In situ secondary structure imaging of protein phase separation and aggregation by hyperspectral stimulated Raman scattering microscopy

Ruifeng Sun, Yanrong Zhuang, Yi Lin () and Fanghao Hu ()
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Ruifeng Sun: Tsinghua University
Yanrong Zhuang: Tsinghua University
Yi Lin: Tsinghua University
Fanghao Hu: Tsinghua University

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract Biomolecular condensates enable the coordination of cellular activities with high spatiotemporal selectivity. Many techniques have been developed to characterize protein condensate. However, direct visualization of protein structure in phase-separated condensate remains underexplored. Here we develop in situ quantitative imaging of secondary structure in protein condensates by stimulated Raman scattering (SRS) microscopy. Characteristic spectra of four secondary structures are obtained from protein amide I vibration analysis. Hyperspectral SRS imaging reveals significant enrichments and disordered to ordered structural changes during phase separation of ALS-related proteins. Time-lapse imaging of protein aging process directly visualizes heterogeneous β-sheet formation on the condensate surface. And secondary structures of mutant proteins are imaged to correlate amino acid sequence to phase separation property. Live-cell label-free imaging of protein structure is further demonstrated to exhibit pronounced heterogeneity in subcellular aggregates. Therefore, our technique provides crucial molecular-level information to investigate protein phase separation and its transition in pathological aggregation.

Date: 2025
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DOI: 10.1038/s41467-025-63894-1

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