Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 78
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This first formulation of the cell theory wasbased on observations made with rather primitive light microscopes. Modern cell biologists have many more-powerfultools for revealing cell architecture. For example, variationsof standard light microscopy permit scientists to view objects that were undetectable several decades ago. Electronmicroscopy, which can reveal extremely small objects, hasyielded much information about subcellular particles andthe organization of plant and animal tissues.
Each techniqueis most suitable for detecting and imaging particular structural features of the cell (Figure 5-41). Digital recording systems and appropriate computer algorithms representanother advance in visualizing cell architecture that hasspread widely in the past decade. Digital systems not onlyAll microscopes produce a magnified image of a small object,but the nature of the images depends on the type of microscope employed and on the way in which the specimen is prepared.
The compound microscope, used in conventionalbright-field light microscopy, contains several lenses thatmagnify the image of a specimen under study (Figure5-42a, b). The total magnification is a product of the magnification of the individual lenses: if the objective lens magnifies 100-fold (a 100 lens, the maximum usually employed)and the projection lens, or eyepiece, magnifies 10-fold, thefinal magnification recorded by the human eye or on filmwill be 1000-fold.However, the most important property of any microscope is not its magnification but its resolving power, orresolution—the ability to distinguish between two veryclosely positioned objects. Merely enlarging the image of aspecimen accomplishes nothing if the image is blurred.
Theresolution of a microscope lens is numerically equivalent toD, the minimum distance between two distinguishable objects. The smaller the value of D, the better the resolution.The value of D is given by the equationD0.61N sin(5-1)where is the angular aperture, or half-angle, of the coneof light entering the objective lens from the specimen; N isthe refractive index of the medium between the specimen andthe objective lens (i.e., the relative velocity of light in themedium compared with the velocity in air); and is thewavelength of the incident light.
Resolution is improved byusing shorter wavelengths of light (decreasing the value of5.6 • Visualizing Cell Architecture(a) Optical microscope(b) Bright-field light path185(c) Epifluorescence light pathDetectorProjectionlensExcitationfilterLampDichroicmirrorObjectiveSpecimenstageCondenserCollectorlensLampMirror▲ EXPERIMENTAL FIGURE 5-42 Optical microscopes arecommonly configured for both bright-field (transmitted) andepifluorescence microscopy.
(a) In a typical light microscope,the specimen is usually mounted on a transparent glass slide andpositioned on the movable specimen stage. The two imagingmethods require separate illumination systems but use the samelight gathering and detection systems. (b) In bright-field lightmicroscopy, light from a tungsten lamp is focused on thespecimen by a condenser lens below the stage; the light travelsthe pathway shown. (c) In epifluorescence microscopy, ultravioletlight from a mercury lamp positioned above the stage is focusedon the specimen by the objective lens.
Filters in the light pathselect a particular wavelength of ultraviolet light for illuminationand are matched to capture the wavelength of the emitted lightby the specimen.) or gathering more light (increasing either N or ). Notethat the magnification is not part of this equation.Owing to limitations on the values of , , and N, thelimit of resolution of a light microscope using visible lightis about 0.2 m (200 nm).
No matter how many times theimage is magnified, the microscope can never resolve objects that are less than ≈0.2 m apart or reveal detailssmaller than ≈0.2 m in size. Despite this limit on resolution, the light microscope can be used to track the locationof a small bead of known size to a precision of only a fewnanometers. If we know the precise size and shape of anobject—say, a 5-nm sphere of gold—and if we use a videocamera to record the microscopic image as a digital image,then a computer can calculate the position of the center ofthe object to within a few nanometers. This technique hasbeen used to measure nanometer-size steps as moleculesand vesicles move along cytoskeletal filaments (see Figures19-17, 19-18, and 20-18).Samples for Microscopy Must Be Fixed,Sectioned, and Stained to ImageSubcellular DetailsLive cells and tissues lack compounds that absorb light andare thus nearly invisible in a light microscope.
Although suchspecimens can be visualized by special techniques to be discussed shortly, these methods do not reveal the fine details ofstructure and require cells to be housed in special glass-facedchambers, called culture chambers, that can be mounted ona microscope stage. For these reasons, cells are often fixed,sectioned, and stained to reveal subcellular structures.Specimens for light and electron microscopy are commonly fixed with a solution containing chemicals that crosslink most proteins and nucleic acids. Formaldehyde, acommon fixative, cross-links amino groups on adjacent molecules; these covalent bonds stabilize protein–protein andprotein–nucleic acid interactions and render the molecules186CHAPTER 5 • Biomembranes and Cell ArchitectureSpecimenholderSpecimenblockBlockSpecimenKnifeKnifeCutsectionSectionsMicroscope slideCoppermeshgrid▲ EXPERIMENTAL FIGURE 5-43 Tissues for microscopyare commonly fixed, embedded in a solid medium, and cutinto thin sections.
A fixed tissue is dehydrated by soaking in aseries of alcohol-water solutions, ending with an organic solventcompatible with the embedding medium. To embed the tissuefor sectioning, the tissue is placed in liquid paraffin for lightmicroscopy or in liquid plastic for electron microscopy; after theblock containing the specimen has hardened, it is mounted onthe arm of a microtome and slices are cut with a knive. Typicalsections cut for electron microscopy 50–100 nm thick; sectionscut for light microscopy are 0.5–50 m thick.
The sections arecollected either on microscope slides (light microscopy) orcopper mesh grids (electron microscopy) and stained with anappropriate agent.insoluble and stable for subsequent procedures. After fixation, a sample is usually embedded in paraffin or plastic andcut into sections 0.5–50 m thick (Figure 5-43).
Alternatively, the sample can be frozen without prior fixation andthen sectioned; such treatment preserves the activity of enzymes for later detection by cytochemical reagents.A final step in preparing a specimen for light microscopyis to stain it so as to visualize the main structural features ofthe cell or tissue.
Many chemical stains bind to moleculesthat have specific features. For example, hematoxylin bindsto basic amino acids (lysine and arginine) on many differentkinds of proteins, whereas eosin binds to acidic molecules(such as DNA and side chains of aspartate and glutamate).Because of their different binding properties, these dyes stainvarious cell types sufficiently differently that they are distinguishable visually. If an enzyme catalyzes a reaction that produces a colored or otherwise visible precipitate from acolorless precursor, the enzyme may be detected in cell sections by their colored reaction products. Such staining techniques, although once quite common, have been largelyreplaced by other techniques for visualizing particular proteins or structures as discussed next.in the refractive index and thickness of cellular materials.These methods, called phase-contrast microscopy and differential interference contrast (DIC) microscopy (orNomarski interference microscopy), produce images thatdiffer in appearance and reveal different features of cell architecture.
Figure 5-44 compares images of live, culturedcells obtained with these two methods and standard brightfield microscopy.In phase-contrast images, the entire object and subcellularstructures are highlighted by interference rings—concentrichalos of dark and light bands. This artifact is inherent in themethod, which generates contrast by interference betweendiffracted and undiffracted light by the specimen. Because theinterference rings around an object obscure many details, thistechnique is suitable for observing only single cells or thin celllayers but not thick tissues.
It is particularly useful for examining the location and movement of larger organelles in livecells.DIC microscopy is based on interference between polarized light and is the method of choice for visualizing extremelysmall details and thick objects. Contrast is generated by differences in the index of refraction of the object and its surrounding medium. In DIC images, objects appear to cast ashadow to one side. The “shadow” primarily represents a difference in the refractive index of a specimen rather than its topography. DIC microscopy easily defines the outlines of largeorganelles, such as the nucleus and vacuole. In addition to having a “relief”-like appearance, a DIC image is a thin opticalsection, or slice, through the object.
Thus details of the nucleusPhase-Contrast and Differential InterferenceContrast Microscopy VisualizeUnstained Living CellsTwo common methods for imaging live cells and unstainedtissues generate contrast by taking advantage of differences5.6 • Visualizing Cell Architecture187▲ EXPERIMENTAL FIGURE 5-44 Live cells can bevisualized by microscopy techniques that generate contrastby interference. These micrographs show live, culturedmacrophage cells viewed by bright-field microscopy (left), phasecontrast microscopy (middle), and differential interferencecontrast (DIC) microscopy (right). In a phase-contrast image, cellsare surrounded by alternating dark and light bands; in-focus andout-of-focus details are simultaneously imaged in a phasecontrast microscope.
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