Confocal laser scanning microscopy

From ArticleWorld

Confocal laser scanning microscopy (CLSM) is the use of a specialized type of microscope to obtain high resolution and 3D images. This microscope is able to produce images that are not blurry or distorted in specimens that are thick at various depths. The images are plotted point-by-point and then reconstructed though computer software as opposed to viewing through an eyepiece. This specialized microscopy method was developed in 1953 by Marvin Minsky but not implemented as a standard until the 1980s when technology was able to provide near-ideal point light sources, namely, lasers.

Image formation

This technology uses a laser beam and projects this through a light source aperture where it is then focused by an objective lens as a small focal volume in a fluorescent specimen. The objective lens then recollects a mixture of fluorescent light and laser light. This mixture of light is then separated by a beam splitter which allows the laser light to pass through and reflects the fluorescent light into the detector.

The detector is a photo-detection device that transforms the light signal into an electrical signal that can be detected by the computer. A 3D picture is then assembled using the 2D images from successive focal planes. Samples must be treated with a fluorescent dye in order to make them visible; however concentrations may be low so as not to disrupt the biological systems.

Resolution enhancement

The resolution provided by confocal laser scanning microscopy is much enhanced as compared to other methods such as the use of the scanning electron microscope. In CLSM the fluorescent specimen is illuminated by a point laser source. The size of the scanning tip is thus determined by the diffraction limit of the optical system as this produces a three-dimensional diffraction pattern.

This diffraction pattern's size is dependent on the numerical aperture of the objective lens and the wavelength of the laser light. This highlights the limitation of the optical microscope as its resolution is limited by the use of wide-field illumination. In CLSM however this limitation is overcomed as only light generated in a small volume element is detected at a time. This thus creates sharper images and hence better resolution than conventional methods.