eChapters

Electron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield information about the topography (surface features of an object), morphology (shape and size of the particles making up the object), composition (the elements and compounds that the object is composed of and the relative amounts of them) and crystallographic information (how the atoms are arranged in the object).

Electron Microscopes were developed due to the limitations of Light Microscopes which are limited by the physics of light to 500x or 1000x magnification and a resolution of 0.2 micrometers. In the early 1930’s this theoretical limit had been reached and there was a scientific desire to see the fine details of the interior structures of organic cells (nucleus, mitochondria...etc.). This required 10,000x plus magnification which was just not possible using Light Microscopes.

Electron microscopy is a specialized branch of science, which uses the electron microscope as a tool. The electron microscope is an instrument which magnify smallest object thousands times. The magnification is not only requirement to see the smallest otherwise invisible object but the details of the object are most important. The details of the object are depending upon the resolving power of the microscope, which is known as the resolution of the microscope. This resolution or power of the microscope is depends upon the wavelength of the illuminating source. The smallest the wavelength of the illuminating sources is the best resolution of the microscope. Electron microscopes use electrons as an illumination source to take advantage of the very short wavelengths attainable in electron beam, which permit a considerable improvement in resolution over the light microscope.

Wilhelm Conrad Roentgen, German physicist, born on November 8, 1895 discovered X-rays and was awarded the first ever Nobel Prize in Physics in 1901 in recognition of the extraordinary services he has rendered by the discovery of the remarkable rays subsequently named after him. These short-wave electromagnetic radiations have the wavelengths that are matching with both the atomic sizes and shortest interatomic distances. But, the index of refraction of X-rays is near unity for all materials and hence they could not be focused by a lens in order to observe small objects such as atoms, as it is done by glass lenses in a visible light microscope or by magnetic lenses in an electron microscope. Thus, in general, X-rays cannot be used to image individual atoms directly. As an alternative to this difficulty, Max von Laue in 1912 proved that the periodicity of the crystal lattice allows atoms in a crystal to be observed with exceptionally high resolution and precision by means of X-ray diffraction for the first time by using a single crystal of hydrated copper sulfate (CuSO₄ 5H₂O).

Raman Spectroscopy

It is a spectroscopic technique based on inelastic scattering of monochromatic light, usually from a laser source. Inelastic scattering means that the frequency of photons in monochromatic light changes upon interaction with a sample. Photons of the laser light are absorbed by the sample and then reemitted. Frequency of the reemitted photons is shifted up or down in comparison with original monochromatic frequency, which is called the Raman effect. This shift provides information about vibrational, rotational and other low frequency transitions in molecules. Raman spectroscopy can be used to study solid, liquid and gaseous samples.

Nano-materials are to be extensively studied and characterized prior to their application in material science, biological science, electrical and electronics. In addition to the basic physical properties of the nano-materials, other characteristics such as paramagnetism, ferromagnetism, supermagnetism, optical properties, quantum size effects and electrical properties have to be studied. With a view to provide the fundamental information on these aspects this chapter is dedicated.

Nano-material possess unique properties such as dimension, shape, specific surface area, surface mass ratio etc., that are highly influenced by the mechanical and physical properties. It is pertinent to understand the basic physical properties of nano-materials in order to apply them in a wide array of functions. In this context, this chapter is devoted to provide basic information on physical, mechanical and photo-luminescence properties of nano-materials.

The history of nanotechnology in the modern world dates back to 1959 when American physicist Richard Feynman inspired the scientific community in his lecture “There is a plenty of rooms at the bottom”. Research on nanosized particles dramatically developed into an important field of modern research with potential effects in electronics to medicine and recently in agriculture and food industry.

Nanoparticles exhibit unique properties in terms of chemical, physical, photoelectrochemical and electronic properties when compared to their respective bulk materials. The applications of these nanosized particles are defined in a bigger ways in the field of Biotechnology. All these applications require custom based synthesis methods for specific applications.

Introduction

The metal particles in nanometer scale shows exciting optical and electronic characteristics when compared with respective bulk metals. Because of the inherent electronic properties of the metal and metal oxide nanoparticles these nanostructure materials are applied in various disciplines in recent days. Mostly these nanoparticles can be synthesized either top down or bottom up approaches. The conversion of bigger size particle or matter into tiny particles is called top down approach where as smaller particles or atoms joined together forming particles in regular shape within nanometer scale dimension is called bottom up approach. The formation of nanoparticles by chemical reduction is an example for the later one and the reduction size with the help of mechanical crusing leads to the formation of fine nanoparticles is named as top down approach. Mostly nanoparticles can be synthesized by chemical method because of its easiness to synthesis particles with uniform size and high purity.

Introduction to Nano-materials

Nano-sized materials are naturally present from forest fires and volcanoes, but are also generated unintentionally from anthropogenic sources as a by-product of combustion and deliberately as manufactured nanomaterials. Nanoparticles have been used for centuries. The coloured glass that we see in many old cathedrals from the middle age was made of gold nanosized clusters that created different colour depending on the size of the nanoparticles. The most prominent example of engineered nanoparticulate material is carbon black which has been around us for decades in applications like printing inks, toners, coatings, plastics, paper, tires and building products.
 
Two of the major factors why nanoparticles have different properties (optical, electrical, magnetic, chemical and mechanical) than bulk material are because in this size-range, the quantum effects start to predominate and the surface area to volume ratio is increased.

Introduction

Nature has been performing wonderful feats for millions of years. All organisms from microbes to humans are powered by highly evolved molecular and cellular machines that operate at nano scale. The capability to manipulate, control, assemble, functionalise, produce and manufacture things at atomic precision provides an opportunity to understand this new and exciting field of science. Nanotechnology is application of science to control the properties and structure of matter at nanometric scale.

Indian Agriculture is facing a wide spectrum of constraints such as burgeoning population, shrinking farm land, restricted water availability, imbalanced fertilization, low soil organic carbon, besides experiencing the fatigue of green revolution and vagaries of climate change. To address all the challenges ahead, we should think of an alternate technology such as “nanotechnology” to precisely detect and deliver the correct quantity of nutrients or other inputs required by crops that promote productivity while ensuring environmental safety. The word “nano” refers to the size of one-billionth of a metre or one-millionth of a millimeter in any one of the dimension. We are aware that all materials are made up of atoms which are the smallest units and for example, ten hydrogen atoms put together will measure one nanometer. As the size gets reduced, the surface area gets increased by several folds.

Introduction

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. The most commonly used sequencing method is the dideoxy method. This method uses dideoxynucleotide triphosphates (ddNTPs) which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). Dideoxynucleotides are chain terminators. In a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3’OH necessary for the addition of the next nucleotide is absent (Sanger et al., 1977).

Introduction

Only after the discovery of restriction endonucleases it became possible to digest any DNA molecule at a specific site and any two pieces of DNA could be joined together with the help of another modifying enzyme, the DNA ligase. Consequently, a new branch of molecular biology, namely genetic engineering has been developed. With the help of genetic engineering techniques, it is now possible to identify and isolate the gene (for a protein of interest and insert it into the genome of another organism with the help of a suitable vehicle. The foreign gene thus cloned, can be amplified to produce millions of its copies. The insert gene can be expressed by the host to synthesize the gene product. The technique can also be used for understanding the regulation of expression of the cloned gene. The possibilities are endless and probably sky is the only limit. In this chapter we will briefly discuss the basic principles and protocol involved in gene cloning.

Southern Blotting
Introduction

Southern hybridization was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. It is also called Southern blotting since the procedure for transfer of DNA from the gel to the nitrocellulose filter resembles blotting. In Southern hybridization, a sample of DNA containing fragments of different sizes is subjected to electrophoresis using either polyacrylamide or agarose gel.

Introduction

Restriction enzymes, recognizes specific base sequences in double-helical DNA and cleaves at specific places, both strands of a duplex containing the recognized sequences. To biochemists, these exquisitely precise scalpels are marvelous gifts of nature. They are indispensable for analyzing chromosome structure, sequencing very long DNA molecules, isolating genes, and creating new DNA molecules that can be cloned. Werner Arber and Hamilton Smith discovered restriction enzymes, and Daniel Nathans pioneered their use in the late 1960s.

Introduction

Gel electrophoresis is a technique for separating charged molecules with different sizes. Two kinds of gels are commonly used: agarose and polyacrylamide. Agarose gels can be applied to a wider range of sizes than polyacrylamide gels.  By using standard agarose electrophoresis, nuclei acids up to 50 kb may be separated. If Pulsed Field Gel Electrophoresis is used, the upper limit can be extended to 10 Mb. Polyacrylamide gels may separate nucleic acids that differ in length by only 1 nucleotide if their length is less than 500 bp (Sambrook et al., 1989).

Introduction

This chapter gives first hand information on PCR for beginners. PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments; usually up to 10 kb (kb stands for kilo base pairs). Certain methods can copy fragments up to 40 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell-for example, a human cell contains about three billion base pairs (Kary Mullis, 1983).

Introduction

In order to use a fine quality of DNA for various molecular biological work like sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (random primer labeling), etc., quality and quantity of DNA is extremely important to have the defined template concentration. Failure to produce results from the above techniques can sometimes be attributed to an incorrect estimate of the DNA template used.

DNA Isolation Protocol
Introduction

Most of the all procedures involved in molecular biology experiments require the purified form of nucleic acid. It forms a fundamental requirements for genome characterization, gene mapping and for identification and isolation of gene for genetic engineering. The most important step involved in removal of proteins which is carried out in different ways. The following protocol is one of the most commonly used protocols in animal, plant and microbial samples.

Plant cell culture
Introduction

As the human population is expanding there has been increasing concern over whether current rates of food productions can support the growing population. In response to the increasing demand, scientists and farmers have been working hard to come up with new ideas to meet these needs. These ideas range from simple procedures including selection of the best seeds to more complex procedures requiring the transplantation of genes into different plant species. This chapter focuses on one of the important aspects of plant science which is the growth of plant cells in an artificial media. This process is known as plant cell culture and can be applied in many different ways.