TY - JOUR
T1 - Resolving the Three-Dimensional Rotational and Translational Dynamics of Single Molecules Using Radially and Azimuthally Polarized Fluorescence
AU - Zhang, Oumeng
AU - Zhou, Weiyan
AU - Lu, Jin
AU - Wu, Tingting
AU - Lew, Matthew D.
N1 - Publisher Copyright:
© 2022 American Chemical Society.
PY - 2022/2/9
Y1 - 2022/2/9
N2 - 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.
AB - 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.
KW - Single-molecule tracking
KW - cholesterol condensing
KW - lateral diffusion
KW - phospholipid bilayer
KW - rotational diffusion
UR - http://www.scopus.com/inward/record.url?scp=85124119933&partnerID=8YFLogxK
U2 - 10.1021/acs.nanolett.1c03948
DO - 10.1021/acs.nanolett.1c03948
M3 - Article
C2 - 35073487
AN - SCOPUS:85124119933
SN - 1530-6984
VL - 22
SP - 1024
EP - 1031
JO - Nano Letters
JF - Nano Letters
IS - 3
ER -