Thursday, 25 February 2016

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Lesson: Introduction to cell  

GLOSSARY

Adenosine tri phosphate (ATP): it is a nucleotide in which adenosine is linked to three phosphate bonds by anhydride bonds. 

Angstrom: a unit of measurement, i.e., 10-10 meters.

 Micrometer: a unit of measurement equal to 10-6m. 

Flow cytometry: it is a technique for automated rapid analysis of cells stained with fluorescent dyes as they pass in a narrow stream through a laser beam. 

X-ray crystallography: it is a technique which is used for determining three dimensional structure of macromolecules based on the pattern produced when a beam of X-ray is passed through the sample.

 Mass spectrometry: a very sensitive high speed technique that uses magnetic and electrical field to separate protein molecules based on difference in their size and net charge. 

Magnetic resonance force microscope: It is an imaging technique that acquires magnetic resonance images (MRI) at nanometer scales. With this technique it is possible to observe protein structure which otherwise cannot be seen with X-ray crystallography.

 Centrifuge: machine for rapidly spinning a tube containing a fluid to subject its contents to centrifugal force. 

Chromatography: the technique that utilizes flow of mobile phase over a stationary phase to separate molecules based on their relative affinities for the two phases.

 Glycoprotein: Protein with one or more carbohydrate groups linked covalently to amino acid side chains. 

Reverse transcription: It is the process in which DNA is synthesized using RNA as a template.

 Retrovirus: Any RNA virus that uses reverse transcription to make a DNA copy of its RNA.

Cell is unit of life 

When you see the world around you, a distinction between living and nonliving can be made immediately. Let us see, how do you distinguish between these two? The organisms, which are living, are able to grow (i.e., there is increase either in size of the organism, or in their mass), they can utilize energy from their surrounding for their growth, and they are able to reproduce i.e. they produce the progeny of their kind.  

Some of the living organisms are so small that you are not able to see them with your unaided eyes, These are smaller than 0.1 mm and are called microorganisms. Others can be as big as 300 meters e.g. redwood trees. Microscopic study of all organisms suggests that they are made up of cells. Like a house, in which bricks are the basic unit of structure, cells are the basic unit of structure and function of all the living beings. Some organisms are made up of single cell and are called unicellular, while others have many cells, so are called multicellular. In this chapter you will learn about: - 

1. The characteristics of a living cell. 
2. Discoveries that led to understanding of cell structure and cell function.
 3. How did the concept of cell biology evolve?  
4. Cell types and cell sizes.
 5. Some of the acellular structures. 

Characteristics of living cells  

All the living cells have following characteristics:- 

 Presence of a membrane around the cell, which restricts entry to only certain molecules, besides allowing free passage to water and to some of the gases such as oxygen and carbon-di-oxide. The membrane is able to separate the inside space of the cell from the surroundings. This helps in keeping the environment of the cell at optimal level suitable for various chemical reactions occurring inside the living cell.
 
 The cell has its own energy generating system. The energy, which is produced by the cell, is conserved in the form of ATP and it is this form of energy, which is utilized for various life functions.
 
 A cell has its own genetic information, which it has received from its parent cell.
 
 The cell has its own machinery by which it can copy, and translate the genetic information, which is present in it in the form of polymer of nitrogen bases. This information is translated to the sequence of amino acids of a protein molecule by the cell machinery. 
 
 The cell is able to produce its own kind i.e. the cell is capable of forming new daughter cells. In plants this capacity of cell division is limited to the meristematic tissues, such as stem meristems, root meristems and intercalary meristems.  

History of Cell Biology 
 
Discovery of the cell was dependent on the invention and improvement of the microscope, the equipment used to observe the structures having dimensions lesser than 0.1 mm. Janssen had invented the first compound microscope in 1590 with the magnification of 9X. Robert Hooke used it in 1665 for the first time to observe a thin section of cork (cork was the piece of bark, which is outer dead layer of tree and it was being used as a stopper of the bottles). He observed that the section was like a honeycomb structure. It had a number of compartments, which were separated by a wall. He considered the wall as a living structure, which was enclosing the empty space. These empty spaces were called as „Cells‟. He thought these cells to be the containers of „noble juices‟ or „fibrous threads‟ of once living  cork trees. He published his observations made with the microscope in the book Micrographia.
Anton Van Leeuwenhoek improved the lens system. Using the improved lens, he observed a number of moving structures in a drop of pond water, which he called ‘animalcules’. Similarly, Nehemiah Grew studied the sections of plant tissues and concluded that all the tissues consist of cells.  

Cell is the basic unit of structure and function
  The cell theory or cell doctrine postulates –  

i) All living substance is concentrated in cells.
 ii) Cells in an organism are all individuals of the same organizational rank.
 iii) Cell is the basic unit of structure and function. 
iv) An organism is an aggregate of cells, which are its building blocks. 
v) The action of an organism is the sum of many action of different kind of collaborating cells.
vi) All cells arise from pre-existing cells.

Simple organisms are unicellular, i.e. they consist of single cell which is capable of performing all the functions of living beings, while more complicated organisms consist of  several cells  and hence are called multicellular. In a multicellular organism, many cells with similar structure and similar functions are organized to make a tissue. Different types of tissues, which are organized to carry out a particular function, are called organs, e.g. leaf is one of the organ, which function to synthesize food for the plant. In leaves similar cells are organized to form the epidermis of leaf, while other types of cells containing chloroplast are organized to from another type of tissue i.e., mesophylls.  Function of epidermal cells in the leaf is the protection of inner tissues, while that of chlorophyll containing mesophyll cells is to harvest the sunlight to prepare food. Xylem cells of vascular bundles (leaf veins) conduct water and minerals from soil to the leaf while function of phloem cells is to conduct sugar form site of its synthesis to different parts of the plant.  Different tissues such as epidermis, mesophylls and vascular bundles together make an organ such as leaf. An organism such as plant has different organs such as leaf, stem roots, flower, which perform specific function. Activities of an organism are the sum of coordinated activities of different organs. So, we see that it is the cell, which is the basic unit of structure and function of an organism.  

Cell size 

A great diversity in size of the cells is observed. Smallest living cell is that of Mycoplasma, with a minimum size of 1000 A in diameter. Size of other bacteria may vary with a minimum size of 5000 A for the cocci bacteria to 20 m in length for some of the filamentous forms. Blue green algae are approximately 10 m in diameter while RBCs of human blood are 7-8 m in diameters. One of the flagellates, Euglena, can have the size up to 0.5 mm in length. The diatoms may be up to 100 m or more in length. Amoeba is one of the largest unicellular organisms, which is about 1000 m in length. Cells of most of the tissues of plant and animals have the size range of 20-30 m. Largest single cell is the yolk of ostrich egg, which is about 5 cm. while size of the ovum in humans is 200 m, and head of the spermatozoa measures 5 m in length, with a tail of 30-50 m in length

Cell Types  

Right from simple unicellular organisms (such as Mycoplasmas) to the cells of complex multicellular organisms such as mammals, cells exhibit the common characteristics of living beings, which you have studied in the preceding text. Earlier all the organisms were classified on the basis of apparent external morphologies. These were classified either as plants or animals. However, some of the organisms could neither be classified as plants nor as animals, such as bacteria or fungi. Though, these organisms did not possess chloroplasts, because of presence of rigid cell wall structure they were still classified along with plants. On the contrary due to absence of rigid cell wall, Euglena was placed in animal kingdom, even when chloroplasts were present like that of plants. So, system of classifying the living beings was modified. In early 1960s, Hans Ris classified all the organisms on the basis of cell structural organization. He used the terms prokaryotes and eukaryotes to describe the organisms on the basis of absence or presence of a well-defined nucleus.  In this lesson you will learn about – 
1. Structure of Prokaryotic and eukaryotic cell'
  2.Comparison of the structure of prokaryotes and eukaryotes. 

Prokaryotic cell  
Characteristics 
 Prokaryotes include the most diverse organisms. Scientists believe that there are more than 5x1030 prokaryotes on earth. These have been found in most diverse habitats, ranging from being parasitic on animals/plants to the cytoplasm of prokaryotes, from distilled water to marine conditions, from Antarctic glaciers to thermal hot.  The term prokaryote is derived from Greek world, i.e. pro – means before and karyon – means nucleus. These are the cells, which have primitive nucleus and lack membrane bound organelles. These are single celled organisms in which genetic material is not separated from rest of the cell by membrane.  The prokaryotes can be classified as Bacteria and the Archaea (The Eukarya forms the third domain in the three domain classification) on the basis of molecular evolution .

Diversity of prokaryotic cell 

Mycoplasma 
 The smallest prokaryote is Mycoplasma genetalium with a size ranging from 0.2 μm to 0.8 μm. These prokaryotes lack cell wall but have tough cytoplasmic membranes. Many contain sterols in their membranes that give rigidity to the membrane. It is the living organism with the smallest known genome. It is believed to possess minimum complement of genes essential for life. There are around 480 protein-coding genes (in the genome of 5,80,070 nucleotide pairs), out of which 100 genes are of unknown function. This organism survives as a parasite in mammals, or survives on many readymade molecules supplied by the environment. However, it can synthesize its own large molecules, i.e., DNA, RNA and proteins, so that it is able to replicate. These are called Mycoplasmas because of their filamentous forms, which resemble filaments of fungi. These are facultative or obligatory anaerobes and colonize in animal or human bodies. 

Bacteria 
 Bacteria include all other prokaryotes except Archaea Cell is generally surrounded by cell wall. The region of the cell where genetic material is present is called „nucleoid‟. Chemical composition of prokaryotic cell wall is different from the cell wall of a plant cell. Cell wall of prokaryotic cell is made up of peptidoglycan, while plant cell wall is consists of mainly cellulose along with other heteropolymers. In prokaryotes, DNA is not complexed with histones. Besides, unlike eukaryotic cell where the DNA present is linear, the DNA present in prokaryotes is circular. No membrane bound organelles are present in prokaryotic cell. Cytoskeletal filaments, if present, are much simpler in their structure and function. Prokaryotic cells have their own protein synthesis machinery, i.e. they have all the enzymes required for DNA duplication, transcription, and protein translation. The ribosomes present in prokaryotic cell are different from that of a eukaryotic cell. These are 70 S types and contain fewer components in comparison to that of eukaryotic cells where cytoplasmic ribosome are 80 S types. 

Prokaryotic cells divide by binary fission. DNA duplication occurs followed by division of the cell into two daughter cells. Each daughter cell receives only one copy of the duplicated DNA. No spindle apparatus is formed.  Prokaryotic cells possess single copy of DNA, so there is no meiosis. Sexual reproduction occurs by means of conjugation, where a part of DNA is transferred from donor bacterium to recipient bacterium, through a tube like structure connecting the two bacteria.Locomotion of a prokaryotic cell is caused by flagellum, which is very simple in structure. It  consists of thin protein filament called „flagellin‟. 

Archaea
 The word, Archaea orginates from a Greek word arachios, which means ancient or primitive. These grow both in moderate and extreme environmental conditions. The most known groups in archaea or archaebacteria are the ones, which grow in extreme temperatures (thermophiles; Pyrolobus fumarii is found at 113 ° C), or in extreme pH (For e.g. Picrophilus sp. is found in acidic soils of Japan existing at pH of 0) or salt condition (halophiles) while, some Archaea generate methane gas (methanogens). 

Archaebacteria differ from other prokaryotes in the following features: i) Membrane lipids of archaebacteria have branched hydrocarbon chain and ether linkages. ii) There is no peptidoglycan in their cell wall. 

Archaebacteria resemble eukaryotes in the characteristic features that the starting codon AUG codes for methionine rather than N-formylmethionine. In other prokaryotes AUG codes for N-formylmethionine. Like bacteria the archaea divide by binary fisson. 

Eukaryotic cell
  The term, eukaryote was first of all used by Hans Ris in early 1960s, which means organisms, which possess true nucleus (Gr. Eu-true, Karyon-nucleus). The cells are characterized by the presence of nuclear membrane, which separates genetic material from rest of the cell. The size of the eukaryotic cell is much larger than that of prokaryotic cell. Between the plasma membrane and nuclear envelope is present the cytoplasm. Many organelles are present in the cytoplasm, which have specific structure and function. The details about eukaryotic cell are discussed in the next chapter. 

Infective particles 
In the previous section, you have studied about the characteristics of a living cell, and also the structure and types of the cells. However, there are certain disease causing agents which are acellular, i.e. these do not meet the characteristics of a living cell and cannot live on their own. They live as parasite on the host cell using the machinery of host cell. These lack cell structures, can neither metabolize, nor can grow or reproduce on their own or respond to the environment. These acellular structures include viruses, viroids and prions.

Viruses 
These are tiny infectious agents with their nucleic acids (DNA/RNA) surrounded by protein coat, known as capsid. Nucleic acid and capsid together are called nucleocapsid. Capsid consists of protein subunits known as capsomeres. Viron is complete virus particle (i.e. nucleic acid and protein coat) when is present outside the cell. A membrane may be present or absent outside the protein coat. Nucleic acid may be single or double stranded DNA or RNA. There may be single or multiple molecules of nucleic acids, which may be present either as linear or circular structures. Virus need host cell for their replication. The host may be plants, animals or bacteria. The viruses that infect a bacterial cell are called bacteriophage.  

The smallest virus is MS2, which is only 20 nm in diameter. It has single stranded RNA genome. Its genome encodes only three genes. The largest virus is mimivirus, which is dsDNA virus. Diameter of its capsid is 400 nm. Its genome contains 1.2 million bases estimated to be encoding 1000 genes. A virus may be called as DNA or RNA virus depending upon the nucleic acid, which makes up its genome. Most of the bacteriophages are DNA viruses while those infecting plants are RNA viruses. Genome of animal viruses may be composed of either DNA or RNA. The genome may be single or double stranded. All animal viruses with RNA genome have envelopes while envelope may be present in some of the DNA viruses. The glycoproteins present on the envelope help in recognizing the host by interacting with the glycoproteins present on the surface of the host cell. Virus genome is replicated inside the host cell using the host enzymes. The genetic information for the synthesis of glycoproteins present in viral envelope is also present in viral genome. Retrovirus are the viruses in which the genome is ssRNA. This RNA, on entering host cell, is transcribed to DNA by reverse transcription. The transcribed DNA gets integrated into host genome and gets replicated along with host genome. This replicated DNA gets transcribed to RNA molecules, which serves as genome for new virus particles and also acts as mRNA, which is translated to capsomeres and glycoproteins. The most common reteroviruse known is HIV (human immunodeficiency virus), the virus that causes AIDS (acquired immunodeficiency syndrome). HIV is enveloped RNA virus that contains 2 identical ssRNA strands and two molecules of reverse transcriptase. Most of the plant viruses are RNA viruses. More than 2,000 types of viral diseases of plants are known. Mode of reproduction of virus in plants is same as that in animals. Mode of transmission of viruses in plants may be horizontal or vertical. Horizontal transmission may be though the grazing animals or through various tools used while the vertical transmission may be due to getting infection from the infected seed or getting from the parent. Infection of a plant by viruses may be facilitated through the injury on the plant and it can get spread through the plasmodesmata connections.  

Viroids 
 Another infectious agents are viroids. Meaning of viroids is virus like. These are smallest infectious agents. These are small RNA viruses without capsid being present. RNA genome is very small. It is 300-400 nucleotides long. It is circular. It does not code for any proteins. It is replicated in the host cell. These were discovered in 1971s by Theodore Diener and his colleagues. Though RNA of viroids is circular, it appears to be linear molecule, because of formation of number of hydrogen bonds within the RNA molecule. These are pathogenic to plants. One of the first Viroid studied was potato spindle tuber Viroid (PSTV). It is a circular RNA molecule, which  consists of 359 nucleotides. 

Prions 
These are another class of infective agents, which are just protein molecules.  These are responsible for a neurodegenerative disease, called “mad cow” in humans. There was an epidemic of disease, which was known as bovine spongiform encephalitis in late 1980s in Great Britain and in 2000s in France. The disease spread in humans who had ingested  prions infected beef. In 1982, Stanley Prusiner reported that protein molecule was responsible for the disease. The infectious protein molecule did not have any nucleic acid. Prusiner named such infectious protein molecules as Prions. It was difficult to accept Prions as disease causing agent, since there was no nucleic acid present. How could a protein molecule be synthesized without the directions of a nucleic acid? Later on, Stanley Prusiner explained role of Prions as disease causing agent and it was in 1997 he was awarded Noble Prize for the same. Prions consist of single protein molecule (PrP). The protein exists in two conformations:- i)  Cellular PrP – This is a normal functional protein. All mammals have genes encoding for the sequence of amino acids of this protein. This can fold in secondary structure with several α–helices only. No β–sheet structure is present. ii) Prion PrP – This is abnormally folded version of normal cellular proteins. This is the disease causing form, which has β–pleated sheet in secondary structure. The normal PrP is required for normal cellular fucntions, which control signal events in brain cells. Prion PrP trigger changes in secondary structure of normal cellular PrP, so that it gets misfolded to Prion PrP. Prion PrP would interrupt the normal function of cellular PrP.  


Summary 
 
 All the living beings consist of cells.

   The cell is characterized by the presence of a membrane with restricted entry, presence of hereditary material and machinery capable of replicating the hereditary material as well as protein translation.

  Depending upon presence or absence of well-defined nuclear membrane, the living beings are classified as prokaryotes and eukaryotes. 

 Diversity in cell size has been observed. 
 Based upon complexity of the cells, diversity of prokaryotic cells is there with the simplest being Mycoplasma.

   Archaea are the organisms, which have been classified under prokaryotes, live under harsh environmental conditions and are different from that of eubacteria. 

 There are acellular structures that are infective yet do not meet the requirements of a living cell. These are infective particles, which include infective RNAs (viroids), infective protein molecules (prions) and nucleoproteins, which are known as viruses.   






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Lesson: Electron Microscopy 

Glossary  
 . Angstron (Ao)- A unit of length usually used to describe molecular dimensions equal to10-8cm .

 Electron Microscope- A microscope in which a focused beam of electrons is used to produce an enlarged image of the object. . 

Freeze-fracture- A technique for preparing material for EM by rapid freezing and fracturing of the tissues ;the exposed faces are used to create a replica which is observed and photographed in EM. .

 Micron(micrometer, μm)- A unit of length used to describe cellular dimensions; it is equal to 10-4cm or 104 Ao. 

. SEM- An electron microscope that permits observation of a specimen’s surface structure .The electron beam is not transmitted through the specimen but causes the release of secondary electrons from the surface of the specimen which forms the image.

. TEM- An electron microscope in which electron beam is transmitted through the specimen and forms the image on fluorescent screen at the bottom of the microscope

Table of Contents

 Introduction  
 Principle of microscopy 
 Comparative account of different types of microscopes 
 Basic components of an electron microscope

Types of Electron Microscope  
Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM) 
 Scanning Transmission Electron Microscope (STEM) 
 Environmental  Scanning Electron Microscope (ESEM)

Techniques for electron microscope  
Negative Staining 
 Freeze -Fracture and Freeze –Etch 
 Shadow Casting

 Summary  


Introduction
 Principle of Microscopy The prokaryotic and eukaryotic cells fall within the size range of 1-100 μm. Unaided human eye cannot resolve objects smaller than 100 μm size. Therefore, microscopes are needed for visualization of subcellular architecture. Microscope not only magnifies the image of objects but also increases the resolution, which refers to ability to distinguish closely adjacent objects as separate entities. The greater is the resolving power of the microscope, the greater is the clarity of the image produced.         
The lower limit of resolution for any optical system can be calculated from the following relationship.
r = 0.61λ/ n sin α
where r, or resolving power, is the minimum distance between two points that can be recognized as separate, λ is the wavelength of light (or other radiation) used to illuminate the object, n is the refractive index of the medium in which the object is placed, and sin α is the sine of half the angle between the specimen and the objective lens. The entire term n sin α is often referred to as the numerical aperture. 
Frequently asked question
What do you understand by numerical aperture? 
The numerical aperture of the objective a microscope is a measure of its resolving power. The value of numerical aperture is given by NA = n sin α. 
n refers to the refractive index (1 for air)
α is half the angle subtended by the rays entering into the objective lens
Higher the NA higher the resolving power.

There are only a small number of variables affect the resolving power of a microscope. The refractive index can be increased by immersing the sample in oil (n = 1.5) rather than air (n = 1.0), and moving the lens closer to the specimen to increase α. The upper theoretical limit of α is 90 °, meaning that the value of sin α cannot exceed 1. Hence the maximum numerical aperture of an optical system employing an oil immersion lens will be 1.5 X 1 = 1.5. A microscope using white light, which has an average wavelength of about 550 nm, will therefore, have a resolving power of 550/1.5, or about 220 nm. This means that objects closer to   one another or smaller than 220 nm cannot be distinguished. A resolving power of 220 nm is adequate to see some details of subcellular structure, but many organelles, such as ribosomes, cellular membranes, microtubules, microfilaments, intermediate filaments, and chromatin fibers, cannot be resolved at this level .The wavelength of an electron is much shorter than that of  

visible light, the electron microscope has a theoretical limit of resolution much lower that of the light microscope—about 0.1-0.2 nm instead of 200-300 nm. Because of problems of specimen preparation of biological samples, the practical limit of resolution is almost about 2 nm which means 100 times more resolution than that of light microscope. Electron microscopes thus offers the possibility of increasing the resolving power many folds. There are two types of electron microscopes:

1.Transmission electron microscope
 2.Scanning electron microscope

The electrostatic and electromagnetic lenses are used in an electron microscope to control the electron beam and focus it to form an image. 
In Transmission electron microscope (TEM), the electrons are transmitted through an object and  then focused by the lenses to form the image.
 In Scanning electron microscope (SEM), the electrons are reflected by the object in a scanned pattern which are then used to form the image. SEM is becoming increasingly popular with cell biologists because of its remarkable ability to study surface topography, along with improved resolution (30-100 Å) and its ability to show 3D structure. 
 

Source of illumination for Image Formation 
Compound  Microscope                                               visible light 
Confocal Microscope                                                        laser light 
Scanning  Electron Microscope  (SEM)                             electrons
  Transmission Electron  Microscope                                      electrons

 Types of cells visualized  
Compound  Microscope                                    Individual cells can be visualised,  even living ones

  Confocal Microscope                                    individual  cells can be visualised,  even living ones. 
Scanning  Electron Microscope  (SEM)                     The specimen is coated with gold  and the electrons  are reflected back and give the details of surface  topography of the specimen.

Transmission Electron  Microscope     Thin sections of the specimen are obtained. The electron beams pass through   the sections and form an image with high magnification and     high resolution. 

Image 
Compound  Microscope                                                                                 2D
Scanning  Electron Microscope  (SEM)                                                           3D 
Transmission Electron  Microscope                                                                    2D   

Nature of Lenses  

Compound  Microscope                                                          glass

   Confocal Microscope                                    glass lenses with dichromatic mirror
Scanning  Electron Microscope  (SEM)       one electrostatic lens with a few electromagnetic lenses

Transmission Electron                  Microscope one electrostatic lense andfew electromagnetic  lenses

Medium
 Air
Air
Vacuum
Vacuum
Specimen mounting  
glass slides
glass slides with dyed samples
mounted on aluminium stubs and are coated in gold
mounted on coated or uncoated copper grid

Focusing and Magnification Adjustments 
changing objectives

digitally    enhanced

electrical

electrical i.e. changing current of the projector lens coil 

Means for obtaining specimen Contrast 
Light Absorption

laser light with dichromatic mirror concentrated at pinhole 

electron scattering 

   Electron scattering 

Basic Components of  an Electron Microscope  

1. The vacuum system—A strong vacuum must be maintained in the entire column along the path of electron beam, since electrons cannot travel very far in air. There are two types of vacuum pumps which work together to create vacuum  
2. The Electron gun----The electron beam is emitted by an electron gun which consist of  
a) The cathode, a filament made of tungsten emits electrons maintained at50-100kv
b) The anode, to shape the beam maintained at 0 kv
The difference in voltage is called accelerating voltage. 

3. Electromagnetic Lenses and image formation—There are many lenses arranged together to control illumination, focus, and magnification 
a) The condenser lens-to control the electron beam
b) The objective lens, intermediate lens and projector lens—in concert with each other produce a final image on the viewing screen
   
4. The photographic system—In addition to viewing, the image can be recorded photographically as an electron micrograph. 
 
5. The cooling system—since a high voltage is used for the emission of electrons, a cooling system is also attached to the column so that it does not get heated up.   

Types Of Electron Microscope

 Transmission Electron Microscope (Tem)  

The prototype electron microscope was invented in 1931 by German physicist E. Ruska and the electrical engineer M. Knoll .In 1933; Ruska built an electron microscope that exceeded the resolution of an optical microscope. E. F.Burton and students C. Hall, J. Hillier, and A. Prebus1938. at the University of Toronto, constructed  the first practical electron microscope.  In 1939, Siemens produced the first commercial Transmission Electron Microscope (TEM).  
In Transmission Electron Microscope (TEM), a beam of highly focused electrons is directed towards a thin section of the specimen (<200 nm) and allowed to pass through it. These highly energetic incident electrons interact with the atoms in the sample and produce characteristic radiation and particles which form image. Images are obtained from transmitted electrons, backscattered and secondary electrons, and emitted photons.
TEM uses a high voltage electron beam which is emitted by electron gun to create an image. The electron gun is made up of a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode and then is  focused by electrostatic and electromagnetic lenses. The electron beam is then transmitted through the specimen. As the electron beam emerges from the specimen, it carries information about the structure of the specimen that is magnified by the objective lens of the microscope. The transmitted electrons hit a fluorescent screen at the bottom of the microscope and give rise to a "shadow image" of the specimen with its different parts displayed in varying darkness according to their density. Image is viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material. 
The image can also be photographically recorded by exposing a photographic film or plate directly to the electron beam or a fibre optic light-guide to the sensor of a CCD camera. The image detected by the CCD may be visualized on a monitor or computer.   There are different ways to prepare the material for TEM. One way is to cut very thin sections of the specimen from a piece of tissue either by fixing it in resin or working with it as frozen material. Another way to prepare the specimen is to isolate it and study a solution after doing negative staining, for example viruses or molecules in the TEM. 

Sample Preparation: Biological material contains large quantities of water. Since the transmission electron microscope works in vacuum, the water must be removed. The tissue is preserved with different fixatives to avoid any disruption due to loss of water. These fixatives also aim to stabilize the specimen's mobile macromolecular structure by chemical crosslinking of proteins with aldehydes such as formaldehyde and glutaraldehyde, and lipids with osmium tetroxide. The tissue is then dehydrated in alcohol or acetone after dehydration. The tissue is then embedded so that it can be sectioned. To do this, the tissue is passed through a 'transition solvent' such as propylene oxide and then infiltrated with an epoxy resin such as Araldite, Epon, or Durcupan;. After the resin has been polymerized (hardened), the sample is thin sectioned (ultrathin sections) by a diamond or glass knife in an instrument called ultramicrotome .Since the sections are very thin it becomes difficult to hold the sections .To pick up sections a boat is made around the glass knife,which is then filled with water .When sections are cut ,they float on the surface of water. The sections are then picked up directly on to surface of copper grid by touching the grid to the surface of water in boat. Once the sections are placed on the copper grid , the staining is done with heavy metals such as lead, uranium or tungsten to scatter imaging electrons and to produce contrast between different structures because many (especially biological) materials are nearly "transparent" to electrons (weak phase objects). The specimens can be stained "en bloc" before embedding or later after sectioning. Typically thin sections are stained for several minutes with uranyl acetate followed by aqueous lead citrate, which can then be studied under the electron microscope. 

AN ULTRA -MICROTOME 
 
A microtome (from the Greek mikros, meaning "small", and temnein, meaning "to cut") is a tool used to cut extremely thin sections. An ultra-microtome is used for the preparation of ultrathin sections (50-100 A) for observation under transmission electron microscope. Glass and diamond knives are used to cut very thin sections for electron microscopy. 
In spite of the enhanced resolution made possible by use of electron microscope, it is not without its inherent limitations .An electron beam is too weak to pass an appreciable distance through air, so a high vacuum is needed inside the internal chamber of electron microscope. This lack of penetrating power also limits specimen thickness to a few hundred nanometers. Such restrictions create many technical problems in preparing biological material for observation.    

Scanning electron microscope (SEM)   
The Scanning Electron Microscope was invented by Manfred von  Ardenne  in 1937 .In Scanning electron microscope the image of the specimen  is produced  with a focused electron beam that is scanned across the area of the specimen. In SEM, a magnetic lens system focuses the beam of electron into an intense spot on the surface of specimen. The spot is moved back and forth across the specimen by charged plates called beam deflectors located between the condenser lens and the specimen. The beam deflectors attract or repel the beam according to signals sent by the deflector circuitory. As the electron beam sweeps rapidly over the specimen molecules in the specimen are excited to high energy level and emit secondary electrons which are then used to form an image of the specimen surface. Secondary electrons are captured by a detector located immediately above and to one side of the specimen. The essential component of the detector is the scintillator, which when excited by electrons incident upon it emit photons of light. These photons are used to generate an electronic signal onto the video screen. As the beam traverses the surface of the object electrons are deflected to varying degrees. The deflected and emitted electrons are detected by a Photomultiplier tube and used to form a 3-D image of the object’s surface features. 
The resolving power of the SEM is less than that of the TEM. However since the image formaion by SEM is dependent of surface properties it can magnify samples up to many centimeters and has a greater depth of field. It can thus produce good representative images of the three dimensional shape of the sample.    
Sample preparation The material is primary fixed by Immersing in 2.8% glutaraldehyde in 0.1M Hepes buffer, pH 7.2 (with 0.02% Triton X-100), for several hours at room temperature or overnight at 4°C. The material is then washed thrice (each 5 to 10 minute duration) in 0.1 M Hepes buffer, pH 7.2. Dehydration is done for 10 min. in 25% ethanol, 10 min. in 50% ethanol, 10 min. in 70% ethanol, 10 min. in 85% ethanol, and 10 min. in 95% ethanol, 2 x 10 min. in 100% ethanol, and 10 min. in 100% ethanol (EM grade). This is followed by Critical Point Drying which is an automated process and takes approximately 40 minutes to complete. The sample is then mounted onto metal stub with double-sided carbon tape. Finally a Sputter Coating is done by apply a thin layer of metals (gold and palladium) over the sample using an automated sputter coater.   

Scanning Transmission Electron Microscope (STEM)  
 
 STEM contains elements of both TEM and SEM. Like SEM, it uses an electron beam that sweeps over the specimen. The image is formed by the electrons transmitted through the specimen as with a TEM. A STEM is capable of distinguishing specific characteristics of the electron that are transmitted by the specimen, thus deriving information about the specimen not obtainable withthe conventional TEM. However a STEM is technically sophisticated and requires a very high vacuum and is much more electronically complex than a TEM or a SEM 

Techniques for preparing tissues for electron microscopy other than sectioning  

Negative Staining
 In contrast to thin sectioning, negative staining method is the easiest technique used in TEM for examining very small objects. The shape and surface appearance of small particles such as intact organelles or viruses can be examined without cutting these into thin sections. In the negative staining technique, such particles are suspended in a small drop of liquid applied to copper grid and allowed to dry in air. After drying, a drop of stain such as phosphotungstic acid or uranyl acetate is applied to the surface.  When viewed in TEM, specimen is visualized against the stained dark background. In the closely related positive staining technique, a specimen is first reacted with the stain and the stain then is removed, producing a stained sample visible.

FREEZE-FRACTURE TECHNIQUE 

The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample along the planes of natural weakness that run through each cell.These planes occur generally between the two layers of lipid molecules which forms part of limiting membrane around various organelles of the cell. A freeze fracture replica is then made by vaccum deposition of platinum and carbon. 
The main steps in making a freeze fracture replica are (i) Pre treatment with glutarladehyde and glycerol for cryoprotection (to reduce ice crystal formation and resulting damage) (ii) rapid freezing, (iii) fracturing, (iv) formation of replica, and (iv) replica cleaning. Images provided by freeze -fracture and other related techniques have profoundly shaped our understanding of the functional morphology of the cell. This technique is used to study membranes and reveal the pattern of integral membrane proteins.   

Freeze- Etching Technique  

The freeze-etching technique of sample preparation is related to freeze fracture, but it adds a further step to freeze -fracture procedure, which makes it more informative.  Instead of employing fixatives to preserve cell structure, specimens are rapidly frozen in liquid Freon, placed in a vacuum and struck with a sharp knife edge as in freeze fracture. At this temperature biological samples are too hard to be cut and instead fracture along lines of natural weakness.  These weak areas are generally associated with biological members. Brief exposure of the broken tissue to vacuum, results in sublimation of water from the fractured surfaces.  This removal of water produces an “etching” effect. This etching will cause small areas of the true cell surface around the periphery of the fracture face to stand out against the background.  A replica of the freeze-etched specimen is made by heavy metal such as platinum, and then backing it with a carbon film.  After dissolving the tissue in strong acid, the remaining metal replica can be viewed with the electron microscope. Such preparations provide a unique picture of cells, particularly where members are studied. Freeze –etching is specifically useful because it avoids exposure to fixatives, embedding agents, and stains, all of which may deform cell ultrastructure. Unlike such treatments, rapid freezing causes minimal tissue distortion and permits immediate arrest of cell function.  

Shadow casting 
 
Shadow-casting is a technique which shows  the surface texture  of  microscopic  material  rather  than the  routine transparent appearances.  Sections or smears may be studied throughout the whole range of microscopic magnification. The method involves the in vacuum deposition of a metallic film on dried specimens.  Metal is deposited from an oblique angle so that it coats some surfaces of specimen more than others. This leaves the area to the "leeward" side of the specimen uncoated producing a "shadow" of the specimen.
The thin metal film is obviously formed on the specimen by condensation after vaporization. It is therefore assumed that the metals with the higher vaporization temperature will condense more quickly after vaporization, and form finer particle sizes. Also, the concurrent evaporation of two or more elements will result in smaller aggregate size by increasing the distance over which any atom must diffuse in order to secure its place within a crystal lattice. The particle size of a film of evaporated gold will therefore be larger than that of evaporated platinum or that of a 60/40 alloy of gold/palladium. The "grain" size of evaporated tungsten is exceedingly fine, but deposition time is very long and temperature is extremely high. Isolated particles can also be visualized by placing them in an evacuated chamber and spraying heavy metal across their surfaces.  The shadow-casting process causes metal to be deposited on one side of the specimen, creating a “shadow” and a resulting three-dimensional appearance.

Summary 
 
Cell biology is an experimental science which is based on the execution and interpretation of experiments designed to provide information about cell structure and function. Our current understanding of the relationship between cell structure and function has been made possible by a combination of microscopic and biochemical techniques. The light microscope was historically helpful in the discovery of cell and the resolution of about 200 nm severely restricts its usefulness for studying the details of cell architecture. By changing the source of illumination from light to electrons, resolving power was enhanced by several orders of magnitude from 200 nm to about 0.5 nm. The invention of the transmission electron microscope therefore revolutionized our view of cell architecture. Diverse set of procedures for specimen preparation, such as thin sectioning, negative staining, positive staining, shadow casting, whole mounting, and freeze-fracture, has opened our eyes to the existence of an exquisite subcellular architecture and the more recent development of the scanning electron microscope has provided the three dimensional view of the cell surface. 

 

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