Far-field fluorescence microscopy with repetitive excitation

Abstract:

We introduce the concept of repeatedly exciting an excited state of a photostable fluorescent entity to generate a nonlinear fluorescence signal which is solely based on the linear susceptibility of the molecule. The excitation cycle between the fluorescent state and another state prolongs the average lifetime of , with emphasis on those molecules that are in the center of the focus. The photons emitted by the long-lived molecules in the center are recorded by a temporal filter and constitute fluorescence that depends nonlinearly on the excitation intensity. Theoretical analysis reveals that this concept can provide three-dimensional imaging and improve the spatial resolution in far-field fluorescence microscopy. We show that despite the presence of diffraction the effective focal waist can in principle be narrowed down to the molecular scale at the expense of signal.