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Traditional microscopy does not provide structural information on protein systems. Routinely used fluorescent protein tags, such as Green Fluorescent Protein (GFP), might perturb native structures. Here, we report a counter\u2010propagating mid\u2010infrared photothermal imaging approach enabling mapping of secondary structure of protein aggregates in live cells modeling Huntington's disease. By comparing mid\u2010infrared photothermal spectra of label\u2010free and GFP\u2010tagged huntingtin inclusions, we demonstrate that GFP fusions indeed perturb the secondary structure of aggregates. By implementing spectra with small spatial step for dissecting spectral features within sub\u2010micrometer distances, we reveal that huntingtin inclusions partition into a \u03b2\u2010sheet\u2010rich core and a \u0251\u2010helix\u2010rich shell. We further demonstrate that this structural partition exists only in cells with the [<jats:italic>RNQ<\/jats:italic><jats:sup>+<\/jats:sup>] prion state, while [<jats:italic>rnq<\/jats:italic><jats:sup>\u2212<\/jats:sup>] cells only carry smaller \u03b2\u2010rich non\u2010toxic aggregates. Collectively, our methodology has the potential to unveil detailed structural information on protein assemblies in live cells, enabling high\u2010throughput structural screenings of macromolecular assemblies.<\/jats:p>","DOI":"10.1002\/anie.202408163","type":"journal-article","created":{"date-parts":[[2024,6,17]],"date-time":"2024-06-17T02:20:05Z","timestamp":1718590805000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Structural Mapping of Protein Aggregates in Live Cells Modeling Huntington's Disease"],"prefix":"10.1002","volume":"63","author":[{"given":"Zhongyue","family":"Guo","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering Boston University  Boston MA 02215 USA"},{"name":"Photonics Center Boston University  Boston MA 02215 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulio","family":"Chiesa","sequence":"additional","affiliation":[{"name":"Department 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