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Over the past decade, substantial progress has been made in the rational design, synthesis, and application of FlAAs, driven by advances in organic synthesis, C\u2013H functionalization, and translation-based incorporation technologies. This review summarizes contemporary strategies for constructing FlAAs, with a particular emphasis on structure\u2013property relationships and modular design principles. Beyond chemical synthesis, we summarize translation-machinery-mediated strategies, including chemical tRNA aminoacylation, flexizyme-based charging, and genetic code expansion, which collectively enable the incorporation of FlAAs into peptides and proteins in vitro and in living cells. Finally, we showcase representative applications of FlAAs in live-cell imaging, super-resolution microscopy, and photodynamic therapy, illustrating how compact fluorescent amino acids can serve as both imaging reporters and functional photoactive units. Together, this review summarizes current molecular design strategies for FlAAs and discusses how chemical modification of amino acid scaffolds can be linked to specific biological applications.<\/jats:p>","DOI":"10.1021\/cbmi.6c00017","type":"journal-article","created":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T13:45:11Z","timestamp":1774359911000},"page":"1665-1677","source":"Crossref","is-referenced-by-count":2,"title":["Design, Synthesis,\nand Applications of Fluorescent\nAmino Acids in Bioimaging"],"prefix":"10.1021","volume":"4","author":[{"given":"Yanyang","family":"Zhao","sequence":"first","affiliation":[{"name":"Nanjing University , , ,","place":["Nanjing, China, 210093"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xu","family":"Wang","sequence":"additional","affiliation":[{"name":"Nanjing University , , ,","place":["Nanjing, China, 210093"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"Wang","sequence":"additional","affiliation":[{"name":"Nanjing University , , ,","place":["Nanjing, China, 210093"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc","family":"Vendrell","sequence":"additional","affiliation":[{"name":"The University of Edinburgh , , ,","place":["Edinburgh, U.K., EH16 4UU"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1585-6760","authenticated-orcid":true,"given":"Huan","family":"Wang","sequence":"additional","affiliation":[{"name":"Nanjing University , , ,","place":["Nanjing, China, 210093"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,3,24]]},"reference":[{"issue":"4","key":"2026083104105965400_cit1","doi-asserted-by":"publisher","first-page":"855","DOI":"10.1021\/cb500078u","article-title":"Bright building blocks for chemical biology","volume":"9","author":"Lavis","year":"2014","journal-title":"ACS Chem. 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