Module 1: Introduction to Cell and Molecular Biology PDF

Republic of the Philippines BICOL UNVERSITY TABACO M.H. del Pilar St., Tayhi, Tabaco City 4511, Albay Email: [email protected] MODULE 1: INTRODUCTION TO...

Republic of the Philippines BICOL UNVERSITY TABACO M.H. del Pilar St., Tayhi, Tabaco City 4511, Albay Email: [email protected] MODULE 1: INTRODUCTION TO CELL AND MOLECULAR BIOLOGY Course Code & Title: Sci Ed 12 / Cell and Molecular Biology Course Placement: Bachelor of Secondary Education – Science III Course Credit: 5.1 units (Lecture and Laboratory) Name of Professor: Dr. Alex P. Camaya Position/Specialization: Assoc. Prof. IV / Cell Structure and Function  MODULE 1: INTRODUCTION TO CELL AND MOLECULAR BIOLOGY WHAT IS THIS MODULE ABOUT? This module mainly aims to introduce Cell and Molecular Biology course as a ‘basic’ or ‘fundamental’ aspect of the entire field of biological sciences. Since mid-16th Century when the first instrument that viewed a smallest living unit had been introduced until now when advanced technology emerged, the studies on cells remains evolving. As what a prominent reference book used in this module said, cell and molecular biology is reductionist; that is, it is based on the view that knowledge of the parts of the whole can explain the character of the whole. When viewed in this way, our feeling for the wonder and mystery of life may be replaced by the need to explain everything in terms of the workings of the “machinery” of the living system. WHAT WILL YOU LEARN HERE? This module contains six (6) lessons namely as follows; Lesson 1: Scope and Field of Cell and Molecular Biology Lesson 2: History and Foundations of the Cell and Molecular Biology Lesson 3: Basic Properties of the Cells Lesson 4: Comparison of Prokaryotic and Eukaryotic Cells Lesson 5: Some Techniques in Cell and Molecular Biology LET’S READ AND STUDY! Lesson 1: Scope and Field of Cell and Molecular Biology Cell and molecular biology is a marriage of two distinct, yet complementary, disciplines. In its traditional sense, the term 'molecular biology' refers to study of the macromolecules essential to life — nucleic acids and proteins. The field of cell biology is a natural extension of this, integrating what we know at the molecular level into an understanding of processes and interactions at the cellular level. Only by combining both fields can we paint a broad picture of essential biological processes such as how cells divide, grow, communicate and die. Cell and molecular biology concerned with the physiological properties, metabolic processes, signaling pathways, life cycle, chemical composition and interactions of the cell with their environment. This is done both on a microscopic and molecular level as it encompasses prokaryotic cells and eukaryotic cells. Knowing the components of cells and how cells work is fundamental to all biological sciences; it is also essential for research in bio-medical fields such as cancer, and other diseases. Research in cell biology is closely related to genetics, biochemistry, molecular biology, immunology and cytochemistry. In details, cell biology is the study of cell structure and function, and it revolves around the concept that the cell is the fundamental unit of life. Focusing on the cell permits a detailed understanding of the tissues and organisms that cells compose. Some organisms have only one cell, while others are organized into cooperative groups with huge numbers of cells. On the whole, cell biology focuses on the structure and function of a cell, from the most general properties shared by all cells, to the unique, highly intricate functions particular to specialized cells. Lesson 2: History and Foundations of the Cell and Molecular Biology Because of their small size, cells can only be observed with the aid of a microscope, an instrument that provides a magnified image of a tiny object. We do not know when humans first discovered the remarkable ability of curved-glass surfaces to bend light and form images. Spectacles were first made in Europe in the thirteenth century, and the first compound (double-lens) light microscopes were constructed by the end of the sixteenth century. By the mid-1600s, a handful of pioneering scientists had used their handmade microscopes to uncover a world that would never have been revealed to the naked eye. The discovery of cells is generally credited to Robert Hooke, an English microscopist who, at age 27, was awarded the position of curator of the Royal Society of London, England’s foremost scientific academy. One of the many questions Hooke attempted to answer was why stoppers made of cork (part of the bark of trees) were so well suited to holding air in a bottle. As he wrote in 1665: “I took a good clear piece of cork, and with a Pen-knife sharpened as keen as a Razor, I cut a piece of it off, and... then examining it with a Microscope, me thought I could perceive it to appear a little porous...much like a Honeycomb.” Hooke called the pores cells because they reminded him of the cells inhabited by monks living in a monastery. In actual fact, Hooke had observed the empty cell walls of dead plant tissue, walls that had originally been produced by the living cells they surrounded. Meanwhile, Anton van Leeuwenhoek, a Dutchman who earned a living selling clothes and buttons, was spending his spare time grinding lenses and constructing simple microscopes of remarkable quality. For 50 years, Leeuwenhoek sent letters to the Royal Society of London describing his microscopic observations—along with a rambling discourse on his daily habits and the state of his health. Leeuwenhoek was the first to examine a drop of pond water under the microscope and, to his amazement, observe the teeming microscopic “animalcules” that darted back and forth before his eyes. He was also the first to describe various forms of bacteria, which he obtained from water in which pepper had been soaked and from scrapings of his teeth. His initial letters to the Royal Society describing this previously unseen world were met with such skepticism that the society dispatched its curator, Robert Hooke, to confirm the observations. Hooke did just that, and Leeuwenhoek was soon a worldwide celebrity, receiving visits in Holland from Peter the Great of Russia and the queen of England. It wasn’t until the 1830s that the widespread importance of cells was realized. In 1838, Matthias Schleiden, a German lawyer turned botanist, concluded that, despite differences in the structure of various tissues, plants were made of cells and that the plant embryo arose from a single cell. In 1839, Theodor Schwann, a German zoologist and colleague of Schleiden’s, published a comprehensive report on the cellular basis of animal life. Schwann concluded that the cells of plants and animals are similar structures and proposed these two tenets of the cell theory, thus “all organisms are composed of one or more cells” and “the cell is the structural unit of life”. Schleiden and Schwann’s ideas on the origin of cells proved to be less insightful; both agreed that cells could arise from non-cellular materials. Given the prominence that these two scientists held in the scientific world, it took a number of years before observations by other biologists were accepted as demonstrating that cells did not arise in this manner any more than organisms arose by spontaneous generation. By 1855, Rudolf Virchow, a German pathologist, had made a convincing case for the third tenet of the cell theory that “cells can arise only by division from a preexisting cell”. Figure 1. The discovery of the cells. (a) One of Robert Hooke’s (c) more ornate compound (double-lens) microscopes. (Inset) Hooke’s drawing of a thin slice of cork, showing the honeycomb-like network of “cells.” (b) Single-lens microscope used by Anton van Leeuwenhoek (d) to observe bacteria and other microorganisms. The biconvex lens, which was capable of magnifying an object approximately 270 times and providing a resolution of approximately 1.35 µm, was held between two metal plates. (images C and D courtesy of Google Images) Table 1. Nobel Prizes Awarded for Research in Cell and Molecular Biology (1958-2012) Lesson 3: The Basic Properties of the Cells The following are the basic properties of the cells which are known uniquely as; a) “Cells are highly complex and organized” The more complex a structure, the greater the number of parts that must be in their proper place, the less tolerance of errors in the nature and interactions of the parts, and the more regulation or control that must be exerted to maintain the system. Cellular activities can be remarkably precise. DNA duplication, for example, occurs with an error rate of less than one mistake every ten million nucleotides incorporated—and most of these are quickly corrected by an elaborate repair mechanism that recognizes the defect. The organization of atoms into small- sized molecules; the organization of these molecules into giant polymers; and the organization of different types of polymeric molecules into complexes, which in turn are organized into subcellular organelles and finally into cells. As will be apparent, there is a great deal of consistency at every level. Each type of cell has a consistent appearance when viewed under a high-powered electron microscope; that is, its organelles have a particular shape and location, from one individual of a species to another. Similarly, each type of organelle has a consistent composition of macromolecules, which are arranged in a predictable pattern. Fortunately for cell and molecular biologists, evolution has moved rather slowly at the levels of biological organization with which they are concerned. Whereas a human and a cat, for example, have very different anatomical features, the cells that make up their tissues, and the organelles that make up their cells, are very similar. b) “Cells possess a genetic program and the means to use it” Organisms are built according to information encoded in a collection of genes, which are constructed of DNA. The human genetic program contains enough information, if converted to words, to fill millions of pages of text. Remarkably, this vast amount of information is packaged into a set of chromosomes that occupies the space of a cell nucleus—hundreds of times smaller than the dot on this i. Genes are more than storage lockers for information: they constitute the blueprints for constructing cellular structures, the directions for running cellular activities, and the program for making more of themselves. The molecular structure of genes allows for changes in genetic information (mutations) that lead to variation among individuals, which forms the basis of biological evolution. Discovering the mechanisms by which cells use and transmit their genetic information has been one of the greatest achievements of science in recent decades. c) “Cells are capable of producing more of themselves” Just as individual organisms are generated by reproduction, so too are individual cells. Cells reproduce by division, a process in which the contents of a “mother” cell are distributed into two “daughter” cells. Prior to division, the genetic material is faithfully duplicated, and each daughter cell receives a complete and equal share of genetic information. In most cases, the two daughter cells have approximately equal volume. In some cases, however, as occurs when a human oocyte undergoes division, one of the cells can retain nearly all of the cytoplasm, even though it receives only half of the genetic material. d) “Cells acquire and utilize energy” Every biological process requires the input of energy. Virtually all of the energy utilized by life on the Earth’s surface arrives in the form of electromagnetic radiation from the sun. The energy of light is trapped by light-absorbing pigments present in the membranes of photosynthetic cells. Light energy is converted by photosynthesis into chemical energy that is stored in energy-rich carbohydrates, such as sucrose or starch. For most animal cells, energy arrives prepackaged, often in the form of the sugar glucose. In humans, glucose is released by the liver into the blood where it circulates through the body delivering chemical energy to all the cells. Once in a cell, the glucose is disassembled in such a way that its energy content can be stored in a readily available form (usually as ATP) that is later put to use in running all of the cell’s myriad energy-requiring activities. Cells expend an enormous amount of energy simply breaking down and rebuilding the macromolecules and organelles of which they are made. This continual “turnover,” as it is called, maintains the integrity of cell components in the face of inevitable wear and tear and enables the cell to respond rapidly to changing conditions. e) “Cells carry out a variety of chemical reactions” Cells function like miniaturized chemical plants. Even the simplest bacterial cell is capable of hundreds of different chemical transformations, none of which occurs at any significant rate in the inanimate world. Virtually all chemical changes that take place in cells require enzymes—molecules that greatly increase the rate at which a chemical reaction occurs. The sum total of the chemical reactions in a cell represents that cell’s metabolism. f) “Cells engage in mechanical activities” Cells are sites of bustling activity. Materials are transported from place to place, structures are assembled and then rapidly disassembled, and, in many cases, the entire cell moves itself from one site to another. These types of activities are based on dynamic, mechanical changes within cells, many of which are initiated by changes in the shape of “motor” proteins. Motor proteins are just one of many types of molecular “machines” employed by cells to carry out mechanical activities. g) “Cells are able to respond to stimuli” Some cells respond to stimuli in obvious ways; a single-celled ‘protist’, for example, moves away from an object in its path or moves toward a source of nutrients. Cells within a multicellular plant or animal respond to stimuli less obviously. Most cells are covered with receptors that interact with substances in the environment in highly specific ways. Cells possess receptors to hormones, growth factors, and extracellular materials, as well as to substances on the surfaces of other cells. A cell’s receptors provide pathways through which external stimuli can evoke specific responses in target cells. Cells may respond to specific stimuli by altering their metabolic activities, moving from one place to another, or even committing suicide. h) “Cells are capable of self-regulation” In recent years, a new term has been used to describe cells: robustness. Cells are robust, that is, hearty or durable, because they are protected from dangerous fluctuations in composition and behavior. Should such fluctuations occur, specific feedback circuits are activated that serve to return the cell to the appropriate state. In addition to requiring energy, maintaining a complex, ordered state requires constant regulation. The importance of a cell’s regulatory mechanisms becomes most evident when they break down. For example, failure of a cell to correct a mistake when it duplicates its DNA may result in a debilitating mutation, or a breakdown in a cell’s growth-control safeguards can transform the cell into a cancer cell with the capability of destroying the entire organism. We are gradually learning how a cell controls its activities, but much more is left to discover. i) “Cells evolve” How did cells arise? Of all the major questions posed by biologists, this question may be the least likely ever to be answered. It is presumed that cells evolved from some type of pre-cellular life form, which in turn evolved from nonliving organic materials that were present in the primordial seas. Whereas the origin of cells is shrouded in near-total mystery, the evolution of cells can be studied by examining organisms that are alive today. If you were to observe the features of a bacterial cell living in the human intestinal tract and a cell that is part of the lining of that tract, you would be struck by the differences between the two cells. Yet both of these cells, as well as all other cells that are present in living organisms, share many features, including a common genetic code, a plasma membrane, and ribosomes. According to one of the tenets of modern biology, all living organisms have evolved from a single, common ancestral cell that lived more than three billion years ago. Because it gave rise to all the living organisms that we know of, this ancient cell is often referred to as the last universal common ancestor (or LUCA). Lesson 4: Comparison of Prokaryotic and Eukaryotic Cells There are known characteristics that distinguish prokaryotic and eukaryotic cells. The similarities and differences between the two types of cells are listed below. Internally, organisms with eukaryotic cells e.g. protists, fungi, plants and animals, are much more complex— both structurally and functionally—than prokaryotic cells e.g. bacteria. The shared properties reflect the fact that eukaryotic cells almost certainly evolved from prokaryotic ancestors. Because of their common ancestry, both types of cells share an identical genetic language, a common set of metabolic pathways, and many common structural features. For example, both types of cells are bounded by plasma membranes of similar construction that serve as a selectively permeable barrier between the living and nonliving worlds. Both types of cells may be surrounded by a rigid, nonliving cell wall that protects the delicate life form within. Although their cell walls of may have similar functions, their chemical composition is very different. Table 2. The Comparison of Prokaryotic and Eukaryotic Cells Figure 2. Prokaryotic cells have a simpler internal organization than eukaryotic cells. (a) Electron micrograph of a thin section of Escherichia coli, a common intestinal bacterium. The nucleoid, consisting of the bacterial DNA, is not enclosed within a membrane. E. coli and some other bacteria are surrounded by two membranes separated by the periplasmic space. The thin cell wall is adjacent to the inner membrane. (b) Electron micrograph of a plasma cell, a type of white blood cell that secretes antibodies. Only a single membrane (the plasma membrane) surrounds the cell, but the interior contains many membrane-limited compartments, or organelles. The defining characteristic of eukaryotic cells is segregation of the cellular DNA within a defined nucleus, which is bounded by a double membrane. The outer nuclear membrane is continuous with the rough endoplasmic reticulum, a factory for assembling proteins. Golgi vesicles process and modify proteins, mitochondria generate energy, lysosomes digest cell materials to recycle them, peroxisomes process molecules using oxygen, and secretory vesicles carry cell materials to the surface to release them. Figure 3. Relative sizes of cells and cell components. These structures differ in size by more than seven orders of magnitude. Figure 4. Origin of a eukaryotic cell. A prokaryotic host cell incorporates another prokaryotic cell. Each prokaryote has its own set of DNA molecules (a genome). The genome of the incorporated cell remains separate (curved blue line) from the host cell genome (curved purple line). The incorporated cell may continue to replicate as it exists within the host cell. Over time, during errors of replication or perhaps when the incorporated cell lyses and loses its membrane separation from the host, genetic material becomes separated from the incorporated cell and merges with the host cell genome. Eventually, the host genome becomes a mixture of both genomes, and it ultimately becomes enclosed in an endomembrane, a membrane within the cell that creates a separate compartment. This compartment eventually evolves into a nucleus. Figure 5. The origin of mitochondria and chloroplasts. Mitochondria and chloroplasts likely evolved from engulfed bacteria that once lived as independent organisms. At some point, a eukaryotic cell engulfed an aerobic bacterium, which then formed an endosymbiotic relationship with the host eukaryote, gradually developing into a mitochondrion. Eukaryotic cells containing mitochondria then engulfed photosynthetic bacteria, which evolved to become specialized chloroplast organelles. Lesson 5: Some Techniques in Cell and Molecular Biology Because of the very small size of the subject matter, cell and molecular biology is more dependent on the development of new instruments and technologies than any other branch of biology. Consequently, it is difficult to learn about cell and molecular biology without also learning about the technology that is required to collect data. In this lesson, we will survey the methods used most commonly in the field without becoming immersed in the details or the many variations that are employed. These are the goals of the present lesson: to describe the ways that selected techniques are used and to provide examples of the types of information that can be learned by using these techniques. We will begin with the instrument that enabled biologists to discover the very existence of cells, providing the starting point for all of the information that has been presented in this text. Light Microscope and its Types Light microscopes are still the central pieces of equipment for cell biologists, because microscopes are instruments that produce an enlarged image of an object. But although these instruments now incorporate many sophisticated improvements, the properties of light itself set a limit to the fineness of detail they can reveal. A light source, which may be external to the microscope e.g. in case of mirror microscope or built into its base e.g. compound microscope, illuminates the specimen. The sub-stage condenser lens gathers the diffuse rays from the light source and illuminates the specimen with a small cone of bright light that allows very small parts of the specimen to be seen after magnification. The light rays focused on the specimen by the condenser lens are then collected by the microscope’s objective lens. From this point, we need to consider two sets of light rays that enter the objective lens: those that the specimen has altered and those that it has not. The latter group consists of light from the condenser that passes directly into the objective lens, forming the background light of the visual field. The former group of light rays emanates from the many parts of the specimen and forms the image of the specimen. These light rays are brought to focus by the objective lens to form a real, enlarged image of the object within the column of the microscope. The image formed by the objective lens is used as an object by a second lens system, the ocular lens, to form an enlarged and virtual image. A third lens system located in the front part of the eye uses the virtual image produced by the ocular lens as an object to produce a real image on the retina. When the focusing knob of the light microscope is turned, the relative distance between the specimen and the objective lens changes, allowing the final image to become focused precisely on the plane of the retina. The total magnification attained by the microscope is the product of the magnifications produced by the objective lens and the ocular lens e.g. 4 (objective lens) x 10 (ocular lens) = 40 x total magnifications (or 40 times larger than the actual specimen size). The basic types of light microscope are bright/dark-field microscope, phase-contrast light microscope, and fluorescence microscope.  Photo A courtesy of Google Images Bright-Field Light Microscopy Specimens to be observed with the light microscope are broadly divided into two categories: whole mounts and sections. A whole mount is an intact object, either living or dead, and can consist of an entire microscopic organism such as a protozoan or a small part of a larger organism. Most tissues of plants and animals are much too opaque for microscopic analysis unless examined as a very thin slice, or section. To prepare a section, the cells are first killed by immersing the tissue in a chemical solution, called a fixative. A good fixative rapidly penetrates the cell membrane and immobilizes all of its macromolecular material so that the structure of the cell is maintained as close as possible to that of the living state. The most common fixatives for the light microscope are solutions of formaldehyde, alcohol, or acetic acid. After fixation, the tissue is dehydrated by transfer through a series of alcohols and then usually embedded in paraffin (wax), which provides mechanical support during sectioning. Paraffin is used as an embedding medium because it is readily dissolved by organic solvents. Slides containing adherent paraffin sections are immersed in toluene, which dissolves the wax, leaving the thin slice of tissue attached to the slide, where it can be stained or treated with antibodies or other agents. After staining, a coverslip is mounted over the tissue using a mounting medium that has the same refractive index as the glass slide and coverslip. Phase-Contrast Microscopy Small, unstained specimens such as a living cell can be very difficult to see with a bright-field microscope. The phase-contrast microscope makes highly transparent objects more visible. We can distinguish different parts of an object because they affect light differently from one another. One basis for such differences is refractive index. Cell organelles are made up of different proportions of various molecules: DNA, RNA, protein, lipid, carbohydrate, salts, and water. Regions of different composition are likely to have different refractive indices. Normally such differences cannot be detected by our eyes. However, the phase- contrast microscope converts differences in refractive index into differences in intensity (relative brightness and darkness), which are visible to the eye. Phase- contrast microscopes accomplish this result by (1) separating the direct light that enters the objective lens from the diffracted light emanating from the specimen and (2) causing light rays from these two sources to interfere with one another. The relative brightness or darkness of each part of the image reflects the way in which the light from that part of the specimen interferes with the direct light. Phase- contrast microscopes are most useful for examining intracellular components of living cells at relatively high resolution. For example, the dynamic motility of mitochondria, mitotic chromosomes, and vacuoles can be followed and filmed with these optics. Simply watching the way tiny particles and vacuoles of cells are bumped around in a random manner in a living cell conveys an excitement about life that is unattainable by observing stained, dead cells. The greatest benefit derived from the invention of the phase-contrast microscope has not been in the discovery of new structures, but in its everyday use in research and teaching labs for observing cells in a more revealing way. The phase-contrast microscope has optical handicaps that result in loss of resolution, and the image suffers from interfering halos and shading produced where sharp changes in refractive index occur. The phase-contrast microscope is a type of interference microscope. Other types of interference microscopes minimize these optical artifacts by achieving a complete separation of direct and diffracted beams using complex light paths and prisms. Another type of interference system, termed differential interference contrast (DIC), or sometimes Nomarski interference after its developer, delivers an image that has an apparent three-dimensional quality. Contrast in DIC microscopy depends on the rate of change of refractive index across a specimen. As a consequence, the edges of structures, where the refractive index varies markedly over a relatively small distance, are seen with especially good contrast.  Fluorescence Microscopy Over the past couple of decades, the light microscope has been transformed from an instrument designed primarily to examine sections of fixed tissues to one capable of observing the dynamic events occurring at the molecular level in living cells. These advances in live-cell imaging have been made possible to a large extent by innovations in fluorescence microscopy. The fluorescence microscope allows viewers to observe the location of certain molecules (called fluorophores or fluorochromes). Fluorophores absorb invisible, ultraviolet radiation and release a portion of the energy in the longer, visible wavelengths, a phenomenon called fluorescence. The light source in a fluorescence microscope produces a beam of ultraviolet light that travels through a filter, which blocks all wavelengths except that which is capable of exciting the fluorophore. The beam of monochromatic light is focused on the specimen containing the fluorophore, which becomes excited and emits light of a visible wavelength that is focused by the objective lens into an image that can be seen by the viewer. Because the light source produces only ultraviolet (black) light, objects stained with a fluorophore appear brightly colored against a black background, providing very high contrast. Photo D courtesy of Google Images Figure 7. A comparison of cells seen with different types of light microscopes. Light micrographs of a ciliated protist as observed under bright-field (a), phase-contrast (b), and differential interference contrast (DIC) (or Nomarski) optics (c). The organism is barely visible under bright-field illumination but clearly seen under phase-contrast and DIC microscopy. An upright fluorescence microscope built with CCD camera (d). Electron Microscopy Transmission Electron Microscopy Transmission electron microscopes (TEMs) form images using electrons that are transmitted through a specimen, whereas scanning electron microscopes (SEMs) utilize electrons that have bounced off the surface of the specimen. The transmission electron microscope can provide vastly greater resolution than the light microscope. The great resolving power of the electron microscope derives from the wave properties of electrons. As indicated in the equation on page 734, the limit of resolution of a microscope is directly proportional to the wavelength of the illuminating light: the longer the wavelength, the poorer the resolution. Unlike a photon of light, which has a constant wavelength, the wavelength of an electron varies with the speed at which the particle is traveling, which in turn depends on the accelerating voltage applied in the microscope. Standard TEMs operate with a voltage range from 10,000 to 100,000 V. At 60,000 V, the wavelength of an electron is approximately 0.05 Å. In actual fact, the resolution attainable with a standard transmission electron microscope is about two orders of magnitude less than its theoretical limit. This is due to serious spherical aberration of electron- focusing lenses, which requires that the numerical aperture of the lens be made very small (generally between 0.01 and 0.001). The practical limit of resolution of standard TEMs is in the range of 3 to 5 Å. The actual limit when observing cellular structure is more typically in the range of 10 to 15 Å. Electron microscopes consist largely of a tall, hollow cylindrical column through which the electron beam passes and a console containing a panel of dials that electronically control the operation in the column. The top of the column contains the cathode, a tungsten wire filament that is heated to provide a source of electrons. Electrons are drawn from the hot filament and accelerated as a fine beam by the high voltage applied between the cathode and anode. Air is pumped out of the column prior to operation, producing a vacuum through which the electrons travel. If the air were not removed, electrons would be prematurely scattered by collision with gas molecules. Just as a beam of light rays can be focused by a ground glass lens, a beam of negatively charged electrons can be focused by electromagnetic lenses, which are located in the wall of the column. The strength of the magnets is controlled by the current provided them, which is determined by the positions of the various dials of the console. The condenser lenses of an electron microscope are placed between the electron source and the specimen, and they focus the electron beam on the specimen. The specimen is supported on a small, thin metal grid (3 mm diameter) that is inserted with tweezers into a grid holder, which in turn is inserted into the column of the microscope. Because the focal lengths of the lenses of an electron microscope vary depending on the current supplied to them, one objective lens provides the entire range of magnification delivered by the instrument. As in the light microscope, the image from the objective lens serves as the object for an additional lens system. The image provided by the objective lens of an electron microscope is only  magnified about 100 times, but unlike the light microscope, there is sufficient detail present in this image to magnify it an additional 10,000 times. By altering the current applied to the various lenses of the microscope, magnifications can vary from about 1000 times to 250,000 times. Electrons that have passed through the specimen are brought to focus on a phosphorescent screen situated at the bottom of the column. Electrons striking the screen excite a coating of fluorescent crystals, which emit their own visible light that is perceived by the eye as an image of the specimen. Image formation in the electron microscope depends on differential scattering of electrons by parts of the specimen. The scattering of electrons by a part of the specimen is proportional to the size of the nuclei of the atoms that make up the specimen. To increase electron scattering and obtain required contrast, tissues are fixed and stained with solutions of heavy metals. These metals penetrate into the structure of the cells and become selectively complexed with different parts of the organelles. Those parts of a cell that bind the greatest number of metal atoms allow passage of the least number of electrons. The fewer electrons that are focused on the screen at a given spot, the darker that spot. Photographs of the image are made by lifting the viewing screen out of the way and allowing the electrons to strike a photographic plate in position beneath the screen. Because photographic emulsions are directly sensitive to electrons, much as they are to light, an image of the specimen can be recorded directly on film. Alternatively, the image can be captured on a CCD video camera. Although a video camera provides an instant image without the need for chemical development, the image lacks the exceptionally high resolution that is available with film. The analogue image captured on film can be converted to a digital image by a process of digitization. Figure 8. Ultrastructure of symbiotic zooxanthellae cells in host coral gastrodermal cell (a) revealed with the aid of transmission electron microscope (TEM). It shows the various major intercellular organelles and fine details of the cortical regions during non-dividing stage and early cleavage formation. A sample image of a TEM (b). Photo of Dr. A. Camaya, working on TEM of zooxanthalle-coral (i.e. Fig. 8a) at Kochi University, Japan in 2016. Fig. 8a is adopted from Camaya, 2020. Figure 9. Preparation of a specimen for observation in the electron microscope. Scanning Electron Microscopy The TEM has been exploited most widely in the examination of the internal structure of cells. In contrast, the scanning electron microscope (SEM) is utilized primarily to examine the surfaces of objects ranging in size from a virus to an animal head. The construction and operation of the SEM are very different from that of the TEM. The goal of specimen preparation for the SEM is to produce an object that has the same shape and surface properties as the living state, but is devoid of fluid, as required for observing the specimen under vacuum. Because water constitutes such a high percentage of the weight of living cells and is present in association with virtually every macromolecule, its removal can have a very destructive effect on cell structure. When cells are simply air dried, destruction results largely from surface tension at air–water interfaces. Specimens to be  examined in the SEM are fixed, passed through a series of alcohols, and then dried by a process of critical-point drying. Critical-point drying takes advantage of the fact that a critical temperature and pressure exist for each solvent at which the density of the vapor is equal to the density of the liquid. At this point, there is no surface tension between the gas and the liquid. The solvent of the cells is replaced with a liquid transitional fluid (generally carbon dioxide), which is vaporized under pressure so that the cells are not exposed to any surface tension that might distort their three-dimensional configuration. Once the specimen is dried, it is coated with a thin layer of metal, which makes it suitable as a target for an electron beam. In the TEM, the electron beam is focused by the condenser lenses to simultaneously illuminate the entire viewing field. In the SEM, electrons are accelerated as a fine beam that scans the specimen. In the TEM, electrons pass through the specimen to form the image. In the SEM, the image is formed by electrons that are reflected back from the specimen (backscattered) or by secondary electrons given off by the specimen after being struck by the primary electron beam. These electrons strike a detector that is located near the surface of the specimen. Image formation in the SEM is indirect. In addition to the beam that scans the surface of the specimen, another electron beam synchronously scans the face of a cathode-ray tube, producing an image similar to that seen on a television screen. The electrons that bounce off the specimen and reach the detector control the strength of the beam in the cathode-ray tube. The more electrons collected from the specimen at a given spot, the stronger the signal to the tube and the greater the intensity of the beam on the screen at the corresponding spot. The result is an image on the screen that reflects the surface topology of the specimen because it is this topology (the crevices, hills, and pits) that determines the number of electrons collected from the various parts of the surface. An SEM can provide a great range of magnification (from about 15 to 150,000 times for a standard instrument). Resolving power of an SEM is related to the diameter of the electron beam. Newer models are capable of delivering resolutions of less than 5 nm, which can be used to localize gold-labeled antibodies bound to a cell’s surface. The SEM also provides remarkable depth of focus, approximately 500 times that of the light microscope at a corresponding magnification. This property gives SEM images their three-dimensional quality. At the cellular level, the SEM allows the observer to appreciate the structure of the outer cell surface and all of the various processes, extensions, and extracellular materials that interact with the environment. There have been many developments in electron microscopy that include shadow casting, immunoelectron microscopy, and freeze-etching and many others. Shadow casting is an important technique that is carried out by depositing a thin layer of platinum or other metal on the microorganism to be examined. This platinum-coated organism, on bombardment with electron beams, scatters the electron and produces an image that is focused on a fluorescent screen.  Immunoelectron microscopy is a method to enhance sensitivity and specificity by reacting the specimen with specific antiviral antibody that results in clumping of viral particles. In this method also, antibody may be conjugated with gold to visualize and determine the location of specific antigenic determinants in a specimen. Freeze-etching is the method by which live organisms can be visualized unlike in traditional methods of electron microscopy in which living cells cannot be examined. This method is useful for the study of cellular ultrastructure of the microorganisms in the living state. This method is based on rapid cooling of specimens by deep-freezing in liquid gas and the subsequent formation of carbon platinum replica of the specimen. Figure 9. Scanning electron microscopy. Scanning electron micrographs of (a) a T4 bacteriophage (x 275,000). Replica of a freeze-fractured onion root cell (b) showing the various organelles. Procedure for the formation of freeze-fracture replicas (c). Examples of negatively stained and metal-shadowed specimens (tobacco rattle virus) (d). The procedure used for shadow casting as a means to provide contrast in the electron microscope. Cell Culture Learning to grow cells outside the organism, that is, in cell culture, has proved to be one of the most valuable technical achievements in the entire study of cell biology. A quick glance through any journal in cell biology reveals that the majority of the articles describe research carried out on cultured cells. The reasons for this are numerous: cultured cells can be obtained in large quantity; most cultures contain only a single type of cell; many different cellular activities, including endocytosis, cell movement, cell division, membrane trafficking, and macromolecular synthesis, can be studied in cell culture; cells can differentiate in culture; and cultured cells respond to treatment with drugs, hormones, growth factors, and other active substances. The early tissue culturists employed media containing a great variety of unknown substances. Cell growth was accomplished by adding fluids obtained from living systems, such as lymph, blood serum, or embryo homogenates. It was found that cells required a considerable variety of nutrients, hormones, growth factors, and cofactors to remain healthy and proliferate. Even today, most culture media contain large amounts of serum. One of the primary goals of cell culturists has been to develop defined, serum-free media that supports the growth of cells. Using a pragmatic approach in which combinations of various ingredients are tested for their ability to support cell growth and proliferation, a growing number of cell types have been successfully cultured in “artificial” media that lack serum or other natural fluids. As would be expected, the composition of these chemically defined media is relatively complex; they consist of a mixture of nutrients and vitamins, together with a variety of purified proteins, including insulin, epidermal growth factor, and transferrin (which provides the cells with iron). Because they are so rich in nutrients, tissue culture media are a very inviting habitat for the growth of microorganisms. To prevent bacteria from contaminating cell cultures, tissue culturists must go to great lengths to maintain sterile conditions within their working space. This is accomplished by using sterile gloves, sterilizing all supplies and instruments, employing low levels of antibiotics in the media, and conducting activities within a sterile hood. The first step in cell culture is to obtain the cells. In most cases, one need only removes a vial of frozen, previously cultured cells from a tank of liquid nitrogen, thaw the vial, and transfer the cells to the waiting medium. A culture of this type is referred to as a secondary culture because the cells are derived from a previous culture. In a primary culture, on the other hand, the cells are obtained directly from the organism. Most primary cultures of animal cells are obtained from embryos, whose tissues are more readily dissociated into single cells than those of adults. Dissociation is accomplished with the aid of a proteolytic enzyme, such as trypsin, which digests the extracellular domains of proteins that mediate cell adhesion. The tissue is then washed free of the enzyme and usually suspended in a saline solution that lacks Ca2+ ions Calcium play a key role in cell– cell adhesion, and their removal from tissues greatly facilitates the separation of cells. A number of laboratories are moving away from traditional two-dimensional culture systems, where cells are grown on the flat surface of a culture dish, to three-dimensional cultures, in which cells are grown in a three-dimensional matrix consisting of synthetic and/or natural extracellular materials. These materials can be purchased as products containing proteins and other components derived from natural basement membranes. Fractionation of a Cell’s Contents by Differential Centrifugation Most cells contain a variety of different organelles. Isolation of a particular organelle in bulk quantity is generally accomplished by the technique of differential centrifugation, which depends on the principle that, as long as they are denser than the surrounding medium, particles of different size and shape travel toward the bottom of a centrifuge tube at different rates when placed in a centrifugal field. To carry out this technique, cells are first broken open by mechanical disruption, typically using a mechanical homogenizer. Cells are homogenized in an isotonic buffered solution (often containing sucrose), which prevents the rupture of membrane vesicles due to osmosis. The homogenate is then subjected to a series of sequential centrifugations at increasing centrifugal forces. Initially, the homogenate is subjected to low centrifugal forces for a short period of time so that only the largest cellular organelles, such as nuclei (and any remaining whole cells), are sedimented into a pellet. At greater centrifugal forces, relatively large cytoplasmic organelles (mitochondria, chloroplasts, lysosomes, and peroxisomes) can be spun out of suspension. In subsequent steps, microsomes (the fragments of vacuolar and reticular membranes of the cytosol) and ribosomes are removed from suspension. This last step requires the ultracentrifuge, which can generate speeds of 75,000 revolutions per minute, producing forces equivalent to 500,000 times that of gravity. Once ribosomes have been removed, the supernatant consists of the cell’s soluble phase and those particles too small to be removed by sedimentation. The initial steps of differential centrifugation do not yield pure preparations of a particular organelle, so that further steps are usually required. In many cases, further purification is accomplished by centrifugation of one of the fractions through a density gradient, which distributes the contents of the sample into various layers according to their density. The composition of various fractions can be determined by microscopic examination, or by measuring the amounts known to be specific for particular organelles. Cellular organelles isolated by differential centrifugation retain a remarkably high level of normal activity, as long as they are not exposed to denaturing conditions during their isolation. Organelles isolated by this procedure can be used in cell-free systems to studies such as synthesis of membrane-bound proteins, the formation of transport vesicles, DNA synthesis, and the transport of solutes and development of ionic gradients. Figure 10. Purification of subcellular fractions by density gradient equilibrium centrifugation. The medium is composed of a continuous sucrose-density gradient, and the different organelles sediment until they reach a place in the tube equal to their own density, where they form bands (a). Due to rotation of the motor, centrifuge can separate supernatant from the larger molecule sediment (b). Liquid Column Chromatography Chromatography is a term for a variety of techniques in which a mixture of dissolved components is fractionated as it moves through some type of porous matrix. In liquid chromatographic techniques, components in a mixture can become associated with one of two alternative phases: a mobile phase, consisting of a moving solvent, and an immobile phase, consisting of the matrix through which the solvent is moving. Liquid chromatography is distinguished from gas chromatography in which the mobile phase is represented by an inert gas. In the chromatographic procedures described below, the immobile phase consists of materials that are packed into a column. The proteins to be fractionated are dissolved in a solvent and then passed through the column. The materials that make up the immobile phase contain sites to which the protein in solution can bind. As individual protein molecules interact with the materials of the matrix, their progress through the column is retarded. Thus the greater the affinity of a particular protein for the matrix material, the slower its passage through the column. Because different proteins in the mixture have different affinity for the matrix, they are retarded to different degrees. As the solvent passes through the column and drips out the bottom, it is collected as fractions in a series of tubes. Those proteins in the mixture with the least affinity for the column appear in the first fractions to emerge from the column. The resolution of many chromatographic procedures has been improved with the development of high performance liquid chromatography (HPLC), in which long, narrow columns are used, and the mobile phase is forced under high pressure through a tightly packed non- compressible matrix composed of exceptionally small (e.g., 5 µm diameter) particles. Ion-Exchange Chromatography Ionic charge is used as a basis for purification in a variety of techniques, including ion-exchange chromatography. Ion-exchange chromatography depends on the ionic bonding of proteins to an inert matrix material, such as cellulose, containing covalently linked charged groups. Two of the most commonly employed ion-exchange resins are diethylaminoethyl (DEAE) cellulose and carboxymethyl (CM) cellulose. DEAE- cellulose is positively charged and therefore binds negatively charged molecules; it is an anion exchanger. CM- cellulose is negatively charged and acts as a cation exchanger. The resin is Figure 11. Ion-exchange chromatography. The packed into a column, and the protein separation of two proteins by DEAE-cellulose. In solution is allowed to percolate through this case, a positively charged ion-exchange the column in a buffer whose composition resin is used to bind the negatively charged promotes the binding of protein to resin. protein. Gel Filtration Chromatography Gel filtration separates proteins (or nucleic acids) primarily on the basis of their effective size (hydrodynamic radius). Like ion-exchange chromatography, the separation material consists of tiny beads that are packed into a column through which the protein solution slowly passes. The materials used in gel filtration are composed of cross-linked polysaccharides (dextrans or agarose) of different porosity, which allow proteins to diffuse in and out of the Figure 12. Gel filtration chromatography. The separation of three globular proteins having beads. different molecular mass. Among proteins of similar basic shape, larger molecules are eluted before smaller molecules. Affinity Chromatography Another purification technique called affinity chromatography takes advantage of the unique structural properties of a protein, allowing one protein species to be specifically withdrawn from solution while all others remain behind in solution. Proteins interact with specific substances: enzymes with substrates, receptors with ligands, antigens with antibodies, and so forth. Each of these types of proteins can be removed from solution by passing a mixture of proteins through a column in which the specific interacting molecule (substrate, ligand, antibody, etc.) is covalently linked to an inert, immobilized material (the matrix). Unlike the other chromatographic procedures that separate proteins on the basis of size or Figure 13. Affinity chromatography. charge, affinity chromatography can Schematic representation of the coated agarose beads to which only a specific protein can achieve a near-total purification of the combine (a). Steps in the chromatographic desired molecule in a single step. procedure (b). Polyacrylamide Gel Electrophoresis Another powerful technique that is widely used to fractionate proteins is electrophoresis. Electrophoresis depends on the ability of charged molecules to migrate when placed in an electric field. The electrophoretic separation of proteins is usually accomplished using polyacrylamide gel electrophoresis (PAGE), in which the proteins are driven by an applied current through a gelated matrix. The matrix is composed of polymers of a small organic molecule (acrylamide) that is cross- linked to form a molecular sieve. A polyacrylamide gel may be formed as a thin slab between two glass plates or as a cylinder within a glass tube. Once the gel has polymerized, the slab (or tube) is suspended between two compartments containing buffer in which opposing electrodes are immersed. In a slab gel, the concentrated, protein- containing sample is layered in slots along the top of the gel (step 1). The protein sample is prepared in a solution containing sucrose or glycerol, whose density prevents the sample from mixing with the buffer in the upper compartment. A voltage is then applied between the buffer compartments, and current flows across the slab, causing the proteins to move toward the oppositely charged electrode (step 2). Separations are Figure 14 Polyacrylamide gel electrophoresis. typically carried out using alkaline The protein samples are typically dissolved in a buffers, which make the proteins sucrose solution whose density prevents the negatively charged and cause them to sample from mixing with the buffer and then migrate toward the positively charged loaded into the wells with a fine pipette as shown in step 1. In step 2, a direct current is applied anode at the opposite end of the gel. across the gel, which causes the proteins to move Following electrophoresis, the slab is into the polyacrylamide along parallel lanes. removed from the glass plates and When carried out in the detergent SDS, which is stained (step 3). usually the case, the proteins move as bands at rates that are inversely proportional to their molecular mass. Once electrophoresis is completed, the gel is removed from the glass frame and stained in a tray (step 3). Mass Spectrometry The input to the detector is converted into a series of peaks of ascending m/z ratio. The emerging field of proteomics Mass spectrometers have been a depends heavily on the analysis of favorite instrument of chemists for many proteins by mass spectrometry. Mass years, but it has only been in the past two spectrometers are analytical instruments decades that biologists have discovered used primarily to measure the masses of their remarkable analytic powers. Now, molecules, determine chemical formulas using mass spectrometry (MS), protein and molecular structure, and identify biochemists are able to rapidly identify unknown substances. the proteins present in a particular type of Mass spectrometers accomplish cell, organelle, or protein complex. these feats by converting the substances in a sample into positively charged, gaseous ions, which are accelerated through a curved tube toward a negatively charged plate. As the ions pass through the tube, they are subjected to a magnetic field that causes them to separate from one another according to their molecular mass [or more precisely according to their mass-to-charge (m/z) ratio]. The ions strike an electronic detector located at the end of the tube. Smaller ions travel faster and strike the Figure 15. Principles of operation of a mass detector more rapidly than larger ions. spectrometer.  LET’S REMEMBER! To summarize our lessons for this module and to give you further details of the discussion, here are the synopsis of all lessons (L1-L7) which you have read. Cell and molecular biology is a foundation field of discipline in biological sciences. As highly significant aspect of biology, this primarily deals with the study of cell structure and function, and it revolves around the concept that the cell is the fundamental unit of life emphasizing the essential biological processes such as how cells divide, grow, communicate and die (L1). Cells are extremely small which were first revealed by earliest microscopists. The discovery of the cell was credited to Robert Hooke, who first observed pores in the dead plant cells using his self-discovered microscope and coined the word ‘cell’ which he compared to the cells being inhabited by the monks in the monastery. The cell theory has three tenets, namely (1) all organisms are composed of one or more cells; (2) the cell is the basic organizational unit of life, proposed by Matthias Schleiden in 1838 and (3) all cells arise from preexisting cells, as concluded by Rudolf Virchow in 1855 (L2). The properties of life, as exhibited by cells, can be described by a collection of properties. Cells are very complex and their substructure is highly organized and predictable. The information to build a cell is encoded in its genes. Cells reproduce by cell division; their activities are fueled by chemical energy; they carry out enzymatically controlled chemical reactions; they engage in numerous mechanical activities; they respond to stimuli; and they are capable of a remarkable level of self-regulation (L3). Cells are either prokaryotic or eukaryotic. Prokaryotic cells are found only among bacteria, whereas all other types of organisms—protists, fungi, plants, and animals—are composed of eukaryotic cells. Prokaryotic and eukaryotic cells share many common features, including a similar cellular membrane, a common system for storing and using genetic information, and similar metabolic pathways. Prokaryotic cells are the simpler type, lacking the complex membranous organelles (e.g., endoplasmic reticulum, Golgi complex, mitochondria, and chloroplasts), chromosomes, and cytoskeleton characteristic of the cells of eukaryotes. The two cell types can also be distinguished by their mechanism of cell division, their locomotor structures, and the type of cell wall they produce (if a cell wall is present). Complex plants and animals contain many different types of cells, each specialized for particular activities (L4). Cells are almost always microscopic in size. Bacterial cells are typically 1 to 5 µm in length, whereas eukaryotic cells are typically 10 to 30 µm. Cells are microscopic in size for a number of reasons: their nuclei possess a limited number of copies of each gene; the surface area (which serves as the cell’s exchange surface) becomes limiting as a cell increases in size; and the distance between the cell surface and interior becomes too great for the cell’s needs to be met by simple diffusion (L5).  Techniques developed in investigating cell and molecular biology are complicated. Because of the very small size of the subject matter, cell and molecular biology is more dependent on the development of new instruments and technologies than any other branch of biology. Some of these modern basic protocols involved microscopy techniques (light and electron microscopy), cell culture, centrifugation, chromatography, electrophoresis, and spectrometry. Similar to these techniques used, other protocols (not discussed in these module) requires instruments and technology, skills, and science to efficiently developed significant findings (L6). HOW MUCH HAVE YOU LEARNED! Let’s answer the following self-assessment tests. Good luck! 1. Why is there a need for studying cells? 2. The cell theories established by the earliest foundation of cell studies are essentially factual, yet remains to be considered as theories and continuously proven even at this time. Why? 3. With the properties of cells mentioned, how will you now describe the cell in a concise yet overall statement? Justify? 4. Compare a prokaryotic and eukaryotic cell on the basis of structural, functional, and metabolic differences. 5. Why are cells almost always microscopic? 6. Why do you think cell and molecular biology studies utilize these complex microtechniques? 7. Referring to the discussion in Lesson 6, create a detailed list of the advantages and disadvantages of light and electron (TEM and SEM) microscopy. Write it in a tabular matrix. 8. You are given experiment to study the cell structure and function of an organism with limited number or volume of available sample collected. How and what are the techniques that should be done to have comprehensive result of your experiments. ANY FEEDBACK! For your feedback to this module, you may write your concern inside the box and let your professor know.  REFERENCES Camaya, A.P. 2020. Stages of the symbiotic zooxanthellae–host cell division and the dynamic role of coral nucleus in the partitioning process: a novel observation elucidated by electron microscopy. Coral Reefs 39: 929-938. doi.org/10.1007/s00338-020-01912-y. Karp, G. 2013. Cell and Molecular Biology, Concepts and Experiment 7 th Edition. John Wiley and Sons, Inc., USA. 874 pp. Lodish, H., Berk, A., Matsudaira, P., Kaiser, CA., Krieger, M., Scott, M.P., Zipursky, L., Darnell, J. 2003. Molecular Cell Biology 5th Edition W.H. Freeman. USA. 975 pp Parija, S.C. 2012. Textbook of Microbiology and Immunology 2nd Edition. Elsevier, India. 682 pp. Rana, SVS 2008. Biotechniques Theory and Practice. Rastogi Publications, India SUGGESTED READINGS AND WEBSITES LINKS https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology https://bio.libretexts.org/Courses/Remixer_University/Download_Center https://www.google.com/webhp https://www.nature.com/nrm/about/aims https://www.nature.com/scitable/ebooks/cntNm-14749010/contents/ https://www.nature.com/scitable/topic/cell-biology-13906536/ https://www.youtube.com/watch?v=SUo2fHZaZCU