Microscopy and its types

Microscopy

Microscope is an inevitable instrument in studying the cell and subcellular structures. It offers scope in studying microscopic organisms therefore it is named as microscope (mikros – small; skipein – to see) in Greek terminology. Compound microscope was invented by Z. Jansen.

Resolution: The term resolving power or resolution refers to the ability of the lenses to show the details of object lying between two points. It is the finest detail available from an object. It can be calculated using the following formula

Resolution = 0.61λ/NA

Where, λ= wavelength of the light and NA is the numerical aperture.

Numerical Aperture: It is an important optical constant associated with the optical lens denoting the ability to resolve. Higher the NA value greater will be the resolving power of the microscope.

Magnification: The optical increase in the size of an image is called magnification. It is calculated by the following formula

Magnification = size of image seen with the microscope / size of the image seen with normal eye

Microscope works on the lens system which basically relies on properties of light and lens such as reflection, magnification and numerical aperture. The common light microscope which has many lenses are called as compound microscope. The microscope transmits visible light from sources to eye or camera through sample, where interaction takes place.

1. Bright field Microscope

Bright field microscope is routinely used microscope in studying various aspects of cells. It allows light to pass directly through specimen and shows a well distinguished image from different portions of the specimen depending upon the contrast from absorption of visible light. The contrast can be increased by staining the specimen with reagent that reacts with cells and tissue components of the object.

The light rays are focused by condenser on to the specimen on a microslide placed upon the adjustable platform called as stage. The light comes from the Compact Flourescent Lamp (CFL) or Light Emitting Diode (LED) light system. Then it passes through two lens systems namely objective lens (closer to the object) and the eye piece (closer to eye). There are four objective lenses (5X, 10X, 45X and 100X) which can be rotated and fixed at certain point to get required magnification. It works on the principle of numerical aperture value and its own resolving power.

The first magnification of the microscope is done by the objective lens which is called primary magnification and it is real, inverted image. The second magnification of the microscope is obtained through eye piece lens called as secondary magnification and it is virtual and inverted image (Figure 6.2 a, b and c).

2. Dark Field Microscope

The dark field microscope was discovered by Z. Sigmondy (1905). Here the field will be dark but object will be glistening so the appearance will be bright. A special effect in an ordinary microscope is brought about by means of a special component called ‘Patch Stop Carrier’. It is fixed in metal ring of the condenser component. Patch stop is a small glass device which has a dark patch at centre of the disc leaving a small area along the margin through which the light passes. The light passing through the margin will travel oblique like a hollow cone and strikes the object in the periphery, therefore the specimen appears glistening in a dark background. (Figure 6.2 d and e).

3. Phase contrast microscope

This was invented by Zernike (1935). It is a modification of light microscope with all its basic principle. The objects observed by increasing the contrast by bringing about change in amplitude of the light waves. The contrast can be increased by introducing the ‘Phase Plate’ in the condenser lens. Phase plate is a circular component with circular annular etching.

Microscopic measurements:

The microscope also has facility to measure microscopic objects through a technique called ‘micrometry’. There are two scales involved for measuring.

1.        Ocular Micrometer

2.        Stage Micrometer

Ocular Micrometer: It is fixed inside the eye piece lens. It is a thin transparent glass disc where there are lines divided into 100 equal units. The scale has no value.

Stage Micrometer: This is a slide with a line divided into 100 units. The line is about 1mm. The distance between two adjacent lines is 10 µm. The known value of the stage micrometer is transferred to the ocular micrometer, thereby the measurements can be made using ocular micrometer.

Light passes with different velocity after coming out of the thinnest and thickest areas of the phase plate thereby increasing the contrast of the specimen. A hollow cone of light passes through the condenser. Direct light pass through thin area of phase plate, whereas light passing from the specimen reaches thick area of phase plate. The light passing through thicker area travel at low speed, on the other hand the light passing through thin area reach fast therefore contrast is increased in the specimen. Phase contrast microscope is used to observe living cells, tissues and the cells cultured invitro during mitosis (Figure 6.2 f and g).

4. Electron Microscope

Electron Microscope was first introduced by Ernest Ruska (1931) and developed by G Binning and H Roher (1981). It is used to analyse the fine details of the cell and organelles called ultrastructure. It uses beam of accelerated electrons as source of illumination and therefore the resolving power is 1,00,000 times than that of light microscope.

The specimen to be viewed under electron microscope is dehydrated and impregnated with electron opaque chemicals like gold or palladium. This is essential  for withstanding electrons and also for contrast of the image.

There are two kinds of electron microscopes namely

1.        Transmission Electron Microscope (TEM)

2.        Scanning Electron Microscope (SEM)

1. Transmission electron microscope: 

Transmission electron microscope: This is the most commonly used electron microscope which provides two dimensional image. The components of the microscope are as follows:

a.        Electron Generating System

b.        Electron Condensor

c.         Specimen Objective

d.        Tube Lens

e.         Projector

A beam of electron passes through the specimen to form an image on fluorescent screen. The magnification is 1–3 lakhs times and resolving power is 2–10 Å. It is used for studying detailed structrue of viruses, mycoplasma, cellular organelles, etc (Figure 6.4 a and b).

Comparison of Microscopes

2. Scanning Electron Microscope:

This is used to obtain three dimensional image and has a lower resolving power than TEM. In this, electrons are focused by means of lenses into a very fine point. The interaction of electrons with the specimen results in the release of different forms of radiation (such as auger electrons, secondary electrons, back scattered electrons) from the surface of the specimen. These radiations are then captured by an appropriate detector, amplified and then imaged on fluorescent screen. The magnification is 2,00,000 times and resolution is 5–20 nm (Figure 6.5 a and b).