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The transition from single molecule to ensemble revealed by fluorescence polarization
Toby D M Bell1, Andrew H A Clayton2
1School of Chemistry, Faculty of Science, Monash University, Clayton, Victoria 3800, Australia.
Scientific Reports
|February 3, 2015
Summary
Molecular number averaging impacts fluorescence polarization measurements. As more molecules are averaged, observed polarization increases due to photo-selection, necessitating a refined definition of limiting polarization.
Area of Science:
- Photophysics
- Macromolecular Science
- Spectroscopy
Background:
- Fluorescence polarization (FP) is crucial for studying molecular dynamics and interactions.
- Limiting polarization (p₀) represents emission polarization without molecular rotation, a key parameter in FP studies.
Purpose of the Study:
- To investigate the effect of molecular number averaging on the observed limiting polarization (p₀).
- To explore the relationship between fluorescence polarization (p) and the number of molecules (N) in condensed phase measurements.
Main Methods:
- Development of a mathematical model for fluorescent dipoles (1-50 molecules).
- Experimental validation using single-molecule polarization measurements of perylene diimide dye in a polymer matrix.
Main Results:
- A model showing fluorescence polarization (p) increases with molecule number (N) via p = p₀(1 - N⁻ᵝ), attributed to photo-selection.
- Experimental data confirmed that average emission polarization significantly rises with increased molecule averaging (1 to 10 molecules).
Conclusions:
- The observed increase in polarization with molecular averaging suggests a need to refine the definition of limiting polarization in the quasi-single molecule regime.
- This study introduces a novel method for quantifying molecular clustering using single-cluster polarization histograms.
