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Chapter_09_Solutions - Chapter 9 Visualizing Cells 9...

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Unformatted text preview: Chapter 9 Visualizing Cells 9 LOOKING AT CELLS IN THE LIGHT MICROSCOPE In This Chapter DEFINITIONS LOOKING AT CELLS IN THE LIGHT MICROSCOPE 9–1 Green fluorescent protein (GFP) 9–2 Limit of resolution 9–3 Bright-field microscope 9–4 Image processing 9–5 Fluorescence microscope 9–6 Confocal microscope 9–7 Fluorescence resonance energy transfer (FRET) 9–8 A213 Microelectrode LOOKING AT CELLS A218 AND MOLECULES IN THE ELECTRON MICROSCOPE TRUE/FALSE 9–9 9–10 9–11 False. Although objects smaller than the limit of resolution can be detected, their images will be blurred because of the unavoidable diffraction of light, which cannot be compensated for by computer-assisted image processing. 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 Nomarski differential-interference-contrast microscopy when they condense during mitosis. Condensed human chromosomes are more than 1 mm in width—well above the resolution limit of 0.2 mm. 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. THOUGHT PROBLEMS 9–12 The components of the light microscope are labeled in Figure 9–16. Magnification of the specimen occurs at two points: in the objective and in the eyepiece. 9–13 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. retina eye eyepiece (ocular) objective specimen condenser light source Figure 9–16 Schematic diagram of a light microscope with components labeled (Answer 9–12). A213 A214 Chapter 9: Visualizing Cells (A) (B) 9–14 The parallel light rays will converge (be focused) by passing through the lens, as shown in Figure 9–17A. The rays will also be focused by the inverted lens (Figure 9–17B). 9–15 In a dry lens a portion of the illuminating light is internally reflected at the interface between the coverslip and the air. By contrast, in an oil-immersion lens there is no interface because glass and immersion oil have the same refractive index; hence, no light is lost to internal reflection. In essence the oil-immersion lens increases the width of the cone of light that reaches the objective, which is a key limitation on resolution. 9–16 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. 9–17 Humans see poorly underwater 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. The image is still distorted by the refractive index changes at the water–glass and glass–air interfaces of the goggles, but the distortion is small enough that the image can still be focused onto the retina, allowing us to see clearly. 9–18 Viewed through a beaker of clear glass beads, the eye chart will be illegible because the light will be refracted and reflected at every glass–air interface. Filling the beaker with water would help somewhat by reducing the refractive index difference (1.33 for water and 1.51 for glass), but there would still be considerable distortion due to refraction and reflection. Filling the beaker with immersion oil would eliminate the refractive index difference since glass and immersion oil have the same refractive index. Thus, the beaker would appear transparent and the eye chart would be fully legible. 9–19 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. 9–20 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. 9–21 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 without significant threat to the DNA. 9–22 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. Figure 9–17 Focusing of parallel rays by a glass lens (Answer 9–14). (A) Lens with curved surface facing incident light. (B) Lens with flat surface toward incident light. Lines normal to the curved surface of the lens are shown by dashed lines. LOOKING AT CELLS IN THE LIGHT MICROSCOPE A215 100 Figure 9–18 Transmission by a commercially available filter set with a dichroic mirror, which is suitable for viewing Hoechst 33342 (Answer 9–23). The first (excitation) filter is optimal for transmission of light in the range that would be absorbed by Hoechst 33342. The second (emission) filter would transmit all the fluorescent light emitted by Hoechst 33342. The beam-splitting mirror would not transmit (would reflect) light that passes through the first filter, but would transmit the light that passes through the second filter. beam-splitter emission transmission (percent) 80 excitation 60 40 20 0 300 350 400 450 500 550 600 650 70 wavelength (nm) 9–23 A. The general idea is to filter the light from the source so that it carries wavelengths that can excite Hoechst 33342 but will not allow passage of longer wavelengths of light that overlap the emission spectrum. Among the listed filters the only choice is filter 1. Similarly, the filter between the sample and the eyepiece should block out wavelengths that are passed by the first filter, but not block wavelengths corresponding to the emission spectrum. This discrimination could be accomplished by two of the listed filters. Filter 2 would capture virtually the entire emission spectrum, whereas filter 3 would capture the main part of the emission spectrum and would likely work just fine, although with lower sensitivity. In practice, filters 1 and 2 are combined in a set for use with Hoechst 33342 (Figure 9–18). B. For the microscope to work properly, you would like to have the beam-splitter reflect the wavelengths from the source and transmit the fluorescent light emitted by the sample. For Hoechst 33342 a mirror that reflected light below 400 nm and transmitted light above 400 nm would be ideal (Figure 9–18). 9–24 9–25 Fluorescently tagged antibodies and enzyme-tagged antibodies each have the advantage of amplifying the initial signal provided by the binding of the primary antibody. For fluorescently tagged secondary antibodies, the amplification is usually severalfold; for enzyme-linked antibodies, amplification can be more than 1000-fold. Although the extensive amplification makes enzyme-linked methods very sensitive, diffusion of the reaction product (often a colored precipitate) away from the enzyme limits the spatial resolution. In the absence of oxygen GFP does not become fluorescent, an observation that suggests oxygen is required for GFP fluorescence. The absence of fluorescence in inclusion bodies, which contain denatured protein, indicates that native protein structure is required for fluorescence. The first-order kinetics of the development of fluorescence and its independence of GFP concentration imply that fluorescence depends only on GFP and oxygen: no other proteins or small molecules are needed. The ability of minor changes in protein sequence to influence brightness and color indicates that the chromophore is very sensitive to the molecular environment provided by the protein. The chromophore of GFP is formed from a stretch of three adjacent amino acids—serine, tyrosine, and glycine. In the presence of oxygen the backbone cyclizes to form a five-membered ring whose double bonds are conjugated with those of the phenol ring of tyrosine (Figure 9–19). The ability of this chromophore to interact with light is sensitive to the surrounding molecular environment of the protein. Figure 9–19 Formation of the chromophore of GFP (Answer 9–25). The bonds in the backbone of the protein are shown as thick lines. O Tyrosine 66 HO H N Glycine 67 N H O N H O Serine 65 OH CYCLIZATION Tyrosine 66 O Glycine 67 HO H N N O N H OH Serine 65 OXIDATION Tyrosine 66 O Glycine 67 HO H N N O N H OH Serine 65 A216 Chapter 9: Visualizing Cells References: Chalfie M, Tu Y, Euskirchen G, Ward WW & Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805. Heim R, Prasher DC & Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl Acad. Sci. U.S.A. 91, 12501–12504. 9–26 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. Reference: Service RF (2004) Immune cells speed the evolution of novel proteins. Science 306, 1457. 9–27 The half-life of 32P is 14.3 days; thus, you had already exposed the film for one half-life in the first two weeks. If you could somehow wait long enough for all the remaining 32P to decay, it would sum to the amount you detected in the first two weeks. You could expose the new film from now to the end of time and not have any brighter band than you had at the end of two weeks. CALCULATIONS 9–28 Substituting values into the equation, the resolution for violet light in air is 0.28 mm and in oil is 0.19 mm. The resolution for red light in air is 0.49 mm and in oil is 0.33 mm. Clearly the best resolution is obtained with violet light (0.4 mm) using oil immersion (n = 1.51), as calculated below. resolution = 0.61 l n sin q = (0.61)(0.4 mm) (1.51)(sin 60°) = 0.19 mm 9–29 When parallel to the interface, the angle of the transmitted light (qt) is 90°. Substituting this angle and the refractive indices into the equation gives an incident angle of 41.5°. ni sin qi = nt sin qt 1.51 sin qi = 1.00 sin 90° sin qi = 1.00/1.51 = 0.66 qi = arcsin 0.66 = 41.5° 9–30 Fluorescence occurs when a molecule absorbs a photon within a narrow energy range (a narrow range of wavelengths), so that an electron is boosted to an allowable higher energy level. When that electron decays back to a lower energy state, it emits a photon that is less energetic (that is, at a longer wavelength). An electron can be boosted to the higher energy state by two lower-energy photons, so long as the second one is absorbed before the partially activated electron decays [within a femtosecond (10–15 sec) or so]. The rule for multiphoton activation is that the total energy of absorbed photons must add up to the amount needed to boost the electron into its allowable higher electronic state. Photons have half the energy at twice the wavelength. Thus, if the sample is illuminated with light of a narrow wavelength, so that all the photons have about the same energy, then the molecule will be activated maximally by two photons when the energy of the photons is about half that required for maximum activation by a single photon. LOOKING AT CELLS IN THE LIGHT MICROSCOPE A217 Reference: Bestvater F, Spiess E, Stobrawa G, Hacker M, Feurer T, Porwol T, Berchner-Phannschmidt U, Wotzlaw C & Acker H (2002) Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microscopy 208, 108–115. 9–31 In order to follow the fluorescent proteins independently, you would ideally like to excite them separately and detect their emissions separately. Thus, the best pairs are those with as little overlap in their excitation and emission spectra as possible. Clearly, of the three possible pairs, CFP and YFP have the smallest overlap in their excitation and emission spectra. 9–32 The increase in FRET depends on phosphorylation of the protein, since no increase occurs in the absence of Abl protein or ATP, or when the phosphate is removed by a tyrosine phosphatase (see Figure 9–11B). Thus, phosphorylation must cause CFP and YFP to be brought closer together. A reasonable explanation is that addition of phosphate to the tyrosine in the substrate peptide allows that segment of the protein to fold back to bind to the adjacent phosphotyrosine-binding domain, thereby decreasing the separation of the CFP and YFP domains (Figure 9–20). 9–33 The data in Figure 9–12 indicate the location of the reporter protein in the cell, and by inference the location of active Abl. The reporter is not phosphorylated (activated) in the nucleus. It is activated in the cytoplasm, but is most highly activated in membrane ruffles. These results are most simply consistent with the idea that active Abl is most prevalent in the membrane ruffles. (You can watch this process in a color movie. Go to the PNAS website, www.pnas.org, type in the volume and first page number of this article, and select ‘supporting movies.’ Movie 2 is spectacular.) Reference: Ting AY, Kain KH, Klemke RL & Tsien RY (2001) Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl Acad. Sci. U.S.A. 98, 15003–15008. There are two keys to how the indicator works. First, emission at 535 nm after excitation at 440 nm means that the indicator depends on fluorescence energy transfer (FRET) between CFP and YFP. As shown in Figure 9–10, CFP is efficiently excited at 440 nm and its emission spectrum overlaps with the excitation spectrum of YFP, which in turn emits light near maximally at 535 nm. Second, the efficiency of FRET depends on the distance between the chromophores in the two fluorescent proteins. In the absence of Ca2+, calmodulin is in its extended form so that the CFP and YFP domains are maximally separated; hence, FRET should be inefficient and emission at 535 nm will be low. In the presence of Ca2+, calmodulin will be folded much more compactly and CFP and YFP will be brought closer together; thus, FRET will occur much more efficiently and emission at 535 nm will increase. References: Miyawaki A, Llopis J, Heim R, McCafferty JM, Adams JA, Ikura M & Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin. Nature 388, 882–887. (A) UNPHOSPHORYLATED (B) PHOSPHORYLATED 434 nm 476 nm 434 nm P ET CF FR 9–34 kinase + ATP CF P substrate peptide phosphatase YF P 526 nm YFP phosphotyrosinebinding protein P Figure 9–20 Conformational change in FRET reporter protein upon tyrosine phosphorylation (Answer 9–32). A218 Chapter 9: Visualizing Cells Nagai T, Yamada S, Tominaga T, Ichikkawa M & Miyawaki A (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl Acad. Sci U.S.A. 101, 10554–10559. 9–35 This problem involves a two-part calculation. If you know the specific activity of the labeled ATP (mCi/mmol), then you can use that value to convert 1 cpm per band (1 dpm/band for 32P) into the number of proteins per band. The specific activity of ATP is 1.1 ¥ 106 mCi/mmol. Since ATP is 0.9 mM in the extract after addition of the label, 6 L specific activity = 10 mCi ¥ ¥ 10 mL 10 mL 0.9 mmol ATP L = 1.11 ¥ 106 mCi/mmol ATP The specific activity of ATP is equal to the specific activity of phosphate on the labeled proteins. Using this specific activity (and various conversion factors shown on the inside of the front cover), you can calculate that 1 dpm/band corresponds to 2.4 ¥ 108 protein molecules per band. 20 proteins = 1 dpm ¥ mmol mCi ¥ 6 ¥ 10 molecules ¥ ¥ Bq band band 3.7 ¥ 104 Bq 60 dpm 1.11 ¥ 106 mCi mmol = 2.4 ¥ 108 molecules/band LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE DEFINITIONS 9–36 Negative staining 9–37 Electron microscope (EM) 9–38 Cryoelectron microscopy 9–39 Scanning electron microscope (SEM) 9–40 Immunogold electron microscopy TRUE/FALSE 9–41 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. THOUGHT PROBLEMS 9–42 The best current approach to preserving the original structures in the living cell is to freeze the sample rapidly before the components have a change 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. 9–43 Biological structures are composed of atoms with similar, low atomic numbers; thus, most structures are marginally different from their surroundings in terms of their ability to scatter electrons. Because electron scattering is proportional to the square of the number of electrons in the atom (which equals the atomic number), heavy metals scatter electrons much more efficiently than biological atoms, enormously enhancing the contrast in the electron LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE A219 Figure 9–21 Bumps and pits (Answer 9–44). Bumps and pits can be distinguished because bumps cast shadows. pits bumps micrographs. Both techniques enhance the ability to see biological structures by providing contrast based on how the structure stands relative to the surface. For negative staining, a solution of uranyl acetate is typically used to coat the low-lying areas adjacent to biological molecules or complexes, providing a dark (electron dense) background against which the less electron dense biological structure stands out. In metal shadowing, a heavy metal such as platinum is sprayed at a low angle across a biological preparation. Biological structures that stand above the surface pile up platinum on one side and cast a platinum-free shadow on the opposite side. In both cases—metal shadowing and negative staining—characteristic features of the biological structure are rendered visible by the adjacent heavy metal atoms. 9–44 Electron microscopists can be sure whether a structure is a pit or a bump. Shadowed structures are unlike shaded circles in a key way: structures that stand above the surface cast a shadow beyond themselves, whereas pits do not. In everyday experience shadows cast by the sun are dark, but in the world of microscopy, where platinum atoms replace sunlight, the shadow is the absence of metal, hence bright, as shown in Figure 9–21. If you examine the micrographs in Figure 9–13, especially in the orientation that looks like pits (look at the lower left hand corner in D), you can see that most of the pits are elongated toward the lower left of the micrograph. Thus, these structures are casting shadows; hence, they are bumps. To have the structures perceived as bumps, microscopists arrange the micrographs as shown in Figure 9–13C, so that the dark portion of each bump is at the bottom. Evidently, this arrangement fits with our hardwired perceptions of the world around us, with the sun coming from above. Note also that we naturally interpret the light areas of such images as structures that reflect light; in reality they are simply the absence of electron dense material and give us no more information about the structure than does a shadow cast by sunlight. 9–45 Averaging structures that are not in the same state will tend to emphasize those parts of the structure that don’t vary. Parts that are moving (that is, in different locations in different images) would be de-emphasized or even eliminated. By way of analogy, imagine combining a series of low-quality snapshots of a cyclist in motion, in order to get a better picture. The frame of the bike and the cyclist’s torso would be clear, but the spokes of the wheels would be invisible and the cyclist’s legs would be a blur. One way to improve the image of the nuclear pore complex is to try to preclassify individual images into similar types, and then combine only those in the same class. This was in fact done for the nuclear pore complexes according to the distribution of mass in the central cavity. Those images with the central mass displaced toward the cytoplasm gave a somewhat different combined image from those that had the central mass more on the nuclear side. The advantages and potential pitfalls of this process can be appreciated by again considering the analogy of the cyclist. In the low-quality images you might notice that the ‘legs’ (of course, you wouldn’t know they were legs) were together in some images and separated in others. Grouping images according to that criterion and combining images within each class would generate two final pictures that each showed a blurred image of ‘legs’ in two positions. In the case of the bicycle, we know that the final image would be crude, at best, because legs occupy a continuum of positions and our classification would combine left and right legs. A220 Chapter 9: Visualizing Cells Reference: Beck M, Forster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W & Medalia O (2004) Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306, 1387–1390. CALCULATIONS 9–46 Substituting numbers into the equation gives a value for q of 1.4°, which is about 43 times (60°/1.4°) smaller than q for a typical light microscope. resolution = 0.61 l n sin q sin q = 0.61 (0.004 nm) (0.1 nm) q = arcsin 0.0244 = 1.4° DATA HANDLING 9–47 The micrograph in Figure 9–14 shows clearly that both proteins localize to the gap junction. Black dots (gold particles) of two different sizes are apparent in the gap junction. And all the black dots except one (near the top righthand corner) are present in the gap junction. Close examination of the single outlier shows that it is also associated with a small patch of membrane that looks exactly like the gap junction. In reality the two proteins labeled in the micrograph are connexins, which are components of the membrane channels that make up gap junctions. Reference: Fujimoto K (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. J. Cell Sci. 108, 3443–3449. 9–48 A. The gold particles in Figure 9–15 are consistently associated with an obvious structure in the membrane, termed square arrays. It is thought that these square arrays represent aggregates of aquaporin water channels. B. There are a few black dots that are not obviously associated with square arrays. These may arise from antibodies that were unbound but not washed off. They may also represent antibodies that are bound to square arrays that are poorly defined in the micrograph or that are small enough to be obscured by the black dot. So long as the proportion of nonassociated dots is reasonably small, it does not affect the principal conclusion. The second issue—some square arrays that were not labeled—may indicate that the antibody is not saturating, or that the denatured aquaporins (denaturation is a consequence of the preparation technique) do not all present appropriate sites for the antibody to recognize. The absence of labeling of all such structures also does not detract from the primary conclusion. Reference: Rash JE, Yasumura T, Hudson CS, Agre P & Nielsen S (1998) Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Proc. Natl Acad. Sci. U.S.A. 95, 11981–11986. ...
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This homework help was uploaded on 04/07/2008 for the course BME 50A taught by Professor Botvinick during the Spring '08 term at UC Irvine.

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