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The advent of high-throughput automated imaging has enabled the capturing of a wide range of morphological features of cells or organisms in response to perturbations at the single-cell resolution. Concurrently, significant advances in machine learning and deep learning, especially in computer vision, have led to substantial improvements in analyzing large-scale high-content images at high throughput. These efforts have facilitated understanding of compound mechanism of action, drug repurposing, characterization of cell morphodynamics under perturbation, and ultimately contributing to the development of novel therapeutics. In this review, we provide a comprehensive overview of the recent advances in the field of morphological profiling. We summarize the image profiling analysis workflow, survey a broad spectrum of analysis strategies encompassing feature engineering\u2013 and deep learning\u2013based approaches, and introduce publicly available benchmark datasets. We place a particular emphasis on the application of deep learning in this pipeline, covering cell segmentation, image representation learning, and multimodal learning. Additionally, we illuminate the application of morphological profiling in phenotypic drug discovery and highlight potential challenges and opportunities in this field.<\/jats:p>","DOI":"10.1093\/bib\/bbae284","type":"journal-article","created":{"date-parts":[[2024,6,18]],"date-time":"2024-06-18T01:12:28Z","timestamp":1718673148000},"source":"Crossref","is-referenced-by-count":43,"title":["Morphological profiling for drug discovery in the era of deep learning"],"prefix":"10.1093","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6726-8728","authenticated-orcid":false,"given":"Qiaosi","family":"Tang","sequence":"first","affiliation":[{"name":"Calico Life Sciences , South San Francisco, CA 94080 , United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ranjala","family":"Ratnayake","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry , Center for Natural Products, Drug Discovery and Development, , Gainesville, FL 32610 , United States"},{"name":"University of Florida , Center for Natural Products, Drug Discovery and Development, , Gainesville, FL 32610 , United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1303-4483","authenticated-orcid":false,"given":"Gustavo","family":"Seabra","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry , Center for Natural Products, Drug Discovery and Development, , Gainesville, FL 32610 , United States"},{"name":"University of Florida , Center for Natural Products, Drug Discovery and Development, , Gainesville, FL 32610 , United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhe","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Computer & Information Science & Engineering, University of Florida , Gainesville, FL 32611 , United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3980-3532","authenticated-orcid":false,"given":"Ruogu","family":"Fang","sequence":"additional","affiliation":[{"name":"Department of Computer & Information Science & Engineering, University of Florida , Gainesville, FL 32611 , United States"},{"name":"J. Crayton Pruitt Family Department of Biomedical Engineering , Herbert Wertheim College of Engineering, , Gainesville, FL 32611 , United States"},{"name":"University of Florida , Herbert Wertheim College of Engineering, , Gainesville, FL 32611 , United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8149-5206","authenticated-orcid":false,"given":"Lina","family":"Cui","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry , Center for Natural Products, Drug Discovery and Development, , Gainesville, FL 32610 , United States"},{"name":"University of Florida , Center for Natural Products, Drug Discovery and Development, , Gainesville, FL 32610 , United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8610-0659","authenticated-orcid":false,"given":"Yousong","family":"Ding","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry , 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