6D single-fluorogen orientation-localization microscopy for elucidating the architecture of beta-sheet assemblies and biomolecular condensates

Tingting Wu; Weiyan Zhou; Jai S. Rudra; Rohit V. Pappu; Matthew D. Lew

doi:10.1117/12.3005992

12 March 2024 6D single-fluorogen orientation-localization microscopy for elucidating the architecture of beta-sheet assemblies and biomolecular condensates

Tingting Wu, Weiyan Zhou, Jai S. Rudra, Rohit V. Pappu, Matthew D. Lew

Author Affiliations +

Proceedings Volume 12853, High-Speed Biomedical Imaging and Spectroscopy IX; 1285302 (2024) https://doi.org/10.1117/12.3005992
Event: SPIE BiOS, 2024, San Francisco, California, United States

Abstract

We develop six-dimensional single-molecule orientation-localization microscopy (SMOLM) to measure the 3D positions and 3D orientations simultaneously of single fluorophores. We show how careful optimization of phase and polarization modulation components can encode phase, polarization, and angular spectrum information from each fluorescence photon into a microscope’s dipole-spread function. We used the transient binding and blinking of Nile red (NR) to characterize the helical structure of fibrils formed by designed amphipathic peptides, KFE8^L and KFE8^D, and the pathological amyloid-beta peptide Aβ42. We also deployed merocyanine 540 to uncover the interfacial architectures of biomolecular condensates.

Conference Presentation

Citation Download Citation

Tingting Wu, Weiyan Zhou, Jai S. Rudra, Rohit V. Pappu, and Matthew D. Lew "6D single-fluorogen orientation-localization microscopy for elucidating the architecture of beta-sheet assemblies and biomolecular condensates", Proc. SPIE 12853, High-Speed Biomedical Imaging and Spectroscopy IX, 1285302 (12 March 2024); https://doi.org/10.1117/12.3005992

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