Microscope

Microscope

The microscope may be defined as an optical instrument consisting of a lens and combination of the lens for making enlarged and magnified images of minute objects.All microscope employed the principle that specific lenses magnify the image of a cell such that detail of its structure is more apparent.Magnification means simply the increase in the size of the image of an object.The compound microscope magnifies the image of an image.The compound microscope magnifies the image of an object twice.The total magnification is the product of the magnifying power of the object and that of the eyepiece.More increase in size without the ability to distinguished structural details and not beneficial.

The smallest distance at which two points can be seen separately is called the resolving power of the lens.The limit of resolution is the smallest distance by which two objects can be separated and still be distinguishable as two separated objects.It is determined by the wavelength of light used and the characteristics of a lens system,the numerical aperture.The limit for resolution of the light microscope is about 2nm i.e two objects closer together than the 0.2nm are not resolvable as distinct and separated.The resolution of electron microscope is to improve over that of light microscope by about 1000 fold

Resolving power(d)=λ/2NA

Where,NA=Numerical aperture

λ =Wavelength of light

Numerical aperture

The numerical aperture can be defined the function of the diameter of the objective lens in relation to its focal length.

The angle subtended by the optical axis and the outermost rays still covered by objective is the measure of the objective and it is half the aperture angle.The magnitude of this angle is expressed as a sine value.

NA=nsinθ

Where n=refractive index of the medium filling the space between the lens and object. With the dry objectives, the value of air.When immersion oil is used as the medium,n is 1.54 and if θ is 58,then

NA=nsin=1.56× sin58=1.33

The degree to which microscopic objects can be altered to increase the NA is limited.

Types of microscope

There are mainly two categories of microscope which are as follows

Light microscope

Electron microscope

• Light microscope

The microscope in which magnification is obtained by a system of optical lenses using waves.They are of following kinds

• Bright field microscope:It is commonly used in microbiology laboratory and consists of two series of lenses(objective lens and ocular lens)which function together to produce the image.In this microscope, the specimen to be examined is illuminated by a core of light focused on it by a substage lens system called the condenser.The microscopic field is brightly lightened while the object under observation because they absorb some of the light,but staining them is a dye increase their light absorbing ability resulting in greater contrast and colour differentiation.Most microorganisms except virus are some very thin bacteria can be clearly observed under bright field microscope.It is often used in counting cells and laboratory identification of microorganism
• Dark field microscope:It is the light microscope in which the lighting system has been modified to reach the specimen from the sides only.The effect produced by dark field technique is that of a dark background against which the object are brightly illuminated.The blank field microscope is equipped with a condenser that blocks light from entering the objective lens directly from the light source instead light reach the specimen from the side only.Thus the specimen appears brightly illuminated on a dark background.A bright object in the dark is much easier to see than a dark object in the bright field.Therefore it can detect the microorganism that is too thin to be seen in a bright field microscope eg Treponema pallidum.It is particularly useful to observe the motility of microorganisms and living unstained cell.
• Fluorescence microscope:It is used to visualise specimens that fluorescence i.e emit large light of one colour when the light of another colour shines upon them many chemical substances can absorb light.After absorbing light of a particular wavelength and energy,some substances will then emit light of longer wavelength and lesser energy content such substances are called fluorescent substances and the phenomena are termed as fluorescence.This microscope is based on the application of this phenomena.Fluorescence occurs either because of the presence of naturally fluorescent substances like chlorophyll with I the cells or the cell being treatment with fluorescent dyes are auramine and rhodamine.It is widely used in the clinical diagnostic microbiology.Eg mycobacterium tuberculosis in sputum can be identified by its characteristics yellow fluorescence after staining with auramine.
• The principle of fluorescence microscopy:In fluorescence microscope highly intensity mercury lamp is used as a light source emits white light.The exciter filter transmits only a blue light to the specimen and blocks out other colours.The blue light is reflected downward to the specimen by dichroic mirrors

The specimen is stained with the fluorescent dye.The stained portion absorbs blue light and emits green light which passes upward,penetrates the dichroic mirror and reaches the barrier filter.This filter blocks out blue light and allows green light to pay through and reach the eye.Thus,the eye perceives the stain portion of the specimen as glowing green against the black background.

• Phase contrast microscope:This type of microscope is the slight modification of bright field microscope and somewhat uses the similar principle.Cells differ in refractive index from their surrounding background and hence bend some of the light rays passing through them.The contrast between the light passing through the specimen and the light passing through the surrounding background is amplified by using specimen arrangements of phase contrast microscope.This will lead to the formation of a darker image on a light microscope.

Application

1. Phase contrast microscopy is extremely valuable for studying living unstained cells and is widely used in applied and theoretical biological studies.
2. It is widely used in the study of living cells under natural condition.
3. It is also used for the visualization of pili.

Electron microscope

In the electron microscope,the radiation enters at the top and specimen is viewed at the bottom.A high voltage such as 50,000 volts is passed through a tungsten filament at the top of the column which releases the stream of electrons.Electromagnets on the column focus the beam.The electrons have the shorter wavelength than light which produces high resolving power and high magnification.A photograph taken with an electron microscope is called an electron micrograph.They are of following types:

• Transmission electron microscope(TEM):It is used to study the internal structure of cells as proteins and nucleic acid due to having greater resolving power.In transmission electron microscope, the electron is used instead of light rays and electromagnetic function as the lense.Since the electrons beams have poor penetration power,even a single cell is cut into many very thin slices with a special knife which is thin examined individually with an electron microscope.The advantage of this microscope is that it has high resolution.The disadvantage of this microscope are:
• Living specimen cannot be viewed.
• It is expensive
• Staining methods are difficult

• Scanning electron microscope(SEM):In scanning electron microscope.the specimen is subjected to a narrow electron beam which rapidly moves over the surface of the specimen.This causes the release of the shower of secondary electrons and other types of radiation from the specimen surface.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.