1. Introduction
1.1 Discovery of Cells
Cells were discovered in 1665 by an English scientist and inventor Robert Hooke. Through his self-designed compound light microscope, he examined structures too small to be seen by the naked eye.
Among the first of structures he examined a thin piece of cork (the outer surface of bark from a tree)/ he described the cork as being made up of hundreds of tiny boxes giving it the appearance of a honeycomb. He called these little boxes cells.
It soon became clear that virtually all living things are made of cells and that cells are the building block of the living organism. Cells come in a wide variety of types and forms.
1.2 The Cell Theory
One of the most important concepts in biology is that the basic unit of structure and function in a living organism is the cell. This is known as the cell theory which was proposed jointly by Schleiden and Schwann in the 1830s.
Cells are the structural units of virtually all organisms. All unicellular organisms as well as multicellular organisms are made of a basic unit called the cell. Cells are the functional units of all organisms. Metabolism takes place within the cell. Cells arise only by division of existing cells. The type of reproduction can either be asexual or sexual reproduction. This means that there is no spontaneous formation. Cells contain hereditary material called nucleic acids (DNA and RNA). The hereditary material allows for special characters to be passed down from parent to daughter cells. Given suitable conditions, cells are capable of independent existence.
It is important to note
that with the discovery of the light microscope, a much more detailed structure
of the cell is now known. This is called the cell ultrastructure.
2. Measuring Cell Structures
2.1 Cell Size
Cells are extremely small and cam only be seen properly when magnified and viewed through the lens of a microscope. Cell dimensions are expressed as micrometres or microns. Cells are in the size range of about 5 – 500 microns but most are between 10 and 150 microns. The angstrom (10 -10 m) is sometimes used to record the thickness of cell membranes and the sizes of certain macromolecules.
Magnification is the number of times larger an image is than its object. It is essential that the same unit be used for the size of the image and the actual size of the object. As a rule of thumb, always express the units for cells in microns.
3. Revealing the Ultrastructure of Cells
Cell biology (Cytology) is the study of the cell structure and function. Today, it incorporates aspects of biology, chemistry and physics. The study of cell structure and function follows the sequence below:
(1) Cell Fractionation, which involves homogenization of tissue by mechanically breaking them down and differential centrifugation, which separated the cell components for electron microscopy of biochemical analysis of the smallest functional components of living cells and tissues.
(2) Studying the Gross and Fine Morphology of the Organelles by using election or light microscopy.
(3) Studying the Functions of an Organelle and its Relationships with other Organelles in the Cell. This is to understand their role either in the synthetic or the degradative pathway in which radioactive isotopes or heavy isotopes can be used to label a precursor compound and trace its movement in the cell through the process of autoradiography.
(4) Studying the Molecular Structure of the Chemicals that make up the Organelle. To do so, the molecules are first isolated by chromatography and electrophoresis. Then to observe the physical structure of these chemicals, the electron microscopy or x-ray diffraction techniques are used.
3.1 Cell Fractionation
3.1.1 Homogenization of Tissues
This technique involves centrifuging disrupted cells at various speeds and duration to isolate components of different sizes, densities and/or shapes. All of the following methods discussed below are carried out at low temperature and in cold buffer solution (e.g. isotonic sucrose solution) This is to maintain the integrity of the organelles. The breaking off of tissues and cells into small fragments is done using pistons, blenders of ultrasound devices. Solution of sucrose of other sugars are generally used to maintain the integrity of the organelles are to counteract the tendency of the organelles freed from the cell to aggregate.
3.1.1.1 Methods of Tissue Disruption
Mechanical means include using a mortar and pestle (with abrasive materials like sand or ground glass. Disadvantages include loss of some cell constituents by adsorption to the sand). Using the blender, pressure cell and ultrasound are some of the other methods.
Chemical means include disrupting by osmotic pressure. For example by being placed in hypotonic solutions, animal cells will burst.
3.1.2 Differential Centrifugation
This method helps to isolate individual organelles to facilitate the study of the structure of each of and every single organelle on its own. Molecules denser than the suspending medium move downwards at a constant rate when the speed of centrifuge is held uniform. The organelles will settle out according to size and density at different centrifugation speeds. Organelles can retain some or all of their biological activity, depending on the ionic composition and pH of the surrounding medium. Larger molecules have larger Svedberg (S) units.
3.2 Studying the Gross & Fine Morphology of Cells
3.2.1 Microscopy
The two most common techniques used to study cell and tissue structure are light and electron microscopy. Three important factors are crucial in microscopy.
3.2.1.1 Resolution, Magnification & Contrast
Resolution is the ability to distinguish between two objects as two separate structures thus determining a microscopic image’s clarity and richness in detail. It measures the extent to which a microscope can distinguish fine details in the specimen as separate distinct image points. If two separate objects cannot be resolved, they will be seen as one object. The smaller the value, the greater the resolution. Resolution of the human eye is 200 microns. The resolution for a light microscope is 0.2 microns. The resolution of an electron microscope is 0.002 to 0.005 microns.
Magnification increases the apparent size of the specimen viewed and makes it appear closer.
Contrast is the ability to distinguish one part of a cell from another. This usually comes about through stainings of the specimen.
Resolution is not the same as magnification. If you take a photograph and keep on magnifying it, you will not eventually be able to see atoms. Magnification can be increased, but the resolution of the photograph stays the same. We enlarge photographs to see them more clearly. But if we go too far, the picture breaks up into separate blurred dots. Another way of explaining the difference is to study a picture of a cell taken with a light microscope and an electron microscope at the same magnification. The resolution is much greater in the electron microscope. Shorter wavelengths of electrons are said to have greater resolving power then those of light.
The resolution of an electron microscope is about 0.5 nm compared with 200 nm for a light microscope. The latter is used for getting the overall view of cells or tissues and preparation of material for them is much quicker and easier. They are used for viewing living material, which is not possible with an electron microscope.
3.2.1.2 Light Microscopy (LM)
Light microscopes are applied in ways that use different light sources and patterns of image formation.
3.2.1.2.1 Types of Light Microscopes
They can be classified as (1) bright-field / compound microscope, (2) dark-field, (3) phase contrast, (4) differential interference contrast and (5) fluorescence microscopes.
3.2.1.2.2 Principles of LM
Light is focused in a specimen by a condenser lens. After passing through the specimen, the rays are focused into a magnified image by two lenses placed either end of a tube. Nearest the specimen is the objective lens, at the opposite end of the tube is the ocular lens. These lenses refract light. The specimen’s image is thus magnified and inverted for the observer. The image appears against a bright background. The image is observed by looking directly into the ocular lens.
The course and fine control knobs function to move the stage to place the specimen in the correct position for focusing by the objective lens.
The condenser’s position can be adjusted so that light focused by this lens converges on the specimen and spreads into a cone of light that exactly fills the objective lens.
The best resolution possible is 200 nm. Using ultraviolet light such as an illumination source pushes this value down to 100 nm. At this level, cell organelles such as nuclei, chloroplasts and mitochondria are clearly resolved but smaller ones such as microtubules and ribosomes remain invisible.
For objects resolved beyond 200 nm, magnification of the microscope beyond 1000x does not improve the resolution of small objects. For this reason, enlargement of the image beyond 1000x is often termed as “empty” magnification.
3.2.1.3 Electron Microscopy (EM)
Electron microscopes use a beam of electrons rather than light as an illumination source. Electron beams have a much smaller wavelength than light rays, so EMs have greater resolving power and can produce much higher effective magnifications. The beams are focused by magnetic fields, generated by massive coils of wire through which precisely controlled electric currents are passed. The focusing of the image is by changing the lens current rather than by moving lenses as in light microscopy. The magnification is adjusted by altering lens current rather than by substituting lenses.
3.2.1.3.1 Types of EM
3.2.1.3.1.1 Transmission EM (TEM)
The TEM is so called because electrons forming the image pass through the specimen. In construction, a TEM resembles an inverted LM. The principle is the same as in a LM. An electron beam with wavelength of 0.005 nm is aimed at an ultra-thin section of specimen, which may be stained with heavy metals to absorb electrons and enhance contrast. A thin tungsten wire is heated to a high temperature by an electric current. The filament and its holder are maintained at a high negative voltage. The anode is grounded and is thus positive with respect to the filament. Consequently, electrons leaving the filament are strongly attracted to the anode. As they travel from the filament to the anode, the electrons are accelerated to a velocity that depends on the voltage difference between the two locations. The wavelength of the electrons in the beam is inversely proportional to the velocity.
The beam leaving the gun is focused by a series of two condenser lenses into a small intense spot of electrons on the specimen. Another series of lenses (objective, intermediate and projector lenses) focuses the electrons passing through the specimen onto a fluorescent viewing screen at the bottom of the microscope. The viewing screen is coated with crystals that respond to electron bombardment by emitting visible light. This process converts the electron image into a visual image. A 2D image is produced.
Airlocks are provided so that specimens and photographic plates can be introduced or removed without disturbing the microscope vacuum. Because electrons cannot travel very far in air, a strong vacuum must be maintained along the entire path of the electron beam. TEM resolves ribosomes, microtubules, microfilaments and proteins.
3.2.1.3.1.2 Scanning EM (SEM)
SEM is used to examine the surfaces of cells or isolated cellular structures. It is only similar to the TEM in its illumination source and condenser lenses.
An electron gun produces an electron beam which is focused into an intense spot on the specimen surface by a magnetic lens system analogous to the condenser lens of a TEM. Rather than being stationary, the focused spot moves rapidly back and forth over the specimen. This scanning is accomplished by beam deflectors, charged plates placed between the condenser lenses and the specimen. The intense spot of electrons scanning the specimen surface excites specimen molecules to a high energy level. The excited molecules release this energy in several forms including secondary electrons. A 3D image is formed from the secondary electrons rather than the illuminating beam.
When the scanning spot in the microscope strikes a point on the specimen surface that responds by emitting large numbers of secondary electrons, the detector adjusts the scanning spot on the television screen at that point and instant to a high level of brightness. At specimen points emitting fewer secondary electrons, the detector current is reduced and the spot scanning the screen at the same point and instant is dimmed. Each point on the screen therefore corresponds in brightness to the number of electrons emitted by the same relative points on the specimen. The scanning is so rapid that an apparently instantaneous image of the specimen surface is produced on the TV screen, with areas of brightness and darkness on the screen corresponding to ridges and valleys on the specimen surface.
3.2.1.4 Comparison of LM, EM, SEM and TEM
Advantages of LM (1) cheap, (2) low cost of operation, (3) portable, (4) not affected by magnetic fields, (5) preparation of material is quick and simple requiring little expertise, (6) specimen rarely distorted to a great extent by preparation, (7) Living as well as dead specimens can be viewed, (8) natural colour can be observed.
Disadvantages of LM (1) only magnifies to 1500x, (2) resolution only 200 nm, (3) restricted depth of field.
Advantages of EM (1) magnifies over 500 000x, (2) resolution is 1 nm apart, (3) possible to investigate a greater depth of field.
Disadvantages of EM (1) expensive, (2) high cost of operation, (3) Huge and must be operated in a special room, (4) affected by magnetic fields, (5) Preparation of specimen is lengthy and requires considerable expertise and sometimes use of complex equipment, (6) Specimen can be distorted, (7) only dead specimens can be used. A high vacuum is required as electrons are easily scattered and absorbed by gas molecules. Specimen must be dry and non- volatile, (8) appears in black and white only.
Advantages of TEM (1) high resolution about 0.5 nm. The short wavelengths of the electron beam greatly improve resolution which is 200 times better than LM.
Disadvantages of TEM (1) specimen must be dead as it is viewed in a vacuum, (2) difficult to be sure that the specimen resembles a living cell in all its details as preservation and staining may change or damage its structure, (3) expensive to run, (4) preparation of material is time-consuming and requires expert training, (5) specimen gradually deteriorates in the electron beam. Photographs must be taken if further study is required.
Advantages of SEM (1) surfaces of structures are shown, (2) great depth of field meaning that a large part of the specimen is in focus at the same time, giving a striking 3D effect, (3) larger samples can be examined than with a TEM.
Disadvantages of SEM. (1) Resolution (5-20 nm) is not as great as with a TEM (0.5 nm), (2) lens system, specimen and detector must be kept in a high vcuum, (3) Specimen must be dry and non-volatile.
3.2.2 Sample Preparation and Techniques for microscopy
3.2.2.1 Fixation
This preserves structural organization of cells, tissues and organs. It prevents bacterial and enzymatic digestion, insolubilizes tissue components to prevent diffusion, and protects against damage from subsequent steps of tissue processing.
Chemical fixation is a common approach. Chemical fixatives are used. Best results are achieved by rapid penetration of living tissue with fixative. Small tissue pieces may be fixed by immersion. Entire organs may be fixed by perfusion (fixative pumped through vessels serving the tissue of interest). The fixative-induced changes in chemical composition and fine structure may produce artifacts.
Freeze fracture / etching may be used for LM or EM. Tissue id embedded in cryprotectant (glycerin). Rapid freezing at low temperatures reduces ice crystal formation and artifacts. This allows tissues to be sectioned or fractured without dehydration of clearing. It is faster than chemical fixation. It avoids dissolving lipids and denaturing fixative-sensitive proteins such as enzymes and antigens. It is not as permanent as chemical fixation. Sections are thicker and resolution is poor.
3.2.2.2 Dehydration
This eases penetration of tissues by clearing agents and prepares fixed tissue for infiltration with embedding medium.
Dehydration replaces water in the tissue with organic solvent (ethanol). Fixed tissue is immersed in a series of alcohol-water mixtures with increasing alcohol concentration, to 100% alcohol.
Alcohol may denature protein of interest. Water loss causes uneven shrinkage of components with different water content. This may create unnatural spaces between cells and tissue layers.
3.2.2.3 Clearing
This prepares fixed tissue for embedding and renders the material transparent.
Alcohol is not miscible with some of the common embedding and mounting media. So, dehydrated tissue is immersed in a clearing agent to replace alcohol. The clearing agent for LM is xylene (paraffin solvent) and the one for EM is propylene oxide (plastic solvent).
Clearing agents may denature proteins of interest. Some components shrink unevenly as their proteins denature.
3.2.2.4 Embedding
This prepares cleared tissue for sectioning. It makes tissue firm and prevents crushing or other tissue disruption during sectioning. Permits thin and uniform sectioning.
The tissue is positioned in a mould filled with embedding medium which hardens into a block. For LM, paraffin is commonly used. For EM, plastics and epoxy rasins are common. It also requires a catalyst to harden or polymerize. Harder embedding media allows thinner sectioning which is a requirement for EM.
The improved sectioning allowed be embedding has limitations associated with dehydration and clearing.
3.2.2.5 Sectioning
Most tissues are too thick and opaque for microscopic analysis of internal structure. Thin slices allow light of electron to penetrate specimen and form an image.
For LM, thin sectioning is used. A microtome with steel blade or glass knife cuts 3-8 microns and 1-5 microns respectively. For EM, thin sectioning is used. The ultramicrotome is used for cutting extremely thin sections (20-100 nm) for EM and a diamond or glass knife must be used.
Sections typically provide only a 2D image of a 3D structure. Chatter results from knife vibrations during sectioning.
3.2.2.6 Staining
Most biological structures are transparent, so some means of obtaining contrast between different structures must be employed. Staining gives the distinctive colour characteristics to different kinds of cellular components. Stains are thus contrasting agents. Certain stains when used in low concentrations are non toxic to living tissue and can be used on living material. These are called vital stains (e.g. methylene blue and neutral red).
For LM, most stain affinities based on reciprocal acid-base characteristics if stain and tissue compounds. Acidic stains (eosin) binds basic structures (cytoplasmic proteins). Basic stains (hematoxylin) bind acidic tissue compounds (nucleic acids in ribosomes). Stain mixtures reveal multiple cell compounds. H and E stains distinguishes nucleus and cytoplasm. Acid-base boundaries may not correspond to boundaries between structures.
For TEM, it uses heavy metal shadowing, negative and positive staining. Most stains are for electron absorbing or scattering ability and affinity for particular cell components. Heavy metal salts (lead citrate and uranyl acetate) are common. The fixative osmium tetroxide interacts with lipids to form electron-dense precipitate and doubles as a stain for cell membranes. TEM stains stop electrons from penetrating. TEM images are shadows of heavy metal deposits. Actual structures are not seen.
For SEM, it uses heavy metal shadowing. Specimens are not stained. Specimen is then mounted on a stub and sputter-coated (sprayed) with fine mist of heavy metal particles (gold) before viewing. SEM reveals surface architecture in exquisite detail, but heavy metal coating prevents electrons form penetrating to reveal internal structure.
3.2.2.7 Mounting
This eases handling and decreases damage to specimen during examination.
For LM, sections are placed on glass slides, either with water or pre-coated with thin layer of albumin, gelatin or polylysine to improve attachment. After staining, sections are covered with glass cover-slips to preserve them for repeated examination. Tissue sections may develop folds, making some regions appear to have more cells and stain darker.
For EM, the specimens are mounted on copper grids as electron beams cannot penetrate glass. For grid-mounted specimens, only portions lying between cross-bars are visible.
3.3 Studying Relationships an Organelle has with others
3.3.1 Autoradiography (Tracing Biological Pathways)
It involves tagging a
specimen molecule with a radioisotope. The tagged sample is then placed in
close contact with a film of photographic emulsion. The rays emitted by the
radioisotope enter the emulsion. The film is then exposed in a similar manner
to visible light and then developed. The location of the radioisotope in the
original sample is determined from the exposure spots on the film. The
radioactive emission can also be detected and measured by a Greiger counter.
