The transition from single molecule to ensemble revealed by fluorescence polarization.
Toby D. M. Bell
Andrew H. A. Clayton
- Publisher: Nature Publishing Group
(issn: 2045-2322, eissn: 2045-2322)
Fluorescence polarization measurements in the condensed phase provide rich information on rotational dynamics and interactions between macromolecules. An important parameter in these studies is the limiting polarization or po which is the emission polarization in the absence of molecular rotation. Here we explore how molecular number averaging affects the observed value of po. Using a simple mathematical model we show that for a collection of fluorescent dipoles (1–50 molecules) the fluorescence polarization (p) increases with the number of molecules (N) due to the progressive onset of photo-selection with a relation of the form p = po(1 − N−β). This concept is demonstrated experimentally using single molecule polarization measurements of perylene diimide dye molecules in a rigid polymer matrix where it is shown that the average emission polarization increases significantly when the number of molecules per averaging window is increased from 1 to 10 molecules. These results suggest that the definition of limiting polarization needs to be refined in the quasi-single molecule regime. Moreover, these results pave a new way for measuring clustering of molecules from single cluster polarization histograms.