Optics far Beyond the Diffraction Limit: Stimulated Emission Depletion Microscopy

Abstract

In this chapter we show that stimulated emission depletion (STED) microscopy and its derivative concepts are able to radically overcome the diffraction barrier in far-field fluorescence imaging, thus disclosing fluorescent details on the macromolecular scale even with diffracted beams of light. The optical microscope is an invaluable tool in the life sciences because it is able to noninvasively image structures within cells and tissues. Unfortunately, due to the fact that light propagates as a wave, the smallest possible size of a focal spot in a far-field light microscope is limited by diffraction, putting a lower limit on the size of the structures which can be observed. Concretely, this means that for a lens of semiaperture angle α, the full-width-half-maximum (FWHM) Δr of the main diffraction maximum of the point-spread function (PSF) in the focal plane of the lens is Δr = λ/(2n sin α), with λ and n denoting the wavelength of light and the refractive index, respectively []. If the distance between two objects is smaller than this FWHM, the objects cannot be readily resolved from one another. The diffraction resolution limit is particularly disadvantageous in the life sciences where about 80% of all microscopy applications are carried out with far-field fluorescence systems.