The following points highlight the nine important types of microscopes. The types are: 1. The Light Microscope 2. The Phase Contrast Microscope 3. The Interference Microscope 4. Dark Field Microscope 5. Polarization Microscope 6. Electron Microscope 7. Transmission Electron Microscope 8. Scanning Electron Microscope 9. Spectrophotometer.
Type # 1. The Light Microscope:
As the name suggests, visible light is used in such microscopes to magnify the image of an object. Visible light is a very small portion of the spectrum of electromagnetic radiation consisting of different wavelengths (Fig. 21.1). The average wavelength of visible light is about 550 nm and it has 7 components, namely, violet, indigo, blue green yellow, orange and red.
Functioning principle of a light microscope is shown in the Fig. 21.2. Wavelength of light becomes a limiting factor in magnification. The light microscope cannot discriminate two objects separated from each other by a distance of 200 nm or less.
The “resolving power” of a microscope signifies the minimum distance required between two points for them to be discriminated from each other. Following formula is used to calculate the lower limit of the resolving power for any optical system.
Where
r = 0.61λ/n sin α…(21.1)
r = resolving power,
λ = wavelength of light being used to illuminate the object,
n = refractive index of the medium in which the object is placed,
sin a = sine of ½ the angle between the object and the objective lens.
The “n sin a” is also called the nuclear aperture (N. A.).
The refractive index of air is (n = 1) and that of the immersion oil is (n = 1.5). The α can be increased by moving the lens closer to the object, and the maximum limit is 90°. Thus sin α cannot increase beyond 1.0 Therefore, the maximum nuclear aperture (n sin α) using an oil immersion lens will be 1.5 x 1.0 = 1.5.
Using the above formula, resolving power of a microscope for white light that has an average wavelength (λ) of about 550 nm can be calculated as;
r = 0.61x 550/1.5 x 1.0 = 223.7 nm (or approximately 220 nm)
The above calculation shows that two objects separated by a distance of more than 220 nm can be distinguished. In other words, the objects with smaller magnitude than about 220 nm cannot be seen by light microscope using the white light.
The blue light, with shorter wavelength can increase the resolving power. The submicroscopic components of the cell are ribosomes, membranes, microtubules and chromatin fibres etc. that require higher resolving power to be visualized.
The material is fixed in fixatives, such as, formaldehyde, Farmer’s fixative (3 : 1 alcohol-acetic acid), Carnoy’s solution (6:3:1 alcohol-chloroform-acetic acid) etc. Thin section is cut for observation. Microtome is used to cut very thin sections. Acetocarmine, aceto-orcein and Feulgen stains are commonly used for staining the chromosomes during cell division.
Type # 2. The Phase Contrast Microscope:
In the light microscope, the transmission of light through the components of cell (a heterogeneous system) is quantitatively equal. Therefore, the cell appears to be homogeneous optically, and the structural details cannot be observed.
Several cellular components have high refractive indices and they alter the phase of the light waves. The phase contrast microscope changes the phase differences into alterations in brightness (Fig. 21.3).
The change occurs due to the “interference”, the process by which two or more light waves combine to reinforce or cancel one another, producing a wave equal to the sum of the two combining waves. The refracted and un-refracted waves are easily separated from each other. The source of illumination in a phase contrast microscope is a cone of light whose rays pass obliquely through the specimen.
The refracted waves (R) deviate away from un-refracted (U) waves. When both rays are focused back into image plane, they undergo interference. The difference of phase between refracted and un-refracted waves is Va in light microscopes.
But in phase contrast microscope, a “phase plate” is used in the path of the refracted rays that retards the refracted waves to additional lA wavelength. The phase contrast microscope changes the differences in refractive index and thickness to contrasting degrees of brightness.
Type # 3. The Interference Microscope:
In this microscope, special mirrors are used to split the light beam into two separate rays, otherwise, it is similar to a phase contrast microscope. One light beam passes through the object, while the other beam passes through the control slide. Both the beams recombine and the change occurring in the phase of the object beam causes it to interfere with the control beam.
“Differential interference microscope” is a special type of microscope in which, the contrast is increased by recombining the split beam in polarized light. This microscope produces a 3-dimensional image of the object.
Type # 4. Dark Field Microscope:
In dark field microscopy, the background appears dark, while the specimen structure appears bright. This type of microscope is also similar to phase contrast microscope. But a special condenser is used to illuminate the specimen at an angle that no direct light enters the lens. Light scatters by interacting with the specimen. Due to scattering of light, the specimen appears bright on the dark background.
Type # 5. Polarization Microscope:
In this technique, polarized light is used to increase the contrast with the light microscope. The structures differing in refractive index in different planes can be visualized by this microscope. Molecular substructure of cell organelles can be viewed through this microscope.
Type # 6. Electron Microscope:
In electron microscope, beam of electrons is used to pass through the object. The image formation occurs on a photographic plate or fluorescent screen. The wavelength of an electron beam is 0.005 nm, that is 105 times shorter than the wavelength of visible light.
So the resolving power of the electron microscope is thousands of times greater than that of light microscope. However, the practical resolving power of an electron microscope is 0.5 nm, much less than the theoretical limit of 0.002 nm.
Electron microscopes are of two types:
(1) Transmission electron microscope, and (
2) Scanning electron microscope.
Type # 7. Transmission Electron Microscope:
This microscope forms an image of the specimen by the electrons that have passed through the specimen (Fig. 21.2). The components of the specimen that scatter electrons appear dark and are called “electrons dense”. The part that have less ability of electron scattering appear light.
The electron scattering ability of the element with higher atomic number, such as, uranium, lead etc. is greater than those of lower atomic numbers. The biological molecules are composed of the elements with comparatively low atomic number, viz., hydrogen, carbon, nitrogen, oxygen, phosphorus and sulphur.
These elements have poor electron scattering ability. Therefore, biological molecules are stained with metals of high atomic number such as uranium, lead and osmium.
The material is fixed in osmium tetrachloride, KMnO4 or phosphotungstic acid. The fixed tissues are then embedded in hard plastic resin. Ultra microtome is used to cut ultrathin sections (50-100 nm) of the material. These section are examined under the electron microscope. Intact organelles and viruses are not sectioned. Followings are some techniques used to observe the materials by electron microscope.
Shadow Casting:
This technique is used to un-sectioned materials e.g., viral particles. The sample dried on a film supported by a grid is placed in an evaporation chamber. The chamber is evacuated. Heavy metal atoms projected from a glowing filament impinge at a predetermined angle on the film (Fig. 21.4).
The metal is deposited as a uniform electron opaque layer on the film. The metal is deposited on one side of the specimen, while the other side lacks the deposition.
Examination under the electron microscope shows the “shadow” of the specimen in the place lacking the deposited metal. The size and shape of shadow provide the information on the 3-dimensional shape of the material.
Positive Staining:
The specimen is stained and than the excess of stain is removed. It gives an unstained background and stained object. Certain viruses can be stained by salts that become absorbed selectively. For example uranyl acetate stains the viral nucleic acid and other components. Abs conjugated to ferritin (electron opaque molecule) stains the protein.
Negative Staining:
Negative staining can be used to study the viral particles and organelles. The viral particles are mixed with salt, such as sodium phos-photungstate which is highly opaque to electrons. The mixture is spread on a carbon membrane and dried.
The regions of the particles which are not penetrated by salt form electron lucent area on an opaque background. Details of the surface structure is revealed by perpetration between protruding parts of the salt.
The Whole Mount Technique:
This technique is also used for the un-sectioned materials, but it does not involve staining or heavy metal deposition. The scattering of electrons from the object produces the image.
The Freeze-etch Technique:
By this technique, a unique picture of cells is viewed, especially where the membrane is involved. The cell is broken along and across the membranes and therefore, it shows the four views of the biological membrane, viz., protoplasmic surface, exoplasmic surface, protoplasmic fracture faces, and exoplasmic fracture faces.
The technique does not involve fixatives, stains and embedding agents and therefore, the cell structure is not deformed.
The material to be studied is frozen in liquid Freon in a vacuum. The cell function is instantly arrested due to rapid freezing. The frozen material becomes very hard, and when struck by a knife, it is broken along the lines of membranes.
Water is evaporated by placing the broken material in a vacuum. Water loss causes the “etching effect” i.e., details become much clearer. A heavy metal (e.g., platinum) is used for shadowing the fractured surface, and a replica is prepared by using a carbon film. A strong acid is used to remove the tissue and to leave the metal replica. This metal replica is viewed with the electron microscope.
Type # 8. Scanning Electron Microscope:
This microscope shows 3-dimensional surface architecture of cells and organelles. The present day scanning electron microscopes have the resolution power of 10 nm which is less than the resolution power of transmission electron microscopes.
However, this resolving power can be increased by making further improvements. In this system (Fig. 21.5), a beam of electrons is used that moves back and forth across the specimen by a canning coil.
It illuminates different points on the surface of specimen at different times. The scan generator synchronizes the movement of this beam in a cathode ray rube (television tube). Electrons are deflected from the specimen and are picked up by a detector that modulates the beam in the cathode ray tube. A 3-dimensional structure of the surface of the cell or organelle is obtained.
X-ray Diffraction:
X-rays are the electromagnetic radiations of the wavelengths ranging from 10-11 to 10-9 m (Fig. 21.1). They bear no electrical charge, so they are less harmful than electrons. Further, X-rays have greater penetrating power than electrons and therefore, they permit observations on relatively thick biological preparations surrounded by water vapour or gas.
But neither conventional glass lenses nor magnetic lenses suitable for focusing the X-rays beams are yet available. This limits the utilization of the full potential of X-rays. Therefore, another technique that does not require a finely focused X-ray beam, is used to study the molecular regular grating, such as, fact present in a crystal.
The image thus formed is the characteristic of the crystal. This phenomenon is called diffraction. A photographic plate is placed at the opposite side of the object and X-ray beam is allowed to pass through the object. Since atoms are opaque to X-rays (0.1 nm wavelength), all atoms larger than hydrogen atom act as separate diffraction edges.
The position of individual atoms within a molecule is determined from the pattern of X- ray diffraction. This technique was used by Wilkins to elucidate the structure of DNA. This technique has also been used for determining the 3-dimensional structure of many important proteins, providing information about protein specificity and the mechanism of enzyme action.
Type # 9. Spectrophotometer:
Different biological molecules absorb light at different wavelengths. The spectrophotometer measures the exact amount of light absorbed by a particular molecule. Concentration of the light absorbing material can be calculated by this method.
The type of the material can be identified with the help of “absorption spectrum” that is determined by measuring the absorption at different wavelengths. For example, proteins show the maximum absorption at 280 nm wavelength (U.V. light), while the nucleic acids show the maximum absorption of U.V. light at 260 nm wavelength (Fig. 21.6).
The absorption occurs due to the presence of ring structures of purine and pyrimidine bases in nucleic acids, and ring structures of certain amino acids in proteins. However, in proteins, peptide bond also absorbs U.V light but at lower wavelengths (220-230 nm).
At this wavelength, the absorption by nucleic acids is minimal, and therefore, protein can be quantitated easily at this wavelength. Ultra Violet microscopes which can reflect such wavelengths have been used to detect the presence of nucleic acids and proteins in various cell structures.
Light microscope can be used as a micro- Fig. 21.7. Functioning system of a micro- spectrophotometer (Fig. 21.7) for the molecular spectrophotometer, system that absorbs light in the visible spectrum (Fig. 21.1). In such micro-spectrophotometers, mirrors are used rather than conventional lenses. Any wavelength of light (from U.V to infra-red) can pass through the specimen without change of focus or lenses.
