MICROSCOPY

1. MICROSCOPY

Microscope is the single most important tool in the study of biology. The existence of cell was known only after the invention of microscope. The cells and all other cellinclusions are so small that they cannot be perceived by naked eye. It was only after the invention of microscope the wonderful world of microorganisms was brought to our notice. The rapid progress in understanding the most complicated biological processes in recent years has been made possible due to application of modern technology in microscopy. Our knowledge about the cell and cell functions are not the same as was known only 10 years back and it will not remain as it is today. Hence there is a direct relationship between the progress in microscopy and the progress and refinement of our knowledge of the science of biology and other branches of science.

TYPES OF MICROSCOPE:----

All earlier microscopes were of the simplest forms of the light microscopes. In modern times many improvements have been made in the light microscope and as a result various types of modern light microscopes have been developed.

There are the following types of microscopes :

1. Dissecting microscopes 2. Light microscopes 3. X-ray microscopes 4. Electron microscopes

1. Dissecting microscopes. A dissecting microscope is actually a simple microscope that consists of only one lens unit (i.e., may be an ordinary magnifying glass). Such microscopes are used to dissect out the material or to observe the materials which require less magnifications (i.e., 6X to 12X, sometimes upto 20X).

A dissecting microscope consists of a short limb attached to a basal foot. A stage of glass plate is attached to the limb. A mirror is attached to limb below the stage to adjust the light. The magnifying lens unit is placed on a folded arm which is attached to the limb. This can be moved vertically with the help of an adjustment screw. The material to be viewed is placed on the stage. It is viewed by placing eye close to the lens and focussing it with the help of adjustment screw.

2. Light microscopes. The microscopes in which ordinary light is used as the source of illumination are generally referred to as light microscopes. Light microscopes mostly used in the biological studies are of the following types :

(a) Compound microscope. In such microscopes three types of lens systems are used,

(i) condenser lens system is located just above the mirror and below the specimen. Its function is to condense the light rays reflected from the mirror so that concentrated light rays can enter the microscope.

LENS

CLIP

STAGE

MIRROR

ADJUSTMENT SCREW

FOOT

Fig. 1.1. Dissecting Microscope

(ii) Objective lens system remains near and above the specimen. It magnifies the specimen and produces a real image. The objective lens system is a combination of convex and concave lens of different types of glasses. Most compound microscopes are equipped with three objective, the low power objective (10x/16 mm), high dry objective (44x/4mm) and an oil immersion objective (95x/1.8mm). 10x, 44x and 95x indicate their power of magnification and 16mm, 4mm and 1.8mm indicate their focal length

(iii) Ocular lens system remains nea the eye of the observer. The real imag produced by the objective lens is furth magnified by the ocular lens. It is comprise of two lenses. The lower lens is called the fie lens which places the real image obtained froz the objective lens into the focal plane of th upper eye lens which further magnifies ar focuses the virtual image of the specimen to the eye. Magnification in such microscope is the product of the magnification of th objective lens and the ocular lens. Thus an ocular lens having magnifying power of 10x when combined with the objective lens having magnifying power of 10x produces a total magnification of 100 times. We can increase magnification by increasing the magnifying

power of the lens. But:can we go on the magnification indefinitely ? No, there is limitation in

magnification in microscopes. The reason for such limitation will be discussed later in this chapter.

such

EYE PIECE

ROUGH ADJUSTMENT

SCREW

BODY TUBE

FINE ADJUSTMENT

SCREW

NOSE PIECE

HANDLE

OBJECTIVES

STAGE

INCLINATION

JOINT

CONDENSER SCREW FOR

DIAPHRAGM MOVING THE SUBSTAGE

PILLAR

MIRROR

BASE

The differences between a simple light microscope and a compound light microscope lie not only in structure but also in lens system. Structurally a compound microscope is complex than a simple microscope in that it has many more mechanical parts (Fig. 1.3) than a simple

Fig. 1.2. A compound microscope

microscope. The optical parts of the simple microscope include only one magnifying glass. Therefore the power of magnification is very much limited to the observation of morphology only.EYE POINT

REAL IMAGE

MECHANICAL TUBE LENGTH (160 mm)

PROJECTION DISTANCE (250) mm)

#

M

OBJECT

VIRTUAL IMAGE

ENTERING PENCILS

OF LIGHT

Fig. 1.3. A compond microscope in sectional view showing the path of light through it.

(6) Ultraviolet microscope. In such microscope, beside the ordinary light, the invisible ultraviolet rays are employed for illumination of objects. Ultraviolet rays are of short wave length and therefore to facilitate easy passage of such rays fused quartz lens are used instead of glass lens. In this microscope image cannot be observed, but can be photographed. This type of microscope has great utility in qualitative as well as in quantitative determination of cellular components.

condenser, light is removed from the field and only the object is illuminated so that the outline of the cell, the nucleus, vacuole, mitochondria, plastids etc. can be detected. Even the spindle and the chromosomes in cell undergoing division can be observed.

(d) Fluorescence microscope. In such microscope fluorescence dyes are used to detect the object and to study their chemical nature. Different fluorescent dyes emit rays of different wave length when ultraviolet rays of higher wave-length is used as sources of illumination. Different dyes are used to stain different objects.

(e) Phase-contrast microscope. It is

(c) Dark field microscope. It is just like an ordinary compound microscope with a special condenser. With the help of thealso

a light compound microscope. Only difference is that it can resolve the minute difference in phases or refractive index of the different cell components and thereby enables to distinguish adjacent structures.

In

X-ray

image plane

of three

rear focal plane

of objective

objective

diffracted ray

ultrastructures of cell and cell inclusions and in measuring the concentration of cell contents. 3. X-ray

microscope. microscopes X-rays are used as source.com illuminations. X-rays possess extremely short wave-length and great penetration power. It is an important tooi in analysis dimensional structure of micro-molecules and macro-molecules of the cell. The molecules must be brought to crystaline form. The image of the object is formed on the film. X-ray microscope has revealed the structural details of lysozyme, haemoglobin, DNA etc.

4. Electron microscope. It has been mentioned earlier that the resolving power of light microscope is very much limited. Object having dimension smaller than that of 220 nm cannot be observed under light microscope. Resolution can be further increased by using ultraviolet rays. Resolution power of the microscope depends upon the source of light used. Shorter the wave-length of the light greater is the resolution. Electrons have very short wave-length. Effort to use electron as a source of illumination was successful in 1938 when Knoll and Ruska could finally develop

electron microscope. Use of electron microscope

started in 1940. At that time transmitted electrons were used and therefore this type of electron microscopes are called transmission electron microscope. This type of electron microscopes are in use even today. The scanning electron microscope is comparatively a recent development. In this type a fine pencil of electrons scans the surface of the objects. In both the types the images are produced on the fluorescence screen which can be photographed, videotaped or produced in motion using a computer. In electron microscopes the magnification upto 2,50,000 times can be achieved. Both living and dead specimens

be

in microscopes. While the transmission electron

electrong microscope produces one dimensional views,

specimen plan

undiffracted ray

an

was

condenser

Fig. 1.4. Diagrammatic flow of rays of phase-contrast.

can

seen

(A) Interference microscope. In this microscope two rays are used. One passes through the object and the other besides the object. Both the rays meet above the object and thereby contrast is created depending upon the refractive index of the object. Thus a true shape of the object can be obtained. (g) Polarizing microscope

used in analysing polarized light. It is

uses

the scanning electron microscope produces three dimensional view of the interior of cells. Scanning electron microscopes were developed.