pcbi.1009242.ref006","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1111\/jmi.12178","article-title":"Pipeline for illumination correction of images for high-throughput microscopy","volume":"256","author":"S Singh","year":"2014","journal-title":"J Microsc"},{"issue":"7","key":"pcbi.1009242.ref007","doi-asserted-by":"crossref","first-page":"703","DOI":"10.1038\/nbt.3626","article-title":"Software tools for single-cell tracking and quantification of cellular and molecular properties","volume":"34","author":"O Hilsenbeck","year":"2016","journal-title":"Nat Biotechnol"},{"key":"pcbi.1009242.ref008","volume-title":"Microscopic Image Analysis with Applications in Biology","author":"M Schwarzfischer","year":"2011"},{"issue":"5","key":"pcbi.1009242.ref009","doi-asserted-by":"crossref","first-page":"404","DOI":"10.1038\/nmeth.3323","article-title":"CIDRE: an illumination-correction method for optical microscopy","volume":"12","author":"K Smith","year":"2015","journal-title":"Nat Methods"},{"key":"pcbi.1009242.ref010","doi-asserted-by":"crossref","first-page":"14836","DOI":"10.1038\/ncomms14836","article-title":"A BaSiC tool for background and shading correction of optical microscopy images","volume":"8","author":"T Peng","year":"2017","journal-title":"Nat Commun"},{"key":"pcbi.1009242.ref011","first-page":"275","volume-title":"Quantification of mechanical forces and physiological processes involved in pollen tube growth using microfluidics and microrobotics","author":"J Burri","year":"2020"},{"key":"pcbi.1009242.ref012","unstructured":"Cardinale J. 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