Steen Hansen, Ph.D., Scientist
Microscopes are used to visualize objects that are too small for the naked eye to see. In biology and medicine, a variety of microscopes exist which are specialized to generate images of everything from individual molecules to entire living organisms. The confocal microscope or, more accurately, the confocal laser scanning microscope is an instrument designed to generate optical sections of cells, subcellular structures, as well as small organisms such as embryos, worms, fruitflies etc.
The confocal microscope utilizes a laser, which excites fluorescent molecules that are either present in cells or attached to molecules inside cells. The emitted fluorescent image is magnified by a fluorescence microscope and measured by a “detector” (i.e. a photomultiplier tube). In front of the detector is a pinhole that only allows light from the focal point of the lens to pass through. In this setup, the pinhole is said to be “conjugate” to the focal point, hence the term “confocal” microscope. As a result, all the light that reaches the detector is always in focus, in contrast to a conventional microscope, where, unless the specimen is completely flat, much of the light is out of focus and therefore creates a blurred image. To generate an image, the laser beam is scanned across the specimen by mirrors, thus the term “scanning” laser confocal microscope. The image, however, doesn’t exist until it is assembled in a computer. By moving the level of the pinhole up and down, it is also possible to generate images from different layers of a thick specimen, which can be reconstructed in 3-D by a computer.
Confocal microscopy has revolutionized biological imaging of thick structures. Whereas previously it was necessary to cut these structures into slices and manually reconstruct images from these slices into 3D, the confocal microscope can do all this for us by computer. Equally important, since the need for cutting sections is avoided, the confocal microscope can also be used to image living organisms. The advantages have made the confocal microscope a household instrument for biomedical research that is widely used.