Ch-9 Visualizing Cells

LOOKING AT CELLS IN THE LIGHT MICROSCOPE:

Definitions:
1. Fluorescent protein (from a jellyfish) that is widely Green fluorescent protein
used as a marker for monitoring the movements of proteins in (GFP)
living cells
2. The minimal separation between two objects at which Limit of resolution
they appear distinct
3. The normal light microscope in which the image is Bright-field microscope
obtained by simple transmission of light through the object
being viewed
4. Computer treatment of images gained from Image processing
microscopy that reveals information not immediately visible
to the eye
5. Similar to a light microscope but the illuminating light Fluorescence microscope
is passed through one set of filters before the specimen, to
select those wavelengths that excite the dye, and through
another set of filters before it reaches the eye, to select only
those wavelengths emitted when the dye fluoresces

6. Type of light microscope that produces a clear image Confocal microscope

of a given plane within a solid object. It uses a laser beam as a
pinpoint source of illumination and scans across the plane to
produce a two-dimensional optical section.

7. Technique for monitoring the closeness of two Fluorescence resonance

fluorescently labeled molecules (and thus their interaction) in energy transfer (FRET)
cells
8. Piece of fine glass tubing, pulled to an even finer tip, Microelectrode
that is used to inject electric current into cells
True/False
9. Because the DNA double helix is only 2 nm wide—well below the limit of resolution of
the light microscope—it is impossible to see chromosomes in living cells without special
stains.
 False. Even though DNA is very thin, chromosomes become thick when they
condense during mitosis. These condensed chromosomes are large enough to be
seen in living cells using special microscopes (like phase-contrast), even without
stains.
 Ans from the book: False: Although it is not possible to see DNA by light microscopy in
the absence of a stain, chromosomes are clearly visible under phase-contrast or Nomar
ski differential-interference-contrast microscopy when they condense during mitosis.
Condensed human chromosomes are more than 1 μm in width—well above the resolution
limit of 0.2 μm.
10. Monoclonal antibodies are superior to the mixture of antibodies in standard antisera
raised against the same protein because they are absolutely specific for that protein.
 False. Monoclonal antibodies bind one small part (epitope) of a protein, but that
same part can sometimes exist on other proteins too. So, they are not always
absolutely specific.
Ans from the book: False: Monoclonal antibodies are homogeneous and recognize the
particular protein against which they were raised, but they may still react with other
unrelated proteins. In general, the specificity of a monoclonal antibody depends on
whether the segment of protein it binds (the epitope, which is usually just a few amino
acids) is present in identical or similar form on another protein. Establishing the
specificity of an antibody probe (whether it is monoclonal or a mixture) is a critical
element in determining its practical usefulness for experimental applications.
11. Caged molecules can be introduced into a cell and then activated by a strong pulse of
laser light at the precise time and cellular location chosen by the experimenter.
 True. Caged molecules stay inactive inside the cell. When a laser light is shone
on a specific spot, the cage is removed and the active molecule is released exactly
where and when the experimenter wants.
Ans from the book: True: Caged molecules are photosensitive precursors of biologically
active substances such as Ca2+, cyclic AMP, and inositol trisphosphate. They are
designed to carry an inactivating moiety attached by a photosensitive linkage. When
exposed to intense light of the correct wavelength, the inactivating group is split off and
the active small molecule is released. Because laser beams can be tightly focused, caged
molecules can be activated at defined locations in a cell. Thus, the time and location of
activation are under the experimenter’s control.
12. Super resolution techniques allow fluorescently tagged molecules to be imaged to
accuracies an order of magnitude below the classic diffraction limit to resolution.
 True. Super-resolution methods (like STED, PALM, STORM) can find the exact
positions of fluorescent tags much more precisely than normal microscopes,
breaking the ~200 nm diffraction limit and giving much higher-detail images.
Ans from the book: True: Super resolution techniques allow localization of fluorescent
molecules to accuracies much below the diffraction limit to resolution, which is about
200 nm. Current super resolution techniques such as STED, PALM, and STORM use
optical methods to illuminate a sparse set of fluorescent tags, whose precise mathematical
centers can then be calculated from the point spread function of the blurred image
representing the tag. By combining multiple such images, these super resolution
techniques build up a high-resolution image of the object that was tagged.
 LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE

36. A contrast-enhancing technique for the Negative staining

electron microscope in which a heavy-metal
salt is used to create a reverse, or negative,
image of the object.
37. Type of microscope that uses a beam of Electron microscope (EM)
electrons to create an image.
38. Electron microscopy technique in which Cryoelectron microscopy
the objects to be viewed, such as
macromolecules and viruses, are rapidly
frozen.
39. Type of electron microscope that produces Scanning electron microscope (SEM)
an image of the surface of an object.

40. Electron microscopy technique in which Immunogold electron microscopy

cellular structures or molecules of interest are
labeled with antibodies tagged with electron-
dense gold particles, which show up as black
spots on the image,
True/False:

41: Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) can
both be used to examine a structure in the interior of a thin section; TEM provides a projection
view, while SEM captures electrons scattered from the structure and gives a more three-
dimensional view.

Ans from the book: False. Although the indicated statements are true with regard to TEM, they
are incorrect for SEM. SEM gathers and analyzes electrons that are scattered from the surface of
the object being viewed, which in this case is the thin section itself. SEM is not useful for
examining internal structures.
Problem Solving
13. Examine the diagram of the light microscope in Figure 9–1. Identify and label the
eyepiece, the condenser, the light source, the objective, and the specimen. At what two
points in the light path is the image of the specimen magnified?
Ans from the book: The components of the light microscope are labeled in Figure 9–17.
Magnification of the specimen occurs at two points: in the objective and in the eyepiece
14. Why is it important to keep dust, fingerprints, and other smudges off the lenses of a light
microscope?
Ans from the book: All such imperfections scatter, refract, and reflect light, reducing the
amount of light that goes through the specimen and adding spurious light rays that add a
background haze to the image. As a result, both contrast and resolution are reduced.
15. When light enters a medium with a different optical density, it bends in a direction that
depends on the refractive indices of the two media. Air and glass, for example, have
refractive indices of 1.00 and 1.51, respectively. When light enters glass—the medium
with the higher refractive index—it bends toward a line drawn normal to the surface
(Figure 9–2A). Conversely, when light exits glass into air, it bends away from the normal
line (Figure 9–2B). Using these principles, draw the paths of two parallel light rays that
pass through the hemispherical glass lens shown in Figure 9–2C. Will the two rays
converge or diverge? Would the result be any different if the glass lens were flipped so
that light entered the flat surface?
Ans from the book:  The two parallel light rays will converge (focus
together) after passing through the hemispherical lens.
 The lens acts as a converging (convex) lens.
 If the lens is flipped, the rays will still converge.
 The focusing effect does not change when the lens is inverted.

16. The diagrams in Figure 9–3 show the paths of light rays passing through a specimen with
a dry lens and with an oil-immersion lens. Offer an explanation for why oil-immersion
lenses should give better resolution. Air, glass, and oil have refractive indices of 1.00,
1.51, and 1.51, respectively.
Ans: Short Exam Answer
In a dry lens, light passes from glass to air, and because their refractive indices are
different, some light is internally reflected and lost. This reduces the amount of light
entering the objective.
In an oil-immersion lens, the oil has the same refractive index as glass, so no light is lost
due to internal reflection. More light enters the objective, increasing the numerical
aperture and improving resolution.
Therefore, oil immersion gives better resolution than a dry lens.
17. Figure 9–4 shows a diagram of the human eye. The refractive indices of the components
in the light path are: cornea 1.38, aqueous humor 1.33, crystalline lens 1.41, and vitreous
 humor 1.38. Where does the main refraction—the main focusing—occur? What role do
you suppose the lens plays?
Ans from the book: The main refraction in the human eye occurs at the interface between
air (refractive index 1.00) and the cornea (refractive index 1.38). Because of the small
differences in refractive index between the cornea and the lens and between the lens and
the vitreous humor, the lens serves to fine-tune the focus in the human eye.
18. Why do humans see so poorly under water? And why do goggles help?
Ans from the book: Humans see poorly under water because the refractive index of water
(1.33) is very close to that of the cornea (1.38), thus eliminating the main refractive
power of the cornea. Goggles improve underwater vision by placing air in front of the
cornea, which restores the normal difference in refractive indices at this interface.
19. Reading through a beaker filled with clear glass balls is impossible (Figure 9–5). Do you
suppose it would help to fill the beaker with water? With immersion oil? Explain your
reasoning.
Ans from the book: Light cannot pass clearly through glass balls in air
because light bends and reflects at each glass–air boundary.
Filling the beaker with water would help a little because water’s refractive index is closer
to glass, but some bending and distortion would still occur.
Filling it with immersion oil would make it clear because oil and glass have the same
refractive index, so light would not bend or reflect.
Therefore, immersion oil would make the image fully visible.
20. Examine the four photomicrographs of the same cell in Figure 9–6. Match each image to
the technique listed below. 1. Bright-field microscopy 2. Dark-field microscopy 3.
Nomarski differential-interference-contrast microscopy 4. Phase-contrast microscopy.

Ans from the book: Image A was taken by bright-field microscopy, image B by phase-
contrast microscopy, image C by Nomarski differential-interference-contrast microscopy,
and image D by dark-field microscopy.
21. Explain the difference between resolution and magnification.
 Ans from the book: Resolution refers to the ability to see two small objects as separate
entities, which is limited ultimately by the wavelength of light used to view the objects.
Magnification refers to the size of the image relative to the size of the object. It is
possible to magnify an image to an arbitrarily large size. It is important to remember that
magnification does not change the limit of resolution.
22. Many fluorescent dyes that stain DNA require excitation by ultraviolet light. Hoechst
33342, for example, binds to DNA and absorbs light at 352 nm and emits at 461 nm. If
you want to use this membrane-permeant dye in living cells, do you have to worry about
ultraviolet light damage to the DNA, which absorbs light maximally at around 260 nm?
Why or why not?
Ans from the book: Ultraviolet light has the potential to damage DNA, whose bases
absorb maximally at around 260 nm. By confining the illuminating beam to wavelengths
well above 260 nm, or by using filters that block the damaging wavelengths, living cells
can be viewed using ultraviolet light without significant threat to the DNA.
23. Why is it, do you suppose, that a fluorescent molecule, having absorbed a single photon
of light at one wavelength, always emits a photon at a longer wavelength?
Ans from the book: Longer wavelengths correspond to lower energies. Because some
energy is lost during absorption and re-emission, the emitted photon is always of a lower
energy (longer wavelength) than the absorbed photon.
24. 25. Antibodies that bind to specific proteins are important tools for defining the locations
of molecules in cells. The sensitivity of the primary antibody—the antibody that reacts
with the target molecule—is often enhanced by using labeled secondary antibodies that
bind to it. What are the advantages and disadvantages of using secondary antibodies that
carry fluorescent tags versus those that carry bound enzymes?
Ans from the book: Secondary antibodies increase the signal of the primary antibody.
Fluorescent-tagged secondary antibodies show the exact location of proteins clearly, but
their signal is only a few times stronger. Enzyme-linked secondary antibodies give very
strong signals, making them highly sensitive, but the reaction product can spread, so the
exact location of the protein is less clear.
27. Figure 9–8 shows a series of modified GFPs that emit light in a range of colors. How
do you suppose the exact same chromophore can fluoresce at so many different
wavelengths?
Ans from the book: The wavelengths at which the chromophore is excited and at which it
emits fluorescent light depend critically on its molecular environment. Using a variety of
mutagenic and selective procedures, investigators have generated mutant GFPs that
fluoresce throughout the visible range. These modified GFPs have a variety of different
amino acids around the chromophore, which subtly influence its ability to interact with
light.
Statement 42: A major challenge to electron microscopists from the beginning was to convince
others that what they observed in micrographs truly reflected structures that were originally
present in the living cell. Outline a current approach to this problem.
Ans from the book: The best current approach to preserving the original structures in the living
cell is to freeze the sample rapidly before the components have a chance to rearrange themselves
and before water can form crystals. The water can then be removed using organic solvents, and
the sample can be embedded in plastic resin, cut into thin sections, stained to provide contrast,
and viewed.

Statement 43: The technique of negative staining uses heavy metals such as uranium to provide
contrast. If the heavy metals don’t actually bind to defined bio logical structures (which they
don’t), how is it that they can help to make such structures visible?
Ans: Biological molecules are made of light atoms (carbon, oxygen,
nitrogen, hydrogen), which scatter electrons very weakly. Because of this,
they look almost the same as their surroundings in an electron microscope.

Heavy metals have large atomic numbers, so they scatter electrons much more strongly than
biological atoms. In negative staining, the heavy metal surrounds the biological structure and
creates a dark background, allowing the less electron-dense structure to stand out clearly.