MICRO SCOPE

 Microscopy
    Microscope is the basic, routine and most characteristic tool or instrument for scientists in laboratories. The main function of any microscope is the magnification of the object. The magnification that is attainable by microscopes ranges from 100 to X 400,000. In addition to the magnification, a microscope must possess good resolution or resolving power which is the ability to distinguish two adjacent points as distinct and separate.
    Different types of microscopes are available today and each of them are having their own advantages and disadvantages. The microscopes used today have evolved significantly from Leevwenhoek's first simple microscope.
    Depending upon the magnification principle involved, microscopes are of two main categories. They are
    1. Light microscope   
    2. Electron microscope.
4.1.1.  Light Microscope
    Light microscopes use optical lenses and visible light for magnification. The light microscope can magnify images up to about 1000 times. To produce an ideal magnified image, the light microscope must possess a good resoving power and suitable numerical aperture. There are two types of light microscopes namely i) Simple microscopes and ii) Compound microscopes.
i) Simple microscope :
     A simple microscopes consists only one biconvex lens.
ii) Compound microscope :
    Compound microscopes are more complex but are common in usage today. A compound microscope consists of atleast two lens systems viz., objective and ocular or eye piece.  The objective lens lies close to the specimen or object and magnifies the specimen. The ocular further magnifies the image produced by the objective lens. So, the total magnification obtained in a compound microscope is equal to the product of the magnifying power of the two sets of lenses. The magnification of the specimen through this compound microscope ranges from 1000 to 2000 times the diameter of the specimen.
    A common compound microscope used today consists a series of optical lenses, mechanical adjustment parts and supportive structures for these components. The optical lenses include ocular, objectives and the substage condenser. The number of objectives is usually three with different magnifying powers. The function of condenser is to collinate the light rays in the plane of the microscopic field. The mechanical adjustment parts comprise the coarse, fine, and condenser adjustment knobs. The supportive structures include the base, arm, pillar, body tube or barrer and resolving nosepiece.
    The most commonly used compound light microscopes are 
1. Bright field microscope.    2. Dark field microscope
3. Phase contrast microscope    4. Fluorescent microscope
1. Bright field microscope :     The bright field microscopes are the most commoly used microscopes in biology and microbiology courses. If forms a dark image of the specimen against a bright background.  As a result, in bright field microscopy, the microscopic field is brightly lighted and the microorganisms appear darker because they absorb some of the light. Generally, most of the microorganisms do not absorbing ability increases. As a result, they absorb more light and exhibit greater contrast and colour differenciation. The basic limitation of the bright - field microscope is the resolving power. Normally, they produce a useful magnification of about 1000 to 2000. At a magnification greater than  2000 the image becomes fuzzy in  their microscopy due to less resolution.  At the best, the resolving power of this bright - field microscope is 0.2mm. That means, a bright field microscope can clearly distinguish two objects separated by distance of 0.2 mm.
2. Dark field microscope :  In this microscope, the specimens or objects are brilliantly illuminated against the dark background of microscopic field.  This dark - field microscope consists a special kind of condenser called as 'Abbecondenser' which is having an opaque disk or dark field stop. This special condenser transmits a hollow cone of light from the source of illumination.  As a result, most of the light directed through the condenser does not enter the objective and thereby the microscopic field becomes dark. In dark - field microscopy, only the light rays deflected or scattered by the specimen cuts on light same for eye piece so the specimen appear as a bright object on a dark backgroud. Dark - field microscopy is particularly valuable for the examination of unstained microorganisms suspended in fluids such as wet mount and hanging - drop preparations.
3. Phase-contrast microscope :  This is special microscope which permits the study of unstained objects  The phase- contrast microscope consists of a phase - contrast objective and a phase. The phase contrast microscope is an ordinary bright field microscope with two additional plates, namely annular diaphram and phase shifting plate. The phase contrast principle was discovered by Fritz zernike who was awarded Noble prize in physics in 1953. According to this principle, light waves have variable character for frequency and amplitude. Human eyes can not perceive a phenomenon when two light rays have similar amplitude and frequency but different phase. This special optical system makes it possible to distinguish unstained structures with in cell that differ slightly in their thickness. The phase of light is altered by different components of a cell e.g. the nucleus and cytoplasmic granules depeding on alterations of phase are converted into optical densities. Thus an unstained object like an animal cell, when viewed through this system, shows a wealth of structural detail not visible through the ordinary microscope.
        Phase - contrast microscopy is extremely useful and valuable for studying living unstained cells is widely used in applied and theoretical biological studies.
4. Fluorescence micro scope : The main principle involved in this fluorescent microscopy is the phenomenon of fluoresence. Some chemical substances absorb light of a particular wave length and energy and then emit light of longer wave lenght and lesser energy. Such substances are called as fluorescent substances. The phenomenon is known as fluoresence. The fluorescent microscope is used to visualize specimens that naturally fluoresence     as they contain fluorescent substances. For example, chloropyll fluoresces brilliant red. Specimens can also be observed in this microscope by treating the specimens with fluorescent dyes like acridine orange R, auramine O, primulin, thiazo - yellow G etc. These fluorescent dye molecules are called as fluorochromes.
    In practice, the microorganisms are stained with a fluorescent dye and the illuminated with blue light. The dye absorbs  the blue light and emit green light.The fluorescence microscope consists of special components like exciter filter and barrier filter for the purpose. The function of the exciter filter is to remove all but the blue light. The barrier filter functions in blocking the blue light and allowing green light to pass through and reach eye. The selection of barrier filters depends on the dye used in microscopy.
    The best adoption of this microscopy is Immuno-fluorescence’. Antibodies can be chemically combined with fluorescent dyes such as fluorescein isothiocyanate and lissamine rhodamine B. The  antibodies. these labelled antibodies when mixed with a suspension of bacteria gets attached to the surface of the bacteria. These bacteria with attached labelled antibodies are clearly visible when observed by fluorescent microscopy. This procedure is known as fluorescent antibody technique and thephenomenon is called as immunofluorescence.
    The fluorescence microscope is an essential and useful tool in medical microbiology and microbial ecology. Bacterial pathogens like Mycobacterium tuberculosis are easily identified by suing fluorochromes or fluorescent antibody technique.
            Inverted Microscopes
    There are two basic types of microscopes. The one most people are familiar with looks down at the specimen with the light source coming from below and is called an upright microscope. An inverted microscope looks up at the specimen with the light source coming from above instead.
    Inverted microscopes were first invented in 1850 by Tulane University's J. Lawrence Smith and debuted at the World's Fair in London in 1852. In the early 20th century, they began to be used for observation of living cells, particularly for aquatic life. It was also used for analysis of heavy metals like iron and steel before World War II.
    As the name suggests, an inverted microscope is upside down compared to a conventional microscope. The light source and condenser are on the top above the stage pointing down. The objectives and turret are below the stage pointing up.
    The only things that are "standard" are that
1) a specimen (as dictated by the laws of gravity) is placed on top of the stage and
2) thank heavens, the binocular or trinocular tube is not upside down but in the standard position pointing at a conventional viewing angle. As a result, one is looking up through the bottom of whatever is holding the specimen and is sitting on the stage rather than looking at the specimen from the top, typically through a cover glass, as on a conventional microscope.
Grades of inverted microscope :
There are two grades of inverted microscopes.
1. A routine inverted microscope is small and comes in low and medium power settings. These can be used in homes and small labs in schools. They are limited in what the can observe as they usually do not allow for fine focus and have relatively low power magnification.
2. A research inverted microscope comes in heavy power settings and can allow for a very fine focus. The major disadvantage to them is that they are extremely expensive and are usually only used by universities and medical institutions. They are usually able to accommodate video cameras and televisions to assist in research documentation. The improvements on the inverted microscope over the course of the 20th and 21st centuries have allowed it to be an integral part of advanced scientific research.
Advantages :
1. An inverted microscope is most helpful when looking at heavy objects or those which are greatly effected by gravity. Material specimens like metal can be large and heavy. They require the large staging areas that inverted microscopes allow for.
2. The materials greatly affected by gravity include living cells and aquatic life that tend to pool and collect on the bottom of specimen containers. An inverted microscope looks at the sample from the bottom, making it easier to see the organisms with ease. It also allows users to see the samples in a more natural environment than a standard glass slide. Petri dishes allow more movement for the samples and are commonly used with inverted microscopes.
3. This type of microscope has been redesigned and improved on to accommodate particular uses. There are stages made particularly for processes like incubation and in vitro fertilization. The nosepieces have been made larger and revolvable, making to make it easier for scientists to identify and rotate objects. They have also been made heavier and sturdier, allowing for less vibration and greater ease of observation.
4.  Inverted microscopes are often used for looking at living organisms and tissue that may be killed by staining, they often provide for "optical staining" through the use of phase contrast or DIC. My CK2 has phase contrast. Rather than use the more sophisticated phase contrast condenser used on a standard microscope, there is a simple slider that goes through the condenser and holds the necessary phase rings. Only the phase rings for the lower power objectives (e.g. 10x and 20x) are centerable by a fairly crude process (although it works). The phase ring for the 40x objective is not but seems to work fine.
  4.1.2 Electron Microscope
     Electron microscopes are of more recent origin and more sophisticated and useful than high microscopes. For the first time, Knoll and Rusca developed the electron microscope in 1932. The electron microscopy markedly differs in many respects from the optical microscopic technique. Electron microscope uses a beam of electrons and magnetic field as light and optical lenses, respectively, and the whole system operates in high vacuum. The electron microscope provide tremendous useful magnifications, because of much high resolution obtained than in light microscope.
    As the electron beam has extremely short wavelength of only 0.05 A it provides much greater resolving power. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to x 40,000. There are two types of electron microscopes namely.
1. Transmission electron microscope ( TEM)
2. Scanning electron microscope (SEM)
1. Transmission electron microscope ( TEM) :  The modern Transmission electron microscope is a complex and sophisticated microscope. The source of illumination in TEM is the electron gun which is made up of by a thin, V- shaped tungsten filament. On heating, the tungsten filament generates a beam electrons which is then focused on the specimen by the condenser. The specimen scatters the electrons passing through it. Then the beam is focused by objective magnet lens and project magnet lens to form an enlarged, visible image of the specimen on fluorescent screen. The magnified image can also be recorded on a photographic plate by a camera built into the instrument. Some substances are denser absorbing more electrons than others.
    Usually, the penetrating power of the electrons through solid matter is weak. So, the specimens are prepared as either thin films or thin sections. To increase the contrast of these thin preparations of the biological specimens, they are usual treated with special electron microscopic stains. The important stains are osmic acid, permanganate, uranium, lanthanum and lead.
Specimen preparation :  In order to observe specimens such as isolated macromolecules of protein or nucleic acid or to study the structural features of cells there are special techniques for specimen preparation. Two important techniques for specimen preparations are shadow casting and freeze etching.
i) Shadow casting technique :  In this, the dried specimen is deposited with electron dense or heavy metal at an angle of about 45 from horizontal. The heavy metals normally used for this purpose are platinum, chromium, nickel, uranium and alloys of gold and platinum or platinum and palladium. This shadow casting technique increases the specimens contrast. This technique is particularly useful in studying virus morphology, bacterial flagella.
ii) Freeze etching technique :  This is the more recent technique which avoids the fixation, embedding and sectioning of specimens. In this technique, the specimen is rapidly frozen in liquid nitrogen and then fractured with a knife pre-cooled with liquid nitrogen. The exposed surfaces of the fractured specimen are shadowed and coated with layers of platinum and carbon to form a replica of the surface. Later, these carbon replicas of the surfaces are used to study the details through TEM. This technique is found more useful in studying cell wall and membrane structures.
Draw backs of TEM :
1. Material to be observed should be dehydrated as TEM operates in vacuum.
2. The dehydration and treatment of specimen with electron dense staining substances may bring about changes in original structure.
3. The specimen preparation should not be thick, as electrons cannot penetrate through dense or thick material. 
2. Scanning electron microscope (SEM) :  The scanning  electron microscope is a comparatively new type of electron microscope. It is quite different in principle and application from transmission electron microscope. The SEM gives a three dimensional quality to specimen images. It is useful in studying the surfaces of microorganisms in great detail. It possess a great depth of focus and can be used to observe even fairly large specimens.
-    With this scanning electron microscope a wide range of magnifications from a low as X 15 up to about 1,00,000 is possible. In this a narrow beam of electrons rapidly move back and forth across the surface of metal coated specimen and scan the surface features. In contrast to the transmission electron microscope, the electrons emitted by the object’s surface produce an image in SEM.
    The SEM consists a detector, photomultiplier, cathode ray tubes for, viewing and photography as special components. In scanning electron microscopy, the specimen preparation is comparatively simple and, the material to be observed is coated with a thin layer of heavy metal such as gold. In SEM, the beam of electrons released from the electronic gun strike the specimen and scans the surface. During this scanning, secondary electrons are released from the specimen. These secondary electrons are collected by a detector where an electronic signal is generated. These signals are amplified by photomultiplier and then sent to a cathode ray rube which produces an image like in a television system. This image can be viewed and photographed. The SEM is especially useful in studies, of bacterial cells, spores, fungi and morphological changes in tissues infected with microorganisms.
Draw backs of SEM : 
1. The resolving power of SEM is comparatively less than that of transmission electron microscope.
2. It gives only the surface characters of the specimen but not of internal components.