Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

DESIGN AND DIFFERENTIATION OF FRET CONSTRUCTS – EASY AS ABEL-PIE

Allison H. Squires 1 ; 1 University of Chicago, Chicago, USA

More than just a “molecular ruler”, the spectroscopic properties of photons emitted from nanoscale systems undergoing FRET depend sensitively on the positions, orientations, dynamic movements, and physical and chemical environments of the fluorophores involved. Using an appropriate combination of wavelengths in Pulsed Interleaved Excitation (PIE) enables direct calibration of donor-acceptor stoichiometry and correction factors for FRET systems, facilitating interpretation of more complex objects and subtle changes in FRET signals. Separately, the extended-duration, isotropic view of particles in solution afforded by an Anti-Brownian ELectrokinetic (ABEL) trap provides high-precision spectroscopic measurements on fluorescent and FRET systems. Here, I will discuss our recent advances in integrating these two powerful technologies into our ABEL-PIE platform, with an emphasis on (1) the technical design, features, and flaws of the system as well as (2) ongoing work to use ABEL-PIE in applications involving natural, biomedical, and engineered FRET samples.

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