Principles of Microscopy
All kind of microscopes use visible light to observe specimens. Light has a number of properties that affect our ability to visualise objects.
Light is a part of the wide spectrum of electromagnetic radiation from the sun. It is a form of energy. The most important property of light is wavelength (the length of light ray) (Figure 2.1).
The sun produces a continuous spectrum of electromagnetic radiation with waves of various lengths (Figure 2.2). Radiation of longer wavelength includes Infrared (IR) and radiowaves, the shorter wavelengths include Ultra Violet (UV) rays and X-rays.
The physical behaviour of light can be caterigorised as either light rays, light waves or light particles. The combined properties of particle and wave enable light to interact with an object in several different ways like transmission, absorption, reflection, refraction, diffraction and scattering (Figure 2.3).
Lenses are optical devices which focus or disperse a light beam by means of refraction. A simple lens consists of a single piece of transparent material. Light rays from a distant source are focused at the focal point F. The focal point lies at a distance f (focal length) from the lens’ centre (Figure 2.4).
When an object is placed outside the focal plane (the plane containing the focal point of the lens.), all the light rays from the object are bent by the lens. The bent rays converge at the opposite focal point. At the focal point, the light rays continue and converge with nonparallel refracted light rays. The resultant reversed and magnified image is formed in the plane of convergence (Figure 2.5).
Objective is the important part in the microscope which is responsible to produce a clear image. The resolution of the objective is most important. Resolution is the capacity of alens to separate or distinguish between small objects that are close together. The major factor in the resolution is the wave length of light used. The greatest resolution obtained with light of the shortest wave length, that is the light at the blue end of the visible spectrum are in the range of 450 to 500nm. The highest resolution possible in compound light microscope is about 0.2μm. That means the two objects closer together than 0.2μm are not resolvable as distinct and separate. The light microscope is equipped with three or four objectives. The working distance of an objective is the distance between the front surface of the lens and the surface of the cover glass or the specimen. Objectives with large numerical apertures and great resolving power have short working distances.
Numerical aperture (NA) is the value representing the light gathering capacity of an objective lens. NA was first described by Ernst Abbe, and is defined by the following expression
Numerical Aperture (NA) =n × sin(θ)
n = the refractive index of the medium between the specimen and objective; θ = half aperture angle or collection angle of the objective. (the maximum half angle of the cone of light that can enter or exit the lens).
The resolving power of a light microscope depends on the wavelength of light used and the NA of the objective lens.
The numerical aperture of a lens can be increased by
· Increasing the size of the lens opening and/or
· Increasing the refractive index of the material between the lens and the specimen.
The larger the numerical aperture the better the resolving power. It is important to illuminate the specimens properly to have higher resolution. The concave mirror in the microscope creates a narrow cone of light and has a small numerical aperture. However, the resolution can be improved with a sub stage condenser. A wide cone of light through the slide and into the objective lens increases the numerical aperture there by improves the resolution of the microscope.
In order to view microorganism and microbial structures of different sizes we require different kinds of microscopes.
· Light microscopes resolve images with the help of light. The specimen is viewed as dark object against a light background in bright field microscope. Dark field microscope uses a special condenser and the specimen appears light against a black background. The other types of mircoscopes are Phase contrast and Fluorescence microscope.
· Electron microscope uses a beam of electrons instead of light. Electrons pass through the specimen and form a two dimensional image in Transmission Electron Microscope (TEM). Electrons are reflected from the specimen and produce a three dimensional image in Scanning Electron Microscope (SEM).