Introduction:

The tools of cytology are very varied and sophisticated depending on level of study. At this level, it is necessary to stress on principles of microscope action and cellular chemistry.

Brief History:

Word Microscope was invented by Grovani Faber in 1625 A.D. Discovery of microscope though, (dating back to 1000 A.D) followed observation that water in the glass bowl magnified objects therein, and that tiny drop of water acted as the magnifier. After that, present optical microscope came out from work of Anthony von Leevwenhoek, and others, between 1673 and 1683 A.D. Such discoveries comprised cautious observations of tiny plano-convex and bi-convex lenses able to magnify objects 30 to 200 times their normal sizes. With such arrangements, bacteria, red blood cells, spermatozoa etc became visible to naked eye for first time. Careful manipulation of two simple lenses to provide compound microscope was attained by Galileo in 1610 A.D. Robert Hook, in 1665 A.D, made the microscope which was able to observe opaque objects. Since then, microscope had suffered alteration to evolve present-day Compound Microscope that is extremely refined and versatile, and could be utilized to view several types of object.

Microscopy:

Many kinds of microscopes are available for study of biological materials. Essentially, they can be categorized by the kinds of light source they use. Light or optical microscope, certainly, utilizes visible light this could be from sun or from electric lamp. There are definite modifications of this, like phase contrast, polarization, interference, and dark-field microscopes. Microscopes that use invisible radiation, like ultraviolet, X-ray, and electron microscope are more current developments.

The Light (or Optical) Microscope:

Light microscope is the system having two magnifying glasses or lenses. The objective lens gives initial magnification, and ocular lens is placed symmetrically so as to magnify main image a second time. An extra condensing lens is usually used beneath stage of microscope to concentrate light from its source into very bright beam illuminating object, therefore giving adequate light for inspection of magnified image.

The Polarizing Microscope:

This instrument was developed by mineralogists who use it in the study of crystalline materials. Several natural objects, comprising crystals and fibres, show the optical property called as double refraction or birefringence. The polarizing microscope is conventional microscope in which Nicol prism (or Polaroid sheet) is introduced in light path below condenser. This Polarizer converts all light passing by instrument in plane polarized light, or light that vibrates in one optical plane only. Second prism, termed analyzer is placed inside barrel of microscope above objective lens. When analyzer is orientated so that its polarizing direction is parallel to that of polarizer below, one sees image. Though, if analyzer is rotated until its axis is perpendicular to that of polarizer, no light can pass by ocular lens and field is black.

Phase Contrast Microscope:

Phase microscopy gives the method whereby contrast is created by simply optical means. Refractive index is a measure of optical density of the object, or speed at which a light wave travels by it. Air, for example has refractive index of about 1.0, water about 1.3, and glass about 1.5. Phase contrast microscope comprises of optical plates placed inside condenser and objective lenses that converts phase differences in amplitude differences. Phase contrast microscope is of no particular assistance in study fixed and stained biological preparation in which transparency differences are not significant. Instrument finds its application mainly in study of living cells and of unstained tissues.

Interference Microscope:

Interference microscope, like phase contrast microscope, depends on ability of object to retard light. Interference microscope sends through specimen two separate beams of light which then are combined in image plane.

The Dark-Field Microscope:

This microscope uses strong oblique light which doesn't enter objective lens. Special dark-field condenser, in which no light passes by centre of lens, is used. Light therefore reaches object to be viewed at angle. Dark-field examination is also observed in the examination of small transparent objects like chylomicrons that are invisible in glare of bright field illumination, and of micro incineration specimens.

The Ultraviolet Microscope:

Ultraviolet light can't pass by ordinary optical lenses; lenses are opaque to ultraviolet (UV) light. Therefore quartz lenses are utilized in UV microscopes. In principle, this system permits improvement in resolution about twice that of ordinary microscope (0.1μ). Ultraviolet light is used in fluorescence microscopy. Several substances have property of emitting visible light when irradiated by invisible rays. UV light is focused on specimen that glows and can be observed but is emitted fluorescence.

The X-Ray Microscope:

X-rays have shorter wavelength than visible or ultraviolet light and thus has a greater penetration and, in theory, a higher resolution power. By using preparation techniques like those utilized in light microscopy, specimen can be placed on a photographic emulsion and exposed to soft X-irradiation. Small X-ray, picture attained is subsequently magnified optically. This procedure is called contact microradiography.

Electron Microscope (EM):

Development of this kind of microscope has revolutionized our complete concept of the cell. EM uses the electron beam instead of light and electromagnets instead of glass lenses. There are two kinds of electron microscopes:

a) Transmission Electron Microscope (TEM)

b) Scanning Electron Microscope (SEM)

Freed from limitations imposed by wavelength of light, electron microscope has the resolving power 500 times as great as optical microscope. Electron microscope can attain magnification of over 500,000 times. The impart on EM on biology has been tremendous.

The Compound Microscope:

Compound microscope is combination of two simple microscopes (i) objective and (ii) ocular (or eyepiece) separated by the long tube called as TUBE or BARREL. In tube objective is lens closer to object, while ocular or eyepiece is lens closer to eye. In more recent times, improvements on microscope have been chiefly on mechanical rather than optical properties.

Optical Principles of the Microscope:

Construction of optical compound microscope is based on principle of effect of light rays passing media of different reflective indices, and of their passing by different thickness of same medium. Objects are distinguished from each other as they reflect light of changeable intensity and color.

This interference effect (the underlying effect in microscope viewing) depends on wavelength (λ) of light utilized for illumination of object under examination. From two objects to be distinguished in microscope field of view, distance between objects should not be less than wavelength (λ) of light used; or else two images will merge and remain as one (i.e unresolved). This minimum distance (approximately 0.3 μm) is known as resolving power of microscope. Ability of objective lens to resolve or distinguish structures depends on numerical aperture of objective lens; higher the numerical aperture of objective the greater the resolution. Relationship between N.A. and resolving power of microscope can be represented by given equation.

 R.P = (λ)/(2 x NA)

Where λ = wavelength of light employed

N.A = numerical aperture

R.P = resolving power

The NA is function of refractive index of medium between objective and object, and angular aperture of object.

NA = nsinμ

Where n = refractive index of medium between object and objective lens

μ = half angular aperture of objective

Magnification:

An object viewed through a light microscope becomes enlarged as a result of the production of a magnified virtual image through the ocular. The degree of magnification is expressed in diameters. The magnification by the objective lens is known as the primary magnification. This is equal to:

λ/f

Where λ = length of microscope tube (generally 160 mm) and f = focal length of objective (generally marked on it). Total magnificent (objective and eye piece lenses) is found by multiplying primary magnification of objective by magnification of eye piece.

I.e. Total Magnification = (λ/f) x e

Where e = magnification of eye piece.

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