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Confocal microscopy offers several advantages over conventional optical
microscopy, including controllable depth of field, the elimination of
image degrading out-of-focus information, and the ability to collect
serial optical sections from thick specimens.
By using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane, confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images. A laser beam scans the specimen pixel by pixel and line by line. A pinhole conjugated to the focal plane obstructs the light emerging from objects outside that plane so that only light from objects that are in focus can reach the detector. The pixel data gathered using this method are then assembled to form an image that represents an optical section of the specimen and is distinguished by high contrast and high resolution in the X, Y and Z planes. Several images generated by means of shifting the focal plane can be combined into a 3D image stack.
One point at a time
In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in light from a light source. All parts of the specimen in the optical path are excited and the resulting fluorescence is detected by the microscope photodetector or camera as background signal. In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information – the name “confocal” stems from this configuration. As only light produced by fluorescence very close to the focal plane can be detected, the image resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However this increased resolution is at the cost of decreased signal intensity, often requiring long exposures.
As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal plane is defined mostly by the inverse of the square of the numerical aperture of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction. The thin optical sectioning possibility allows 3D imaging of samples.