List of top seven types of microscopes:- 1. Phase Contrast Microscope 2. Interference Contrast Microscope 3. Ultraviolet Microscope 4. Fluorescence Microscope 5. Immunofluorescence 6. Dark-Field Microscope 7. Electron Microscope.
Type # 1. Phase Contrast Microscope:
This microscope was developed by Fritz Zernikes (1935), a Dutch physicist who was awarded Nobel Prize in 1953 for this contribution. It is a conventional light microscope fitted with a phase- contrast objective and a phase-contrast condenser (Fig. 15.7).
Phase contrast microscope is based on the fact that the rate at which light travels through objects is inversely related to their refractive indices. Thus the light passing through one object into another object of a slightly different refractive index undergoes a change in phase.
These differences in phase are translated into variation in brightness of the objects and hence the objects differing even slightly in refractive index are viewed by the eye. Phase contrast microscope helps viewing living unstained structures of microbial cells. Unlike interference contrast microscope, the phase contrast microscope relies upon a single beam of light.
Type # 2. Interference Contrast Microscope:
This microscope (developed by Merton et al., 1947) relies upon the two beams of light illuminating the specimen and they combine after passing the specimen.
Nomarski Differential Interference Contrast (NDIC) Microscopes (Fig. 15.8A) are the most frequently used interference microscopes by the microbiologists. These microscopes produce high contrast images of unstained, transparent specimens in what appear to be three- dimensional.
The three-dimensional image is produced because the two beams of light travelling very close to each other through the specimen produce a stereoscopic effect. Although these microscopes are very useful for qualitative observations of unstained cells, they do not produce clear images if the object viewed is very thin.
Type # 3. Ultraviolet Microscope:
As we know that the resolving power of a light microscope is related to the wavelength of the light used; longer the wavelength lower the resolving power. Therefore, resolution can be improved by reducing the wavelength of the light. The UV microscope has this advantage.
Since glass is opaque to ultraviolet light, the lens system must be made of appropriate quality quartz and the microscopes should have filters to eliminate ultraviolet light from reaching the eyes. This microscopy is complicated and expensive thus a modification known as fluorescence microscopy has come into use.
Type # 4. Fluorescence Microscope:
Fluorescence microscope (Fig. 15.8B), developed by Coons (1945), is that in which a specimen stained with fluorescent dye is viewed. A fluorescent substance is that which absorbs light of one wavelength (the excitation wavelength) and emits light at a different wavelength (the emission wavelength).
For example, when the fluorescent dye “fluorescein isothiocynate” is illuminated with blue light, it emits green light. Fluorescent microscopes are equipped with excitation filters that permit the selection of the wavelength used to illuminate the specimen and barrier filters that prevent all but the emission wavelength from reaching the ocular lens if the dye is exposed to ultraviolet light, the emitted light to be viewed must be in the visible range.
Fluorescent microscopy has become important in microbiology as the fluorescent dyes can be linked with antibodies. When such antibodies combine with specific antigen, they become fluorescent. Thus, this technique makes possible to detect specially the cells of a particular type of bacterium in a mixed microbial population.
Type # 5. Immunofluorescence (The Fluorescent Antibody Technique):
Immunofluorescence or the fluorescent antibody technique is a rapid procedure used to identify an unknown bacterium with the help of fluorescence microscopy. This technique is based on the behaviour of certain dyes (e.g. fluorescencein isothiocyanate, rhodamine isothiocyanate) which fluoresce (glow) when exposed to certain wavelengths of light. These dyes are called fluorescent dyes.
In immunofluorescence the fluorescent dyes are chemically combined with antibodies; the antibodies to which a fluorescent dye is attached are called labelled or fluorescent antibodies. Labelled or fluorescent antibodies are mixed with a suspension of unknown bacteria.
When a sample of the mixture is placed on a slide and the smear examined by fluorescence microscope, only those bacteria (particularly those antigens are present on their surface) that reacted with the specific labeled or fluorescent antibodies become visible.
Thus only a few bacteria need to be present in the smear of the mixture to be observed. This is the direct method in which the fluorescent dye is combined with the antibody specific for the antigen present on bacterial cell surface (Fig. 15.9A).
There is an indirect method of immunofluorescence in which the initially applied antibody is not labelled. Instead, a second labelled antibody against the globulin of the animal species used for the preparation of the initial specific antibody is applied.
This binds the fluorescent label to the specific antibody that has already reacted with antigen in the smear (Fig. 15.9B). The indirect method is more sensitive as two or more anti-globulin molecules can be attached to each antibody bound to its antigen.
Type # 6. Dark-Field Microscope:
Dark-field microscopy permits the detection of unstained small biological objects which otherwise provide insufficient contrast. In a dark- field microscope (Fig. 15.10) the normal condenser of a light microscope is replaced with a dark-field condenser that does not permit light to be transmitted directly through the object and thus through the objective lens.
The dark-field condenser focuses light on the specimen at an oblique angle in such a way that the light does not impinge on the object and, does not enter the objective lens.
Thus, only light that reflects-off the object is seen, and in the absence of the object the entire field appears dark. This microscope helps viewing even small bacteria and large viruses but it is not helpful in distinguishing the internal structure of any bacterium being viewed.
Type # 7. Electron Microscope:
EM was invented by Knoll and Ruska (1932). It works on the principle similar to that of a light microscope except that an electro-magnetic field and a beam of electrons act in a way similar to the action of a glass lens and a beam of light (Fig. 15.11). An electron beam when accelerated through an electric field of 100 KV, has a wavelength of only 0.04 nm which is about 10,000 times shorter than the wave-length of visible light.
The resolving power and magnification of an electron microscope is therefore much higher than any light microscope.
In an electron microscope (Fig. 15.12), a beam of electrons is projected from a cathode (electron gun) and is passed through a series of electro-magnetic lenses. The condenser lens collimates the electron beam on the specimen and an enlarged image is produced by a series of magnifying lenses.
The specimens which are focused cannot be directly seen, their image is rendered visible by projection on a phosphorescent screen. Since the penetrating power of the electrons through solid matter is weak, only very thin sections of specimen can be examined.
Two types of electron microscopes are in use today.
Transmission Electron Microscope (TEM):
TEM is used to see the fine structure of cells; an object as small as 1 nm may be viewed. Ultra-thin sections of the objects are prepared, by embedding or freezing the specimen and sectioning it with a diamond of glass knife. Sections are floated in water and picked up on a wire grid.
They are stained with a heavy metal (gold or pallidium) to make certain part dense, and inserted in the vacuum chamber of the microscope. A 100,000 volt electron beam is focussed on the section and manipulated by magnetic lenses. A photograph prepared from the image may be enlarged with enough resolution to achieve magnification of 500,000 to 1,000,000 times at 1 nm resolution.
Scanning Electron Microscope (SEM):
SEM (Fig. 15.13) allows surfaces of objects to be seen in their natural state without staining (Fig. 15.14). The specimen is put into the vacuum chamber and covered with a thin coating of gold to increase electrical conductivity and thus forms a less blurred image. The electron beam then sweeps across the object building an image line by line as in a TV camera.
As electrons strike the object, they knock loose showers of electrons that are captured by a detector to form the image. Magnification with this microscopy are limited to about 10,000- 1,000,000 times at 1-10 nm resolution. 
