Confocal microscopy
Scanning microscopy that focuses light to a spot and places a pinhole in the conjugate image plane, so only light from the focal volume reaches the detector. It gives optical sectioning: at 488 nm with a 1.4 NA oil objective, about 180 nm lateral and 460 nm axial resolution (FWHM) with a one-Airy-unit pinhole.
A conventional widefield microscope collects light from the whole thickness of a specimen, so structures above and below the plane of focus add blur. A confocal microscope illuminates one point at a time with a focused laser spot and images that point onto a pinhole in front of the detector. Light from the focus passes the pinhole; light from other depths is out of focus in the pinhole plane and is mostly blocked. Scanning the spot across the sample, usually with galvanometer mirrors, builds an image of a thin optical section, and a stack of sections at different depths gives a three-dimensional image. The principle was patented by Marvin Minsky in 1957; laser-scanning instruments became standard in biology in the late 1980s.
Resolution and the pinhole
Because the image is the product of the illumination spot and the detection spot, a confocal microscope with a pinhole much smaller than one Airy unit resolves somewhat better laterally than a widefield one. With the pinhole set to one Airy unit, the diameter of the Airy disk projected onto the pinhole plane, the commonly used estimates for the full width at half maximum are laterally and axially. For 488 nm excitation and a 1.4 NA oil objective ( = 1.518), these give about 180 nm and 460 nm; at one Airy unit the lateral value is close to the widefield one, and the gain is mainly the axial sectioning. Closing the pinhole below about one Airy unit sharpens the image only slightly while discarding signal; opening it passes more light but thickens the optical section. The pinhole is the same device as the one in a spatial filter, placed in an image plane.
Detectors and variants
Signal levels are low, so point-scanning confocals use photomultiplier tubes, GaAsP photocathodes or hybrid detectors, and more recently small GaAsP or SPAD detector arrays that let the pinhole be replaced by a small detector array whose pixels are reassigned computationally to improve resolution. Spinning-disk confocals scan thousands of pinholes in parallel for speed. Two-photon microscopy reaches a similar sectioning without a pinhole, because excitation only occurs at the focus. In industry, chromatic confocal sensors use the same principle to measure distance and surface profile, with the wavelength that passes the pinhole encoding height.
Measuring performance
Resolution is measured by imaging sub-resolution fluorescent beads, typically 100 nm or smaller, and fitting the lateral and axial profiles of the resulting point spread function. The axial response is also checked by scanning through a thin reflective or fluorescent layer. Refractive-index mismatch between the immersion medium and the sample elongates the axial response and shifts apparent depths, so measurements are made in a medium matched to the specimen.
References: M. Minsky, US Patent 3,013,467 (filed 1957); J. B. Pawley (ed.), Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006); S. Wilhelm et al., Confocal Laser Scanning Microscopy: Principles (Carl Zeiss).