H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 78
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Cells in which this recombinant DNA has been introduced will synthesize the chimeric protein whose greenfluorescence reveals the subcellular location of the protein ofinterest. This GFP-tagging technique, for example, has been▲ EXPERIMENTAL FIGURE 5-47 Fura-2, a Ca2+-sensitiveflurochrome, can be used to monitor the relative cytosolicCa2+ concentrations in different regions of live cells. (Left) In amoving leukocyte, a Ca2+ gradient is established. The highestlevels (green) are at the rear of the cell, where corticalcontractions take place, and the lowest levels (blue) are at thecell front, where actin undergoes polymerization.
(Right) When apipette filled with chemotactic molecules placed to the side ofthe cell induces the cell to turn, the Ca2+ concentrationmomentarily increases throughout the cytoplasm and a newgradient is established. The gradient is oriented such that theregion of lowest Ca2+ (blue) lies in the direction that the cell willturn, whereas a region of high Ca2+ (yellow) always forms at thesite that will become the rear of the cell. [From R.
A. Brundage etal., 1991, Science 254:703; courtesy of F. Fay.]5.6 • Visualizing Cell Architecturediffuse from the medium across the plasma membrane intocells. Within the cytosol, esterases hydrolyze fura-2 ester,yielding fura-2, whose free carboxylate groups render themolecule nonlipophilic, and so it cannot cross cellular membranes and remains in the cytosol. Inside cells, each fura-2molecule can bind a single Ca2 ion but no other cellularcation.
This binding, which is proportional to the cytosolicCa2 concentration over a certain range, increases the fluorescence of fura-2 at one particular wavelength. At a secondwavelength, the fluorescence of fura-2 is the same whether ornot Ca2 is bound and provides a measure of the totalamount of fura-2 in a region of the cell. By examining cellscontinuously in the fluorescence microscope and measuringrapid changes in the ratio of fura-2 fluorescence at these twowavelengths, one can quantify rapid changes in the fractionof fura-2 that has a bound Ca2 ion and thus in the concentration of cytosolic Ca2 (Figure 5-47).Similarly to fura-2, fluorescent dyes (e.g., SNARF-1) thatare sensitive to the H concentration can be used to monitor the cytosolic pH of living cells.Confocal Scanning and DeconvolutionMicroscopy Provide Sharp Imagesof Three-Dimensional ObjectsConventional fluorescence microscopy has two major limitations.
First, the physical process of cutting a section destroys material, and so in consecutive (serial) sectioning a(a) Conventional fluorescence microscopy189small part of a cell’s structure is lost. Second, the fluorescentlight emitted by a sample comes from molecules above andbelow the plane of focus; thus the observer sees a blurredimage caused by the superposition of fluorescent imagesfrom molecules at many depths in the cell. 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.
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