Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 80
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The blurring effectmakes it difficult to determine the actual three-dimensionalmolecular arrangement (Figure 5-48a). Two powerful refinements of fluorescence microscopy produce much sharper images by reducing the image-degrading effects of out-of-focuslight.In confocal scanning microscopy, exciting light from a focused laser beam illuminates only a single small part of asample for an instant and then rapidly moves to differentspots in the sample focal plane.
The emitted fluorescent lightpasses through a pinhole that rejects out-of-focus light,thereby producing a sharp image. Because light in focus withthe image is collected by the pinhole, the scanned area is anoptical section through the specimen.
The intensity of lightfrom these in-focus areas is recorded by a photomultipliertube, and the image is stored in a computer (Figure 5-48b).Deconvolution microscopy achieves the same imagesharpening effect as confocal scanning microscopy butthrough a different process. In this method, images from consecutive focal planes of the specimen are collected.
A separate focal series of images from a test slide of subresolutionsize (i.e., 0.2 m diameter) bead are also collected. Each beadrepresents a pinpoint of light that becomes an object blurredby the imperfect optics of the microscope. Deconvolution(b) Confocal fluorescence microscopy40 mFocal planeImagedvolume▲ EXPERIMENTAL FIGURE 5-48 Confocal microscopyproduces an in-focus optical section through thick cells. Amitotic fertilized egg from a sea urchin (Psammechinus) waslysed with a detergent, exposed to an anti-tubulin antibody, andthen exposed to a fluorescein-tagged antibody that binds to thefirst antibody.
(a) When viewed by conventional fluorescencemicroscopy, the mitotic spindle is blurred. This blurring occursFocal planeImagedvolumebecause background fluorescence is detected from tubulin aboveand below the focal plane as depicted in the sketch. (b) Theconfocal microscopic image is sharp, particularly in the center ofthe mitotic spindle. In this case, fluorescence is detected onlyfrom molecules in the focal plane, generating a very thin opticalsection. [Micrographs from J. G. White et al., 1987, J.
Cell Biol. 104:41.]190CHAPTER 5 • Biomembranes and Cell Architecture▲ EXPERIMENTAL FIGURE 5-49 Deconvolutionfluorescence microscopy yields high-resolution opticalsections that can be reconstructed into one threedimensional image. A macrophage cell was stained withfluorochrome-labeled reagents specific for DNA (blue),microtubules (green), and actin microfilaments (red).
The series offluorescent images obtained at consecutive focal planes (opticalsections) through the cell were recombined in three dimensions.(a) In this three-dimensional reconstruction of the raw images,the DNA, microtubules, and actin appear as diffuse zones in thecell. (b) After application of the deconvolution algorithm to theimages, the fibrillar organization of microtubules and thelocalization of actin to adhesions become readily visible in thereconstruction.
[Courtesy of J. Evans.]reverses the degradation of the image by using the blurredbeads as a reference object. The out-of-focus light is mathematically reassigned with the aid of deconvolution algorithms. Images restored by deconvolution display impressivedetail without any blurring (Figure 5-49). Astronomers usedeconvolution algorithms to sharpen images of distant stars.oretically 0.005 nm (less than the diameter of a single atom),or 40,000 times better than the resolution of the lightmicroscope and 2 million times better than that of the unaided human eye.
However, the effective resolution of thetransmission electron microscope in the study of biologicalsystems is considerably less than this ideal. Under optimalconditions, a resolution of 0.10 nm can be obtained withtransmission electron microscopes, about 2000 times betterthan the best resolution of light microscopes. Several examples of cells and subcellular structures imaged by TEM areincluded in Section 5.3.Because TEM requires very thin, fixed sections (about50 nm), only a small part of a cell can be observed in any onesection.
Sectioned specimens are prepared in a manner similar to that for light microscopy, by using a knife capable ofproducing sections 50–100 nm in thickness (see Figure5-43). The generation of the image depends on differentialscattering of the incident electrons by molecules in the preparation. Without staining, the beam of electrons passesthrough a specimen uniformly, and so the entire sample appears uniformly bright with little differentiation of components.
To obtain useful images by TEM, sections arecommonly stained with heavy metals such as gold or osmium. Metal-stained areas appear dark on a micrograph because the metals scatter (diffract) most of the incidentResolution of Transmission Electron MicroscopyIs Vastly Greater Than That of Light MicroscopyThe fundamental principles of electron microscopy are similar to those of light microscopy; the major difference is thatelectromagnetic lenses, rather than optical lenses, focus ahigh-velocity electron beam instead of visible light. In thetransmission electron microscope (TEM), electrons are emitted from a filament and accelerated in an electric field. Acondenser lens focuses the electron beam onto the sample;objective and projector lenses focus the electrons that passthrough the specimen and project them onto a viewingscreen or other detector (Figure 5-50, left).
Because electronsare absorbed by atoms in air, the entire tube between theelectron source and the detector is maintained under anultrahigh vacuum.The short wavelength of electrons means that the limitof resolution for the transmission electron microscope is the-5.6 • Visualizing Cell ArchitectureTEMSEMTungsten filament(cathode)AnodeCondenser lensBeam of electronsScanningcoils191natured and nonfunctional. However, the technique of cryoelectron microscopy allows examination of hydrated, unfixed, and unstained biological specimens directly in atransmission electron microscope. In this technique, anaqueous suspension of a sample is applied in an extremelythin film to a grid.
After it has been frozen in liquid nitrogen and maintained in this state by means of a special mount,the sample is observed in the electron microscope. The verylow temperature (196 C) keeps water from evaporating,even in a vacuum, and the sample can be observed in detailin its native, hydrated state without fixing or heavy metalSpecimen(a)Electromagneticobjective lensAntibody Protein AGoldAntigen(catalase)Projector lensFc domain(b)DetectorPeroxisomesSpecimen▲ EXPERIMENTAL FIGURE 5-50 In electron microscopy,images are formed from electrons that pass through aspecimen or are released from a metal-coated specimen. In atransmission electron microscope (TEM), electrons are extractedfrom a heated filament, accelerated by an electric field, andfocused on the specimen by a magnetic condenser lens.Electrons that pass through the specimen are focused by aseries of magnetic objective and projector lenses to form amagnified image of the specimen on a detector, which may be afluorescent viewing screen, photographic film, or a chargedcouple-device (CCD) camera.
In a scanning electron microscope(SEM), electrons are focused by condensor and objective lenseson a metal-coated specimen. Scanning coils move the beamacross the specimen, and electrons from the metal are collectedby a photomultiplier tube detector. In both types of microscopes,because electrons are easily scattered by air molecules, theentire column is maintained at a very high vacuum.electrons; scattered electrons are not focused by the electromagnetic lenses and do not contribute to the image.
Areasthat take up less stain appear lighter. Osmium tetroxide preferentially stains certain cellular components, such as membranes (see Figure 5-2a). Specific proteins can be detected inthin sections by the use of electron-dense gold particlescoated with protein A, a bacterial protein that binds antibody molecules nonspecifically (Figure 5-51).Standard electron microscopy cannot be used to studylive cells because they are generally too vulnerable to the required conditions and preparatory techniques.
In particular,the absence of water causes macromolecules to become de-0.5 µm▲ EXPERIMENTAL FIGURE 5-51 Gold particles coatedwith protein A are used to detect an antibody-bound proteinby transmission electron microscopy. (a) First antibodies areallowed to interact with their specific antigen (e.g., catalase) in asection of fixed tissue. Then the section is treated with acomplex of protein A from the bacterium S. aureus and electrondense gold particles. Binding of this complex to the Fc domainsof the antibody molecules makes the location of the targetprotein, catalase in this case, visible in the electron microscope.(b) A slice of liver tissue was fixed with glutaraldehyde,sectioned, and then treated as described in part (a) to localizecatalase. The gold particles (black dots) indicating the presence ofcatalase are located exclusively in peroxisomes.
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