WOC 6e Guide to Microscopy

More documents

Recommendations

Info

(a) Confocal microscropy (a) Multiphoton microscopy (a) (b) Figure A-19 Digital Deconvolution Microscopy. A fission yeast cell stained with a dye specific for DNA (red) and a membrane-specific dye (green). The image on the left is an unprocessed optical section through the center of the cell. The image on the right is a projection of all the sections following three-dimensional image processing. The ring of the developing medial septum (red) is forming between the two nuclei (red) that arose by nuclear division during the previous mitosis. of the same cell 30 times per second, making it possible to monitor rapid changes in the appearance and behavior of subcellular components. This has allowed scientists to obtain information on the changes in concentration and subcellular distribution of such cytosolic components as second messengers during cellular signaling, and to study the role of cytoskeletal structures in intracellular movements. Thus, digital video microscopy has greatly expanded our ability to monitor events as they occur within living cells. A-14 Appendix Principles and Techniques of Microscopy Figure A-18 Multiphoton Excitation Microscopy. (a) In a standard laser scanning confocal microscope, the laser results in fluorescence in an hourglass-shaped path throughout the specimen. Because a large area fluoresces, photodamage is much more likely to occur than in multiphoton excitation microscopy. (b) In a multiphoton excitation microscope, fluorescence is limited to a spot at the focus of the pulsed infrared laser beam, resulting in much less damage. The infrared illumination also penetrates more deeply into the specimen than visible light. 4 m Digital microscopy is not only useful for examining events in one focal plane. In a variation of this technique, a computer is used to control a focus motor attached to a microscope. Images are then collected throughout the thickness of a specimen. When such a series of images is collected at specific time intervals, such microscopy is called four-dimensional microscopy (this phrase is borrowed from physics; the four dimensions are the three dimensions of space plus the additional dimension of time). Analyzing four-dimensional data
(a) (b) (c) (d) Figure A-20 Computer-Enhanced Digital Video Microscopy. This series of micrographs shows how computers can be used to enhance images obtained with light microscopy. In this example, an image of several microtubules, which are too small to requires special computer software that can navigate between focal planes over time to display specific images. OPTICAL METHODS CAN BE USED TO MEASURE THE MOVEMENTS AND PROPERTIES OF PROTEINS AND OTHER MACROMOLECULES Optical microscopy can be used to help us visualize where molecules reside within cells and to study the dynamic movements and properties of biological molecules. We briefly consider these modern techniques in this section. Photobleaching and photoactivation. When fluorescent molecules are irradiated with light at the appropriate excitation wavelength for long periods of time, they undergo photobleaching (i.e., the irradiation induces the molecules to cease fluorescing). If a cell is only exposed to intense light in a small region, such bleaching results in a characteristic decrease in fluorescence (Figure A-21a). As unbleached molecules move into the bleach zone, the fluorescence gradually returns to normal levels. Such fluorescence recovery after photobleaching (FRAP) is therefore one useful measure of how fast molecules diffuse or undergo directed transport (see Figure 7-11 on p. 167 for a classic example of the use of FRAP). In other cases, fluorescent molecules are chemically modified so that they do not fluoresce until they are irradiated with a specific wavelength of light, usually ultraviolet light. The UV light induces the release of the chemical modifier, allowing the molecule of interest to fluoresce. Both chemically modified dyes and forms of GFP have been produced that behave in this way. Such compounds are often called caged compounds, because they are only “freed” by this light-induced cleavage. Uncaging, or photoactivation, produces the converse situation to photobleaching: local uncaging of a fluorescent molecule produces a bright spot of fluorescence that can be followed as the molecules make their way throughout the cell (Figure A-21b). Uncaging via be seen with unenhanced light microscopy, are processed to make them visible in detail. (a) The image resulting from electronic contrast enhancement of the original image (which appeared to be empty). (b) The background of the enhanced image in (a), 2.5 m which is then (c) subtracted from image (a), leaving only the microtubules. (d) The final, detailed image resulting from electronic averaging of the separate images processed as shown in a–c. photoactivation can also be used to convert other kinds of inert molecules to their active state. For example, “caged calcium” is actually a calcium chelator that is bound to calcium ions. When the caged compound is irradiated, it gives up its calcium, causing a local elevation of calcium within the cell. Total internal reflection fluorescence microscopy. An even more spatially precise method for observing fluorescent molecules involves total internal reflection fluorescence (TIRF) microscopy. TIRF relies on a useful property of light: when light moves from a medium with a high refractive index (such as glass) to a medium with a lower refractive index (such as water or a cell), if the angle of incidence of the light exceeds a certain angle (called the critical angle), the light is reflected. You may be familiar with fiber-optic cables, which rely on the very same physical principle. The curvature of the fiber causes almost all of the light that passes into the cable to be internally reflected, allowing such fibers to serve as “light pipes.” Microscopists can use special lenses to exploit total internal reflection as well. When fluorescent light shines on a cell such that all of the light exceeds the critical angle, it will undergo total internal reflection. If all of the light is reflected, why is TIRF useful? It turns out that a very small layer of light, called the “evanescent field,” extends into the water or cell (Figure A-21c). This layer is very thin, about 100 nm, making TIRF up to ten times better than a confocal microscope for resolving small objects very close to the surface of a coverslip. This makes TIRF extremely useful for studying the release of secretory vesicles, or for observing the polymerization of actin. Fluorescence resonance energy transfer. Fluorescence microscopy is useful not only for observing the movements of proteins, but also for measuring their physical interactions with one another. When two fluorescent molecules whose fluorescence properties are matched are brought very close to one another, it is possible for them to experience fluorescence resonance energy transfer (FRET). In FRET, illuminating a The Light Microscope A-15
Page 1 and 2: Principles and Techniques of Micros
Page 3 and 4: (a) (b) (c) (d) (e) Wave form Wavel
Page 5 and 6: of the lens being used. Since numer
Page 7 and 8: 50 m Figure A-7 Phase-Contrast Micr
Page 9 and 10: Visible light Ultraviolet light Vis
Page 11 and 12: (a) Traditional fluorescence micros
Page 13: (a) To computer Image plane Detecto
Page 17 and 18: Figure A-23 “Optical tweezers.”
Page 19 and 20: The Vacuum System and Electron Gun.
Page 21 and 22: Sample Preparation Techniques for E
Page 23 and 24: gap between dynamic imaging using t
Page 25 and 26: Specimen support Cryoprotected spec
Page 27 and 28: structures on the surfaces of the t
Page 29 and 30: Figure A-39 X-Ray Diffraction. X-ra

WOC 6e Guide to Microscopy

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?