CONFOCAL MICROSCOPE

The confocal microscope is a microscope that employs an optical imaging technique to increase contrast and/or reconstruct three-dimensional images using a spatial "pinhole" to eliminate out-of-focus light or lens flare in specimens that are more thicker than the focal plane.1 This technique has become increasingly popular in the scientific and industrial communities. It is typically applied in life sciences and semiconductor inspection.

Basic concept

The confocal imaging concept was patented by Marvin Minsky in 1957.2 In a conventional (eg, widefield) fluorescence microscope, the entire specimen is supersaturated with light from the illumination source. Due to the conservation of light intensity in its path, all parts of the specimen along its optical path will be excited and the fluorescence detected by a photodetector or camera. In contrast, a confocal microscope uses point illumination and a "pinhole" in a conjugate optical plane in front of the detector to remove information that is outside the focal plane.

Only light that is within this plane can be detected, so the image quality is much better than wide field. Since only one spot is illuminated at a time in the confocal microscope, scanning over a regular raster in the specimen is required to obtain two- or three-dimensional images. Focal plane thinness 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 refractive index of the environment.

Types

There are three types of commercially available confocal microscopes: the scanning laser confocal microscope, the spinning disk (Nipkow disk) confocal microscope, and programmable array microscopes (PAM). Laser scanning confocal microscopy yields higher image quality performance than disk or PAM, but the frame rate was very slow (less than three frames/second) until recently. Disk microscopes can achieve speeds compatible with video creation—a desirable trait for dynamic observations, such as live cell imaging. Scanning laser confocal microscopy has now been improved to obtain rates higher than video (60 frames/second) using MEMS based on scanning mirrors.

Partial profile of the surface of a 1 Euro coin measured with a Nipkow disk confocal microscope.

Information on the reflection of a 1 Euro coin.

Cross section through a 1 Euro coin (x - in the plane of the coin, z - towards the inside of the coin).