Confocal microscopy is an optical imaging technique in common use in laboratories around the world for a variety of purposes. It is particularly important in the life sciences, semiconductor inspection and material science industries.
It increases the optical resolution and contrast by using point illumination and a spatial pinhole, which eliminate out-of-focus light and ensure a clear image of the specimen is formed. Confocal microscopes enable the reconstruction of three-dimensional structures from the obtained images.
The principle behind confocal microscopy was patented by Marvin Minsky in 1957. The method was devised to overcome some of the limitations of traditional wide-field fluorescence microscopes. Because they flood specimens in light from one source, all parts are detected, including the unfocused background. Confocal microscopy eliminates this problem, and results in clearer, more defined images. The point illumination means only the light produced by fluorescence very close to the focal plane is detected, meaning better optical resolution. However, this does mean decreased signal intensity, so longer exposures are often required.
Furthermore, because only one point in the sample is illuminated, 2D or 3D imaging requires scanning over a regular raster. Achievable thickness is defined mostly by the wavelength of the used light, divided by the numerical aperture of the lens, but it also depends on the properties of the specimen.
Confocal microscopes are particularly good at 3D imaging and surface profiling, thanks to the thin optical sectioning.
There are three types of confocal microscope:
Confocal laser scanning microscopes
Spinning-disk confocal microscopes
Programmable Array Microscopes
Each one has its own advantages and disadvantages and may be particularly beneficial in different areas of scientific study.