Dual-color STED microscopy at 30-nm focal-plane resolution.

Owing to its sensitivity and noninvasiveness, far-field fluorescence microscopy would be almost ideal for biological imaging if the resolution of its established variants were not limited by diffraction toDr l=ð2n sinaÞ, with l denoting the wavelength of light, n the index of refraction, and a the aperture angle of the objective lens. The first concept to overcome these limits in a fundamental way is stimulated emission depletion (STED) microscopy. In most current implementations, a typical STED microscope utilizes a focused beam of light exciting the fluorophore to the fluorescent state S1 (Figure 1a) and a red-shifted doughnutshaped beam of light, the so-called STED beam, for quenching the dye back to its ground state S0. Overlapping the two beams confines the population of S1 and hence fluorescence emission to the proximity of the central minimum of the doughnut, which is ideally at zero. Since the population of the S1 decreases nearly exponentially with the intensity of the STED beam, elevating the doughnut intensity I decreases the region in which the molecule can effectively reside in the S1 far below the diffraction barrier. The full width at half-maximum (FWHM) of the resulting fluorescent spot can be described by a modification of Abbe’s equation: