Confocal microscopy

Although conventional light and fluorescence microscopy allow the examination of both living and fixed specimens, certain problems exist with these techniques. One of the main problems is out-of-focus blur degrading the image by obscuring important structures of interest, particularly in thick specimens. In conventional microscopy, not only is the plane of focus illuminated, but much of the specimen above and below this point is also illuminated resulting in out-of-focus blur from these areas. This out-of-focus light leads to a reduction in image contrast and a decrease in resolution. In the confocal microscope all out-of-focus structures are suppressed at image formation. This is obtained by an arrangement of diaphragms, which, at optically conjugated points of the path of rays, act as a point source and as a point detector respectively. The detection pinhole does not permit rays of light from out-of-focus points to pass through it. The wavelength of light, the numerical aperture of the objective and the diameter of the diaphragm (wider detection pinhole reduces the confocal effect) affect the depth of the focal plane. To obtain a full image, the point of light is moved across the specimen by scanning mirrors. The emitted/reflected light passing through the detector pinhole is transformed into electrical signals by a photomultiplier and displayed on a computer monitor.

Major improvements offered by a confocal microscope over the performance of a conventional microscope may be summarised as follows:

1. Light rays from outside the focal plane will not be recorded.

2. Defocusing does not create blurring, but gradually cuts out parts of the object as they move away from the focal plane. The practical consequence is that these parts become darker and eventually disappear. This feature is called optical sectioning.

3. True, three-dimensional data sets can be recorded.

4. Scanning the object in x/y-direction as well as in z-direction (along the optical axis) allows viewing the object from all sides.

5. Due to the small dimension of the illuminating light spot in the focal plane, stray light and photo bleaching are minimized.

6. By image processing, many slices can be superimposed giving an extended focus image. This can only be achieved in conventional microscopy by reduction of the aperture and thus sacrificing resolution.