CHAPTER 18

Techniques in Celland 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 light-

gathering 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 lightand 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 observationin 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 andmetal-shadowed specimens

The procedure used for shadow casting

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.

Procedure for the formation of freeze-

fracture 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 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.

Scanning electron microscopy

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.

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