{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,8,2]],"date-time":"2025-08-02T17:10:22Z","timestamp":1754154622529,"version":"3.41.2"},"reference-count":26,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2025,1,27]],"date-time":"2025-01-27T00:00:00Z","timestamp":1737936000000},"content-version":"vor","delay-in-days":26,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2025,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Competitive fitness is a fundamental concept in evolutionary biology that captures the ability of organisms to survive, reproduce, and compete for resources in their environment. Competitive fitness is typically assessed in the lab by growing two or more competitors together and measuring the frequency of each at multiple time points. Traditional microbial competitive fitness assays are labor intensive and involve plating on solid medium and counting colonies. Here, we describe a method to quantitatively measure competitive fitness using fluorescence microscopic imaging and machine\u2010learning\u2010enabled image analysis to directly count the number of cells from each competitor in the mixed population. This high\u2010throughput, primarily automated, and efficient process gives accurate and reproducible results for competitive fitness. Here, we describe the entire process, from sample preparation through microscopy to quantification, and provide instructions and scripts for the image analysis, fitness calculations, and sample data visualizations. \u00a9 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Sample preparation<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Photographing fluorescing and non\u2010fluorescing cells using an EVOS microscope<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Counting fluorescing and non\u2010fluorescing cells with Orbit Image Analysis<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Getting the average cell counts per well and changing the file names<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Calculating competitive fitness using R<\/jats:p>","DOI":"10.1002\/cpz1.70093","type":"journal-article","created":{"date-parts":[[2025,1,27]],"date-time":"2025-01-27T10:09:46Z","timestamp":1737972586000},"source":"Crossref","is-referenced-by-count":0,"title":["Quantifying Competitive Fitness in Yeast with High\u2010Throughput Fluorescence Microscopy Imaging"],"prefix":"10.1002","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2154-3875","authenticated-orcid":false,"given":"Aruni S.","family":"Sumanarathne","sequence":"first","affiliation":[{"name":"Department of Microbiology University of Manitoba  Winnipeg Manitoba Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0781-9356","authenticated-orcid":false,"given":"Aleeza C.","family":"Gerstein","sequence":"additional","affiliation":[{"name":"Department of Microbiology University of Manitoba  Winnipeg Manitoba Canada"},{"name":"Department of Statistics University of Manitoba  Winnipeg Manitoba Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,1,27]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuaa001"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.18637\/jss.v066.i05"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1098\/rsbl.2010.0580"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2019.00471"},{"volume-title":"An introduction to population genetics theory","year":"2017","author":"Crow J. 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