3 Light microscopy
Light and its properties
Retardation and refraction
Media through which light is able to pass will slow down or retard the speed of the light in proportion to the density of the medium. The higher the density, the greater the degree of retardation. Rays of light entering a sheet of glass at right angles are retarded in speed but their direction is unchanged (Fig. 3.3a). If the light enters the glass at any other angle, a deviation of direction will occur in addition to the retardation, and this is called refraction (Fig. 3.3b). A curved lens will exhibit both retardation and refraction (Fig. 3.3c), the extent of which is governed by:
(a) the angle at which the light strikes the lens – the angle of incidence,
(b) the density of the glass – its refractive index, and
Image formation
Parallel rays of light entering a simple lens are brought together by refraction to a single point, the ‘principal focus’ or focal point, where a clear image will be formed of an object (Fig. 3.4c). The distance between the optical center of the lens and the principal focus is the focal length. In addition to the principal focus, a lens also has other pairs of points, one either side of the lens, called conjugate foci, such that an object placed at one will form a clear image on a screen placed at the other. The conjugate foci vary in position, and as the object is moved nearer the lens the image will be formed further away, at a greater magnification, and inverted. This is the ‘real image’ and is that formed by the objective lens of the microscope (Fig. 3.5).
If the object is placed yet nearer the lens, within the principal focus, the image is formed on the same side as the object, is enlarged, the right way up, and cannot be projected onto a screen. This is the ‘virtual image’ (Fig. 3.6), and is that formed by the eyepiece of the microscope of the real image projected from the objective. This appears to be at a distance of approximately 25 cm from the eye – around the object stage level. Figure 3.7 illustrates the formation of both images in the upright compound microscope, as is commonly used in histopathology.

Figure 3.6 A virtual image is viewed through the lens. It appears to be on the object side of the lens.
The components of a microscope
Objectives

Figure 3.11 Diagram of achromatic and apochromatic objectives.
Some examples of the latter may have as many as 15 separate lens elements.
You may also need

Full access? Get Clinical Tree

