Resolving the Three-Dimensional Rotational and Translational Dynamics of Single Molecules Using Radially and Azimuthally Polarized Fluorescence

Oumeng Zhang, Weiyan Zhou, Jin Lu, Tingting Wu, Matthew D. Lew

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

We report a radially and azimuthally polarized (raPol) microscope for high detection and estimation performance in single-molecule orientation-localization microscopy (SMOLM). With 5000 photons detected from Nile red (NR) transiently bound within supported lipid bilayers (SLBs), raPol SMOLM achieves 2.9 nm localization precision, 1.5° orientation precision, and 0.17 sr precision in estimating rotational wobble. Within DPPC SLBs, SMOLM imaging reveals the existence of randomly oriented binding pockets that prevent NR from freely exploring all orientations. Treating the SLBs with cholesterol-loaded methyl-β-cyclodextrin (MβCD-chol) causes NR's orientational diffusion to be dramatically reduced, but curiously NR's median lateral displacements drastically increase from 20.8 to 75.5 nm (200 ms time lag). These jump diffusion events overwhelmingly originate from cholesterol-rich nanodomains within the SLB. These detailed measurements of single-molecule rotational and translational dynamics are made possible by raPol's high measurement precision and are not detectable in standard SMLM.

Original languageEnglish
Pages (from-to)1024-1031
Number of pages8
JournalNano Letters
Volume22
Issue number3
DOIs
StatePublished - Feb 9 2022

Keywords

  • Single-molecule tracking
  • cholesterol condensing
  • lateral diffusion
  • phospholipid bilayer
  • rotational diffusion

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