Principle of confocal microscopy

Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane.¹ This technique has gained popularity in the scientific and industrial communities. Typical applications include life sciences and semiconductor inspection.

Basic concept

The principle of confocal imaging was patented by Marvin Minsky in 1957.² In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in light from a light source. Due to the conservation of light intensity transportation, all parts of the specimen throughout the optical path will be excited and the fluorescence detected by a photodetector or a camera. 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. Only the light within the focal plane can be detected, so the image quality is much better than that of wide-field images. As only one point is illuminated at a time in confocal microscopy, 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 square of the numerical aperture of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction. They also are able to see into the image by taking images at different depths.

Types

Three types of confocal microscopes are commercially available: Confocal laser scanning microscopes, spinning-disk (Nipkow disk) confocal microscopes and Programmable Array Microscopes (PAM). Confocal laser scanning microscopy yields better image quality than Nipkow and PAM, but the imaging frame rate was very slow (less than 3 frames/second) until recently; spinning-disk confocal microscopes can achieve video rate imaging—a desirable feature for dynamic observations such as live cell imaging. Confocal laser scanning microscopy has now been improved to provide better than video rate (60 frames/second) imaging by using MEMS based scanning mirrors.

Images

β-tubulin in Tetrahymena (a ciliated protozoan).

References

External links

• Molecular Expressions: Laser Scanning Confocal Microscopy
• Nikon's MicroscopyU. Comprehensive introduction to confocal microscopy.
• Emory’s Physics Department. Introduction to confocal microscopy and fluorescence.
• The Science Creative Quarterly's overview of confocal microscopy - high res images also available.
• Programmable Array Microscope - Confocal Microscope Capabilities.