Tool And Techniques

Cell biology is the branch of biology that deals with the study of structure, biochemistry, physiology, reproduction, evolution and genetics of cells. The living organisms consist of small and complex cells, which are not visible with unaided eye. Ordinary microscope could not observe details of tissues and cells. Now–a–days a number of tools and techniques are available to study the detailed structure of cell and biomolecules.

History

• Term ‘microscope’ – Faber.

• Father of microscopy – Antony von Leeuwenhoek.

• First compound microscope – F.H. Janssen and Z. Janssen.

• Binocular microscope – Rheita.

• Ocular lens — Two lens eye pieces – Huygens.

• Compound microscope with rock and pinion system – Wilson.

• Modern classroom compound microscope – Kepler and Galileo.

• Oil immersion lens (objective lens of 100 x  resolution 0.25 µm) – Tolles. [It works in immersion oil (Cedarword oil, Liquid paraffin and its R.I. is 1.5 equal to glass slide)].

• Substage condenser to focus light on object – Abbe.

• Apochromatic objective lenses free from chromatic and spherical abberations – Abbe.

Linear units of measurement

• 1 meter (m) = 100 cm = 1000 mm = 1,000,000 μm = 1000,000,000 nm = 10¹⁰ Å.

• 1 centimeter (cm) = 1.01m = 10^-2 m = 10 mm =10,000μm = 10,000,000 nm = 108 Å.

• 1 millimeter (mm) = 0.001 m = 0.1 cm = 1000μm = 1000,000 nm = 107 Å.

• 1 micron (μ) = 1/1000 mm = 10^-3 mm = 0.000,001 m = 0.0001 cm

  • =0.001 mm = 1,000 nm = 10,000 Å

• The new symbol for micron is μm (micrometer)

• 1 millimicron (mµ) or 1 nanometer (nm) – equal to one thousand of a micron

MICROSCOPY (GK : mikros – small, skipein – to see)

The cells of most animals, plants and bacteria have microscopic size and these are measured by a unit of measurements, the micron or µ which is equal to 1/1,000 mm (1000 µ = 1 mm). The various components of the cells (organelles) are still smaller, therefore, are measured by millimicron or angstrom Å) which is equal to 1/10,000 mm or 1,000 Å = 0.1 µ or 10,000 Å = 1 µ. The diameter of majority of cells ranges from 0.1 micron to 1 millimeter.

Fundamental Qualities of Microscope

A. MAGNIFICATION/POWER OF ENLARGEMENT

• It is the degree of enlargement or the ratio of size of the object as seen under the microscope to the actual size observe with unaided eyes. It has no unit and no limit.

• Magnification =

• It depends on (a) Focal length of lenses (b) Length of body tube.

B. RESOLVING POWER (LIMIT OF RESOLUTION)

• The ability of a microscope to distinguish two close point as two separate points is known as resolving power.

• It depends on (a) Wave length of light used for illumination(lambda, l)  (b) Numerical aperture of the first lens (objective lens)(NA).

• Greatest resolutionis obtained with the shortest wavelength of light and larger value of numerical apertureof the objective lens. Higher resolution makes image clear.

TYPES OF MICROSCOPE

LIGHT MICROSCOPE / bright–field microscope

• The image is formed due to selective absorption of light by different parts of the specimen.

• The image is inverted and virtual and can be seen directly with naked eyes as well as screened.

• These microscopes use 2 – 3 lens systems.

• Each lens system is made up of minimum  2 ordinary convex glass lenses acting together as one unit, simply called lens.  These are - Ocular lens, Objective lensand Condenser lens.

• These optical (light) compound microscopes provide useful magnification of 1000 to 2000 X and resolving power of average 3000 Å (0.3 µm).

DARK FIELD MICROSCOPE OR ULTRAMICROSCOPE

• Invented by Zsigmondy(1905). Dark field microscope is constituted by adding a disc called ‘stop’ to condenser in bright field microscope. The stop withheld the light in central field.

• Now object is provided light by oblique beam of light. The object gets illuminated by oblique beam. This light is reflected in the margins of specimens. It is based on the fact that light is attached at boundaries between regions having different refractive indexes. This leads to bright illumination of specimen against dark background.

POLARISING MICROSCOPE

• Invented by Talbot (1834). It uses polarised light.

• The polarization microscope differs from the ordinary microscope having two rotatable polarizing devices, the Polariser(condenser) and the analyser(colour).

• A material (anisotropic) has two different index of refraction in two perpendicular directions.

• Polarization microscopy is useful mainly for viewing highly ordered objects such as crystals or bundles of parallel filaments.

ULTRAVIOLET MICROSCOPE - CASPERSEN

• Helpful for quantitative analysis of chemical constituents of the cells

• Used to locate nucleic acids in the cell due to there strong capacity to absorb UV rays which are the source of illumination in this microscope.

• The wavelength of UV rays is 1500-3500-A. The lenses are made of quartz and the image is taken on a film sensitive to UV rays. Resolving power of UV microscope is 0.1 μ= 1000 Å.

FLUORESCENT MICROSCOPE – HAITINGER AND COONS

Lehmann(1911) described the first fluorescence microscope. Max Haitinger(1935) developed fluorescence microscope by staining histological preparations and smears with fluorescent dyes.

Fluorescence microscope uses UV light with higher wavelengths (3500-400 Å). Instead of visible light. Here the object is coated with fluorochrome dyes. When illuminated with UV radiations, the stained specimen releases fluorescent wave-lengths like red, orange, yellow or green against dark field.

X- RAY MICROSCOPE – KIRKAPTRICK

X – ray microscope help in the study of molecular structure of substances in solid state and in the analysis of three - dimensional structure of different substances.

C. MICROSCOPES USING ELECTRONS AS SOURCE OF LIGHT

Most powerful tool in biological studies because of useful magnification of 1 – 4 lakh and high resolution of 2 – 10 Å.

                                                TABLE : DIFFERENT TYPES OF MICROSCOPES

Type of Microscope	Maximum useful magnification	Resolution	Comments
Bright field Microscope	1,500 x	100 – 200 nm	Extensively used for visualization of Micro organism, usually necessary to stain
Darkfield Microscope	1,500 x	100 – 200 nm	Used for viewing living Micro organism particularly those with characteristic morphology, stain not required
UV Ultra Vilot Microscope	2,500 x	100 nm	Improved resolution over normal light microscope, largely replaced by electron microscope
Fluorescence	1,500 x	100 – 200 nm	Usefluorescence staining useful in many diagnostic procedures for identifying Microorganism
Phase contrast Microscope	1,500 x	100 – 200 nm	Does not require staining used to examine microbes
Interference Microscope	1,500 x	100 – 200 nm	Use to examine Microorganism produces sharp multi colour image
TEM (Transmission Electron Microscope)	5,000-1000,000 x	1 nm	Used to view ultrastructure of micro organism including viruses
SEM (Scan Electron Microscope)	10,000-1,000,000 x	1 – 10 nm	Used for showing detailed surface structure of micro organism 3D external surface study.

                        Difference Between Light Microscope and Electron Microscope

S. No.	Light Microscope	Electron Microscope
1.	Illuminating source is light.	Illuminating source is beam of electrons
2.	Both living and fixed specimens can be studied.	Only fixed specimens are studied
3.	The object is 5 μm or thicker.	The object is ultra thin 0.1μm or below.
4.	The specimen need not be dehydrated.	Only dehydrated specimens are used.
5.	A vacuum is not required.	Vacuum is essential for its operation.
6.	There is no need for high voltage electricity.	High voltage electric current (50,000 volts and above) is required.
7.	A filament is not used.	Tungsten filament is used to produce electrons.
8.	There is no cooling system.	It has a cooling system to take out heat generated by high voltage electric current.
9.	Radiation risk is absent.	There is a risk of radiation leakage.
10.	Light microscope used glass lenses are used.	It employs electromagnetic lens.
11.	Image is formed due to absorption of light waves.	Image is formed due to scattering of electrons.
12.	Image can be seen directly.	Image is seen only on a flouorescent screen.
13.	Speciman is stained by coloured dyes .	Specimen is coated with heavy metals in order to reflect electrons.
14.	Resolving power is 0.25μm or 250nm (2500 ).	Resolving power is 0.5-5.0, though theoretically it can be 0.25.
15.	Magnification is up to 2000.	Magnification is up to 400,000.
16.	It is used for the study of detailed gross internal structure	Electron microscope is used in the study of external surface (SEM), ultrastructure of cell and very small organisms (TEM).

CHROMATOGRAPHY

Developed by M S. Tswett, a botanist, who in 1906, isolated the principle plant pigments by passing them in solution, in petroleum ether through a column of powdered chalk. Chromatography based no differential partition between two immiscible solvents was first described in 1941, by A. T. P. Martin and R. L. M. Synge. Chromotography is a technique employed for the separation of mixtures into their components. It is a technique of separating and analyzing different components of a mixture due to differences in the rate of their passage and adsorption while moving through absorbent medium / stationary phase.

             There are different types of chromatography depending up on the nature of stationary medium :

Types of Chromatography	Application
Adsorption or column chromatography	Separation of mixture of tissue lipids
Thin layer and paper chromatography	Separation of amino acids, nucleotides and low molecule products
Ion exchange chromatography	Purification of insulin and plasma fractionation
Gel filtration chromatography	Determination of molecular weight of proteins
Affinity chromatography	Separation of immunoglobulins cellular enzymes and m-RNA.

ELECTROPHOReSiS – Reuiss and Tiselius

It is the migration of charged particles in an electrolyte solution between two poles when an electric current is passed through it. The process is used for the separation of different protein of a mixture, nucleotide, nucleic acid etc. Electrophoresis is of two types. Nondenaturing- The charged particles are allowed to separate on the basis of their net charge. Denaturing - The charges of the solutes are nullified and the solutes are allowed to separate according to their molecular weight.

TYPES

1.GEL ELECTROPHORESIS

Electrophoretic separations on paper, cellulose acetate and agar are based on a simple electrophoretic effect namely the migration of ions under the influence of an electric current. The medium acts as an inert support for buffer in which the separation occurs but if the medium does exert any effect it is often adverse eg, the slight denaturing of proteins by paper causing tailing.

2.  TWO DIMENSIONAL ELECTROPHORESIS

This technique involves a combination of two different electrophoretic separations in single dimension.

The two dimensional electrophoresis is performed in two directions. In one direction, the molecule may be separated in non-denaturing conditions and in the second direction, at right angles to the first, under denaturing conditions.

IMMUNO ELECTROPHORESIS

• The separating power of a gel can be greatly enhanced by combining it with techniques based on the ability of a ligand such as an antibody, to precipitate a complex molecule from a complex mixture.

• Immuno electrophoresis is based on electrophoresis of antigenic proteins into an antibody containing gel which results in the precipitation of antigen antibody complexes.

SOUTHERN, NORTHERN AND WESTERN BLOTTING

• A mixture of DNA, RNA or protein fragments can be separated by gel electrophoresisand the separated bands can be stained and visualized directly in the gel.

• To confirm the identity of these bands or to find similarity of one or more of these bands with a known and available molecular probe, it is possible to hybridize these bands with a labelled probe. To facilitate this hybridization the bands are often transferred to a nitrocellulose membrane through blotting.

• When DNA bands are blotted, it is called Southern blotting(after the name of E.M. Southern).

• When RNA bands are transferred it is described as Northern blotting.

• When protein bands are transferred, the technique is described as Western blotting.

DNA FINGER PRINTING

The DNA of every individual has distinctive characteristics like the fingerprint which is not the same for two humans except monozygotic (identical) twins.

All segments of DNA do not code for proteins. Some DNA segments have a regulatory function, while others are intervening sequences (Introns) and still  others are repeated DNA sequences.

The non-repetitive or unique DNA sequences form almost half of the haploid genome (DNA in one set of 23 chromosomes) and except identical twins, no two humans have genomes with same DNA nucleotides sequences.

SPECTROPHOTOMETRY

It is the technique of producing and studying spectra formed by various chemicals. It is of several types-light spectroscopy, ultra-violet, infra-red, nuclear magnetic resonance, emission spectroscopy. It is based on absorption of specific wavelengths of electromagnetic radiations by substances present in gaseous, liquid, solution or solid state.

The method is used for determining the chemicals inside the cells, their extract and emissions. NMR or nuclear magnetic resonance spectroscopy is used in analysing the structure of small but complex compounds like proteins in a solution.