3.2. Practical use of confocal microscopes

The above principle describes the confocal imaging of a single point on the specimen. For practical use of confocal microscopy, however, a method is needed to image successive points to build up an image. This can be done by either scanning the illuminated spot across the specimen in a raster fashion (scanned beam design) or the specimen can be moved through a stationary light path (stage scanning design). The stage scanning design has the advantage of a simple and accurate optical design, but suffers from lack of speed in scanning an image, particularly at low magnification. The first confocal microscopes built were of this stage scanning type.

However, all current biological confocal microscopes used the scanned beam design. An angular deflection of the light beam at a diffraction plane becomes transformed into a translation in the specimen / image planes. Thus, a system of two deflecting mirrors scanning back and forth about two axes positioned at or near a diffraction plane can be used to scan the light beam in a 2D raster across the specimen. The scan driving and measurement circuitry are interfaced together so that light intensity measurements are taken which cover the specimen area in a regular raster. These intensities are digitized into a computer to produce a digital image.

In order to increase the scanning to video frame rates, slit scanning design has been developed. Instead of a pin hole aperture, a narrow slit of light is scanned in a direction at right angles to its length across the specimen. The emitted light is then passed through a narrow detector slit. This is of advantageous, since only one dimensional scan is required; the scanning rate can be much faster than a point scanning system. Also, because a line of specimen is imaged at one time, the rate of light accumulation from the specimen is much higher. However, this design is not capable of producing clean optical sections as a point scanning system because a proportion of the out – of – focus light is also detected.

Confocal microscopes are also designed with an extended light source and an array of many pin holes on a disc called the Nipkow disc which is placed in a conjugate image plane. The full image is obtained by spinning the disc rapidly, so that pin holes that are usually arranged in a spiral pattern, scan across the whole image area. The major hurdle of this design is that the light source has to be spread out over the whole of the disc, and so is less bright than single hole / laser arrangement. Moreover, only a very small proportion of light passes back through the disc to be detected resulting in the difficulty in recording enough light for a satisfactory image.

 

3.3. Objective lenses in confocal imaging

Although any objective can be used, objectives with higher numerical aperture are preferred as the brightness of the image is strongly dependent on the numerical aperture of the objectives.

 

3.4. Specimen preparation for confocal imaging

Generally, confocal imaging (confocal fluorescence microscopy) simply requires a fluorescent specimen. However, since confocal microscopy are capable of producing clean optical sections from thick, three dimensionally well preserved specimens, care should be taken to preserve the three dimensional structure of the specimen. For living specimens, methods that keep the tissue or cells alive and active will almost certainly preserve the three dimensional structure. For dead specimens, fixatives such as formaldehyde solutions with small percentage of glutaraldehyde which is consistent with the labelling method should be used. The fixed tissues also often need extra permeabilization to allow penetration of probes. In case of plant material, the cell wall is partially digested with cellulose or other cell wall degrading enzymes. Producing good specimen depends on maintaining a balance between preservation of the structures of interest but at the same time disrupting them so as to allow probes in to visualize the structures.

 

3.5. Uses of confocal microscopy

Since confocal microscopy possesses the advantage of elimination of out - of - focus light, it is mainly used for observing specimens with substantial thickness.