CHAPTER 18
Techniques in Cell
and Molecular Biology
Introduction
• Research in cell biology requires complex
instrumentation and techniques.
• Understanding the technology helps in
understanding the cell.
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.
The paths taken by light rays to form an image
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 lightgathering qualities of a lens.
• The limit of resolution depends on the wavelength of
light.
• Optical flaws, or aberrations, affect resolving power.
Resolution
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.
The Feulgen stain
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.
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.
A comparison of cells
seen with different
types of light
microscopes
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.
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.
Use of GFP variants to follow the dynamic interactions
between neurons and target cells in vivo
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.
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.
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.
Laser scanning confocal fluorescence
microscopy
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 <20
nm.
– Fluorescent images can be positioned with greater
accuracy.
Breaking the light microscope limit of resolution
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.
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.
A comparison of the lens system of a light
and electron microscope
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.
Preparation of a specimen for observation
in the electron microscope
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.
Examples of negatively stained and
metal-shadowed specimens
The procedure used for shadow casting
Transmission Electron Microscope (5)
• Freeze-Fracture Replication and FreezeEtching
– 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.
Procedure for the
formation of freezefracture replicas
Freeze-fracture and freeze-etching
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 criticalpoint 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.
Scanning electron microscopy
Scanning Electron Atomic Force
Microscopy (2)
• Atomic Force Microscopy
– The atomic force microscope (AFM) is a highresolution scanning instrument.
– AFM provides an image of each individual
molecule as it is oriented in the field.
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.
Properties of a variety of radioisotopes
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.
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.
Preparation of
light microscopic
autoradiograph
Examples of autoradioagraphs
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.
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.
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.
A comparison of cell morphology of cells
growing in 2D versus 3D cultures
18.6 The Fractionation of a Cell’s
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