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In wide-field microscopy the contrast of the focused image can sometimes be reduced by light from out of the focus plane - a phenomenon called blur. This is especially problematic for thick fluorescent specimens such as tissue or small organisms. Confocal microscopes are designed to reduce the amount of out-of-focus light reaching the detector.

Laser scanning confocal microscopes illuminate very small volumes within the sample with a focused laser spot so that the entire sample is not illuminated at the same time. The light emitted from the spot travels back through the microscope and through a pinhole in front of the detector. This pinhole is in a conjugate plane to the focused laser spot in the sample plane, so an image of the spot is formed at the pinhole. Light from above and below the plane of focus will come into focus above and below the pinhole plane and most will not go through the pinhole. So most of the light reaching the detector is from the focused spot.

Since a confocal image represents a single ‘slice’ from a three-dimensional sample with next to no light coming from adjacent slices, a motorised focus mechanism can be used to acquire every slice from the sample in sequence. These slices can then be reconstructed into a stack that represents the entire 3D structure. This is called optical sectioning.

Another technique called deconvolution can be used to computationally reassign light back to its correct plane of focus in both wide-field and confocal images by applying information known about how the microscope distorts the image of the relative to the object (the point spread function) to a reconstruction algorithm.