Techniques in Cell

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CHAPTER 18

• Techniques in Cell
• and Molecular Biology

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Introduction

• Research in cell biology requires complex
  instrumentation and techniques.
• Understanding the technology helps in
  understanding the cell.

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18.1 The Light Microscope (1)

• The light microscope uses the refraction of light
  rays to magnify an object.
• A condenser directs light toward the specimen.
• The objective lens collects light from the
  specimen.
• The ocular lens forms an enlarged, virtual image.
  

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The paths taken by light rays to form an image
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The Light Microscope (2)

• Resolution
• Resolution is the ability to see two nearby
  points as distinct images.
• The numerical aperture is a measure of the
  light-gathering qualities of a lens.
• The limit of resolution depends on the wavelength
  of light.
• Optical flaws, or aberrations, affect resolving
  power.

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Resolution
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The Light Microscope (3)

• Visibility
• Visibility deals with factors that allow an
  object to be observed.
• It requires that the specimen and the background
  have different refractive indexes.
• Translucent specimens are stained with dyes.
• A bright-field microscope a light that
  illuminates the specimen is seen as a bright
  background it is suited for specimens of high
  contrast such as stained sections of tissues.

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The Feulgen stain
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The Light Microscope (4)

• Preparation of Specimens for Bright-Field Light
  Microscopy
• A whole mount is an intact object, either living
  of dead.
• A section is a very thin slice of an object.
• To prepare a section, cells are immersed in a
  chemical called a fixative.
• The rest of the procedures minimize alteration
  from the living state.

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The Light Microscope (5)

• Phase-Contrast Microscopy
• The phase-contrast microscope makes highly
  transparent objects more visible by converting
  differences in the refractive index of some parts
  of the specimen into differences in light
  intensity.
• Differential interference contrast (DIC) optics
  gives a three-dimensional quality to the image.

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A comparison of cells seen with different types
of light microscopes
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The Light Microscope (6)

• Fluorescence Microscopy (and Related
  Fluorescence-Based Techniques)
• Fluorescence microscopy has made possible
  advances in live-cell imaging.
• Fluorochromes are compounds that release visible
  light upon absorption of UV rays.
• Fluorochrome stains cause cell components to
  glow, a phenomenon called fluorescence.

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The Light Microscope (7)

• Fluorescence microscopy (continued)
• Fluorochrome-conjugated antibodies are used to
  locate specific cellular structures
  (immunofluorescence).
• The gene for green fluorescent protein (GFP) from
  jellyfish can be recombined with genes of
  interest in model organisms.
• GFP is expressed with the host gene of interest.
• GFP is used to follow a gene of interest.

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Use of GFP variants to follow the dynamic
interactions between neurons and target cells in
vivo
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The Light Microscope (8)

• Fluorescence microscopy (continued)
• A GFP variant is called fluorescence resonance
  energy transfer (FRET), which uses fluorochromes
  to measure changes in distance between labeled
  cellular components.

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The Light Microscope (9)

• Video Microscopy and Image Processing
• Video microscopy is used to observe living cells.
• Video cameras offer several advantages for
  viewing specimens.
• They can detect and amplify very small
  differences in contrast.
• Images produced by video cameras can be converted
  to digital electronic images and processed by a
  computer.

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The Light Microscope (10)

• Laser Scanning Confocal Microscopy
• A laser scanning confocal microscope produces an
  image of a thin plane located within a much
  thicker specimen.
• A laser beam is used to examine planes at
  different depths in a specimen.

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Laser scanning confocal fluorescence microscopy
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The Light Microscope (11)

• Super-Resolution Fluorescence Microscopy
• STORM (stochastic optical reconstruction
  microscopy) allows the localization of a single
  fluorescent molecule within a resolution of lt20
  nm.
• Fluorescent images can be positioned with greater
  accuracy.

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Breaking the light microscope limit of resolution
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18.2 Transmission Electron Microscope (1)

• Transmission electron microscopes (TEMs) use
  electrons instead of light to form images.
• The limit of resolution is about 10-15 Å.

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Transmission Electron Microscope (2)

• The components of an electron microscope
• An electron beam from a tungsten filament
  accelerated by high voltage, and focused with a
  magnetic field.
• A condenser lens is placed between the electron
  source and the specimen.
• Differential scattering of electrons by the
  specimen creates the image.
• Proportional to the thickness of the specimen.
• Tissues are stained with heavy metals for
  contrast.

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A comparison of the lens system of a lightand
electron microscope
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Transmission Electron Microscope (3)

• Specimen Preparation for Electron Microscopy
• Specimens must be fixed, embedded, and sectioned
  thinly.
• Glutaraldehyde and osmium tetroxide are common
  fixatives.
• Specimens are dehydrated prior to embedding.
• Epon or Araldite are common embedding resins.
• Thin sections cut with glass or diamond knives
  are collected on grids.

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Preparation of a specimen for observationin the
electron microscope
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Transmission Electron Microscope (4)

• Specimen preparation (continued)
• Chemicals used may cause an artifact, which may
  be disproved by using other techniques.
• In negative staining, heavy metal diffuses into
  spaces between specimen molecules.
• Shadow casting coats a specimen with metal to
  produce a three-dimensional effect.

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Examples of negatively stained andmetal-shadowed
specimens
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The procedure used for shadow casting
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Transmission Electron Microscope (5)

• Freeze-Fracture Replication and Freeze-Etching
• In freeze-fracture replication, frozen tissue is
  fractured with a knife.
• A heavy-metal layer is deposited on fractured
  surface.
• A cast of the surface is formed with carbon.
• The metal-carbon replica is viewed in the TEM.
• In freeze-etching, a layer of ice is evaporated
  from the surface of the specimen prior to coating
  it with heavy metal.

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Procedure for the formation of freeze-fracture
replicas
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Freeze-fracture and freeze-etching
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18.3 Scanning Electron Atomic Force Microscopy
(1)

• Scanning electron microscopes (SEMs) form images
  from electrons that have bounced off the surface
  of a specimen.
• Specimens for SEM are dehydrated by
  critical-point drying.
• Specimens are coated with a layer of carbon, then
  gold.
• The image in SEM is indirect.
• SEM has a wide range of magnification and focus.

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Scanning electron microscopy
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Scanning Electron Atomic Force Microscopy (2)

• Atomic Force Microscopy
• The atomic force microscope (AFM) is a
  high-resolution scanning instrument.
• AFM provides an image of each individual molecule
  as it is oriented in the field.

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18.4 The Use of Radioisotopes (1)

• Radioisotopes can be easily detected and
  quantified.
• Properties of radioisotopes
• An isotope refers to atoms that differ in the
  number of neutrons.
• Isotopes with an unstable combination of protons
  and neutrons are radioactive.
• The half-life of a radioisotope measures its
  instability half of the radioactive material
  disintegrates in a given amount of time.

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Properties of a variety of radioisotopes
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The Use of Radioisotopes (2)

• Liquid scintillation spectrometry
• Scintillants absorb the energy of an emitted
  particle and release it in the form of light.
• Radiation of a tracer in a sample can be detected
  by measuring light emitted by a scintillant.

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The Use of Radioisotopes (3)

• Autoradiography is a technique to where a
  particular isotope is located.
• A particle emitted from a radioactive atom
  activates a photographic emulsion.
• The location of the radioisotope in the specimen
  is determined by the positions of the overlying
  silver grains in a photographic emulsion.

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Preparation of light microscopic autoradiograph
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Examples of autoradioagraphs
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18.5 Cell Culture (1)

• Most of the study of cells is carried out using
  cell culture.
• Cells can be obtained in large quantities.
• Most culture contain a single type of cell.
• Many different types of cells can be grown in
  culture.
• Cell differentiation can be studied in a cell
  culture.
• Cells in a culture require media that includes
  hormones and growth factors.

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Cell Culture (2)

• A primary culture is when cells are obtained
  directly from the organism.
• A secondary culture is derived from a previous
  culture.
• A cell line refers to cells with genetic
  modifications that allow them to grow
  indefinitely.
• Many types of plant cells can be grown in culture.

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Cell Culture (3)

• A two-dimensional culture system is when cells
  are grown on the flat surface of a dish.
• Labs are moving to three-dimensional cultures in
  which cells are grown in a 3D matrix consisting
  of extracellular materials.
• 3D cultures are better suited to study cell-cell
  interactions.

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A comparison of cell morphology of cells growing
in 2D versus 3D cultures
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18.6 The Fractionation of a Cells Contents by
Differential Centrifugation

• Differential centrifugation facilitates the
  isolation of particular organelles in bulk
  quantity.
• Prior to centrifugation, cells are broken by
  mechanic disruption in a buffer solution.
• The homogenate is subjected to a series of
  sequential centrifugations.
• Organelles isolated can be used in a cell-free
  system to study cellular activities