Fluorescence microscopy and spectroscopy of subsurface layer dynamics of polymers with nanometer resolution in the axial direction.

We studied the dynamics in ultrathin subsurface layers of an amorphous polymer by the spectra of single fluorescent molecules embedded into the layer by vapor deposition and subsequent controlled diffusion to the desired depth in ≈0.5 nm steps. The spectral trails of single molecules were recorded at 4.

Conformational changes in complex macromolecules studied by single donor-acceptor pair fluorescence.

A single donor-acceptor (D-A) pair with a fluctuating FRET rate F is studied theoretically in the frame of a "two-state model" in which the FRET rate F fluctuates taking two values, F1 and F2, with average rates B for the forward and b for the backward transitions. Theoretical expressions are derived for the autocorrelation function g((2))(DA)(τ) and for the Mandel parameter Q(D,A)(τ) allowing for background emission. Fluctuating intensities ID,A(t) and FRET efficiency E(t) = IA(t)/[IA(t) + ID(t)] are calculated with the help of the Monte Carlo technique.