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Blueprint Series

Complete Solution
Chapter 4

4
CULTURING AND
VISUALIZING CELLS

REVIEW THE CONCEPTS
1
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2
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Being able to clearly
distinguish between two closely spaced points at even the highest total magnification
is the ultimate goal because if the two objects are already blurred and cannot be discriminated at a lower magnification, simply increasing the magnification will have no
effect
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61l N sin a, where l is the wavelength of
light used to illuminate the specimen, N is the refractive index of the medium (usually
air) between the front face of the objective lens and the specimen, and a is the halfangle of the cone of light entering the face of the objective lens
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Since only three of the values can be altered to achieve the best resolution (the smallest D possible), one has to either decrease the wavelength of light or
increase the numerical aperture by gathering more light into the front face of the
objective lens
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Increasing the numerical aperture is accomplished by placing a
drop of oil or water, which have greater refractive indices (1
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3, respectively)
relative to that of air (1), between the specimen and the objective lens
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Chemical stains are required for visualizing cells and tissues with the basic light
microscope because most cellular material does not absorb visible light and
therefore cells are essentially invisible in a light microscope
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For example, certain stains may reveal where proteins are in a cell but not
where a specific protein is located
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Only light emitted by the sample will form an image, so
the location of the fluorescence indicates the location of the molecule of interest
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As a result, these techniques facilitate
optical sectioning of thick specimens as opposed to physical sectioning and associated techniques that may alter the specimen
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Certain electron microscopy methods rely on the use of metal to coat the specimen
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Methods that use
this approach include metal shadowing, freeze fracturing, and freeze etching
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5
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Since these cells are not transformed, they have a limited lifespan in
culture
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Such cells are said to be immortal
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6
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However, such cells have a finite lifespan in culture
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Such cells, called hybridoma cells, retain characteristics
of both parent cells, allowing for production of a single-type, or monoclonal,
antibody
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Specific types of cells in suspension may be isolated by a fluorescence-activated
cell sorter (FACS) machine in which cells previously “tagged” with a fluorescentlabeled antibody are separated from cells not recognized by the antibody
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Specific organelles
are generally separated by centrifugation of lysed cells
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This is often combined with density-gradient separations to purify specific organelles on the basis of their buoyant density
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FACS (see Figure 9-2), whereby labeled cells pass through a laser light beam and
the fluorescent intensity of light emitted is measured, allowing the computer to
assign each cell with an electric charge proportional to the fluorescence
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