Confocal microscopy

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 in specimens that are thicker than the focal plane.^[1] This technique has gained popularity in the scientific and industrial communities and typical applications are in life sciences and semiconductor inspection.

Basic concept

The principle of confocal imaging was patented by Marvin Minsky in 1957^[2] and aims to overcome some limitations of traditional wide-field fluorescence microscopes. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in light from a light source. All parts of the specimen in the optical path are excited and the resulting fluorescence is detected by the microscope photodetector or camera as background signal. 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 - the name "confocal" stems from this configuration. As only light produced by fluorescence very close to the focal plane can be detected the image resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However as much of the light from sample fluorescence is blocked at the pinhole this increased resolution is at the cost of decreased signal intensity so long exposures are often required.

As only one point in the sample is illuminated at a time, 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 inverse of 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. The thin optical sectioning possible make these types of microscopes particularly good at 3D imaging of samples.

Types

Three types of confocal microscopes are commercially available:

• Confocal laser scanning microscopes
• Spinning-disk (Nipkow disk) confocal microscopes
• Programmable Array Microscopes (PAM)

Each of these classes of confocal microscope have particular advantages and disadvantages, most systems are either optimised for resolution or high sensitivity for video capture. Confocal laser scanning microscopes generally yield better image quality than Nipkow and PAM but imaging frame rates are typically very slow (less than 3 frames/second). Spinning-disk confocal microscopes can achieve video rate imaging - a desirable feature for dynamic observations such as live cell imaging - but at lower resolution.

Cutting edge development of confocal laser scanning microscopy now allows better than video rate (60 frames/second) imaging by using multiple 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.

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