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, KFE8L and KFE8D, and the pathological amyloid-beta peptide Aβ42. We also deployed merocyanine 540 to uncover the interfacial architectures of biomolecular condensates.

Original languageEnglish
Title of host publicationHigh-Speed Biomedical Imaging and Spectroscopy IX
EditorsKevin K. Tsia, Keisuke Goda
ISBN (Electronic)9781510669659
StatePublished - 2024
EventHigh-Speed Biomedical Imaging and Spectroscopy IX 2024 - San Francisco, United States
Duration: Jan 27 2024Jan 28 2024

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceHigh-Speed Biomedical Imaging and Spectroscopy IX 2024
Country/TerritoryUnited States
CitySan Francisco


  • Environment-sensitive fluorophores
  • Fluorescence
  • Polarization-sensitive imaging
  • Transient binding
  • super-resolution microscopy


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