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If a pixel is shifted by a distance d from the optical axis, the image this pixel records will be shifted by d/2 with respect to an image recorded on the optical axis ( Theory of Image-Scanning Microscopy). For this, we need to consider the offset from the optical axis of each pixel and its effect on the image that this pixel records. To obtain a final image that contains all the light from every pixel, the confocal images gathered by every pixel must be combined. In this fashion, every pixel records a “perfect” confocal image. In the case of a camera, however, in which there are many pixels, we do not lose light because every (detectable) photon from the sample will be recorded on the camera chip. Although it has been known for a while that a very small pinhole enhances the resolution in confocal microscopy ( 9), this approach is not practical, because almost all the fluorescent light is rejected by the pinhole. Here we show how the CSD can be upgraded easily to be used for ISM. The principle behind the use of CSD microscopy for ISM is based on the use of a camera and the inherent presence of many excitation foci that rapidly scan the sample.
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The beauty of CSD-based ISM (CSD-ISM) as a 3D superresolution technique lies in the simplicity of the optical setup. If the pixels of the camera are small enough, they may be considered “infinitely small pinholes” for the purpose of enhancing the lateral resolution. In ISM, the sample is excited by a tightly focused laser spot and the fluorescence light emerging from the focus is imaged by means of a camera. The disadvantages of SIM are its technical complexity, reflected in the rather large cost of the commercially available systems, and its sensitivity to optical imperfections and aberrations, which are unavoidable in biological samples. Meanwhile, several commercial instruments for SIM have become available. The most prominent example of this class is structured illumination microscopy (SIM) ( 5), in which one scans a sample with a structured illumination pattern while taking images with a wide-field imaging system. Although these methods do not reach the resolution achievable with STED, PALM, STORM, and related techniques, they do not require any specialized labels or high excitation intensities, and they may be applied to any fluorescent sample at any excitation/emission wavelength.
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Although still bound to light diffraction, increased spatial resolution can be achieved in a class of advanced resolution methods that exploit a clever combination of excitation and detection modalities ( 4– 7).