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Chapter 1:
Confocal Fluorescence Microscopy
Confocal fluorescence microscopy is a microscopic technique that provides true three-dimensional (3D) optical resolution. In microscopy, 3D resolution is generally realized by designing the instrument so that it is primarily sensitive to a specimen's response coming from an in-focus plane, or by subsequently removing the contributions from out-of-focus planes. Several techniques have been developed to achieve this. For instance, 3D deconvolution [Agard and Sedat, 1983] uses both in- and out-of-focus information from a stack of images, taken at various focal planes, to reconstruct the 3D image. Another example is two- and three-photon absorption microscopy [Denk et al., 1990; Hell et al., 1996], where a nonlinear interaction with the specimen is used to confine the specimen's response to the focal plane only. In confocal fluorescence microscopy, true 3D resolution is accomplished by actively suppressing any signal coming from out-of-focus planes. This is achieved by using a pinhole in front of the detector as schematically depicted in Fig. 1.1. Light originating from an in-focus plane is imaged by the microscope objective such that it freely passes the pinhole, whereas light coming from out-of-focus planes is largely blocked by the pinhole.
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