Confocale

Confocal microscopy is an optical imaging technique that uses point illumination and a spatial pinhole to reject out-of-focus light, producing sharp images from selected depths within a specimen. The term confocal refers to the alignment of illumination and detection optically in the same focal plane, enabling optical sectioning of thick samples without physical slicing.

In a typical confocal system, a laser or other light source generates a narrow beam that is scanned across the sample. Light from the illuminated point is collected through a pinhole placed in front of the detector. Only light from the focal plane passes through the pinhole, while out-of-focus light is largely excluded. The detected signal is assembled into an image; changing the focal plane yields 3D information.

There are several implementations, most notably laser scanning confocal microscopy (LSCM) and spinning-disk confocal microscopy. LSCM uses a single pinhole and point-scanning with a moving mirror; spinning-disk confocal uses a rotating disk with many pinholes, enabling faster imaging. Detectors include photomultiplier tubes and hybrid detectors. The technique can image fluorescence and reflectance signals, and there are label-free confocal methods such as reflectance confocal microscopy.

Confocal methods have broad applications in biology, medicine, and materials science, including 3D reconstructions from z-stacks, live-cell imaging, and tissue imaging. Its advantages include improved contrast and axial resolution, while limitations include reduced signal leading to longer acquisition times, photobleaching, and limited depth in scattering specimens. The technology has matured into widely used commercial systems since the late 20th century.