We demonstrate far-field fluorescence microscopy with a focalplane resolution of 15-20 nm in biological samples. The 10-to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitationdepletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to Ϸ140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffractionunlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.imaging ͉ stimulated emission depletion illumination ͉ subdiffraction ͉ triplet state F or more than a century, the resolution of a lens-based (far-field) optical microscope has been limited by diffraction (1). However, in the 1990s it became evident that the limiting role of diffraction can be broken in lens-based fluorescence microscopy if certain fluorophore properties are judiciously integrated into the image formation (2). The first viable concept of this kind is stimulated emission depletion (STED) microscopy (3), which, since its experimental validation (4, 5), has been key to solving a number of problems in biophysics (6) and cell biology (7,8).STED microscopy typically uses a scanning excitation spot that is overlapped with a doughnut-shaped counterpart for deexcitation of fluorophores by light, a phenomenon referred to as stimulated emission (9, 10). Oversaturating the deexcitation squeezes the fluorescence spot to subdiffraction dimensions (Fig. 1a) so that superresolved images emerge by scanning this spot through the object (5).The rate for deexcitation by stimulated emission is given by k STED ϭ I STED , with denoting the fluorophore cross-section and I STED denoting the intensity of the stimulating beam. Oversaturating the deexcitation requires k STED be much larger than the fluorescence decay given by the inverse of the lifetime, Fl Ϸ 1-5 ns, of the fluorescent state S 1 . With Ϸ 10 Ϫ17 cm 2 , it follows that I STED Ͼ Ͼ 1͞( Fl ) ϭ 10 26 photons per second per squared centimeter, which, at a wavelength of ϭ 600 nm, amounts to Î STED Ͼ Ͼ 33 MW͞cm 2 . This intensity value is at least 10 3 -fold lower than what is required for multiphoton excitation (11), but still 10 2 -fold larger than what is used ...

We undertake a comprehensive study of the inverse square root dependence of spatial resolution on the saturation factor in stimulated emission depletion (STED) microscopy and generalize it to account for various focal depletion patterns. We used an experimental platform featuring a high quality depletion pattern which results in operation close to the optimal optical performance. Its superior image brightness and uniform effective resolution <25 nm are evidenced by imaging both isolated and self-organized convectively assembled fluorescent beads. For relevant saturation values, the generalized square-root law is shown to predict the practical resolution with high accuracy.

The exchange bias coupling at ferromagnetic/antiferromagnetic interfaces in epitaxially grown Co/CoO layers can intentionally be increased by a factor of up to 3 if the antiferromagnetic CoO layer is diluted by nonmagnetic defects in its volume part away from the interface. Monte Carlo simulations of a simple model of a ferromagnetic layer on a diluted antiferromagnet show exchange bias and explain qualitatively its dilution and temperature dependence. These investigations reveal that diluting the antiferromagnet leads to the formation of volume domains, which cause and control exchange bias.

For a model system consisting of a ferromagnetic layer coupled to a diluted, antiferromagnetic layer extensive Monte Carlo simulations are performed. Exchange bias is observed as a result of a domain state in the antiferromagnetic layer which develops during fiel cooling, carrying an irreversible domain state's magnetization. In agreement with recent experimental observations on Co/CoO bilayers a strong dependence of the exchange bias fiel on dilution of the antiferromagnet is found and it is shown that a variety of typical effects associated with exchange bias, such as positive bias, temperature, and time dependencies as well as the dependence on the thickness of the antiferromagnetic layer can be explained within our model.