Light microscopy

Introduction

This is an introduction to the theory of light microscopy. The subject is dealt with in more depth in the previous editions of this book and further information may be found in dedicated texts to the subject.

The light microscope is an essential part of the histopathology laboratory as it is the device with which histological preparations are studied. The designs and specifications of modern microscopes vary widely, but the basic principle is the same as the original simple microscope which used sunlight as its light source ( Fig. 3.1 ). Electric bulbs or light emitting diodes (LEDs) are now used to produce a beam of light which is focused on the tissue section or sample, and then the transmitted light passes through a set of objectives, along the tube and through the eyepiece into the eye of the microscopist.

The lens system of the light microscope allows the eye to see an image of the target tissue at varying magnifications depending on the objectives used. The varying lenses seen in the modern microscope are present within the substage condenser below the slide, as well as above it. The additional objective lenses above the sample can be brought into position depending on the tissue magnification required. The objectives are usually mounted in a rotating disc and are brought into alignment with the main body tube of the microscope to select higher or lower magnifications.

The different magnifications required are achieved by altering three variables; firstly, the angle at which the light strikes the lens, the angle of incidence; secondly, the curvature of the lens and finally, the density of the glass or refractive index (RI). Parallel light entering a lens from a small object is brought to a sharp focus at a point behind the lens, then the eyepiece allows a magnified real image to be formed below the eyepiece ( Fig. 3.2 ). This is the basic principle of light microscopy.

Light and its properties

Visible light occupies a narrow portion of the electromagnetic spectrum and can be detected by the human eye, although the full spectrum extends from radio and microwaves through to gamma rays. Electromagnetic energy is complex, having both wave and particle-like properties.

It is common practice to illustrate the light in the electromagnetic spectrum as a sine wave. The distance from one wave peak to another is the wavelength of light ( Fig. 3.3 ).

Light with a single wavelength is monochromatic, but the majority of light sources are composed of many different colors and wavelengths which are refracted in different directions. The pan-spectral distortion which can occur to an image can be corrected by different types of lenses within a microscope.

The human eye responds to a complex mixture of light of different wavelengths and when this approximates to the mixture of light derived from the sun, it is known as ‘white’ light. By definition, white light is a mixture of light which contains a percentage of wavelengths from all of the visible portions of the electromagnetic spectrum. One measure of the mixture of light given off by a light source is color temperature . The higher the color temperature, the closer the light is to natural daylight derived from the sun.

Light sources produce light in all directions and usually consist of a complex mixture of wavelengths which define the color temperature of the light source. Some sources, e.g. tungsten filament and xenon lamps provide a relatively uniform mixture of wavelengths, although of different amplitudes or intensities. Others, e.g. mercury lamps, provide discrete wavelengths scattered over a broad range, but with distinct gaps of no emissions between these peaks.

Most light sources are non-coherent, but standard optical diagrams draw light rays as straight lines even though the actual light is emitted from the source in all directions. Another property important in understanding microscope optics is that some of the light is absorbed by the media (lens and air) through which it passes ( Fig. 3.4 ). This produces a reduction in the amplitude, or energy level, of the light. The media can also have an effect on the actual speed of the light passing through the microscope, this is known as retardation .

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. 3.5a ). When light enters the glass at any other angle, a change of direction also occurs and this is called refraction ( Fig. 3.5b ). A curved lens will exhibit both retardation and refraction ( Fig. 3.5c ) and this is governed by:

• The angle at which the light strikes the lens, the angle of incidence.

• The density of the glass, its refractive index.

• The curvature of the lens.

The angle by which the rays are deviated within the glass or other transparent medium is the angle of refraction and the ratio of the sine values of the angles of incidence (i) and refraction (r) gives a figure known as the refractive index (RI) of the medium ( Fig. 3.6a ). The greater the RI, the higher the density of the medium. The RI of transparent substances is important in the computation and design of lenses, microscope slides, coverslips and mounting media. Air has a refractive index of 1.00, water 1.30 and glass has a range of values depending on the type, mostly averaging 1.50.

Usually, light passing from one medium into another of higher density is refracted towards the normal, and when passing into a less dense medium it is refracted away from the normal. The angle of incidence may increase to the point where the light emerges parallel to the surface of the lens – beyond this angle of incidence total internal reflection will occur, and no light will pass through ( Fig. 3.6a ).

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. 3.6b ). The distance between the optical center of the lens and the principal focus is the focal length . A lens has an additional pair of points, one either side of the lens, called conjugate foci , and an object placed at one will form a clear image on a screen placed at the other. The conjugate foci vary in position – 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 formed by the objective lens of the microscope ( Fig. 3.7 ).

If the object is placed nearer the lens, within the principal focus, the image is formed on the same side as the object and is enlarged, the correct way up, and cannot be projected onto a screen. This is the virtual image ( Fig. 3.8 ) and is formed by the eyepiece of the microscope from the real image projected by the objective. This appears to be at a distance of approximately 250mm from the eye, near the object stage level . This may be illustrated as in Fig. 3.2 with the formation of both images in the upright compound microscope.

Image quality

White light is composed of all the visible spectral colors and on passing through a simple lens, each wavelength is refracted differently – blue is brought to a shorter focus than red. This lens defect is chromatic aberration ( Fig. 3.9a ) and results in an unsharp image with colored fringes. It is possible to construct compound lenses of different glass elements to correct this fault. An achromat corrected for blue and red produces a secondary spectrum of yellow and green; this in turn can be corrected by adding more lens components, the more expensive apochromat .

Microscope objectives of both achromatic and apochromatic types are usually overcorrected for longitudinal chromatic aberration and must be combined with matched compensating eyepieces to form a good quality image. This restriction on changing lens combinations is overcome by using chromatic, aberration free (CF) optics which correct for both longitudinal and lateral chromatic aberrations, and remove all color fringes. This is useful for fluorescence and interference microscopes.

Other distortions in the image may be due to coma, astigmatism, curvature of field and spherical aberration; they are due to the lens shape and quality. Spherical aberration is caused when light rays entering a curved lens at its periphery are refracted more than those rays entering the center of the lens and are not brought to a common focus ( Fig. 3.9b ). These faults are corrected by making lenses of different glass components, e.g. fluorite, and of differing shapes.

The components of a microscope