Subdiffraction resolution in far-field fluorescence microscopy

TLDR

The method uses a spatially offset near‑UV pulse to quench outer‑focus fluorescence via stimulated emission, thereby reducing the effective point‑spread function. The technique achieves sub‑diffraction resolution, improving lateral resolution from ~150 nm to ~106 nm and enabling separation of adjacent Pyridine 2 nanocrystals that were previously indistinguishable.

Abstract

We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150 +/- 8 nm to 106 +/- 8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.