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We present a fluorescent bioimaging approach for detecting\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    , based on aptamer F23. Conjugated with fluorescent dye, its detection efficacy was evaluated on 15 Gram\u2010negative and \u2010positive bacteria, including fixed and live cells, as homogeneous and heterogeneous populations. We developed an automated, open\u2010access software for quantifying microscopy images. Its high sensitivity enables accurate quantification of bacteria labeled with aptamers. For example, it successfully detected 1122\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    cells labeled with aptamer F23 out of a total of 1123\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    cells in a single image. With 200,000 analyzed bacteria, we demonstrated that the aptamer effectively detects various reference and clinical strains of\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    , while failing to detect Gram\u2010positive\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    ,\n                    <jats:italic>Staphylococcus haemolyticus<\/jats:italic>\n                    ,\n                    <jats:italic>Staphylococcus epidermidis<\/jats:italic>\n                    , and\n                    <jats:italic>Corynebacterium striatum<\/jats:italic>\n                    , as well as Gram\u2010negative\n                    <jats:italic>Klebsiella pneumoniae<\/jats:italic>\n                    ,\n                    <jats:italic>Acinetobacter baumannii<\/jats:italic>\n                    , and\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    . This aptamer is therefore a promising tool to distinguish\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    from different strains of the skin microbiota. However, our quantitative method also revealed partial labeling to other bacterial cells, highlighting the issue of refining aptamer selection to improve selectivity.\n                  <\/jats:p>","DOI":"10.1002\/mbo3.70202","type":"journal-article","created":{"date-parts":[[2025,12,23]],"date-time":"2025-12-23T07:10:59Z","timestamp":1766473859000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Bioimaging With Fluorescent Nucleic\u2010Acid Aptamers for the Specific Detection and Quantification of\n                    <i>Pseudomonas aeruginosa<\/i>\n                    Alone and in Heterogeneous Bacterial Populations"],"prefix":"10.1002","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4167-3987","authenticated-orcid":false,"given":"Chaimae","family":"Mezouarhi","sequence":"first","affiliation":[{"name":"UMR7021 Laboratory of Bioimaging and Pathologies CNRS University of Strasbourg Illkirch Strasbourg France"},{"name":"UMR7242 Laboratory of Biotechnology and Cell Signaling CNRS University of Strasbourg Illkirch Strasbourg France"},{"name":"Medical Biotechnology Laboratory, Medical and Pharmacy School Mohammed V University Rabat Morocco"},{"name":"Prevention and Therapeutics Center, MAScIR Mohammed VI Polytechnic University Ben Guerir Morocco"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-2413-8325","authenticated-orcid":false,"given":"Romain","family":"Vauchelles","sequence":"additional","affiliation":[{"name":"UMR7021 Laboratory of Bioimaging and Pathologies CNRS University of Strasbourg Illkirch Strasbourg France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1359-3170","authenticated-orcid":false,"given":"Basma","family":"Abdallah","sequence":"additional","affiliation":[{"name":"UMR7021 Laboratory of Bioimaging and Pathologies CNRS University of Strasbourg Illkirch Strasbourg France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6682-8551","authenticated-orcid":false,"given":"R\u00e9gine","family":"Janel","sequence":"additional","affiliation":[{"name":"UMR7242 Laboratory of Biotechnology and Cell Signaling CNRS University of Strasbourg Illkirch Strasbourg France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2306-9364","authenticated-orcid":false,"given":"Mouna","family":"Ouadghiri","sequence":"additional","affiliation":[{"name":"Medical Biotechnology Laboratory, Medical and Pharmacy School Mohammed V University Rabat Morocco"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5815-3531","authenticated-orcid":false,"given":"Hassan Ait","family":"Benhassou","sequence":"additional","affiliation":[{"name":"Prevention and Therapeutics Center, MAScIR Mohammed VI Polytechnic University Ben Guerir 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