Accurate and Robust Measurement of Single-Molecule Fluorescence Spectral Heterogeneity.
- 1Molecular Analytics and Photonics (MAP) Lab, Integrative Sciences Initiative, Department of Textile Engineering, Chemistry and Science, North Carolina State University, 1020 Main Campus Drive, Raleigh, North Carolina 27606, United States.
- 2Frost Institute for Chemistry and Molecular Science, University of Miami, 1201 Memorial Drive, Coral Gables, Florida 33146, United States.
- 3Lampe Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, 4310 Engineering Building III, Raleigh, North Carolina 27695, United States.
Abstract
Understanding single-molecule fluorescence spectral heterogeneity (smFLUSH) is vital for developing new probes for multidimensional super-resolution microscopy and decoding nanoscopic environments. However, traditional analysis methods distort the data and fail to distinguish random background noise for actual spectral differences between the single molecules. To overcome this bottleneck, we developed a Monte Carlo simulation and an adaptive signal processing framework inspired by balanced detection in spectral domain optical coherence tomography that employs a dynamically scaling, molecule-specific spectral boundary to effectively decouple intrinsic molecular fluctuations from instrumental noise, achieving an exceptional subnanometer precision floor. We experimentally validated this framework using a tailor-designed library of Boron Dipyrromethene (BODIPY) dyes engineered with systematically tuned degrees of conformational freedom. This adaptive pipeline revealed a distinct structural bifurcation: in a subseries of dyes, targeted molecular rigidification monotonically suppressed smFLUSH, whereas steric crowding and core predistortion in a phenyl- and ethyl-containing subseries caused spectral variance to plateau at an elevated baseline regardless of additional rotor count. Furthermore, correlating smFLUSH with excited-state relaxation dynamics revealed that spectral heterogeneity aligns with shorter fluorescence lifetimes and larger Stokes shifts, demonstrating that matrix-coupled structural adjustments simultaneously accelerate nonradiative deactivation. Ultimately, this framework establishes a robust workflow for single-molecule photophysics and delivers a rational synthetic blueprint for designing single-molecule spectrally stable probes.
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BibTeX
@article{gong2026accurate,
title = {{Accurate and Robust Measurement of Single-Molecule Fluorescence Spectral Heterogeneity.}},
author = {Xiayi Gong and Amrita Singh and Hongjing Mao and Ambarish Kumar Singh and Md Abul Shahid and Françisco M Raymo and Yang Zhang},
journal = {ACS measurement science au},
year = {2026},
month = oct,
doi = {10.1021/acsmeasuresciau.6c00211},
url = {https://europepmc.org/article/MED/42921430},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Accurate and Robust Measurement of Single-Molecule Fluorescence Spectral Heterogeneity.
AU - Xiayi Gong
AU - Amrita Singh
AU - Hongjing Mao
AU - Ambarish Kumar Singh
AU - Md Abul Shahid
AU - Françisco M Raymo
AU - Yang Zhang
PY - 2026
DA - 2026/10/12
JO - ACS measurement science au
DO - 10.1021/acsmeasuresciau.6c00211
UR - https://europepmc.org/article/MED/42921430
AB - Understanding single-molecule fluorescence spectral heterogeneity (smFLUSH) is vital for developing new probes for multidimensional super-resolution microscopy and decoding nanoscopic environments. However, traditional analysis methods distort the data and fail to distinguish random background noise for actual spectral differences between the single molecules. To overcome this bottleneck, we developed a Monte Carlo simulation and an adaptive signal processing framework inspired by balanced detection in spectral domain optical coherence tomography that employs a dynamically scaling, molecule-specific spectral boundary to effectively decouple intrinsic molecular fluctuations from instrumental noise, achieving an exceptional subnanometer precision floor. We experimentally validated this framework using a tailor-designed library of Boron Dipyrromethene (BODIPY) dyes engineered with systematically tuned degrees of conformational freedom. This adaptive pipeline revealed a distinct structural bifurcation: in a subseries of dyes, targeted molecular rigidification monotonically suppressed smFLUSH, whereas steric crowding and core predistortion in a phenyl- and ethyl-containing subseries caused spectral variance to plateau at an elevated baseline regardless of additional rotor count. Furthermore, correlating smFLUSH with excited-state relaxation dynamics revealed that spectral heterogeneity aligns with shorter fluorescence lifetimes and larger Stokes shifts, demonstrating that matrix-coupled structural adjustments simultaneously accelerate nonradiative deactivation. Ultimately, this framework establishes a robust workflow for single-molecule photophysics and delivers a rational synthetic blueprint for designing single-molecule spectrally stable probes.
SN - 2694-250X
ER -