Types of microscopes
(Gk. Micros = small ; Skopein = to see) It is practice of using microscopes for the study of finer details of small objects including cells and tissues. Microscope are instruments consisting of lenses (made of glass / Lithium fluoride / electromagnetic lens) which magnify and resolve small objects not visible to unaided eye for the study of their details.
Types of microscopes
1. Simple microscope
It is also known as magnifying glass and consists of a convergent lens. The image formed by it is virtual, enlarged and on same side of lens.
Robert Hooke designed a primitive microscope and discovered cells with it. It was the first tool ever used to observe biological objects. Its magnification power was 14 – 42 times only, so it is considered as simple microscope.
2. Compound microscope or Light microscope
The first compound microscope was assembled by Zacharias Janseen and J. Janseen, the Dutch spectacles makes in 1590. The first compound microscope was prepared by Kepler and Galileo in 1611. However, it was not used for laboratory study. It is simplest, widely used microscope having three lens i.e. condensor, which collects the light rays and precisely focuses them on the objects; objective lens, which magnifies the image by three objective lenses, i.e. low power, high power and oil immersion lenses. In a compound microscope an object can be magnified upto 1000 times and the magnification is independent of intensity of light, size of microscope and numerical aperture. The light microscope is also called bright field microscope because it forms the image when light is transmitted through the object. The specimen formed is darker than the surrounding bright field. The light microscope can be used to examine the live, unstained materials as well as the preserved and stained specimens.
3. Fluorescent microscope
It was developed by Coons (1995). It is observed that when ultraviolet light is irradiated on certain chemical substances, they absorb it and emit visible light. These chemical substances are called fluoro-chromes. The fluorescent substances e.g., quinine sulphate, rhodamine and auramine are used to stain the cellular objects and these objects are easily visible as fluorescent areas when illuminated with ultraviolet light. Property of emission of long wave radiation soon after getting excited by shorter wave called, fluorescence.
4. Polarizing microscope
It was invented by Tolbart. In this microscope the plane polarised light is used as a source of illumination. Unlike the ordinary light, plane polarised light vibrates only in one direction and the cellular objects are easily visible as they appear bright against the dark ground. Polarizing microscope is helpful in studying the spindle fibres in the cells.
5. Ultraviolet microscope
It was invented by Caspersson. In this microscope the source of illumination is ultraviolet radiations having shorter wavelengths (1500 Å – 3500 Å) as compared to ordinary visible light. In this microscope, the lenses are made of fluoride, lithium fluoride or quartz instead of glass.Ultraviolet microscope is helpful in quantitative determination of all those cell components which absorb ultraviolet rays, e.g., those places where high concentration of nucleic acids is found, appear as darker regions than the remaining cell components.
6. Phase contrast microscope
(a) Discovered by Dutch man Fredericke Zernicke (1935).
(b) Source of illumination is visible light.
(c) It is used to study living cells and tissues without staining and effect of chemical and physical agents on the living cells.
(d) The optical system of the phase contrast microscopy converts these phase variations into visible variations in light intensity or contrast.
(e) It also used to study spindle formation, pinocytosis, karyokinesis, cytokinesis etc.
(f) It is draw back is low magnification power so subcellular organelles smaller than like ribosomes, lysosomes, ER, cannot be visualised.
7.Interference microscope (Morten et.al.)
(a) It’s principle is similar to that of the phase contrast microscope and gives / studies quantitative data.
(b) Nomarski interference contrast microscope is useful to study mitosis /cell components in living state.
(c) It gives better image of living structures. It also used to measure thickness of the cell and determination of several light absorbing chemicals like nucleic acid, proteins, lipids etc.
(d) It helps to measure dry weight and water contents of the specimen.
8. Dark field microscope
(a) Zsigmondy (1905) invented this microscope.
(b) It is based on the fact that light is scattered at boundaries between regions having different refractive index.
(c) The object smaller than those seen with ordinary light microscope can be detected but can not be resolved.
(d) It makes use of visible light.
9. Electron microscope
This was developed by M. Knoll and E. Ruska (1931) in Germany. It is a large sized instrument which has an internal vacuum, high voltage (50,000 – 1,00,000 volts), a cooling system, a fast beam of electrons (0.54 Å wavelength), a cathod filaments of tungsten and electromagnetic lens (which having a coil of wire enclosed in soft iron casing) for focusing.Thus an electron microscope essentially comprises an electron gun and electron lenses. The electron gun is the source of electrons consisting of a heated tungsten filament. It is preferred because it can be heated upto 3000°C. The electron beam can be reflected by magnetic field. Therefore, a very powerful magnetic coil acts as lens. The focal length of the electromagnetic lenses change with the wavelength of illumination. Since the wavelength is controlled by the voltage, it should be controlled and made constant. Three types of magnetic lenses are used namely projector, objective and condenser. The magnetic field produced is concentrated by soft iron casing. When the filament is heated to incandescence, it emits electron. The electrons then move to positively charged anode. The entire microscope column operates under conditions of high vacuum. It is due to this fact that we can not observe living objects through an electron microscope (EM). For viewing objects under EM, ultrathin sections are prepared through an ultramicrotone.Electron microscope can magnify the objects upto 2,00,000 times (now possible upto 2,50,000 – 4,00,000) and direct study of objects is possible on this microscope. The resolving power of electron microscope is 10 Å which is 100 times more than the light microscope. The images obtained in electron microscope have usually black gray and white shades. Computer is used to enhance contrast and develop colour. The most recent technique for examining objects through electron microscope is freeze fracture. The material is frozen quickly in liquid nitrogen (–196°C). This material during microtomy tends to along lines of weakness.
Electron microscope are of two types: –
(a) Transmission electron microscope (TEM) : It was the first microscope developed by Ruska (1932). It produces two dimensional images. Study of living cells can not be done through this microscope because of high voltage, which is required to operate it, kills the living materials.Magnification of TEM is 1–3 lakh and resolving power is 2–10Å. Because of them transmission electron microscope has helped in the discovery of a number of small cell organelles e.g., ER, ribosomes, centrioles, microtubules etc. Details structure of larger cell organelles could also be known only with the help of TEM. e.g., chloroplast (thylakoids), mitochondria (elementary particles, DNA, ribosomes) etc. Study of virus, mycoplasma and other small entities could also be made possible with the advent of electron microscope.
(b) Scanning electron microscope (SEM) : This microscope was invented by Knoll (1935). It is used to see the surface view of structures by forming an image with the secondary electrons reflected by those structures and it gives three dimensional image. The specimen to be studied is first super cooled (in liquid propane at –180°C) and dehydrated in alcohol (at –70°C). It is then coated with gold, platinum or some other metal for creating a reflecting surface for electrons. Magnification of SEM varies from 15 – 2,00,000. Resolution power is 5 – 20 nm.
10. Advanced high power microscope
(a) Scanning probe microscope : The microscope is capable of resolving the outer texture of the material to the minutest detail since it has the potential to image even a single atom. Magnification is upto 100 million.
(b) Scanning tunnelling microscope : It has a tiny tungsten probe for moving over the surface of specimen. The microscope is used to detect defect in electrical conductors and computer chips.
(c) Atomic force microscope : It has an extremely fine diamond probe for moving over the surface of biochemicals. Oscillations produced in the probe are changed into images by a computer. The microscope is useful in viewing detailed structure of biological molecules, e.g., DNA, proteins, etc.