What is a Confocal Microscope?

A confocal microscope is a particular kind of fluorescent microscope used to obtain high-quality 2D and 3D images. With a basic (wide-field) fluorescent microscope, light of a particular wavelength, is shined on a specimen, and the specimen absorbs some of that light, and reflects the rest of it at a longer wavelength. The longer wavelength light passes through a dichroic mirror, which allows the longer wavelength light, but not the original (shorter) wavelength light to pass through. The person viewing the sample sees only the light which has passed through the dichroic mirror. Basic fluorescent microscopes capture both in-focus and out-of-focus light, which works well for large specimens where very high resolution is not required. It does not work so well for viewing fine detail. A confocal microscope is needed to capture high resolution fluorescent images.

The confocal microscope uses a pinhole to reject out of focus light. Instead of light passing only through a dichroic mirror, light gets passed through both a dichroic mirror, and a pinhole, which means that the person viewing the sample sees only a single point of the sample. In order to view a whole sample, movable mirrors scan light across a rectangular plane of the sample, and a computer collects the emitted light as the specimen gets scanned. A complete scan will result in an optical slice, or cross-section, of the specimen. This type of microscopy is called confocal laser scanning microscopy, in part because a laser is used as the light source.

To create a 3D image, several scans are done, creating several "slices" of the specimen, and a computer combines each slice to create a three dimensional image. The microscope can be programmed to vary the space between slices, depending on how crisp a 3D image the user wants to obtain. The microscope can also vary the speed at which it scans an image, with slow scanning giving higher resolution than fast scanning.

The confocal microscope has become a very commonly used piece of equipment in many life-science laboratories. They are expensive, and somewhat large, and often have rooms dedicated to their use, because the room needs to be dark in order to obtain a good fluorescent image. Although confocal microscopes are most commonly used to view cells and structures within cells, they can be used to obtain an image of any three dimensional fluorescent specimen, and are sometimes used to view semiconductor materials.