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Investigating Single-Molecule Fluorescence Spectral Heterogeneity of Rhodamines Using High-Throughput Single-Molecule
The Journal of Physical Chemistry Letters
|April 16, 2021
Summary
Structural flexibility in fluorophores, specifically N,N-dialkylated groups, significantly impacts single-molecule fluorescence spectral heterogeneities (smFSH). This finding helps explain variations in fluorescence signals from rigid dyes like Rhodamine B.
Area of Science:
- Photochemistry
- Spectroscopy
- Materials Science
Background:
- Single-molecule fluorescence spectral heterogeneities (smFSH) present challenges in interpreting fluorescence data.
- Understanding the origins of smFSH is crucial for advancing single-molecule spectroscopy techniques.
Purpose of the Study:
- To experimentally investigate intramolecular and environmental factors causing smFSH.
- To identify the primary drivers of high smFSH in commonly used fluorophores.
Main Methods:
- Developed a high-throughput single-molecule spectroscopy method.
- Analyzed over 5000 single-molecule emission spectra for 9 different fluorophores.
- Varied fluorophore structural rigidity and substrate polarity.
Main Results:
- Observed unexpectedly high smFSH in structurally rigid Rhodamine B compared to flexible Cyanine dye-Alexa Fluor 647.
- Ruled out system noise, spectral diffusion, and environmental polarity as primary causes.
- Identified rotational flexibility of N,N-dialkylated groups as a key contributor to smFSH.
Conclusions:
- Rotational flexibility of specific molecular groups is a significant factor in smFSH.
- Further investigation into other intramolecular and environmental factors is warranted for rigid fluorophores like Rhodamines.
