The first of these socalled superresolution approaches, structured illumination microscopy (SIM), is a fluorescence imaging method with a resolution of about 100 nm, or twice the resolution of conventional brightfield and confocal microscopy. SIM overcomes the diffraction limit by using a grated or structured pattern of light to illuminate the sample. The microscope’s physical setup and operation is quite complex, but the general principle can be thought of as similar to creating a moiré pattern, an interference pattern created by overlaying two grids with different angles or mesh sizes (Figure 9–34). In a similar way to creating a moiré pattern, the illuminating grid and the sample features combine into an interference pattern, from which the original highresolution contributions to the image of features beyond the classical resolution limit can be calculated. Illumination by a grid means that the parts of the sample in the dark stripes of the grid are not illuminated and therefore not imaged, so the imaging is repeated several times (usually three) after translating the grid through a fraction of the grid spacing between each image. As the interference effect is strongest for image components close to the direction of the grid bars, the whole process is repeated with the grid pattern rotated through a series of angles to obtain an equivalent enhancement in all directions. Finally, mathematically combining all these separate images by computer creates an enhanced superresolution image. SIM is versatile because it can be used with any fluorescent dye or protein, and combining SIM images captured at consecutive focal planes can create threedimensional data sets (Figure 9–35).
Figure 9–34 Structured illumination microscopy. the principle, illustrated here, is to illuminate a sample with patterned  light and measure the moiré pattern. shown are (a) the pattern from an unknown structure and (B) a known pattern.
(C) When these are combined, the resulting moiré pattern contains more information than is easily seen in (a), the original pattern. if the known pattern (B) has higher spatial frequencies, then better resolution will result. however, because the spatial patterns that can be created optically are also diffraction-limited, siM can only improve the resolution by about a factor of two. (From B.o. Leung and k.C. Chou, Appl. Spectrosc. 65:967–980, 2011.)
Figure 9–35 Structured illumination microscopy can be used to create three-dimensional data. these three-dimensional projections of the meiotic chromosomes at pachytene in a maize cell show the paired lateral elements of the synaptonemal complexes. (a) the chromosome set has been stained with a fluorescent antibody to cohesin and is viewed here by conventional fluorescence microscopy. Because the distance between the two lateral elements is about 200 nm, the diffraction limit, the two lateral elements that make up each complex are not resolved. (B) in the three-dimensional siM image, the improved resolution enables each lateral element, about 100 nm across, to be clearly resolved, and the two chromosomes can clearly be seen to coil around each other. (C) Because the complete three-dimensional data set for the whole nucleus is available, the path of each separate pair of chromosomes can be traced and artificially assigned a different color. (Courtesy of C.J. rachel Wang, peter Carlton and Zacheus Cande.) approximates a gaussian distribution, whose width at half-maximum height under ideal conditions is about 200 nm. (C) two point sources that are about 200 nm apart
To get around the diffraction limit, the other two superresolutiontechniques exploit aspects of the point spread function, a property of the optical system mentioned earlier. The point spread function is the distribution of light intensity within the threedimensional, blurred image that is formed when a single point source of light is brought to a focus with a lens. Instead of being identical to the point source, the image has an intensity distribution that is approximately described by a Gaussian distribution, which in turn determines the resolution of the lens system (Figure 9–36). Two points that are closer than the width at halfmaximum height of this distribution will become hard to resolve because their images overlap too much (see Figure 9–36C).