The World of the Cell
Seventh Edition

Wayne M. Becker Lewis J. Kleinsmith Jeff Hardin Gregory Paul Bertoni

Chapter 1 A Preview of the Cell
Copyright © 2009 Pearson Education, Inc..

Chapter 1--Cell
The basic structural and functional unit of living organisms The smallest structure capable of performing the essential functions characteristic of life

The study of cells
•Began with the invention of microscopes in the 17th century •Using a microscope to look at cork, Robert Hooke described little box-like compartments and named them cellulae (little rooms) in 1665 (Micrographia) •Actually, the cellulae were dead plant cells •Limited by the microscope resolution

Antonie van Leeuwenhoek
Developed superior lenses that had 10-fold better resolution than Hooke’s. Looked at algae, protists, minerals, fossils, animals using a microscope. First to describe bacteria:
"I then most always saw, with great wonder, "an unbelievably great there wereof living that in the said matter company many very animalcules, a-swimming more nimbly than little living animalcules, very prettily a-moving. any Ibiggest sort. . . up to this time. The The had ever seen had a very strong and biggest sort. .and shot through into water (or swift motion, . bent their body the curves in going forwards. . . does through the water. The spittle) like a pike Moreover, the other animalcules were in suchspun round numbers, second sort. . . oft-times enormous like a top. that allthese were.far more in number." . . and the water. . seemed to be alive."

Modern Cell and Molecular Biology
Cytology the study of cell structure

Genetics the study of the behavior of genes

Biochemistry the study of the chemistry of living systems

Cell and Molecular Biology

Figure 1-2 Resolving Power of the Human Eye, the Light Microscope, and the Electron Microscope
?

The study of cells
•1830’s microscopes had higher magnification and improved resolution (1 micrometer= µm) magnification – the ability to make images of objects larger resolution – minimum distance that can separate two points that still remain identifiable as separate points when viewed through a microscope

The study of cells
•1830’s microscopes had higher magnification and improved resolution •Robert Brown saw rounded structure in every plant cell he looked at •Nucleus (kernal) •1838/9 – Matthias Schleiden and Theodor Schwann concluded all plant and animal tissues were composed of cells

The study of cells
1839 – Schwann’s cell theory 1. All organisms consist of one or more cells 2. The cell is the basic unit of structure for all organisms 1855 –third component added by Rudolf Virchow 3. All cells arise only from pre-existing cells (omnis cellula e cellula)

The World of the Micrometer
a.k.a. micron, µm 10-6 meter

The World of the Nanometer nm = 10-9 meter

Angstrom (Å) = 0.1 nm

Modern Cell and Molecular Biology
Cytology the study of cell structure

Genetics the study of the behavior of genes

Biochemistry the study of the chemistry of living systems

Scientific

Method

Scientific Method

Imagination

Scientific Method
Hypothesis – a statement or explanation that is consistent with most of the observational and experimental evidence to date (must be testable) Theory – a hypothesis that has been tested critically under many different conditions – usually by many different investigators using a variety of approaches - and is consistently supported by the evidence Law – a theory that has been so thoroughly tested and confirmed over a long period of time by a large number of investigators that virtually no doubt remains as to its validity

Modern Cell and Molecular Biology
Cytology the study of cell structure

Genetics the study of the behavior of genes

Biochemistry the study of the chemistry of living systems

Important Advances in Biochemistry
1828 – Frederich Wöhler urea – could be synthesized from a synthetic reactant Living organisms governed by laws of nonliving world (chemistry & physics)

Important Advances in Biochemistry
Late 1800’s – Louis Pasteur yeast could ferment sugar into alcohol Eduard & Hans Buchner yeast extracts could also ferment sugar Activities in living organisms are linked to specific processes

Important Advances in Biochemistry
1920’s-1930’s – Metabolic pathways began to be delineated glycolysis, TCA cycle 1939-1941 – Fritz Lipmann demonstrated that ATP was the principle energy storage molecule in most cells (1953-Nobel Prize in Medicine with Hans Krebs)

Important Technological Advances in Biochemistry
Late 1940’s-1950’s – Calvin and colleagues used radioactive carbon dioxide to elucidate the Calvin cycle 1st use of isotope to understand a metabolic pathway

Important Technological Advances in Biochemistry
Centrifugation Subcellular fractionation – technique for isolating organelles from cell homogenates using various types of centrifugation (size, shape, density) differential centrifugation equilibrium density centrifugation

Ultracentrifugation extremely high speeds can separate organelles & macromolecules

Important Technological Advances in Biochemistry
Differential centrifugation

http://fig.cox.miami.edu/~cmallery/255/255tech/mcb5.36.fuge.jpg

Important Technological Advances in Biochemistry
Density gradient centrifugation
Solvent: H20 plus Sucrose or CsCl

http://bio.winona.edu/berg/ILLUST/gradient.jpg

Important Technological Advances in Biochemistry
Equilibrium density centrifugation

Additional important biochemical techniques
Chromatography a group of related techniques that utilize the flow of a fluid phase over a non-mobile absorbing phase to separate molecules based on their relative affinities for the two phases, which in turn reflect differences in size, charge, hydrophobicity, or affinity for a particular chemical group

Additional important biochemical techniques
Electrophoresis - a group of related techniques that utilize an electrical field to separate electrically charged molecules
AGE (Agarose gel electrophoresis) SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)

Modern Cell and Molecular Biology
Cytology the study of cell structure

Genetics the study of the behavior of genes

Biochemistry the study of the chemistry of living systems

Important Advances in Genetics
Late 1860’s Gregor Mendel Principles of segregation & Independent assortment Father of Transmission Genetics

Important Advances in Genetics
Walther Flemming - Chromosomes during dividing cells (mitosis) Roux and Weissman (independently) -theorized that the natural basis of heredity was on the chromosomes Walter Sutton – chromosome theory of heredity hereditary factors are located on the chromosomes within the nucleus Thomas Hunt Morgan – linked specific traits to specific chromosomes

Important Advances in Genetics
Chemistry of genetics 1869 -Johann Friedrich Miescher – isolated nuclear (DNA) from salmon sperm & pus 1914 – Robert Feulgen – DNA component of chromosomes 1944 - Avery, MacLeod & McCarty; 1952 – Hershey & Chase – DNA is the hereditary material 1940’s – Beadle & Tatum – one gene – one enzyme 1953 – (Rosalind Franklin and Maurice Wilkins) Watson & Crick – double helix model of DNA 1960’s – DNA & RNA polymerases identified, genetic code deciphered Jacob & Monod – prokaryotic transcription regulation

Important Technological Advances in Genetics
Separation of DNA (ultracentrifugation & electrophoresis) Nucleic acid hybridization (e.g. Southern & northern blots) Recombinant DNA technology – group of laboratory techniques for joining DNA fragments derived from two or more sources DNA sequencing – technology used to determine the linear order of bases in DNA molecules or fragments Bioinformatics - using computers to analyze the vast amounts of data generated by sequencing and expression studies on genomic, proteomic, and lipidomic, ….omic

Modern Cell and Molecular Biology
Genetics the study of the behavior of genes

Cytology the study of cell structure

Biochemistry the study of the chemistry of living systems

Cytology - the study of cell structure

Microscopy

Microscopy
Light microscope (LM) – instrument consisting of a source of visible light and a system of glass lenses that allows an enlarged image of a specimen to be viewed Can be used to view living and non-living cells
Image seen by viewer Eyepiece Ocular lens

Objective lens Specimen Condenser lens Light source
Figure 4.1A

• Widespread use of electron microscope (EM) began in the 1930s • They use a beam of electrons instead of light • The greater resolving power of electron microscopes
– allows greater magnification – reveals cellular details

• Cannot be used to view live samples

Electron Microscope (EM)
Photons vs electrons
12V

Fig. A1

Microscopy – optical principles
Need 3 elements to form an image
1. Illumination source 2. Specimen 3. Lens system to focus light on the specimen

Microscopy (M)
Light microscope (LM) – instrument consisting of a source of visible light and a system of glass lenses that allows an enlarged image of a specimen to be viewed Can be used to view live and non-living cells
Image seen by viewer Eyepiece Ocular lens

Objective lens Specimen Condenser lens Light source
Figure 4.1A

Microscopy – optical principles
Resolution – minimum distance that can separate points when viewed through a microscope Dependent upon: 1. Wavelength of light used to illuminate specimen 2. Lens angular aperture 3. Refractive index

Microscopy – optical principles
1. Wavelength of light used to illuminate specimen

Wavelength – distance between the crests of two successive waves

Electron wavelengths much shorter than visible light

Electron-magnetic spectrum

Microscopy – optical principles

Illumination source Specimen size Perturbations (interference)

Amplitude ?

Fig. A.2

Microscopy – optical principles
Image is formed by interference – the process by which two or more waves combine to reinforce or cancel on another

The pattern of either additive or canceling interference by light waves is called diffraction

Microscopy – optical principles
2. Lens angular aperture
Objective Lens

α
Specimen Image

α – angular aperture A measure of the amount of light that enters the objective lens from the specimen

Determines sharpness of the interference pattern (best is 70°)

Microscopy – optical principles
2. Lens angular aperture -is one property of lenses focal length

Microscopy – optical principles
Properties of lenses – focal length

Microscopy – optical principles
Properties of lenses – focal length angular aperture

Microscopy – optical principles
Resolution – minimum distance that can separate points when viewed through a microscope Dependent upon: 1. Wavelength of light used to illuminate specimen 2. Lens angular aperture 3. Refractive index

Microscopy – optical principles
3.Refractive index - The change in velocity of light as it passes from one medium to another (air to glass).

Microscopy – optical principles
Resolution – minimum distance that can separate points when viewed through a microscope Dependent upon: 1. Wavelength of light used to illuminate specimen 2. Lens angular aperture 3. Refractive index

How do these three factor affect resolution?

Microscopy – optical principles
Resolution
Abbé equation 0.61λ r= n sin α r = resolution λ = wavelength n = refractive index of medium between specimen and objective lens 0.61 = degree to which image points can overlap and be recognized as separate points by an user α – angular aperture Objective lens numerical aperture (NA) = n sin α Smaller r, better resolution (Limit of resolution for light microscope ~200 to 300 nm)

• Widespread use of electron microscope began in the 1930s • They use a beam of electrons instead of light • The greater resolving power of electron microscopes
– allows greater magnification – reveals cellular details

• Cannot be used to view live samples

Transmission electron microscope (TEM) • Specimens cut into thin sections (0.1 um; microtome) and the TEM aims an electron beam through the section Result: image of the internal architecture
Figure 4.1C

(Limit of resolution for Electron microscope ~1 to 2 nm)

Transmission electron microscope (TEM)

Transmission electron micrograph of cilia
Figure 4.1C

Scanning electron microscope (SEM)
Electrons scan the surface of cells that have been coated with metal Result: Detailed picture of the cell surface
Figure 4.1B

Scanning electron microscope (SEM)

Scanning electron micrograph of cilia

Figure 4.1B

Compound Light Microscopy
Image seen by viewer Eyepiece Ocular lens

Objective lens Specimen Condenser lens Light source

Overall magnification = product of enlarging powers of ocular lens, objective lens (& intermediate lens) Brightfield microscopy

Phase-Contrast Microscopy

Phase-Contrast Microscopy

Differential Interference Microscopy

A.9

Fluorescence Microscopy

Fluorescence Microscopy

Fluorescence Microscopy – Immunostaining

Fluorescence Microscopy

Fluorescence Microscopy

Fluorescence Microscopy Problem – out of focus light Confocal microscopy Digital Deconvolution Microscopy

Confocal microscopy