Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

65-POS Board 33 HIGH-PRECISION FRET MEASUREMENTS FOR MULTIPLEXING, CHARACTERIZATION AND SENSING USING ABEL-PIE Kavya Vaidya 1 ; Ayesha Ejaz 1 ; Ryan Febryanto 1 ; Kyle Lin 1 ; Samantha Riesenfeld 1 ; D. Allan Drummond 1 ; Allison H Squires 1 ; 1 University of Chicago, Chicago, IL, USA Single molecule spectroscopy, when paired with Förster Resonance Energy Transfer (FRET) measurements, brings rich insights into biomolecular assemblies, conformations and interactions beyond averaging in ensemble measurements. The Anti-Brownian ELectrokinetic (ABEL) trap is a powerful platform for analyzing single molecules and their interactions in free solution, enabling high-precision fluorescence spectroscopy and hydrodynamic profiling with seconds long observation times. We have introduced pulsed interleaved excitation (PIE) to the ABEL trap allowing for quantitative two-color stoichiometric information and high-precision FRET measurements while detangling the excitation sources for each detected photon. ABEL-PIE has enabled new multiparameter measurements that provide an improved view of nanoscale energy transfer for both biological and engineered systems. We demonstrate this improvement by expanding our previous multiplexing results using FRET-encoded DNA sensors, FRETfluors, with additional parameters made possible by the excitation modulation. Here, we highlight advances in our measurement capabilities through two applications: 1. we leverage the FRETfluors to develop an assay for multiplexed single-molecule sensing for multi-target visualization and low abundance biomarker detection and, 2. we probe heterogenous stress induced protein condensates to gain insight into their organization and mechanistic functions using the multi-parameter high-precision spectroscopy made possible with ABEL-PIE.

95