Fluorescence Microscopy Can Localize and Quantify Specific Molecules in Fixed and Live Cells

Perhaps the most versatile and powerful technique for localizing proteins within a cell by light microscopy is fluorescent staining of cells and observation by fluorescence microscopy. A chemical is said to be fluorescent if it absorbs light at one wavelength (the excitation wavelength) and emits light (fluoresces) at a specific and longer wavelength. Most fluorescent dyes, or flurochromes, emit visible light, but some (such as Cy5 and Cy7) emit infrared light. In modern fluorescence microscopes, only fluorescent light emitted by the sample is used 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).

Immunological Detection of Specific Proteins in Fixed Cells

The common chemical dyes just mentioned stain nucleic acids or broad classes of proteins. However, investigators often want to detect the presence and location of specific proteins. A widely used method for this purpose employs specific antibodies covalently linked to flurochromes. Commonly used flurochromes include rhodamine and Texas red, which emit red light; Cy3, which emits orange light; and fluo-rescein, which emits green light. These flurochromes can be chemically coupled to purified antibodies specific for almost any desired macromolecule. When a flurochrome-antibody complex is added to a permeabilized cell or tissue section, the complex will bind to the corresponding antigens, which then are surrounded by alternating dark and light bands; in-focus and out-of-focus details are simultaneously imaged in a phase-contrast microscope. In a DIC image, cells appear in pseudorelief. Because only a narrow in-focus region is imaged, a DIC image is an optical slice through the object. [Courtesy of N. Watson and J. Evans.]

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 at different wavelengths allows multiple proteins to be localized within a cell (see Figure 5-33).

▲ EXPERIMENTAL FIGURE 5-45 One or more specific proteins can be localized in fixed tissue sections by immunofluorescence microscopy. A section of the rat intestinal wall was stained with Evans blue, which generates a nonspecific red fluorescence, and with a yellow green-fluorescing antibody specific for GLUT2, a glucose transport protein. As evident from this fluorescence micrograph, GLUT2 is present in the basal and lateral sides of the intestinal cells but is absent from the brush border, composed of closely packed microvilli on the apical surface facing the intestinal lumen. Capillaries run through the lamina propria, a loose connective tissue beneath the epithelial layer. [See B. Thorens et al., 1990, Am. J. Physio. 259:C279; courtesy of B. Thorens.]

▲ EXPERIMENTAL FIGURE 5-46 Expression of fluorescent proteins in early and late mouse embryos is detected by emitted blue and yellow light. The genes encoding blue fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) were introduced into mouse embryonic stem cells, which then were grown into early-stage embryos (top) and late-stage embryos (bottom). These bright-field (left) and fluorescence (right) micrographs reveal that all but four of the early-stage embryos display a blue or yellow fluorescence, indicating expression of the introduced ECFP and EYFP genes. Of the two late-stage embryos shown, one expressed the ECFP gene (left) and one expressed the EYFP gene (right). [From A.-K. Hadjantonakis et al., 2002, 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 specific proteins within them. This 238-residue protein, called green fluorescent protein (GFP), contains a serine, tyrosine, and glycine sequence whose side chains have spontaneously cyclized to form a green-fluorescing chromophore. With the use of recombinant DNA techniques discussed in Chapter 9, the GFP gene can be introduced into living cultured cells or into 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 used to localize the cells within a tissue. Figure 5-46 illustrates the results of this approach, in which a variant of GFP that emits blue 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 particular cellular protein, producing a recombinant DNA encoding one long chimeric protein that contains the entirety of both proteins. Cells in which this recombinant DNA has been introduced will synthesize the chimeric protein whose green fluorescence reveals the subcellular location of the protein of interest. This GFP-tagging technique, for example, has been used to visualize the expression and distribution of specific proteins that mediate cell-cell adhesion (see Figure 6-8).

In some cases, a purified protein chemically linked to a fluorescent dye can be microinjected into cells and followed by fluorescence microscopy. For example, findings from careful biochemical studies have established that purified actin "tagged" with a flurochrome is indistinguishable in function from its normal counterpart. When the tagged protein is microinjected into a cultured cell, the endogenous cellular and injected tagged actin monomers copolymerize into normal long actin fibers. This technique can also be used to study individual microtubules within a cell.

Determination of Intracellular Ca2+ and H+ Levels with Ion-Sensitive Fluoresent Dyes Flurochromes whose fluorescence depends on the concentration of Ca2+ or H+ have proved useful in measuring the concentration of these ions within live cells. As discussed in later chapters, intracellular Ca2+ and H + concentrations have pronounced effects on many cellular processes. For instance, many hormones or other stimuli cause a rise in cytosolic Ca2+ from the resting level of about 10~7 M to 10~6 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 linkages with ethanol. The resulting fura-2 ester is lipophilic and can j w

▲ EXPERIMENTAL FIGURE 5-47 Fura-2, a Ca2+-sensitive flurochrome, can be used to monitor the relative cytosolic Ca2+ concentrations in different regions of live cells. (Left) In a moving leukocyte, a Ca2+ gradient Is established. The highest levels (green) are at the rear of the cell, where cortical contractions take place, and the lowest levels (blue) are at the cell front, where actin undergoes polymerization. (Right) When a pipette filled with chemotactic molecules placed to the side of the cell induces the cell to turn, the Ca2+ concentration momentarily increases throughout the cytoplasm and a new gradient is established. The gradient is oriented such that the region of lowest Ca2+ (blue) lies in the direction that the cell will turn, whereas a region of high Ca2+ (yellow) always forms at the site that will become the rear of the cell. [From R. A. Brundage et al., 1991, Science 254:703; courtesy of F Fay.]

t diffuse from the medium across the plasma membrane into cells. Within the cytosol, esterases hydrolyze fura-2 ester, yielding fura-2, whose free carboxylate groups render the molecule nonlipophilic, and so it cannot cross cellular membranes and remains in the cytosol. Inside cells, each fura-2 molecule can bind a single Ca2+ ion but no other cellular cation. This binding, which is proportional to the cytosolic Ca2+ concentration over a certain range, increases the fluorescence of fura-2 at one particular wavelength. At a second wavelength, the fluorescence of fura-2 is the same whether or not Ca2+ is bound and provides a measure of the total amount of fura-2 in a region of the cell. By examining cells continuously in the fluorescence microscope and measuring rapid changes in the ratio of fura-2 fluorescence at these two wavelengths, one can quantify rapid changes in the fraction of 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) that are sensitive to the H+ concentration can be used to monitor the cytosolic pH of living cells.

Confocal Scanning and Deconvolution Microscopy Provide Sharp Images of Three-Dimensional Objects

Conventional fluorescence microscopy has two major limitations. First, the physical process of cutting a section destroys material, and so in consecutive (serial) sectioning a

▲ EXPERIMENTAL FIGURE 5-48 Confocal microscopy produces an in-focus optical section through thick cells. A

mitotic fertilized egg from a sea urchin (Psammechinus) was lysed with a detergent, exposed to an anti-tubulin antibody, and then exposed to a fluorescein-tagged antibody that binds to the first antibody. (a) When viewed by conventional fluorescence microscopy, the mitotic spindle is blurred. This blurring occurs small part of a cell's structure is lost. Second, the fluorescent light emitted by a sample comes from molecules above and below the plane of focus; thus the observer sees a blurred image caused by the superposition of fluorescent images from molecules at many depths in the cell. The blurring effect makes it difficult to determine the actual three-dimensional molecular arrangement (Figure 5-48a). Two powerful refinements of fluorescence microscopy produce much sharper images by reducing the image-degrading effects of out-of-focus light.

In confocal scanning microscopy, exciting light from a focused laser beam illuminates only a single small part of a sample for an instant and then rapidly moves to different spots in the sample focal plane. The emitted fluorescent light passes through a pinhole that rejects out-of-focus light, thereby producing a sharp image. Because light in focus with the image is collected by the pinhole, the scanned area is an optical section through the specimen. The intensity of light from these in-focus areas is recorded by a photomultiplier tube, and the image is stored in a computer (Figure 5-48b).

Deconvolution microscopy achieves the same image-sharpening effect as confocal scanning microscopy but through 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 subresolution size (i.e., 0.2 ^m diameter) bead are also collected. Each bead represents a pinpoint of light that becomes an object blurred by the imperfect optics of the microscope. Deconvolution because background fluorescence Is detected from tubulin above and below the focal plane as depicted In the sketch. (b) The confocal microscopic Image Is sharp, particularly In the center of the mitotic spindle. In this case, fluorescence Is detected only from molecules In the focal plane, generating a very thin optical section. [Micrographs from J. G. White et al., 1987, J. Cell Biol. 104:41.]

▲ EXPERIMENTAL FIGURE 5-49 Deconvolution fluorescence microscopy yields high-resolution optical sections that can be reconstructed into one three-dimensional image. A macrophage cell was stained with fluorochrome-labeled reagents specific for DNA (blue), microtubules (green), and actin microfilaments (red). The series of fluorescent images obtained at consecutive focal planes (optical reverses the degradation of the image by using the blurred beads as a reference object. The out-of-focus light is mathematically reassigned with the aid of deconvolution algorithms. Images restored by deconvolution display impressive detail without any blurring (Figure 5-49). Astronomers use deconvolution algorithms to sharpen images of distant stars.

Resolution of Transmission Electron Microscopy Is Vastly Greater Than That of Light Microscopy

The fundamental principles of electron microscopy are similar to those of light microscopy; the major difference is that electromagnetic lenses, rather than optical lenses, focus a high-velocity electron beam instead of visible light. In the transmission electron microscope (TEM), electrons are emitted from a filament and accelerated in an electric field. A condenser lens focuses the electron beam onto the sample; objective and projector lenses focus the electrons that pass through the specimen and project them onto a viewing screen or other detector (Figure 5-50, left). Because electrons are absorbed by atoms in air, the entire tube between the electron source and the detector is maintained under an ultrahigh vacuum.

The short wavelength of electrons means that the limit of resolution for the transmission electron microscope is the-

sections) 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 the cell. (b) After application of the deconvolution algorithm to the images, the fibrillar organization of microtubules and the localization of actin to adhesions become readily visible in the reconstruction. [Courtesy of J. Evans.]

oretically 0.005 nm (less than the diameter of a single atom), or 40,000 times better than the resolution of the light microscope and 2 million times better than that of the unaided human eye. However, the effective resolution of the transmission electron microscope in the study of biological systems is considerably less than this ideal. Under optimal conditions, a resolution of 0.10 nm can be obtained with transmission electron microscopes, about 2000 times better than the best resolution of light microscopes. Several examples of cells and subcellular structures imaged by TEM are included in Section 5.3.

Because TEM requires very thin, fixed sections (about 50 nm), only a small part of a cell can be observed in any one section. Sectioned specimens are prepared in a manner similar to that for light microscopy, by using a knife capable of producing sections 50-100 nm in thickness (see Figure 5-43). The generation of the image depends on differential scattering of the incident electrons by molecules in the preparation. Without staining, the beam of electrons passes through a specimen uniformly, and so the entire sample appears uniformly bright with little differentiation of components. To obtain useful images by TEM, sections are commonly 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 incident

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