The Aaron Klug Centre for Imaging and Analysis

Lecture 5: Confocal laser scanning microscopy

Minski (1961) was the first to propose the technique of confocal microscopy used by laser scanning confocal microscopes. The principle is quite simple and is illustrated in the light paths in Figure 1. The image seen through a microscope includes the in-focus portion and the out-of-focus portion above and below the plane of focus. The smear or blur produced by the out-of-focus planes is a natural consequence of the optics of the microscope. Confocal microscopy removes out-of-focus haze by passing the light through one or more small apertures, leaving only a thin, highly focused plane. The light from this focused plane can be digitized and stored on a computer. The distance between the specimen and the microscope objective is then changed producing a new focal plane. The new focal plane is digitized and stored. After a series of planes has been collected, individual slices can be examined or the whole specimen can be digitally reconstructed by a computer as a three-dimensional volume. A confocal microscope consists of a standard microscope with a number of complex attachments to direct and process the beam of light. Most confocal microscopes use an intense laser light to scan the specimen. This intense light source is needed to compensate for the light loss which occurs as the light passes through small apertures.