Far-field fluorescence microscopy with repetitive excitation
High Resolution Optical Microscopy Group, Max-Planck-Institute for Biophysical
Chemistry 37070 Göttingen, Germany
Published online: 15 June 1999
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.
PACS: 42.90.+m – Other topics in optics / 42.30.-d – Imaging and optical processing / 42.65.-k – Nonlinear optics
© EDP Sciences, Società Italiana di Fisica, Springer-Verlag, 1999