Author's School

McKelvey School of Engineering

Document Type

Conference Proceeding

Language

English (en)

Publication Date

3-4-2026

Abstract

Fluorescence anisotropy and single-molecule orientation-localization microscopy (SMOLM) are powerful techniques that quantify the rotational diffusion of dipole-like emitters, which is important for sensing molecular interactions and chemical environments at the nanoscale. Numerous theoretical and experimental studies have thoroughly characterized single-molecule rotations even when those rotations are much faster than the detector integration time. Here, we extend the theory of measuring rotational diffusion to situations where a single dipole rotates uniformly everywhere outside of an isotropic cone of a certain size, termed a negative cone. This scenario corresponds to negative fluorescence anisotropy π‘Ÿ and has been observed in emitters exhibiting strong homo-FRET or those composed of two degenerate perpendicular dipoles. We extend existing theory to model the negative wobble and discover fundamental challenges in determining whether a cone is positive or negative. Difficulties in discriminating positive vs. negative wobble are further exaggerated by severe photon shot noise. Moreover, due to photon shot noise, we find that all emitters, i.e., for both positive and negative wobble, appear more constrained than they actually are. To address these challenges, we identify optimal experimental conditions for accurate rotational diffusion measurements. Finally, we demonstrate experimental measurements of negative cone rotational diffusion (negative wobble) by inducing orientation hole-burning in fluorescent beads.

Comments

(2026) Published by SPIE. For personal use only. Β  Yuanxin Qiu, Kaizhi A. Nie, and Matthew D. Lew "Sensing negative-cone rotational diffusion of dipole-like emitters", Proc. SPIE 13858, Single Molecule Spectroscopy and Superresolution Imaging XIX, 1385804 (4 March 2026); https://doi.org/10.1117/12.3078973

Download

Share

COinS