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While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their <jats:italic>in\u2005vivo<\/jats:italic> uses are often limited. Here, we report a near\u2010infrared fluorescent <jats:sc>D<\/jats:sc>\u2010amino acid (FDAA) probe, Cy7ADA, for <jats:italic>in\u2005situ<\/jats:italic> labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria <jats:italic>in\u2005vitro<\/jats:italic> and pathogenic <jats:italic>Staphylococcus aureus<\/jats:italic> in mice after intraperitoneal injection. Because of Cy7\u2019s high tissue penetration and the quick excretion of free probes via urine, real\u2010time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA\u2032s capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three\u2010dimensional pathogen imaging in tissue\u2010cleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating near\u2010infrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections <jats:italic>in\u2005vivo<\/jats:italic>.<\/jats:p>","DOI":"10.1002\/cbic.202400283","type":"journal-article","created":{"date-parts":[[2024,5,7]],"date-time":"2024-05-07T23:28:40Z","timestamp":1715124520000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["<i>In vivo<\/i> Labeling and Intravital Imaging of Bacterial Infection using a Near\u2010infrared Fluorescent <scp>D<\/scp>\u2010Amino Acid Probe"],"prefix":"10.1002","volume":"25","author":[{"given":"Yixuan","family":"Li","sequence":"first","affiliation":[{"name":"Department of Anesthesiology Renji Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China Key Laboratory of Anesthesiology (Shanghai Jiao Tong University) Ministry of Education  China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingjun","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Genetic Engineering Department of Microbiology Microbiome Center School of Life Sciences Fudan University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yahui","family":"Du","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology College of Chemistry and Chemical Engineering Xiamen University  Xiamen China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Po","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology Renji Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China Key Laboratory of Anesthesiology (Shanghai Jiao Tong University) Ministry of Education  China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liqun","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology Renji Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China Key Laboratory of Anesthesiology (Shanghai Jiao Tong University) Ministry of Education  China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5539-4532","authenticated-orcid":false,"given":"Wei","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Genetic Engineering Department of Microbiology Microbiome Center School of Life Sciences Fudan University  Shanghai China"},{"name":"Beijing National Laboratory for Molecular Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,6,24]]},"reference":[{"key":"e_1_2_8_1_1","unstructured":"Antimicrobial Resistance Collaborators Lancet2022 399 629\u2013655."},{"key":"e_1_2_8_2_1","unstructured":"J. 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