Studying Cells

Microscopy

Microscopes allow for magnification and visualization of cells and cellular components that cannot be seen with the naked eye.

Learning Objectives

Compare and contrast light and electron microscopy.

Key Takeaways

Key Points

  • Light microscopes allow for magnification of an object approximately up to 400-1000 times depending on whether the high power or oil immersion objective is used.
  • Light microscopes use visible light which passes and bends through the lens system.
  • Electron microscopes use a beam of electrons, opposed to visible light, for magnification.
  • Electron microscopes allow for higher magnification in comparison to a light microscope thus, allowing for visualization of cell internal structures.

Key Terms

  • resolution: The degree of fineness with which an image can be recorded or produced, often expressed as the number of pixels per unit of length (typically an inch).
  • electron: The subatomic particle having a negative charge and orbiting the nucleus; the flow of electrons in a conductor constitutes electricity.

Microscopy

Cells vary in size. With few exceptions, individual cells cannot be seen with the naked eye, so scientists use microscopes (micro- = “small”; -scope = “to look at”) to study them. A microscope is an instrument that magnifies an object. Most photographs of cells are taken with a microscope; these images can also be called micrographs.

The optics of a microscope’s lenses change the orientation of the image that the user sees. A specimen that is right-side up and facing right on the microscope slide will appear upside-down and facing left when viewed through a microscope, and vice versa. Similarly, if the slide is moved left while looking through the microscope, it will appear to move right, and if moved down, it will seem to move up. This occurs because microscopes use two sets of lenses to magnify the image. Because of the manner by which light travels through the lenses, this system of two lenses produces an inverted image (binocular, or dissecting microscopes, work in a similar manner, but they include an additional magnification system that makes the final image appear to be upright).

Light Microscopes

To give you a sense of cell size, a typical human red blood cell is about eight millionths of a meter or eight micrometers (abbreviated as eight μm) in diameter; the head of a pin of is about two thousandths of a meter (two mm) in diameter. That means about 250 red blood cells could fit on the head of a pin.

Most student microscopes are classified as light microscopes. Visible light passes and is bent through the lens system to enable the user to see the specimen. Light microscopes are advantageous for viewing living organisms, but since individual cells are generally transparent, their components are not distinguishable unless they are colored with special stains. Staining, however, usually kills the cells.

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Light and Electron Microscopes: (a) Most light microscopes used in a college biology lab can magnify cells up to approximately 400 times and have a resolution of about 200 nanometers. (b) Electron microscopes provide a much higher magnification, 100,000x, and a have a resolution of 50 picometers.

Light microscopes, commonly used in undergraduate college laboratories, magnify up to approximately 400 times. Two parameters that are important in microscopy are magnification and resolving power. Magnification is the process of enlarging an object in appearance. Resolving power is the ability of a microscope to distinguish two adjacent structures as separate: the higher the resolution, the better the clarity and detail of the image. When oil immersion lenses are used for the study of small objects, magnification is usually increased to 1,000 times. In order to gain a better understanding of cellular structure and function, scientists typically use electron microscopes.

Electron Microscopes

In contrast to light microscopes, electron microscopes use a beam of electrons instead of a beam of light. Not only does this allow for higher magnification and, thus, more detail, it also provides higher resolving power. The method used to prepare the specimen for viewing with an electron microscope kills the specimen. Electrons have short wavelengths (shorter than photons) that move best in a vacuum, so living cells cannot be viewed with an electron microscope.

In a scanning electron microscope, a beam of electrons moves back and forth across a cell’s surface, creating details of cell surface characteristics. In a transmission electron microscope, the electron beam penetrates the cell and provides details of a cell’s internal structures. As you might imagine, electron microscopes are significantly more bulky and expensive than light microscopes.

Crystallographic Analysis

Crystallographic analysis reveals the arrangement of atoms in solids that help build the three-dimensional model of molecules.

Learning Objectives

Distinguish between the three methods of crystallography: X-ray, neturon and electron crystallography

Key Takeaways

Key Points

  • Crystallographic analysis is a comprehensive mutlistep process that involves different molecular disciplines.
  • In the late 1950s the first crystal structure resolved was that of myoglobin. Today, a massive database of protein crystal structures has been developed.
  • Crystallographic analysis is important in understanding the morphology of a protein and facilitates the development of inhibitors and antagonists.

Key Terms

  • recombinant protein: Manipulated form of protein produced in specialized vehicles known as vectors.
  • bioinformatics: A field of science in which biology, computer science, and information technology merge into a single discipline to analyse biological information using computers and statistical techniques.

Atomic Methods in Bacterial Analysis

Crystallography is the scientific study of the arrangement of atoms in a solid. The field has greatly advanced with the development of x-ray diffraction methods, where the matter analyzed is usually in its crystal form. Nuclear magnetic resonance spectroscopy and x-ray crystallography have become the methods of choice for understanding three-dimensional protein structures.

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Insulin crystals: insulin crystals

METHODS OF CRYSTALLOGRAPHY

  • X-ray crystallography is the primary method for determining the molecular conformation of biological macromolecules, particularly proteins and nucleic acids such as DNA and RNA. Indeed, the double-helical structure of DNA was deduced from crystallographic data.
  • Neutron crystallography is often used to help refine structures obtained by x-ray methods or to solve a specific bond; the methods are often viewed as complementary, as x-rays are sensitive to electron positions and scatter most strongly off heavy atoms, while neutrons are sensitive to nucleus positions and scatter strongly off many light isotopes, including hydrogen and deuterium.
  • Electron crystallography has been used to determine some protein structures, most notably membrane proteins and viral capsids.

ANALYSIS OF DATA

Studies of protein crystallography help determine the three dimensional structure of proteins and analyze their function alone or within multimolecular assemblies. The structure-function analysis is completed by biochemical and biophysical studies in solution. The protocol for completing a successful crystallographic analysis requires production of proteins (cloning, mutagenesis, bacterial culture, etc.), purification of recombinant proteins (such as chromatography of affinity and gel filtration), enzymatic tests and inhibition measurement (spectrophotometry), crystallization, x-rays crystallography and structural analysis, interactions determination (microcalorimetry, fluorescence, BIAcore), conformational analyses (circular dichroism, ultracentrifugation, light scattering), modifications analysis (mass spectrometry), bioinformatics, and molecular modelisation.

The Protein Data Bank (PDB) is a freely accessible repository documenting the structures of proteins and other biological macromolecules. It stores information about crystals and crystal structures. Computer programs like RasMol or Pymol can be used to visualize biological molecular structures.

Genetic Analysis

Genetic analysis is a growing field in microbiology that provides information about specific adaptations and the evolution of organisms.

Learning Objectives

Summarize the techniques used to study genomes: PFGE. ordered clone approach, direct shotgun sequencing and microarray hybridization

Key Takeaways

Key Points

  • Bacterial cells obey the laws of genetics and the central dogma of life ( DNA – RNA – Protein ).
  • Genetic analysis of a cell can be achieved using DNA sequencing, polymerase chain reactions, microarray hybridization, and gel electrophoresis.
  • Analysis of microbial mutations and genetic make up help understand mechanisms of resistance to antibiotics.

Key Terms

  • mutation: Any heritable change of the base-pair sequence of genetic material.
  • genome: The complete genetic information (either DNA or, in some viruses, RNA) of an organism, typically expressed in the number of basepairs.

The study of microbial pathogens with genetic methods is a new and explosive field set to dominate microbiology in the next decade. Microbes provide an excellent starting point for genetic studies because they have a relatively simple genomic structure compared to higher, multicellular organisms.

Microbial Genomes

Studies on microbial genomes may provide crucial starting points for understanding the genomics of higher organisms. There are 32 microbial genomes sequenced to date and published (25 domain Bacteria, 5 Domain Archaea, 1 domain Eukarya).

Relationship between Bacteria and Plasmids

Bacteria obey the laws of genetics and the central dogma of life. DNA that carries genetic information is transcribed to RNA polypeptides, which are translated into protein. Viruses differ from other microbes as they can carry either DNA or RNA. Cell function depends on specific polypeptides, proteins, and enzymes encoded by genes. Bacterial chromosomes contain double stranded molecules of DNA arranged in a circular form called plasmids.

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Circular Plasmid DNA: Plasmid DNA that can replicate independently of chromosomal DNA.

Plasmids are located in the cytoplasm of bacteria, are capable of autonomous replication, and transfer genes from parent cell to daughter cell. Bacteria possess extra chromosomal genetic elements that encode for antibiotic resistance, toxins, virulence determining genes, and reduced sensitivity to mutagens such as heavy metals. Plasmid profiling using molecular, biochemical, and microbial techniques is essential to understanding the mechanism of pathogenicity and to fuel genetic engineering.

Techniques to Study Genomes

Many of the techniques used to study whole genomes are conventional molecular biology techniques adapted to operate effectively with DNA in a much larger size range. Pulsed field gel electrophoresis (PFGE) is an adaptation of conventional agarose gel electrophoresis that allows extremely large DNA fragments to be resolved (up to megabase size fragments). PFGE is essential for estimating the sizes of whole genomes/chromosomes prior to sequencing and is necessary for preparing large DNA fragments for large insert DNA cloning and analysis of subsequent clones. It is a commonly used and extremely powerful tool for genotyping and epidemiology studies for pathogenic microorganisms. DNA cloning is another technique fundamental to molecular biology that requires adaptation in order to be useful in studying DNA at a whole genome scale.

There are two main approaches to sequencing microbial genomes – the ordered clone approach and direct shotgun sequencing both require large and small insert genomic DNA libraries in order to be effective.

Microarray hybridization is another technique used to characterize the dynamic nature of gene expression within a microbial cell. Microarray technology allows whole organism gene expression to be investigated. PCR products of every gene from a complete genome sequence are bound in a high-density array on a glass slide. These arrays are probed with fluorescently labeled cDNA prepared from whole RNA under specific environmental conditions. The level of cDNA is then quantified using high-resolution image scanners.

These techniques allow the identification of the genotype (i.e.-the genetic makeup) of a cell. Genotypic variations exist in microbes and these include mutations, gene transfer by transformation, conjugation, and transduction.

Mutation

Mutation is random, undirected, heritable variation caused by alteration in nucleotide sequence at some point of DNA. Mutations can take the form of deletion, addition, or substitution of one or more bases. All genes are susceptible to mutations, but not all mutations are expressed. Genetic analysis of microbes allows the characterization of genes implicated in microbial pathogenesis.