Light Microscope : Main Parts of Light Microscope!
It is a simple ordinary microscope which is most popularly used in biological studies.
The first light microscope was made by Janssen and Hans in 1590. The light microscope can magnify objects upto 2000 times.
Its limits of resolution are about 0.2 µ, about one-half the wavelength of the light used. The wavelengths of visible spectrum of light range from 4000 A to 8000 A. The resolving power of light microscope can be 3000 A if 6000 A is taken as an average wavelength.
The structures are visible as a result of differences in light absorption by the different portions of the object under examination. In untreated cells, the differences in light absorption are very small. Therefore, fixation and staining methods are used to differentiate the structural components of the material.
The compound light microscope uses visible light for illuminating the object and contains lenses that magnify the image of the object and focus the light on the retina of the observer’s eye. In its simplest form, the compound microscope consists of two lenses, one at each end of a hollow tube (Fig. 1).
The lens closer to eye is called the eyepiece, and the lens closer to the object being viewed is called the objective. The object, supported by a glass slide under the objective lens, is illuminated by light beneath it.
In some microscopes, a third lens, called a condenser is located between the object and the light source and it serves to focus the light on the object. In practice, the eyepiece and objective lenses are each composed of a number of lenses that are combined in such a way as to overcome certain optical aberrations that occur when single lenses are used.
The limit of the resolving power of the best compound light microscope is about 0.2µm. Thus, it will just resolve all but the smallest bacteria, however the image will not obviously distinguish any internal detail in these cells.
Lens system in light microscope:
In light microscope, the lens system is the most important. The lens systems parts are as follows —
It collects and focusses light on the material.
2. Objective lens:
It produces the image and also magnifies it.
3. Eyepiece or ocular lens:
It forms the image of the image formed by the objective lens and thus magnifies the image again.
The resolution of the microscope is based on the capacity of the lenses to distinguish detail and to present distinct images of objects that are very close together. More specifically, the limit of resolution is dependent upon the ability of the lens system to collect light and the wavelength of light utilized. The limit of resolution is determined according to the relationship—
R= 0- 61 λ / NA
Where 0.61 is a constant representing the minimum detectable difference in contrast and λ is the wavelength of light. The limit of resolution of the light microscope does not exceed 0.17 mµ. When the wavelength is 4000 A (the shortest λ, of visible light) and the NA is 1.4. With white light having wavelength of 5500 A, the limit of resolution is only about 0.25 mµ.
The higher the NA, the better the resolution, but there are limits here, since the glass lenses ordinarily used in a light microscope prohibit the passage of shorter wavelengths of light. In general, the smallest detectable detail in the light microscope is equal to about one-half the wavelength of light with which it is observed. Thus, the smaller the object, the shorter the wavelength of light necessary.
In the compound light microscope, the quality of image (its contrast) depends primarily upon differences in the absorption of light by various regions of the object. Since, living cells are largely transparent, it is not always easy to see much internal structure when viewing even very large cells in the ordinary light microscope.
Consequently, various staining methods have been developed that use coloured dyes having affinity for specific cellular structures. Some of these dyes or stains, do not kill the cells when added to the medium at low concentrations. Such dyes are called vital stains. Two widely used vital stains are neutral red, which stains cytoplasm, and Janus green B, which selectively stains mitochondria.