WOC 6e Guide to Microscopy

More documents

Recommendations

Info

RADIOISOTOPES AND ANTIBODIES CAN LOCALIZE MOLECULES IN ELECTRON MICROGRAPHS In our discussion of light microscopy, we described how microscopic autoradiography can be used to locate radioactive molecules inside cells. Autoradiography can also be applied to transmission electron microscopy, with only minor differences. For the TEM, the specimen containing the radioactively labeled compounds is simply examined in ultrathin sections on copper specimen grids instead of in thin sections on glass slides. We also described how fluorescently labeled antibodies can be used in conjunction with light microscopy to locate specific cellular components. Antibodies are likewise used in the electron microscopic technique called immunoelectron microscopy (immunoEM); fluorescence cannot be seen in the electron microscope, so antibodies are instead visualized by linking them to substances that are electron dense and therefore visible as opaque dots. One of the most common approaches is to couple antibody molecules to colloidal gold particles. When ultrathin tissue sections are stained with gold-labeled antibodies directed against various proteins, electron microscopy can reveal the subcellular location of these proteins with great precision (Figure A-29). CORRELATIVE MICROSCOPY CAN BE USED TO BRIDGE THE GAP BETWEEN LIGHT AND ELECTRON MICROSCOPY Immunostaining of fixed specimens and the imaging of living cells expressing proteins tagged with GFP are powerful tools in the cell biologist’s arsenal, because they provide important information about the location of cellular components. Figure A-30 Correlative microscopy. (Left) A thin section through the pharynx (a muscular feeding structure) of an adult C. elegans (a nematode worm) visualized using confocal microscopy. The green signal is a protein tagged with the Green Fluorescent Protein A-22 Appendix Principles and Techniques of Microscopy (GFP) expressed at junctions between epithelial cells in the pharynx. The red is a colorized image of the light reflected from the section. (Right) The same section after processing for immunoEM, using an antibody that recognizes GFP. The gold particles localize to the same 0.25 m Figure A-29 The Use of Gold-Labeled Antibodies in Electron Microscopy. Cells of the bacterium E. coli were stained with goldlabeled antibodies directed against a plasma membrane protein. The small dark granules distributed around the periphery of the cell are the gold-labeled antibody molecules. Because the wavelength of visible light is long, however, optical microscopy has inherent limits. Conversely, transmission electron microscopy provides incredibly detailed views of cells, but the cells must be chemically fixed or rapidly frozen prior to processing them for TEM. A powerful approach that unites these ways of examining cellular structures is correlative microscopy. In correlative microscopy, dynamic images of a cell are acquired using the light microscope, often using antibodies and/or GFP. The very same cell is then processed and viewed using EM. Commonly, immunoEM is used to determine where a protein is found at very high resolution (Figure A-30). Correlative microscopy thus bridges the 0.5 m regions as the fluorescent signal. A higher magnification view (inset) shows where the gold particles localize relative to junctions between cells (TEM).
gap between dynamic imaging using the light microscope and the detailed images that can only be acquired via EM. NEGATIVE STAINING CAN HIGHLIGHT SMALL OBJECTS IN RELIEF AGAINST A STAINED BACKGROUND Although cutting tissues into ultrathin sections is the most common way of preparing specimens for transmission electron microscopy, other techniques are suitable for particular purposes. For example, the shape and surface appearance of very small objects, such as viruses or isolated organelles, can be examined without cutting the specimen into sections. In the negative staining technique, which is one of the simplest techniques in transmission electron microscopy, intact specimens are simply visualized in relief against a darkly stained background. To carry out negative staining, the copper specimen grid must first be overlaid with an ultrathin plastic film. The specimen is then suspended in a small drop of liquid, applied to the overlay, and allowed to dry in air. After the specimen has dried on the grid, a drop of stain such as uranyl acetate or phosphotungstic acid is applied to the film surface. The edges of the grid are then blotted in several places with a piece of filter paper to absorb the excess stain. This draws the stain down and around the specimen and its ultrastructural features. When viewed in the TEM, the specimen is seen in negative contrast because the background is dark and heavily stained, whereas the specimen itself is lightly stained (Figure A-31). SHADOWING TECHNIQUES USE METAL VAPOR SPRAYED ACROSS A SPECIMEN’S SURFACE Isolated particles or macromolecules can also be visualized by the technique of shadowing (Figure A-32), which involves 25 nm Figure A-31 Negative Staining. An electron micrograph of a bacteriophage as seen in a negatively stained preparation. This specimen was simply suspended in an electron-dense stain, allowing it to be visualized in relief against a darkly stained background (TEM). 0.1 m Figure A-32 Shadowing. An electron micrograph of tobacco mosaic virus particles visualized by shadowing. In this technique, heavy metal vapor was sprayed at an angle across the specimen, causing an accumulation of metal on one side of each virus particle and a shadow region lacking metal on the other side (TEM). spraying a thin layer of an electron-dense metal such as gold or platinum at an angle across the surface of a biological specimen. Figure A-33a illustrates the shadowing technique. The specimen is first spread on a clean mica surface and dried (step 1 ). It is then placed in a vacuum evaporator, a bell jar in which a vacuum is created by a system similar to that of an electron microscope (Figure A-33b). Also within the evaporator are two electrodes, one consisting of a carbon rod located directly over the specimen and the other consisting of a metal wire positioned at an angle of about 10°–45° relative to the specimen. After a vacuum is created in the evaporator, current is applied to the metal electrode, causing the metal to evaporate from the electrode and spray over the surface of the specimen (Figure A-33a, 2 ). Because the metal-emitting electrode is positioned at an angle to the specimen, metal is deposited on only one side of the specimen, generating a metal replica of the surface. The opposite side of the specimen remains unstained; it is this unstained region that creates the “shadow” effect. An overhead carbon-emitting electrode is then used to coat the specimen with evaporated carbon, thereby provid- ing stability and support to the metal replicas ( 3 ). Next, the mica support containing the specimen is removed from the vacuum evaporator and lowered gently onto a water surface, causing the replica to float away from the mica surface. The replica is transferred into an acid bath, which dissolves away remaining bits of specimen, leaving a clean metal replica of the specimen ( 4 ). The replica is then transferred to a Sample Preparation Techniques for Electron Microscopy A-23
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 and 14: (a) To computer Image plane Detecto
Page 15 and 16: (a) (b) (c) (d) Figure A-20 Compute
Page 17 and 18: Figure A-23 “Optical tweezers.”
Page 19 and 20: The Vacuum System and Electron Gun.
Page 21: Sample Preparation Techniques for E
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

Create successful ePaper yourself

Delete template?

Save as template?