Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

TLDR

The method uses stimulated emission to quench excited molecules at the rim of the focal spot, breaking the diffraction limit. The resulting focal spot is 90–110 nm in diameter, a six‑fold axial and two‑fold radial reduction that shrinks the volume to 0.67 attoliters—18 times smaller than confocal microscopy—and provides higher‑resolution images of live cells.

Abstract

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90–110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.