Several types of microscopes are at hand for study of biological materials. Their classification is based on the types of light source used and consists of two main categories; optical microscopes utilizing visible light and microscopes that utilize sources other than visible light.
Microscopes utilizing a visible light source:
Microscopes that utilize the visible light as their source of illumination are of the following types;
Light (Optical) Microscope:
Basically it acts as a two stage magnifying device. An objective lens provides the initial enlargement and an ocular lens is placed so as to magnify the primary image a second time. Total magnification is obtained by multiplying the magnifying power of the objective and ocular lenses. An additional condensing lens is normally employed beneath the stage of microscopes to concentrate the light from its source into a very bright beam illuminating the object, thus providing sufficient light for inspection of the magnified image.
Many natural objects including crystals & fibers exhibit special optical property known as double refraction or birefringence. In histological material, birefringence is caused by asymmetric particles, too small to be resolved even by best possible lenses. The polarizing microscope is a conventional microscope in which a nickel prism or Polaroid sheet is interposed in the light path below the condenser. This “Polarizer” converts all the light passing through the instrument into plain polarized light (light which vibrates in one optical plane only). A similar second prism termed “analyzer” is placed within the barrel of the microscope above the objective lens. When the analyzer is rotated until its axis is perpendicular to that of polarizer, no light can pass through the ocular lens, resulting in a dark field effect. The field will remain black if an isotropic or singly refractive object is placed on the stage. A birefringent object, however, will appear bright upon a dark background when examined in this manner.
Phase contrast Microscope:
Lack of contrast has always been a problem in biological work because the refractive indices of cytoplasm and its inclusions are similar. In normal microscopy the problem is solved by staining differentially but this is subject to numerous limitations. Phase microscopy provides a method whereby contrast is created by purely optical means. Refractive index is the measure of optical density of an object or the speed with which it is traversed by the light wave. Air e.g. has a refractive index of approximately 1.0, Water 1.3 and a glass about 1.5. In other words, light traverses fastest in air, more slowly in water and slower still in glass.
Light waves traversing equal distance through air, water and glass will not emerge at the same time; they will emerge out of the phase with each other. The phase contrast apparatus consist of optical plates within the condenser and objective lens which converts the phase differences into amplitude differences, so that differences in refractive indices are rendered directly visible. Objects ordinarily transparent become visible through contrast difference.
The phase contrast microscope is of no practical assistance in the study of fixed and stained preparations in which transparency differences are not important. The instrument finds its application chiefly in the study of living cells, tissues and of unstained, plastic embedded sections.
It depends upon the ability of an object to retard light. However, unlike the phase microscope, which depends upon the specimen diffracting light, the interference microscope send two separate beams of light through the specimens, which are then combined in the image plane. After recombination, difference in retardation of light results in interference that can be used to measure the thickness or refractive index of the object under investigation.
Dark field Microscope:
This microscope utilizes a strong, oblique light that does not enter the objective lens. A special dark field condenser, in which no light passes through the center of the lens, is employed. Light thus reaches the object to be viewed at an angle so oblique that none of it can enter the objective lens. The field is therefore dark. However small particles present in the specimen will reflect some light into the objective lens and will appear as glistening spots. Thus, it is possible to visualize particles far below the limits of bright light resolution. The effect is similar to phenomenon of dust particles seen in a beam of sunlight entering a darkened room.
It is useful in the examination of small transparent objects such as chylomicron (particles of fat in the blood) which are invisible in the glare of bright field examination.
Microscopes utilizing a non-visible light source:
Images can be formed by rays other than visible light and in this instance, since the images can be viewed directly; they are made visible by means of a suitably sensitized photographic film. In general, the rays used in these special microscopes have a shorter wave length than that of visible light, which permit higher resolution.
Since ordinary optical lenses are practically opaque to ultraviolet rays of light, quartz lenses are used throughout the lens system of this microscope.
This microscope depends upon the differential absorption of ultraviolet light by molecules within the specimen and the results are recorded photographically. In principle, this system allows an improvement in resolution about twice that of light microscope. This system is useful for detecting proteins that contain certain amino acid and in detecting nucleic acids.
Ultraviolet light is also employed in fluorescence microscopy. Many substances have the property of emitting visible light when irradiated by invisible rays. When ultraviolet light is focused upon such a specimen it glows and can be observed by its emitted fluorescence. Fluorescence may be naturally occurring within the specimens or it may result from the introduction of fluorescent dyes that bind to certain specific components of these specimens.
Commonly, two types of electron microscopes are in use:
• TEM (Transmission Electron Microscope)
• SEM (Scanning Electron Microscope)
Transmission Electron Microscope (TEM):
The transmission electron microscope utilizes a system which in principle is analogous to that of light microscope. In electron microscope, the illuminating source is a beam of high velocity electrons, accelerated in vacuum. The beam is passed through the specimens and is focused upon a fluorescent screen or photographic plate by series of electromagnetic or electrostatic fields. The wave length of the electron depends upon the acceleration voltage used. At the voltage used routinely, the wavelength of electrons is of the order of 0.05 A° (Angstroms).
The electric or magnetic field used as lenses are imperfect and do not have the numerical apertures of optical lenses. Thus the practical limit of resolution of electron microscope is about 2 A°. The electron microscope permits the observation of cell and tissue structures beyond that seen with light microscope.
Scanning Electron Microscope (SEM):
It is a more recent development and unlike TEM, it does not depend upon electrons passing through the specimen under examination. The SEM bombards the surface of a specimen with a finely focused beam of electron. As the beam strikes a point on the specimen, deflected primary and emitted secondary electrons which originate from the surface are collected by a detector. The resulting signals are accumulated from many points to build up an image that is displayed on a cathode ray tube. Since the scanning electron microscope is characterized by a great depth of focus, it gives a three dimensional image of the surface of a bulky specimen. The electron microscopes (TEM and SEM) require special technique for preparing specimens for examination.