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Updated: Mar 7, 2026

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
Published on: April 25, 2025
Single molecule optical measurements of orientation and rotations of biological macromolecules
Deborah Y Shroder1,2, Lisa G Lippert1,2, Yale E Goldman1,3
1Department of Biochemistry and Biophysics, Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Abstract:
Subdomains of macromolecules often undergo large orientation changes during their catalytic cycles that are essential for their activity. Tracking these rearrangements in real time opens a powerful window into the link between protein structure and functional output. Site-specific labeling of individual molecules with polarized optical probes and measurement of their spatial orientation can give insight into the crucial conformational changes, dynamics, and fluctuations of macromolecules. Here we describe the range of single molecule optical technologies that can extract orientation information from these probes, review the relevant types of probes and labeling techniques, and highlight the advantages and disadvantages of these technologies for addressing specific inquiries.
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