2013
In Fluorescence Microscopy: From Principles to Biological Applications. John Wiley & Sons

STED microscopy

Authors:

Gould, T. J., Pellett, P. A., & Bewersdorf, J.

Abstract:

The consensus for over a century was that resolution in far-field microscopy is fundamentally limited by diffraction. Ernst Abbe’s famous formula was considered to be the resolution limit, with λ being the wavelength, n the refractive index of the medium, and α the half aperture angle of the objective lens. Equation (10.1) represents the full width at half maximum (FWHM) of the point spread function (PSF) and is commonly used as a quantitative definition of resolution in far-field light microscopy. Identical fluorescent emitters separated by less than Δr, which is typically ∼200–250 nm, appear merged into one image and therefore cannot be resolved. Fortunately, this long-standing barrier has not prevented researchers from pursuing new techniques that significantly improve resolution in the far field.Over the last two decades, the field of fluorescence imaging has seen a number of developments that have pushed resolution beyond the limits imposed by diffraction (Hell, 2009). The concept of stimulated emission depletion (STED) microscopy was first introduced by Stefan Hell in 1994 and demonstrated that the diffraction barrier could, in fact, be surpassed (Hell and Wichmann, 1994). In STED microscopy, the diffraction limit is overcome by exploiting the inherent photophysical properties of the fluorescent molecules to reduce the size of the effective focal volume of a laser scanning microscope (LSM).
In concert with other super-resolution microscopy methods, STED microscopy is currently revolutionizing the field of biomedical imaging, offering resolutions that previously have been limited to the realm of electron microscopy.