Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.

Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.super resolution ͉ moiré ͉ resolution extension ͉ saturation T he fluorescence microscope has become a ubiquitous imaging tool in cell biology through its unique ability to image the 3D interior of a living specimen with multicolor molecular labels of extreme specificity, a combination of strengths not shared by higher-resolution techniques such as electron microscopy and scanned-probe methods. It is therefore unfortunate that its spatial resolution is subject to a hard limit caused by diffraction.Recently, ways have been found to bypass the diffraction limit. 2D resolution in the 30-nm range has been realized by using stimulated emission depletion (STED) (1). STED is based on saturated stimulated emission using two synchronized ultrafast laser sources (2, 3); the underlying concept has been generalized to encompass a class of reversible saturable phenomena (4). STED and other proposed methods (5-7) were conceived in the context of laser-scanning microscopy and are designed to directly minimize the size of a scanned focal point. This article demonstrates an alternative approach that brings theoretically unlimited resolution to a wide-field (nonscanning) microscope by using a nonlinear fluorescence response together with a periodic illumination pattern that fills the field of view.Both structured illumination light and optical nonlinearity, of course, are established ideas. Patterned light, for example, has been used for measuring surface shapes (8) and deformations (9) and for enhancing the sensitivity of fluorescence-recovery-afterphotobleaching experiments (10). Axially structured light has been used to enhance axial resolution in standing-wave fluorescence microscopy (11), 4Pi microscopy (12), and I 5 M (13). Lukosz and Marchand (14) suggested in 1963 that lateral light patterns could be used to enhance resolution, and such patterns have been used for both axial (15) and lateral (16-19) resolution enhancement. They can be more effective than point scanning at retrieving high-resolution information (17,19). Nonlinear fluorescence is the basis for multiphoton fluorescence microscopy (20), and several other c...

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.Light microscopy is a key technology in modern cell biology and, in combination with immunofluorescence, fluorescent protein fusions, or in situ hybridization, allows the specific localization of nearly all cellular components. A fundamental limitation of optical microscopy is its low resolution relative to the scale of subcellular structures. This limitation occurs because light traveling through a lens cannot be focused to a point but only to an Airy disk (1) with a diameter of about half the wavelength of the emitted light (2,3). Because the wavelengths of visible light range from 400 to 700 nm, objects closer than 200 to 350 nm apart cannot be resolved but appear merged into one.