H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 77
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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. In a DIC image, cells appear inpseudorelief. Because only a narrow in-focus region is imaged, aDIC image is an optical slice through the object. [Courtesy ofin thick specimens (e.g., an intact Caenorhabditis elegansroundworm) can be observed in a series of such optical sections, and the three-dimensional structure of the object can bereconstructed by combining the individual DIC images.light up when illuminated by the exciting wavelength, a technique called immunfluorescence microscopy (Figure 5-45).Staining a specimen with two or three dyes that fluoresce atdifferent wavelengths allows multiple proteins to be localizedwithin a cell (see Figure 5-33).N.
Watson and J. Evans.]Fluorescence Microscopy Can Localizeand Quantify Specific Moleculesin Fixed and Live CellsLaminapropiaLateralmembraneBrushborderPerhaps the most versatile and powerful technique for localizing proteins within a cell by light microscopy is fluorescentstaining of cells and observation by fluorescence microscopy.A chemical is said to be fluorescent if it absorbs light at onewavelength (the excitation wavelength) and emits light (fluoresces) at a specific and longer wavelength. Most fluorescentdyes, or flurochromes, emit visible light, but some (such asCy5 and Cy7) emit infrared light.
In modern fluorescence microscopes, only fluorescent light emitted by the sample isused to form an image; light of the exciting wavelength induces the fluorescence but is then not allowed to pass the filters placed between the objective lens and the eye or camera(see Figure 5-42a, c).20 mImmunological Detection of Specific Proteins in Fixed CellsThe common chemical dyes just mentioned stain nucleicacids or broad classes of proteins. However, investigatorsoften want to detect the presence and location of specificproteins. A widely used method for this purpose employsspecific antibodies covalently linked to flurochromes. Commonly used flurochromes include rhodamine and Texas red,which emit red light; Cy3, which emits orange light; and fluorescein, which emits green light.
These flurochromes can bechemically coupled to purified antibodies specific for almostany desired macromolecule. When a flurochrome–antibodycomplex is added to a permeabilized cell or tissue section, thecomplex will bind to the corresponding antigens, which then▲ EXPERIMENTAL FIGURE 5-45 One or more specificproteins can be localized in fixed tissue sections byimmunofluorescence microscopy.
A section of the rat intestinalwall was stained with Evans blue, which generates a nonspecificred fluorescence, and with a yellow green–fluorescing antibodyspecific for GLUT2, a glucose transport protein. As evident fromthis fluorescence micrograph, GLUT2 is present in the basal andlateral sides of the intestinal cells but is absent from the brushborder, composed of closely packed microvilli on the apicalsurface facing the intestinal lumen.
Capillaries run through thelamina propria, a loose connective tissue beneath the epitheliallayer. [See B. Thorens et al., 1990, Am. J. Physio. 259:C279; courtesy ofB. Thorens.]188CHAPTER 5 • Biomembranes and Cell Architectureused to visualize the expression and distribution of specificproteins that mediate cell–cell adhesion (see Figure 6-8).In some cases, a purified protein chemically linked to afluorescent dye can be microinjected into cells and followedby fluorescence microscopy. For example, findings fromcareful biochemical studies have established that purifiedactin “tagged” with a flurochrome is indistinguishable infunction from its normal counterpart. When the tagged protein is microinjected into a cultured cell, the endogenous cellular and injected tagged actin monomers copolymerize intonormal long actin fibers.
This technique can also be used tostudy individual microtubules within a cell.Determination of Intracellular Ca2 and H Levels withIon-Sensitive Fluoresent Dyes Flurochromes whose fluo-▲ EXPERIMENTAL FIGURE 5-46 Expression of fluorescentproteins in early and late mouse embryos is detected byemitted blue and yellow light. The genes encoding bluefluorescent protein (ECFP) and yellow fluorescent protein (EYFP)were introduced into mouse embryonic stem cells, which thenwere grown into early-stage embryos (top) and late-stageembryos (bottom).
These bright-field (left) and fluorescence (right)micrographs reveal that all but four of the early-stage embryosdisplay a blue or yellow fluorescence, indicating expression ofthe introduced ECFP and EYFP genes. Of the two late-stageembryos shown, one expressed the ECFP gene (left) and oneexpressed the EYFP gene (right). [From A.-K.
Hadjantonakis et al.,rescence depends on the concentration of Ca2 or H haveproved useful in measuring the concentration of these ionswithin live cells. As discussed in later chapters, intracellularCa2 and H concentrations have pronounced effects onmany cellular processes. For instance, many hormones orother stimuli cause a rise in cytosolic Ca2 from the restinglevel of about 107 M to 106 M, which induces various cellular responses including the contraction of muscle.The fluorescent dye fura-2, which is sensitive to Ca2,contains five carboxylate groups that form ester linkageswith ethanol.
The resulting fura-2 ester is lipophilic and can2002, BMC Biotechnol. 2:11.]Expression of Fluorescent Proteins in Live Cells A naturally fluorescent protein found in the jellyfish Aequorea victoria can be exploited to visualize live cells and specificproteins within them. This 238-residue protein, called greenfluorescent protein (GFP), contains a serine, tyrosine, andglycine sequence whose side chains have spontaneouslycyclized to form a green-fluorescing chromophore. With theuse of recombinant DNA techniques discussed in Chapter 9,the GFP gene can be introduced into living cultured cells orinto specific cells of an entire animal. Cells containing the introduced gene will express GFP and thus emit a green fluorescence when irradiated; this GFP fluorescence can be usedto localize the cells within a tissue.
Figure 5-46 illustrates theresults of this approach, in which a variant of GFP that emitsblue fluorescence was used.In a particularly useful application of GFP, a cellular protein of interest is “tagged” with GFP to localize it. In this technique, the gene for GFP is fused to the gene for a particularcellular protein, producing a recombinant DNA encoding onelong chimeric protein that contains the entirety of both proteins.
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