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Photoelectron imaging and photoelectron labeling
U l~,-amicroscopy 24 (I 988) 299- 312 North-Holland, Amsterdam
299
PHOTOELECTRON IMAGING AND PHOTOELECTRON LABELING O. Hayes G R I F F I T H Zn,,'titute of Molecular Biology and Department of Chemist~. , University of Oregon, Eugene, Oregon 97403, USA
and Gertrude F. R E M P F E R Department of Physics, Portland State University, Portland, Oregon 97207, USA Work presented August 1986; manuscript received 22 June 1987
Photoelectron imaging involves the photoejection of low-energy electrons from a specimen surface exposed to UV light. The electrons are then accelerated and focused by an electron-optics system in much the same way fluorescent light is focused in an optical microscope. Thus, photoelectron imaging is the electron-optical analog of fluorescence microscopy. In combination with photoemissive labels it serves to extend the range of studies possible by fluorescence, for example in work on cell surfaces and internal structures of cells that have been exposed by detergent extraction of membranes.
I. Introduction Research in cell biology focuses on the structure, biochemistry, and genetics of single cells, which are both enormously complex and fragile. Beginning with the thin plasma membrane that forms the outside boundary, continuing into the cytoplasm with the cytoskeletal elements, mitochondria, and endoplasmic reticulum, and finally past the nuclear membrane to the chromosomes, the variation of structure with position is of a magnitude unparalleled in even the most sophisticated man-made devices. Often there is very little difference between the elemental composition of structures. For example, cell surface receptors have similar elemental compositions and dimensions but very different functions. One of ./
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without excessive damage and to identify components of the structures in the micrographs as a step toward understanding their combined functions. Many imaging devices are being developed for this purpose (see other articles in this issue, and Echlin [1]). These techniques tend to be complementary in application and information con-
tent, and can be grouped by the nature of the excitation source. At one end of the scale are the high-energy electron beams and X-rays capable of exciting core level electrons and providing information on elemental distributions. At the other end of the scale are the very low-energy excitation radiations, such as ultraviolet light, that probe only valence electrons. It is the latter group that we are concerned with here. Fig. 1 shows some of the the processes that can occur upon absorption of a UV photon. Of these, the most widely used in cell biology is fluorescence, both in spectroscopy and microscopy [2]. The pr;ncipal reason for its popularity is that fluores ent dye molecules can be easily incorporated into biological systems, and the emitted light ;- ,eadily detected against the background. To . . . . . . . i;.,,~
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of great comrast and, vhen used iv_ combination with site-specific labels, provides a useful way of observing the location and organizatiop of biological macromolecules. Fluorescence microscopy is not the only possible emission technique. Phosphorescence, for example, can also be observed by similar instruments. Another method,
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photoelectron labeling, the analog to using fluorescent dyes in a fluorescence microscope. By attaching photoemissive labels to antibodies, information regarding the distribution of specific structures on the surface of and inside cells is being obtained.
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one with much No,her resolution, is tile electronoptical equivalent of fluorescence microscopy. This is called photoelectron imaging (photoelectron microscopy, PEM, or photoemission electron microscopy, PEEM). Fig. 2 shows simplified drawings comparing the essential features of a fluorescence microscope and a photoelectron microscope. The F o.~" principle involved in the latter is the photoelectric effect. Essentially all materials will emit photoelectrons when illuminated with light of the appropriate wavelength. The electrons can be accelerated and focused by an electron-optical system to form a magnified image of the specimen. The photoelectron image contains useful information regarding the exposed surface structures. Recent reviews of photoelectron imaging are available [3-6]. Our purpose here is tu discuss the present status of this tecknique, including basic principles and specimen preparation. As an example of an application in cell biology, we discuss
The photoelectrons released from the specimen by UV light have very small kinetic energies. Prior to being focused by the lens system they are accelerated to high energies by an electric field between the specimen and the anode. The anode has an aperture which allows the accelerated electrons to pass from the accelerating region into the electron-optical system which forms an image of the specimen. The arrangement for accelerating the electrons is shown in fig. 3. (Radial distances have been exaggerated for clarity.) An accelerating voltage ~(d is applied across the gap / between the specimen and the anode. The electric field is essentially uniform except in the immediate vicinity of the anode aperture. The perturbation caused by the aperture acts as a thin diverging lens (aperture lens). The accelerating field can be considered equivalent to a uniform field terminated by the aperture lens. In the uniform field the electrons follow parabolic paths, as illustrated in fig. 3 for an emitting point on the axis. At the termination of the field the paths are diverged by the aperture lens and become the incident rays for the objective lens of the imaging system. In the process of accelerating the electrons, a virtual image of the specimen is formed, which serves as the object for the objective lent, The virtual image has aberrations introduced during acceleration. These aberrations, when combined with those introduced by the objective lens, determine the resolution limit due to geometrical aberrations. The electron-optics of the uniform acceleration field is illustrated in fig. 4 for the situation where the emission velocity ue of the electrons is fixed and the emission angle ae varies. The tangents to
O.H. Griffith, G.F. Rempfer ./Photoelectro,7 imaging and photoelectron labeling
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the parabolic paths at the termination of the field, when extrapolated backwards, form a virtual image of the specimen (virtual specimen) at unit lateral magnification, approximately twice as far from AN()DE
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the anode as is the specimen. As shown in fig. 4, parabolas corresponding to electrons emitted at right angles to the axis ( a e = 9 0 °) have their vertices at the emitting point, and the tangent rays focus at a distance 21 from the anode (i.e., twice the distance of the specimen from the anode). However, the trajectories of electrons leaving the specimen at angles % < 90 ° do not have their vertices at the emitting point, and the image distance 1" determined by the tangent rays is not exactly 21. The bundle of tangent rays does not characteristic of spherical aberration. The smallest cross-section of the bundle is called the circle of least confusion. The radius of this circle is often used as a measure of resolution although it does not represent the best resolution. The virtual specimt.n is the object for the aperture lens. The aperture lens forms a virtual image of the virtual specimen at a distance of ~1 from
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the anode and at a lateral magnification of 3-The 2 angles of the tangeat rays are increased by the 3 diverging action of the aperture lens to ~1 = ~,a.,. The demagnified virtual image formed by the aperture lens is in turn the object for the objective lens. The objective lens forms a real image, which contains the aberrations due to both the accelerating field and the objective lens. The combined aberrations due to the accelerating field and the objective lens can ag;in be expressed in terms of a circle of least conJ asion. However, if there is a substantial amount o~ chromatic aberration, due to a distribution of e~,ergies in the emitted beam, the circle of least con.~usion is not meaningful in describing the aber ation. since its location and size depend on the en ission energy. The approach which we have useQ is to calculate the intensity distribution due to the spherical aberration in a given image plane for each of a series of energies throughout the energy distributien range. The individual intensity distributions are summed to obtain the intensity profile for the beam as a whole. The position of the image
plane can be varied to find the plane of best focus. From the intensity profile for the beam as a whole, the resolution limit due to the combined ~;hro.,natic and spherical aberrations of the microscope was found to be about 2 nm. This result was obtained for parameters typical of a modern photoelectron microscope: an accelerating gap l of 3 mm, an accelerating voltage of 3 x 10 4 V, and an angular aperture at the objective lens of al = 3 mrad. In addition, a parabolic emission energy distribution ranging from 0 to 0.5 eV, and a Lambertian angular distribution were assumed. The calculations showed that, except for the contributions due to the objective lens aberrations, the resolution limit is v e r y nearly nrc~ncwlinnal tc~ t V / V , where V. is the average emission enero, v. For the values of F. and a~ ( = 2a 1) used in the calculations, the diffraction error is also about 2 nm. The resultant resolution due the geometrical and the diffraction errors is about 3 nm. If the energy distribution is greater than the 0 to 0.5 eV assumed for this calculation, the resolution limit will be larger than 3 nm. In principle the resolu__
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tion can be improved further using corrected electron optics to reduce the aberrations of the accelerating field and of the objective lens [71. The fundamental limit then would be the diffraction error associated with the wavelength of the emitted electrons.
2.2. Contrast mechanisms Surface imaging requires two contrast mechanisms; one is topographical so that a view of the surface can be obtained, and the other is a material contrast mechanism to distinguish the sites or molecules of interest. In PEM, the topographical contrast is a consequence of the very low kinetic energies of the emitted electrons and the fact that the specimen is in an electric field [8]. Electrons emitted from slopes of negative or positive surface relief are deflected by the microfields at the surface of the specimen, causing some of them to be stopped out by the aperture of the electron-optical system. Electrons emitted from flat areas are undeflected and most of them pass through the aperture stop. The overall effect is that ~he images of sides of a protrusion are darker than the top and surrounding flat areas. This effect enhances the contrast of small fibers and other topographical features, making them m~ch more easily visible in the photoelectron imag~.. The material contrast mechanism in PEM is the dependence of the photoelectron quantum yield (electrons emitted per incident photon) on the electronic structure of molecules. Photoelectron quantum yields in the thresh,,ld region (180-230 nm) have been measured for amino acids, lipids, saccharides, and other biomolecules (for references, see ref. [4]). The material contrast is somewhat dependent on the length of exposure to UV radiation, so the measurement should be considered quafitative rather than quantitative unless special calibration curves have been generated, a step not required in the applications described here. In geaeral, highly conjugated moieculcb ,,,uch as the carcinogen, benzo(a)pyrene, and chlorophyll are the most photoemissive. Also very photoemissive are certain metals such as silver (see discussion of labeling below). As in fluorescence microscopy, the goal is to image the distribution
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of a known label or marker. The material contra~r~ of PEM is very good for this purpose.
2.3. Depth of information, depth of field, and comparison with SEM All methods of imaging, even those classified as surface techniques, probe a finite volume of the specimen. The depth of information is defined as the distance below the surface from which information is contributed at a given resolution [9]. Another term that refers to this important physical parameter is depth resolution. For surface techniques in general, the depth of information is determined by a combination of the depth of penetration of the excitation source, the escape depth of the electrons (or X-rays, ions, or other detected signal) and the surface-etching rate, if any. "It PEM, there is no appreciable surface etching, and the UV photons penetrate relatively deeply into the specimen, so the determining factor is the escape depth of the photoelectrons. Measured escape depths for organic and biological surfaces are relatively short, from 1 to 10 nm [9]. Thus, the escape depths are on the order of the lateral resolution of PEM, and the depth of information is approximately equal to the electron escape depth. Because of the short escape depths of photoelectrons, PEM is among the most surface sensitive of the imaging techniques being applied in the biological sciences. Another parameter with a similar sounding name but very different meaning is the depth of field. The depth of field is the axial distance the object can be traversed and still appear to be in focus. The greater the depth of field, the larger is the range of topography on an object that will be in focus at the same lens settings. The depth of field in all microscopes is inversely proportional to . . . . times the hall .... v . . . . ,,.~ of thc magmficat~on -" an~le of accer~lance of the objective lens. TEM and SEM have the same expression for the depth of field, although SEM micrographs often give the impression of a greater depth of field because they are usually recorded at lower magnification. For PEM, the equation for the depth of field for two planar regions at different heights being brought into focus is similar to that ot TEM and SEM [10].
O.H. Griffith, G.F. Rempfer / Photoelectron imaging and photoelectron labeling
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However, the working depth of field in PEM for specimens with rapidly varying topography is greatly reduced because of the electron-optical effect of the microfields at the surface. In practice the depth of field is not usually a limiting factor in PEM. It is adequate for a wide range of cell surface and cytoskeletal studies. Photoelectron micrographs sometimes appear similar to scanning electron micrographs, but these two techniques have some important differences as diagrammed in fig. 5. PEM is not a scanning technique; the image is formed by collecting photoelectrons simultaneously from the entire portion of the specimen being viewed. The depth resolution of PEM is inherently greater than in SEM because of the method of image formation. In PEM, a Gaussian image is formed of electrons emerging from the surface of the specimen. Since topography is purely a surface effect, fine topographic detail is always imaged at the full instrument resolution in PEM. The farther below the surface the electrons originate, the more diffuse the information. Since the electron escape depths are short, this does not normally degrade the
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image. But even if there were a deeper source of electrons, analogous to the bloom area of an SEM, this would only superimpose a blurred image on the sharp image of the surface. The two images would not be averaged together. In SEM, however, the information is collected sequentially point by point, and the detector records all secondary electrons from the entire bloom area, thus averaging the image information and increasing the depth of information. This is consistent with the observations of Bode et al. [11] on bronze and cemented carbide samples, where surface detail appeared sharper in the PEM than in the SEM. The SEM has other advantages that make it a valuable and versatile technique, for example, the ability to detect many different signals. We view the PEM and SEM as complementary techniques that can be used together in studies of biological surfaces.
3. Specimen preparation Specimen preparation for photoelectron imaging parallels that for ether forms of electron microscopy except that two steps are omitted: the specimens are usually not stained as for TEM or coated with a conductive layer as for SEM. The specimen mount is a commercially available (Bellco Glass Inc., Vineland, NJ, USA) glass microscope cover slip, 5 mm in diameter. Solid metal supports as commonly employed in SEM would be acceptable specimen mounts. The advantage of glass coverslips is that the specimens can be viewed first in an optical mic:o.~cope. This makes possible rapid screening of specimens before selection for photoelectron imaging. The coverslips are coated on both sides with a thin conducting layer sinc~ electrons lost througi~ photoemission must be replenished from the power supply via the cathode stage and sample support. The conducting layer to the coatings used on the inside face of cathode ray osciloscopes and TV tubes, or a ,ran evaporated metal layer such as chromium, which we are currently using. For experiments involving cultured cells, the conductive glass discs are sterilized, coated with serum proteins, and placed into the b~-,ttom of tissue culture dishes. The ceils then t 1
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O.t1 Griffith. G.F. Rempfer / Photoelectron imaging and photoelectron labeling
grow on the coated glass discs, and the extent of coverage can be observed with a conventional inverted-stage microscope. For cells free in suspension, for example red cells or sperm, a few drops of suspension are placed on the conductive glass disc. Adhesion of cells to these discs is facilitated by first dipping the discs in 1 m g / m l aqueous solutions of alcian blue, cytochrome c, or poly-L-lysine, and air drying before use. Chromosomes, D N A a n d other cell c o m p o n e n t s can be examined by similar procedures, with adaptations as required to insure good spreading of the specimens. Whole cell preparations and cytoskeletal preparations must be fixed to preserve the delicate structures. For immunolabeling studies, there is a wel| known trade-off between preserving antigenic integrity and preserving physical structures, irrespective of the type of trficroscopy used. Treatments that preserve structures well, e.g. prolonged gluteraldehyde fixation, tend to abolish the antigenicity. Also, detergent extraction procedures to remove m e m b r a n e s and provide access for the antibodies to the cell interior c'~.n affect cytoskeletal structure and composition. One solution is to use a water-soluble crosslinker, suG, as DTSP, to gently prefix and stabilize structure- [13]. Another prefixation procedure involves a brief exposure to a very dilute solution of glutaralde-
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hyde (0.025%) prior to detergent extraction [~i4]. Labelling with photoemissive molecules is ,,"iscussed in the next section. Another considerat,on is that the structures of interest must be expoaed after treatment because PEM is a surface technique. For example, fragments of membranes remaining on the surface after detergent treatment would not be visible in a fluorescent micrograph of cytoskeletal elements but could obscure details in a photoelectron micrograph of the same preparation. Several types of sample preparation procedures have been tested for use in photoelectron imaging [15]. T h e photoelectron microscope requires, as do all electron microscopes, a vact~um to prevent air molecules from colliding with the information-carrying electrons. The specimens must therefore be either quick-frozen or dehydrated before insertion into the instrument. Currently all samples are dried by conventional procedures similar to those used in TEM and SEM. Typically, the fixed cell preparations are washed with glass-distilled water and then dehydrated through a series of aqueous ethanol mixtures beginning with 70% ethanol followed by critical-point drying trom CO, or vacuum drying from Freon 113. Alternatively, the newer cryofixation methods should also be applicable. The final specimens on the conductive glass discs are mounted on a metal rod as s':c,wn in fig. 6,
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O.H. Griffith, G. F Rempfer / Photo~..'ctron imaging and photoelectron labeling
and inserted with the specimen facing down in the photoelectron microscope. The photoeletron microscope used for this work is an ultrahigh vacuum instrument designed and built at the University of Oregon and Portland State University [16]. In order to eliminate specimen contamination, ion pumps and sorb pumps are used in place of oil diffusion and fore pumps, and bellows seals are used in place of dynamic O-rings. The instrument is constructed primarily of stainless steel and utilizes copper sealed flanges. The electron lenses are of the electrostatic unipotential type [17]. Typical operating conditions are: acceleration voltage of 30 kV, illumination provided by two OSRAM HBO 100 W / 2 Hg short-arc lamps, and an objective aperture of 50
pointed out in the early work of Coons, who introduced the technique of chemically attaching dyes such as fluorescein to antibodies [19]. The antibodies then bound to their specific antigenic sites, and produced a bright fluorescence pattern in the modified optical microscope. Immunofluorescence microscopy is now one of the most widely used techniques in cell biology, and it has made possible, for example, much of our understanding of the cytoskeietal structures of cells. The same concept of labeling has been extended to TEM and SEM through the use of electron-dense markers, with good success in specific cases. 4.1. An overoiew of markers in electron microscopy
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4. Fhotoelectron labeling As an application of photoelectron imaging, we discuss the use of photoemissive markers in combination with site-specific antibody molecules. By analogy to immunofluorescence microscopy, this combination of methods is called immunophotoelectron microscopy [18]. This is not the only area of application, as photoelectron imaging can be used without labels. All four fundamental building blocks of life (amino acids, saccharides, lipids, and nucleic acids) exhibit some photoemission under the action of UV light. The topography of an exposed surface can be photographed without labels, and in certain cases where naturally photoemissive molecules are present (e.g. chlorophyll or hemes), labeling may not be needed or desirable. However, in most applications the goal is to distinguish the location of one protein in the presence of other proteins of similar amino acid content, and no significant material contrast is pre,;cat. Here, labeling provides the only sure way of ........ _y,,,~ ............. ,~ of proteins such as specific enzymes or receptors. The situation is reminiscent of fluorescence microscopy. ,.,.'here even without labeling some fluorescence (autofluorescence) is present. Only in special cases is autofluorescence useful in relating structure and function. The way to overcome this difficulty was
In the search for markers for photoelectron microscopy, it is instructive to step back and view the wide range of markers that have been used in various forms of optical and electron microscopy, iacluding TEM and SEM. Fig. 7 shows several of l hese markers, drawn so that the markers are scaled correctly relative to the dimensions of the Y-shaped antibody molecule. One of the largest
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O.H. Griffith. G.F. Rempfer / Photoelectron imaging and photoelectron labeling
markers used in SEM is tobacco mosaic virus (TMV). TMV contains no heavy metal atoms so there is essentially no material contrast:. This is purely a topographical marker and because of its very large length relative to the antibody, TMV has not been w~dely used. Hemocyanin shown below TMV in fig. 7 is much smaller and is frequently used in SEM. There are no significant concentrations of heavy metals (other than a low density of copper atoms) and so the label is a topographical marker, recognized solely by its size and shape. On the other hand the third marker shown, ferritin, contains electron-dense iron atoms and is used as a marker in TEM. Labeling with these markers and others, such as latex spheres, has been reviewed by Molday [20]. Another type of marker, and one that is gaining in popularity in all areas of microscopy, is colloidal gold [20-22]. It has many advantages starting with the uniform shape, near-perfect spheres of gold that cannot be confused with biological surface structures. Gold probes are electron dense as required in TEM, and exhibit sufficient emission of backscattcred and secondary electrons to be useful in SEM. They also are visible in light microscopy in higher concentrations or after silver enhancement [23]. Colloidal gold can be prepared by a wide selection of reducing agents, and procedures are available for generating relatively homogeneous preparations of just about any desired diameter, from ross than 5 nm to 150 nm. The smallest colloidal gold particles used have about the same dimensions as the antibody itself (see fig.
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Fig. 8. Diagram showing the basic scheme of cell surface labeling for photoelectron imaging (immunophotoelectron microscopy). Requirements for PEM markers (M): (a) high photoelectron quantum yield, (b) distinctive size and shape, (c) stable, (d) high affinity binding to ligand (e.g., antibody, lectin, biotin), (e) minimal nonspecific binding to cell surfaces.
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7). The attachment of the colloidal gold to the antibody is achieved by mixing the two solutions together with an appropriate stabilizer. The antibody is adsorbed onto the gold particles (clean metal surfaces of any geometry will, in general, bind protein in the appropriate pH range). A significant fraction of the antibody bound to gold retains its binding activity, and the preparations are stable. These advantages combined with the commercial availability of a selection of gold probes have led to a large and growing literature on colloidal gold labeling.
4.2. The choice of photoelectron labels The ideas of immunofluorescence microscopy can also be extended to photoelectron microscopy as outlined in fig. 8. A photoemissive marker (M) is coupled to an antibody, and then allowed to diffuse into the specimen and bind to the specihc antigen. In practice, attaching a marker to a specific site is usually a two-antibody process (socalled indirect imnmnolabeling). The first antibody binds to the specific antigen in the specimen, and the second ap.tlbody, carrying the marker, binds to the first antibody. The ideal PEM marker would: (1) have a very hio~ pbmoelectron quantum yield (electrons emitted per incident photon), (2) be of small size, (3) be stable, and (4) exhibit no nonspecific binding to the cell surface. A natural starting point in the search for photoelectron labels was to try the first fluorescent probes introduced by Coons in 1941. In many ways these are ideal markers, being much smaller than the antibody molecule itself (fig. 7). Photoelectron quantum yields measured in the conventional way at very low light levels indicated that the dyes are much more photoemissive than cell surface components in the 230 nm to 180 nm wavelength range. However, under the more intense illumination necessary to achieve high magnifications in the photoelectron microscope, the Fhotoemission of proteins and other biological surface coniponents increases so that the contrast between label and background decreases with exposure to UV light [24]. The net result is that for the low labeling ratios typically used in fluorescence microscopy (e.g. one dye per antibody), the
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contras~ is not sufficient far a photoelectron labeling experiment. Much higher labeling ratios can be u::ed, but there are now better alternatives. Curr mt research in photoelectron imaging uses colloidal gold markers and silver-enhanced colloidal gold. Colloidal gold has been shown to be photoemissive and easily detected in photoelectroll images of lectin binding sites on erythrocyte ghosts [25] and in antibody labeling of cytoskeletal elements on cultured cells [18]. Silver-enhanced gold exhibits even higher contrast [26].
The reasons for the improved contrast are larger particle size and, more importantly, the higher q u a n t u m yield of silver and silver oxides and the generation of a relatively clean surface from which the photoelectrons can escape.
4,3. Photoelectron micrographs of cytoskeletal elements and cell surface fibronectin Cells of higher organisms contain comple× networks of protein filaments collectively called " t h e
Fig. 9. Immunofluorescence (insert) and immunophotoeh:ctron micrcgraphs of per'Sans of the same rat embryo fibroblast labeled for actin visualization, using rhodarnine as the fluorescer ce marker and colloidal gold as the photoelectron marker. Bar = 10 # m.
O.H. Griffith, G.F. Rempfer / Photoelectron imaging and photoelectron labeling
cytoskeleton', which are involved in the shape, internal organization and movement of cellular components. The three main components are actin filaments, microtubules, and the intermediate filaments. The results of a double !~befing experiment for the visualization of actin are shown m fig. 9. The specimen is a rat embryo fibroblast cell that was grown directly on the type of serum-coated conductive glass disc diagrammed in fig. 6. The membranes of the cell were removed by brief treatment with a dilute aqueous sohLtion of the mild detergent, Triton X-100. The specimen was then washed in detergent-free buffer and fixed in - 2 0 ° C methanol for 5 min. The resulting cytoskeletal preparation was exposed to a monoclonal antibody recognizing actin followed by a rhodamine-labeled second antibody recognizing the fir~:, and a 20 nm colloidal gold-labeled antibody recognizing the second antibody. After a final fixation in 2% glutaraldehyde, the fluorescence micrograph (insert, fig. 9) was recorded and the specimen was then dehydrated and inserted into the PEM to obtain the photoelectron micrograph. Thus, the fluorescence and photoelectron micrographs of fig. 9 are of portions of the same cell, and many of the actin-containing fibers can be traced in both micrographs. Clearly, however, the fibers are imaged in the photoelectron micrograph at much higher resolution. Many of the lightly labeled fibers do not show up in the fluorescence
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300
micrograph, whereas essentially all labeled structures are visible in the photoelectron micrograph. The higher resolution permits the imaging of single antibodies, which appear as bright dots along the fibers in the photoelectron micrographs [15,18]. Photoelectron imaging using photoelectron labels can also be performed on cell surfaces and the extracellular matrix, as diagrammed in fig. 10. One ,rroblem of interest is the effect of tumor promoters on cells. Tumor promotors are molecules that are not in themselves cancer-causing agents, but greatly increase the incidence of tumors when present with carcinogens [27]. One effect of the potent tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate~ is to cause an increased release of fibronectin from fibroblasts. Fibronectin is a major component of the extracellular matrix that promotes cell adhesion and is present in reduced amounts on fibroblasts derived from tumors [28-30]. Photoelectron micrographs of normal human foreskin fibroblasts as a function of exposure to TPA show a loss of the fibronectin from the upper cell surface (fig. 11, ref. [31]). The cells were labeled for fibronectin visualization by a three-step immunolabeling procedure with a colloidal gold-antibody complex and subsequent silver enhancement. Before treatment, the labeled fibronectin appeared as both dense networks and ha numerous fibrillar streaks against the less photoemissive uncoated cell surface (fig. ]]a). After
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Fig. 10. Cro~,,,-~ectiona] drawing representing enhanced e~nisslon from labeled fibronectin (dotted bands) on cell surfaces. More ,qectron emission (arrows) comes from the silver-coated ~,old markers bound to the fibronectin than from the unlabeled cell surface, giving rise to corresponding bright areas in the image. Labeled fibronectin beneath the cell does not contribute to the image. From [31] with permission.
310
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Fig, 1i. Photoelectron micrograph illus~ 'ating the effect of 100 ng/ml TPA on the amount and distribution of fibronectin remaining on human foreskin fibroblast (FS-2) celi surfaces. (a), exposed to medium contairfing 0.02% DMSO only for 40 rain, (b-d) exposed to I'PA, 0.02% DMSO i.n medium for (b) 5 rrdn, (c) 10 rain, (d) 40 min. (a) and (b) show overlapping Fortions of several cells. From ref. [3!] with permission.
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• • v - o" ul f i n-tln of e x p o s u r e to ~n,, , u v , ~__ / , u,__, -~,-,, ~ r ~ in the c u l t u r e m e d i u m , t h e r e w a s a visible d e c r e a s e in the u p p e r cell surfa.-e f i b r o n e c t i n (figs. l l b a n d l l c ) . A f t e r 60 m i n n . ' a r l y all of the u p p e r cell s u r f a c e f i b r o n e c t i n w a s ~ o n e (fig. i l d ) . H o w e v e r , the ex-
t r a c e l l u l a r m a t r i x f i b r o n e c t i n b e t w e e n cells ap p e a r s to be m o r e r e s i s t a n t to release a n d c a n still
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O.H. Griffith, G. 1:. Rempfer / Photoelectron imaging and photoelectron labeling
5. Advantages, limitations, and future directions It is essential to establish the limitations ,,s well as the advantages of each new technique in order to know which combination of methods is most appropriate for a particular application. We have found that virtually every specimen examined emitted a sufficient current of photoelectrons to produce an image, so the range of potential applications is large. The main limitation, other than lack of commercial instrumentation, is the roughness of the surface of the specimen. In this respect PEM is complementary to SEM. Many specimens that are very difficult or impossible to view by SEM can be seen clearly in PEM. In this category are relatively flat cell surfaces, cell membranes, fine fibers, and nucleic acids. Conversely, rounded up cells present no problem for SEM but are not suitable specimens for PEM. This is because the height and curvature of the cell affects the electric field at the surface, and consequently a significant proportion of the slow-moving photoelectrons emerging from the surface are deflected too much to be included in the angular aperture of the optical system. This results in dark areas in the image corresponding to areas of extremes in sampie topography. On the other hand, it is this same sensitivity to topography that makes possible the detection of very fine topographical detail not easily visualized by SEM. To summarize, the advantages of PEM are (1) a direct extension of the immunofluorescence technique using similar labeling methods and the advantages of emission (detecting a bright object against a darker background), (2) high sensitivity to fine topographic detail on relatively flat surfaces, (3) high depth resolution (short escape depth of the photoelectrons), and (4) lower damage to biological specimens than is caused by electron beams since UV light causes less mass loss than does charged particle radiation. There also appcu~,,, to be a lower specimen conductivity requirement since it has not been necessary to coat thin biological specimens with a conductive metal or carbon layer. Part of this increased conductivity' may be due to photoconductivity produced by the UV and visible light striking the sample, but another factor is that the electrons are drawn off
311
at a low and continuous rate from the entire specimen surface being viewed, and not from a Ic,c'aliT~d spo! as in an SEM or scanning electron microprobe. Looking towards the future is always difficult, but it is useful to speculate. There are two general areas of application. One involves the UV excitation described above. We expect that wi& continued improvements in instrumentation, photoelectron imaging with UV excitation will be increasingly used in c,o,,,o,,,~,on . . . . '-: .... " with immunofluorescence microscopy and photoemissive labels to provide new information regarding the distribution of binding sites on biological structures. The other area involves X-ray excitation. By equipping the photoelectron microscope with a suitable intense X-ray source and energy analyzer. it should be possible to perform microanalysis of biological surfaces. In addition to elemental distributions, the images would contain information about chemical bonding since the binding energies of the inner shell electrons are influenced by their chemical surroundings, just as X-ray photoelectron spectroscopy (XPS or ESCA) provides this information but without the imaging capability. The first steps towards imaging photoelectrons released by X-rays have already been taken [32-34]: see also the review [6]. PEM has a different set of advanlages and disadvantages from SEM and the microprobe methods, so it is likely that ail of these instruments will be used in combination to study biological specimens. The current resolution of PEM is 10 nm with a possibility of eventually reaching 2 nm. TEM will remain the highest resolution technique but there are many cases where this high resolution is not realized, or where contrast factors, information content, or specimen damage become more important. In the final analysis, Mother Nature provides a range of challenges far greater then the capabilities of any one technqiue, and all of these methods are needed ili gi ~rt,au-spcctrum approach to so~xang structure-function problems in cell bioloov I
I
.
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Acknovdedgements We are pleased to acknowledge the many contributions of our colleagues Drs. G. Bruce Birreii
312
O.H. Griffith, G.F. Rempfer / Photoelectron imaging and photoelectron labeling
and Karen K. Hedberg, and Douglas L. Habliston, Walter P. Skoczylas and Loreene Evans. This work was supported by PHS grant CA 11695 from the National Cancer Institute and a grant from the M.J. Murdock Charitable Trust.
References [1] P. Echlin, Ed., Analysis of Organic and Biological Surfaces (Wiley, New York, 1984) pp. 1-646. [2] D.L. Taylor, A.S. Wag,goner, R.F. Murphy, F. Lanni and R.R. Birge, Eds., Applications of Fluorescence in the Biomedical Sciences (Liss, New York, 1986) pp. 1-639. [3] R.A. Schwarzer, Microsc. Acta 84 (1981) 51. [4] O.H. Griffith, K.K. Nadakavukaren and P.C. Jost, in: Scanning Electron Microscopy/1982, Ed. O. Johari (SEM, AMF O'Hare, IL, 1984) p. 633. [5] O.H. Griffith and G.F. Rempfer, Ann. Rev. Biophys. Biophys. Chem. 14 (1985) 113. [6] O.H. Griffith and G.F. Rempfer, in: Advances in Optical and Electron Microscopy, Vol. 10, Eds. R. Barer and V.E. Cosslett (Academic Press, London, 1987). [7] G.F. Rempfer and M.S. Mauck, in: Proc. 43rd Annual EMSA Meeting, Louisville, KY, 1985, Ed. G.W. Bailey (San Francisco Press, San Francisco, CA, 1985) p. 132. [8] G.F. Rempfer, K.K. Nadakavukaren and O.H. Griffith, Ultramicroscopy 5 (1980) 437. [9] W.A. Houle, W. Engel, F. Willig, G.F. Rempfer and O.H. Griffith, Ultramicroscopy 7 (1982) 371. [10] G.F. Rempfer, K.K. Nadakavukaren and O.H. Gfiffith, Ultramicre~copy 5 (1980) 449. [11] M. Bode, G. Pfefferkom, K. Schur and L. Wegmann, J. Microscopy 95 (1972) 323. [12] O.H. Griffith, G.F. Rempfer and K.K. Nadakavukaren, in: Electron Microscopy, Proc. 10th Intern. Congr. on Electron Microscopy, Hamburg, 1982, pp. 59-68. [13] P. Bell, Jr., in: Scanning Electron Microscopy/1981, Voi. II, Ed. O. Johari (SEM, AMF O'Hare. iL, 1981) p. 130. [14] L.B. Chen, I.C. Summerhayes, K.K. Nadakavukaren, T.J. Lampidis, S.D. Bemal and E.L. Shepherd, in: Cancer
Cells, Vol. 1, The Transformed Phenotype (Cold Spring Harbor Laboratory, 1984) pp. 75-86. [151 K.K. Nadakavukaren and O.H. Griffith, Ultramicroscopy 17 (1985) 31. [16] O.H. Griffith, G.F. Rempfer and G.H. Lesch, in: Scanning Electron Microscopy/1981, Vol. II, Ed. O. Johafi (SEM, AMF O'Hare, IL, 1981) p. 123. [171 G.F. Rempfer, J. Appl. Phys. 57 (1985) 2385. [181 G.B. Birrell, D.L. Habliston, K.K. Nadakavukaren and O.H. Gdffith, Proc. Natl. Acad. Sci. USA 82 (1985) 109. [191 A.H. Coons, H.J. Creech and R.N. Jones, Proc. Scc. Expti. Biol. Med. 47 (1941) 200. [2o] R.S. Molday, in: Techniques in Immunocytochemistry, Vol. 2, Eds. G.R. Bullock and P. Petrusz (Academic Press, London, 1983) pp. 117-154. [211 J. De Mey, in" lmmunocytochemistry: Practical Applications in Pathology and Biology, Eds. J.M. Polak and S. Van Noorden (Wright, Bristol, 1983) p. 82. [221 J. Roth, in: Techniques in Immunocytochemistry, Vol. 2, Eds. G.R. Bullock and P. Petrusz (Academic Press, London, 1983) pp. 217-284. [231 G. Danscher and J.O.R. Norgaard, J. Histochem. Cytochem. 31 (1983) 1394. 1241 O.H. Griffith, D.L. Hoimbo, D.L. Habliston and K.K. Nadakavukaren, Ultramicroscopy 6 (1981) 149. [251 G.B. Birrell, S.M. Rose and O.H. Griffith, Ultramicroscopy 12 (1983) 213. [26] G.B. BirreU, D.L. Habliston, K.K. Hedberg and O.H. Griffith, J. Histochem. Cytochem. 34 (1986) 339. [271 P.M. Blumberg, CRC Critical Rev. Toxicology 8 (1980) 153. I281 R.O. Hynes, So.. Am. 254 (1986) 42. [29] K.M. Yamada, in: Cell Interactions and Development, Ed. K.M. Yamada (Wiley, New York, 1983) pp. 231-249. [301 E. Ruoslahti, M. Pierschbacher, E.G. Hayman and E. Engvall, Trends Biochem. Sci. 7 (1982) 188. [311 D.L. Habliston, G.B. Birrell, K.K. Hedberg and O.H. Griffith, European J. Cell Biology 41 (1986) 222. [32] J. Cazaux, Rev. Physique Appl. 8 (1973) 371. [33] J. Cazaux, in: Scanning Electron Microscopy/1984, Vol. III, Ed. O. Johari (SEM, AMF O'Hare, IL, 1984) p. 1193. [341 G. Beamson, H.Q. Porter and D,W. Turner, Nature 290 (1981) 556.
299
PHOTOELECTRON IMAGING AND PHOTOELECTRON LABELING O. Hayes G R I F F I T H Zn,,'titute of Molecular Biology and Department of Chemist~. , University of Oregon, Eugene, Oregon 97403, USA
and Gertrude F. R E M P F E R Department of Physics, Portland State University, Portland, Oregon 97207, USA Work presented August 1986; manuscript received 22 June 1987
Photoelectron imaging involves the photoejection of low-energy electrons from a specimen surface exposed to UV light. The electrons are then accelerated and focused by an electron-optics system in much the same way fluorescent light is focused in an optical microscope. Thus, photoelectron imaging is the electron-optical analog of fluorescence microscopy. In combination with photoemissive labels it serves to extend the range of studies possible by fluorescence, for example in work on cell surfaces and internal structures of cells that have been exposed by detergent extraction of membranes.
I. Introduction Research in cell biology focuses on the structure, biochemistry, and genetics of single cells, which are both enormously complex and fragile. Beginning with the thin plasma membrane that forms the outside boundary, continuing into the cytoplasm with the cytoskeletal elements, mitochondria, and endoplasmic reticulum, and finally past the nuclear membrane to the chromosomes, the variation of structure with position is of a magnitude unparalleled in even the most sophisticated man-made devices. Often there is very little difference between the elemental composition of structures. For example, cell surface receptors have similar elemental compositions and dimensions but very different functions. One of ./
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without excessive damage and to identify components of the structures in the micrographs as a step toward understanding their combined functions. Many imaging devices are being developed for this purpose (see other articles in this issue, and Echlin [1]). These techniques tend to be complementary in application and information con-
tent, and can be grouped by the nature of the excitation source. At one end of the scale are the high-energy electron beams and X-rays capable of exciting core level electrons and providing information on elemental distributions. At the other end of the scale are the very low-energy excitation radiations, such as ultraviolet light, that probe only valence electrons. It is the latter group that we are concerned with here. Fig. 1 shows some of the the processes that can occur upon absorption of a UV photon. Of these, the most widely used in cell biology is fluorescence, both in spectroscopy and microscopy [2]. The pr;ncipal reason for its popularity is that fluores ent dye molecules can be easily incorporated into biological systems, and the emitted light ;- ,eadily detected against the background. To . . . . . . . i;.,,~
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of great comrast and, vhen used iv_ combination with site-specific labels, provides a useful way of observing the location and organizatiop of biological macromolecules. Fluorescence microscopy is not the only possible emission technique. Phosphorescence, for example, can also be observed by similar instruments. Another method,
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photoelectron labeling, the analog to using fluorescent dyes in a fluorescence microscope. By attaching photoemissive labels to antibodies, information regarding the distribution of specific structures on the surface of and inside cells is being obtained.
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one with much No,her resolution, is tile electronoptical equivalent of fluorescence microscopy. This is called photoelectron imaging (photoelectron microscopy, PEM, or photoemission electron microscopy, PEEM). Fig. 2 shows simplified drawings comparing the essential features of a fluorescence microscope and a photoelectron microscope. The F o.~" principle involved in the latter is the photoelectric effect. Essentially all materials will emit photoelectrons when illuminated with light of the appropriate wavelength. The electrons can be accelerated and focused by an electron-optical system to form a magnified image of the specimen. The photoelectron image contains useful information regarding the exposed surface structures. Recent reviews of photoelectron imaging are available [3-6]. Our purpose here is tu discuss the present status of this tecknique, including basic principles and specimen preparation. As an example of an application in cell biology, we discuss
The photoelectrons released from the specimen by UV light have very small kinetic energies. Prior to being focused by the lens system they are accelerated to high energies by an electric field between the specimen and the anode. The anode has an aperture which allows the accelerated electrons to pass from the accelerating region into the electron-optical system which forms an image of the specimen. The arrangement for accelerating the electrons is shown in fig. 3. (Radial distances have been exaggerated for clarity.) An accelerating voltage ~(d is applied across the gap / between the specimen and the anode. The electric field is essentially uniform except in the immediate vicinity of the anode aperture. The perturbation caused by the aperture acts as a thin diverging lens (aperture lens). The accelerating field can be considered equivalent to a uniform field terminated by the aperture lens. In the uniform field the electrons follow parabolic paths, as illustrated in fig. 3 for an emitting point on the axis. At the termination of the field the paths are diverged by the aperture lens and become the incident rays for the objective lens of the imaging system. In the process of accelerating the electrons, a virtual image of the specimen is formed, which serves as the object for the objective lent, The virtual image has aberrations introduced during acceleration. These aberrations, when combined with those introduced by the objective lens, determine the resolution limit due to geometrical aberrations. The electron-optics of the uniform acceleration field is illustrated in fig. 4 for the situation where the emission velocity ue of the electrons is fixed and the emission angle ae varies. The tangents to
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the parabolic paths at the termination of the field, when extrapolated backwards, form a virtual image of the specimen (virtual specimen) at unit lateral magnification, approximately twice as far from AN()DE
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the anode as is the specimen. As shown in fig. 4, parabolas corresponding to electrons emitted at right angles to the axis ( a e = 9 0 °) have their vertices at the emitting point, and the tangent rays focus at a distance 21 from the anode (i.e., twice the distance of the specimen from the anode). However, the trajectories of electrons leaving the specimen at angles % < 90 ° do not have their vertices at the emitting point, and the image distance 1" determined by the tangent rays is not exactly 21. The bundle of tangent rays does not characteristic of spherical aberration. The smallest cross-section of the bundle is called the circle of least confusion. The radius of this circle is often used as a measure of resolution although it does not represent the best resolution. The virtual specimt.n is the object for the aperture lens. The aperture lens forms a virtual image of the virtual specimen at a distance of ~1 from
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the anode and at a lateral magnification of 3-The 2 angles of the tangeat rays are increased by the 3 diverging action of the aperture lens to ~1 = ~,a.,. The demagnified virtual image formed by the aperture lens is in turn the object for the objective lens. The objective lens forms a real image, which contains the aberrations due to both the accelerating field and the objective lens. The combined aberrations due to the accelerating field and the objective lens can ag;in be expressed in terms of a circle of least conJ asion. However, if there is a substantial amount o~ chromatic aberration, due to a distribution of e~,ergies in the emitted beam, the circle of least con.~usion is not meaningful in describing the aber ation. since its location and size depend on the en ission energy. The approach which we have useQ is to calculate the intensity distribution due to the spherical aberration in a given image plane for each of a series of energies throughout the energy distributien range. The individual intensity distributions are summed to obtain the intensity profile for the beam as a whole. The position of the image
plane can be varied to find the plane of best focus. From the intensity profile for the beam as a whole, the resolution limit due to the combined ~;hro.,natic and spherical aberrations of the microscope was found to be about 2 nm. This result was obtained for parameters typical of a modern photoelectron microscope: an accelerating gap l of 3 mm, an accelerating voltage of 3 x 10 4 V, and an angular aperture at the objective lens of al = 3 mrad. In addition, a parabolic emission energy distribution ranging from 0 to 0.5 eV, and a Lambertian angular distribution were assumed. The calculations showed that, except for the contributions due to the objective lens aberrations, the resolution limit is v e r y nearly nrc~ncwlinnal tc~ t V / V , where V. is the average emission enero, v. For the values of F. and a~ ( = 2a 1) used in the calculations, the diffraction error is also about 2 nm. The resultant resolution due the geometrical and the diffraction errors is about 3 nm. If the energy distribution is greater than the 0 to 0.5 eV assumed for this calculation, the resolution limit will be larger than 3 nm. In principle the resolu__
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tion can be improved further using corrected electron optics to reduce the aberrations of the accelerating field and of the objective lens [71. The fundamental limit then would be the diffraction error associated with the wavelength of the emitted electrons.
2.2. Contrast mechanisms Surface imaging requires two contrast mechanisms; one is topographical so that a view of the surface can be obtained, and the other is a material contrast mechanism to distinguish the sites or molecules of interest. In PEM, the topographical contrast is a consequence of the very low kinetic energies of the emitted electrons and the fact that the specimen is in an electric field [8]. Electrons emitted from slopes of negative or positive surface relief are deflected by the microfields at the surface of the specimen, causing some of them to be stopped out by the aperture of the electron-optical system. Electrons emitted from flat areas are undeflected and most of them pass through the aperture stop. The overall effect is that ~he images of sides of a protrusion are darker than the top and surrounding flat areas. This effect enhances the contrast of small fibers and other topographical features, making them m~ch more easily visible in the photoelectron imag~.. The material contrast mechanism in PEM is the dependence of the photoelectron quantum yield (electrons emitted per incident photon) on the electronic structure of molecules. Photoelectron quantum yields in the thresh,,ld region (180-230 nm) have been measured for amino acids, lipids, saccharides, and other biomolecules (for references, see ref. [4]). The material contrast is somewhat dependent on the length of exposure to UV radiation, so the measurement should be considered quafitative rather than quantitative unless special calibration curves have been generated, a step not required in the applications described here. In geaeral, highly conjugated moieculcb ,,,uch as the carcinogen, benzo(a)pyrene, and chlorophyll are the most photoemissive. Also very photoemissive are certain metals such as silver (see discussion of labeling below). As in fluorescence microscopy, the goal is to image the distribution
303
of a known label or marker. The material contra~r~ of PEM is very good for this purpose.
2.3. Depth of information, depth of field, and comparison with SEM All methods of imaging, even those classified as surface techniques, probe a finite volume of the specimen. The depth of information is defined as the distance below the surface from which information is contributed at a given resolution [9]. Another term that refers to this important physical parameter is depth resolution. For surface techniques in general, the depth of information is determined by a combination of the depth of penetration of the excitation source, the escape depth of the electrons (or X-rays, ions, or other detected signal) and the surface-etching rate, if any. "It PEM, there is no appreciable surface etching, and the UV photons penetrate relatively deeply into the specimen, so the determining factor is the escape depth of the photoelectrons. Measured escape depths for organic and biological surfaces are relatively short, from 1 to 10 nm [9]. Thus, the escape depths are on the order of the lateral resolution of PEM, and the depth of information is approximately equal to the electron escape depth. Because of the short escape depths of photoelectrons, PEM is among the most surface sensitive of the imaging techniques being applied in the biological sciences. Another parameter with a similar sounding name but very different meaning is the depth of field. The depth of field is the axial distance the object can be traversed and still appear to be in focus. The greater the depth of field, the larger is the range of topography on an object that will be in focus at the same lens settings. The depth of field in all microscopes is inversely proportional to . . . . times the hall .... v . . . . ,,.~ of thc magmficat~on -" an~le of accer~lance of the objective lens. TEM and SEM have the same expression for the depth of field, although SEM micrographs often give the impression of a greater depth of field because they are usually recorded at lower magnification. For PEM, the equation for the depth of field for two planar regions at different heights being brought into focus is similar to that ot TEM and SEM [10].
O.H. Griffith, G.F. Rempfer / Photoelectron imaging and photoelectron labeling
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However, the working depth of field in PEM for specimens with rapidly varying topography is greatly reduced because of the electron-optical effect of the microfields at the surface. In practice the depth of field is not usually a limiting factor in PEM. It is adequate for a wide range of cell surface and cytoskeletal studies. Photoelectron micrographs sometimes appear similar to scanning electron micrographs, but these two techniques have some important differences as diagrammed in fig. 5. PEM is not a scanning technique; the image is formed by collecting photoelectrons simultaneously from the entire portion of the specimen being viewed. The depth resolution of PEM is inherently greater than in SEM because of the method of image formation. In PEM, a Gaussian image is formed of electrons emerging from the surface of the specimen. Since topography is purely a surface effect, fine topographic detail is always imaged at the full instrument resolution in PEM. The farther below the surface the electrons originate, the more diffuse the information. Since the electron escape depths are short, this does not normally degrade the
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Fig. 5. A schematic comparison of depth of information of I'EM (a) and 5EM (b). (a) In PEM, hght ~s absorbed over a relatively large raa~c of depdas hi ~hc specimen, but only those electrons that are emitted v e ~ close to the surface, within a depth D 1 approximately equal to the escape depth L, can contribute to the image, tb) In SEM, the backscattered dectrons (BE) carry information from below the escape depth, and some of this signal is converted into secondary electrons (SE) near the surface, so that in either the BE or SE mode the deptil of information D3 is larger than D I
image. But even if there were a deeper source of electrons, analogous to the bloom area of an SEM, this would only superimpose a blurred image on the sharp image of the surface. The two images would not be averaged together. In SEM, however, the information is collected sequentially point by point, and the detector records all secondary electrons from the entire bloom area, thus averaging the image information and increasing the depth of information. This is consistent with the observations of Bode et al. [11] on bronze and cemented carbide samples, where surface detail appeared sharper in the PEM than in the SEM. The SEM has other advantages that make it a valuable and versatile technique, for example, the ability to detect many different signals. We view the PEM and SEM as complementary techniques that can be used together in studies of biological surfaces.
3. Specimen preparation Specimen preparation for photoelectron imaging parallels that for ether forms of electron microscopy except that two steps are omitted: the specimens are usually not stained as for TEM or coated with a conductive layer as for SEM. The specimen mount is a commercially available (Bellco Glass Inc., Vineland, NJ, USA) glass microscope cover slip, 5 mm in diameter. Solid metal supports as commonly employed in SEM would be acceptable specimen mounts. The advantage of glass coverslips is that the specimens can be viewed first in an optical mic:o.~cope. This makes possible rapid screening of specimens before selection for photoelectron imaging. The coverslips are coated on both sides with a thin conducting layer sinc~ electrons lost througi~ photoemission must be replenished from the power supply via the cathode stage and sample support. The conducting layer to the coatings used on the inside face of cathode ray osciloscopes and TV tubes, or a ,ran evaporated metal layer such as chromium, which we are currently using. For experiments involving cultured cells, the conductive glass discs are sterilized, coated with serum proteins, and placed into the b~-,ttom of tissue culture dishes. The ceils then t 1
*
O.t1 Griffith. G.F. Rempfer / Photoelectron imaging and photoelectron labeling
grow on the coated glass discs, and the extent of coverage can be observed with a conventional inverted-stage microscope. For cells free in suspension, for example red cells or sperm, a few drops of suspension are placed on the conductive glass disc. Adhesion of cells to these discs is facilitated by first dipping the discs in 1 m g / m l aqueous solutions of alcian blue, cytochrome c, or poly-L-lysine, and air drying before use. Chromosomes, D N A a n d other cell c o m p o n e n t s can be examined by similar procedures, with adaptations as required to insure good spreading of the specimens. Whole cell preparations and cytoskeletal preparations must be fixed to preserve the delicate structures. For immunolabeling studies, there is a wel| known trade-off between preserving antigenic integrity and preserving physical structures, irrespective of the type of trficroscopy used. Treatments that preserve structures well, e.g. prolonged gluteraldehyde fixation, tend to abolish the antigenicity. Also, detergent extraction procedures to remove m e m b r a n e s and provide access for the antibodies to the cell interior c'~.n affect cytoskeletal structure and composition. One solution is to use a water-soluble crosslinker, suG, as DTSP, to gently prefix and stabilize structure- [13]. Another prefixation procedure involves a brief exposure to a very dilute solution of glutaralde-
305
hyde (0.025%) prior to detergent extraction [~i4]. Labelling with photoemissive molecules is ,,"iscussed in the next section. Another considerat,on is that the structures of interest must be expoaed after treatment because PEM is a surface technique. For example, fragments of membranes remaining on the surface after detergent treatment would not be visible in a fluorescent micrograph of cytoskeletal elements but could obscure details in a photoelectron micrograph of the same preparation. Several types of sample preparation procedures have been tested for use in photoelectron imaging [15]. T h e photoelectron microscope requires, as do all electron microscopes, a vact~um to prevent air molecules from colliding with the information-carrying electrons. The specimens must therefore be either quick-frozen or dehydrated before insertion into the instrument. Currently all samples are dried by conventional procedures similar to those used in TEM and SEM. Typically, the fixed cell preparations are washed with glass-distilled water and then dehydrated through a series of aqueous ethanol mixtures beginning with 70% ethanol followed by critical-point drying trom CO, or vacuum drying from Freon 113. Alternatively, the newer cryofixation methods should also be applicable. The final specimens on the conductive glass discs are mounted on a metal rod as s':c,wn in fig. 6,
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Fig. 6. Sketch of th,: specimen suppert. The standard specimen mount is a round glass coverslip, 5 mm in diameter. The coverslip is coated on both sides with a thin layer of conductive metal (e.g. chromium) or tin oxide. For tissue culture preparations the support is then coated with serum on which the cells grow. The treated glass coverslip with the attached cell culture is placed on tile end of a metal rod, secured in place by a retaining ring, and inserted into the photoelectron microscope cathode. The cell and la~'ers are sketched orders of magnitude larger than actual size.
306
O.H. Griffith, G. F Rempfer / Photo~..'ctron imaging and photoelectron labeling
and inserted with the specimen facing down in the photoelectron microscope. The photoeletron microscope used for this work is an ultrahigh vacuum instrument designed and built at the University of Oregon and Portland State University [16]. In order to eliminate specimen contamination, ion pumps and sorb pumps are used in place of oil diffusion and fore pumps, and bellows seals are used in place of dynamic O-rings. The instrument is constructed primarily of stainless steel and utilizes copper sealed flanges. The electron lenses are of the electrostatic unipotential type [17]. Typical operating conditions are: acceleration voltage of 30 kV, illumination provided by two OSRAM HBO 100 W / 2 Hg short-arc lamps, and an objective aperture of 50
pointed out in the early work of Coons, who introduced the technique of chemically attaching dyes such as fluorescein to antibodies [19]. The antibodies then bound to their specific antigenic sites, and produced a bright fluorescence pattern in the modified optical microscope. Immunofluorescence microscopy is now one of the most widely used techniques in cell biology, and it has made possible, for example, much of our understanding of the cytoskeietal structures of cells. The same concept of labeling has been extended to TEM and SEM through the use of electron-dense markers, with good success in specific cases. 4.1. An overoiew of markers in electron microscopy
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4. Fhotoelectron labeling As an application of photoelectron imaging, we discuss the use of photoemissive markers in combination with site-specific antibody molecules. By analogy to immunofluorescence microscopy, this combination of methods is called immunophotoelectron microscopy [18]. This is not the only area of application, as photoelectron imaging can be used without labels. All four fundamental building blocks of life (amino acids, saccharides, lipids, and nucleic acids) exhibit some photoemission under the action of UV light. The topography of an exposed surface can be photographed without labels, and in certain cases where naturally photoemissive molecules are present (e.g. chlorophyll or hemes), labeling may not be needed or desirable. However, in most applications the goal is to distinguish the location of one protein in the presence of other proteins of similar amino acid content, and no significant material contrast is pre,;cat. Here, labeling provides the only sure way of ........ _y,,,~ ............. ,~ of proteins such as specific enzymes or receptors. The situation is reminiscent of fluorescence microscopy. ,.,.'here even without labeling some fluorescence (autofluorescence) is present. Only in special cases is autofluorescence useful in relating structure and function. The way to overcome this difficulty was
In the search for markers for photoelectron microscopy, it is instructive to step back and view the wide range of markers that have been used in various forms of optical and electron microscopy, iacluding TEM and SEM. Fig. 7 shows several of l hese markers, drawn so that the markers are scaled correctly relative to the dimensions of the Y-shaped antibody molecule. One of the largest
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Fig. 7. A schemalic represen|ation of the relatiw :,ize~ of macromolecular markers u.,,ed it, electron m~croscopy.
O.H. Griffith. G.F. Rempfer / Photoelectron imaging and photoelectron labeling
markers used in SEM is tobacco mosaic virus (TMV). TMV contains no heavy metal atoms so there is essentially no material contrast:. This is purely a topographical marker and because of its very large length relative to the antibody, TMV has not been w~dely used. Hemocyanin shown below TMV in fig. 7 is much smaller and is frequently used in SEM. There are no significant concentrations of heavy metals (other than a low density of copper atoms) and so the label is a topographical marker, recognized solely by its size and shape. On the other hand the third marker shown, ferritin, contains electron-dense iron atoms and is used as a marker in TEM. Labeling with these markers and others, such as latex spheres, has been reviewed by Molday [20]. Another type of marker, and one that is gaining in popularity in all areas of microscopy, is colloidal gold [20-22]. It has many advantages starting with the uniform shape, near-perfect spheres of gold that cannot be confused with biological surface structures. Gold probes are electron dense as required in TEM, and exhibit sufficient emission of backscattcred and secondary electrons to be useful in SEM. They also are visible in light microscopy in higher concentrations or after silver enhancement [23]. Colloidal gold can be prepared by a wide selection of reducing agents, and procedures are available for generating relatively homogeneous preparations of just about any desired diameter, from ross than 5 nm to 150 nm. The smallest colloidal gold particles used have about the same dimensions as the antibody itself (see fig.
%
Fig. 8. Diagram showing the basic scheme of cell surface labeling for photoelectron imaging (immunophotoelectron microscopy). Requirements for PEM markers (M): (a) high photoelectron quantum yield, (b) distinctive size and shape, (c) stable, (d) high affinity binding to ligand (e.g., antibody, lectin, biotin), (e) minimal nonspecific binding to cell surfaces.
307
7). The attachment of the colloidal gold to the antibody is achieved by mixing the two solutions together with an appropriate stabilizer. The antibody is adsorbed onto the gold particles (clean metal surfaces of any geometry will, in general, bind protein in the appropriate pH range). A significant fraction of the antibody bound to gold retains its binding activity, and the preparations are stable. These advantages combined with the commercial availability of a selection of gold probes have led to a large and growing literature on colloidal gold labeling.
4.2. The choice of photoelectron labels The ideas of immunofluorescence microscopy can also be extended to photoelectron microscopy as outlined in fig. 8. A photoemissive marker (M) is coupled to an antibody, and then allowed to diffuse into the specimen and bind to the specihc antigen. In practice, attaching a marker to a specific site is usually a two-antibody process (socalled indirect imnmnolabeling). The first antibody binds to the specific antigen in the specimen, and the second ap.tlbody, carrying the marker, binds to the first antibody. The ideal PEM marker would: (1) have a very hio~ pbmoelectron quantum yield (electrons emitted per incident photon), (2) be of small size, (3) be stable, and (4) exhibit no nonspecific binding to the cell surface. A natural starting point in the search for photoelectron labels was to try the first fluorescent probes introduced by Coons in 1941. In many ways these are ideal markers, being much smaller than the antibody molecule itself (fig. 7). Photoelectron quantum yields measured in the conventional way at very low light levels indicated that the dyes are much more photoemissive than cell surface components in the 230 nm to 180 nm wavelength range. However, under the more intense illumination necessary to achieve high magnifications in the photoelectron microscope, the Fhotoemission of proteins and other biological surface coniponents increases so that the contrast between label and background decreases with exposure to UV light [24]. The net result is that for the low labeling ratios typically used in fluorescence microscopy (e.g. one dye per antibody), the
308
O.H Griffith, G.F. Rempfer / Photoelectron imaging and photoelectron labeling
contras~ is not sufficient far a photoelectron labeling experiment. Much higher labeling ratios can be u::ed, but there are now better alternatives. Curr mt research in photoelectron imaging uses colloidal gold markers and silver-enhanced colloidal gold. Colloidal gold has been shown to be photoemissive and easily detected in photoelectroll images of lectin binding sites on erythrocyte ghosts [25] and in antibody labeling of cytoskeletal elements on cultured cells [18]. Silver-enhanced gold exhibits even higher contrast [26].
The reasons for the improved contrast are larger particle size and, more importantly, the higher q u a n t u m yield of silver and silver oxides and the generation of a relatively clean surface from which the photoelectrons can escape.
4,3. Photoelectron micrographs of cytoskeletal elements and cell surface fibronectin Cells of higher organisms contain comple× networks of protein filaments collectively called " t h e
Fig. 9. Immunofluorescence (insert) and immunophotoeh:ctron micrcgraphs of per'Sans of the same rat embryo fibroblast labeled for actin visualization, using rhodarnine as the fluorescer ce marker and colloidal gold as the photoelectron marker. Bar = 10 # m.
O.H. Griffith, G.F. Rempfer / Photoelectron imaging and photoelectron labeling
cytoskeleton', which are involved in the shape, internal organization and movement of cellular components. The three main components are actin filaments, microtubules, and the intermediate filaments. The results of a double !~befing experiment for the visualization of actin are shown m fig. 9. The specimen is a rat embryo fibroblast cell that was grown directly on the type of serum-coated conductive glass disc diagrammed in fig. 6. The membranes of the cell were removed by brief treatment with a dilute aqueous sohLtion of the mild detergent, Triton X-100. The specimen was then washed in detergent-free buffer and fixed in - 2 0 ° C methanol for 5 min. The resulting cytoskeletal preparation was exposed to a monoclonal antibody recognizing actin followed by a rhodamine-labeled second antibody recognizing the fir~:, and a 20 nm colloidal gold-labeled antibody recognizing the second antibody. After a final fixation in 2% glutaraldehyde, the fluorescence micrograph (insert, fig. 9) was recorded and the specimen was then dehydrated and inserted into the PEM to obtain the photoelectron micrograph. Thus, the fluorescence and photoelectron micrographs of fig. 9 are of portions of the same cell, and many of the actin-containing fibers can be traced in both micrographs. Clearly, however, the fibers are imaged in the photoelectron micrograph at much higher resolution. Many of the lightly labeled fibers do not show up in the fluorescence
"
300
micrograph, whereas essentially all labeled structures are visible in the photoelectron micrograph. The higher resolution permits the imaging of single antibodies, which appear as bright dots along the fibers in the photoelectron micrographs [15,18]. Photoelectron imaging using photoelectron labels can also be performed on cell surfaces and the extracellular matrix, as diagrammed in fig. 10. One ,rroblem of interest is the effect of tumor promoters on cells. Tumor promotors are molecules that are not in themselves cancer-causing agents, but greatly increase the incidence of tumors when present with carcinogens [27]. One effect of the potent tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate~ is to cause an increased release of fibronectin from fibroblasts. Fibronectin is a major component of the extracellular matrix that promotes cell adhesion and is present in reduced amounts on fibroblasts derived from tumors [28-30]. Photoelectron micrographs of normal human foreskin fibroblasts as a function of exposure to TPA show a loss of the fibronectin from the upper cell surface (fig. 11, ref. [31]). The cells were labeled for fibronectin visualization by a three-step immunolabeling procedure with a colloidal gold-antibody complex and subsequent silver enhancement. Before treatment, the labeled fibronectin appeared as both dense networks and ha numerous fibrillar streaks against the less photoemissive uncoated cell surface (fig. ]]a). After
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Fig. 10. Cro~,,,-~ectiona] drawing representing enhanced e~nisslon from labeled fibronectin (dotted bands) on cell surfaces. More ,qectron emission (arrows) comes from the silver-coated ~,old markers bound to the fibronectin than from the unlabeled cell surface, giving rise to corresponding bright areas in the image. Labeled fibronectin beneath the cell does not contribute to the image. From [31] with permission.
310
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Fig, 1i. Photoelectron micrograph illus~ 'ating the effect of 100 ng/ml TPA on the amount and distribution of fibronectin remaining on human foreskin fibroblast (FS-2) celi surfaces. (a), exposed to medium contairfing 0.02% DMSO only for 40 rain, (b-d) exposed to I'PA, 0.02% DMSO i.n medium for (b) 5 rrdn, (c) 10 rain, (d) 40 min. (a) and (b) show overlapping Fortions of several cells. From ref. [3!] with permission.
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• • v - o" ul f i n-tln of e x p o s u r e to ~n,, , u v , ~__ / , u,__, -~,-,, ~ r ~ in the c u l t u r e m e d i u m , t h e r e w a s a visible d e c r e a s e in the u p p e r cell surfa.-e f i b r o n e c t i n (figs. l l b a n d l l c ) . A f t e r 60 m i n n . ' a r l y all of the u p p e r cell s u r f a c e f i b r o n e c t i n w a s ~ o n e (fig. i l d ) . H o w e v e r , the ex-
t r a c e l l u l a r m a t r i x f i b r o n e c t i n b e t w e e n cells ap p e a r s to be m o r e r e s i s t a n t to release a n d c a n still
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t_,~ seeii in the p_in.u. .t .o. ~ l 1~.u. .t .r .u i l ifliages. ,v,l_: lnl~. . . ~tuuy . . j_. d e m o n s t r a t e d t h a t t h e initial f i b r c m e e 6 n rele.a~e c a u s e d by the t o m o r p r o m o t e r is at ~he e x p e n s e o f p r e - e x i s t i n g c e l l - s u r f a c e fibror~ectin. It is a l s o a good example a c h i e v a b l e in surfaces.
of the resolution and photoelectron imaging
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O.H. Griffith, G. 1:. Rempfer / Photoelectron imaging and photoelectron labeling
5. Advantages, limitations, and future directions It is essential to establish the limitations ,,s well as the advantages of each new technique in order to know which combination of methods is most appropriate for a particular application. We have found that virtually every specimen examined emitted a sufficient current of photoelectrons to produce an image, so the range of potential applications is large. The main limitation, other than lack of commercial instrumentation, is the roughness of the surface of the specimen. In this respect PEM is complementary to SEM. Many specimens that are very difficult or impossible to view by SEM can be seen clearly in PEM. In this category are relatively flat cell surfaces, cell membranes, fine fibers, and nucleic acids. Conversely, rounded up cells present no problem for SEM but are not suitable specimens for PEM. This is because the height and curvature of the cell affects the electric field at the surface, and consequently a significant proportion of the slow-moving photoelectrons emerging from the surface are deflected too much to be included in the angular aperture of the optical system. This results in dark areas in the image corresponding to areas of extremes in sampie topography. On the other hand, it is this same sensitivity to topography that makes possible the detection of very fine topographical detail not easily visualized by SEM. To summarize, the advantages of PEM are (1) a direct extension of the immunofluorescence technique using similar labeling methods and the advantages of emission (detecting a bright object against a darker background), (2) high sensitivity to fine topographic detail on relatively flat surfaces, (3) high depth resolution (short escape depth of the photoelectrons), and (4) lower damage to biological specimens than is caused by electron beams since UV light causes less mass loss than does charged particle radiation. There also appcu~,,, to be a lower specimen conductivity requirement since it has not been necessary to coat thin biological specimens with a conductive metal or carbon layer. Part of this increased conductivity' may be due to photoconductivity produced by the UV and visible light striking the sample, but another factor is that the electrons are drawn off
311
at a low and continuous rate from the entire specimen surface being viewed, and not from a Ic,c'aliT~d spo! as in an SEM or scanning electron microprobe. Looking towards the future is always difficult, but it is useful to speculate. There are two general areas of application. One involves the UV excitation described above. We expect that wi& continued improvements in instrumentation, photoelectron imaging with UV excitation will be increasingly used in c,o,,,o,,,~,on . . . . '-: .... " with immunofluorescence microscopy and photoemissive labels to provide new information regarding the distribution of binding sites on biological structures. The other area involves X-ray excitation. By equipping the photoelectron microscope with a suitable intense X-ray source and energy analyzer. it should be possible to perform microanalysis of biological surfaces. In addition to elemental distributions, the images would contain information about chemical bonding since the binding energies of the inner shell electrons are influenced by their chemical surroundings, just as X-ray photoelectron spectroscopy (XPS or ESCA) provides this information but without the imaging capability. The first steps towards imaging photoelectrons released by X-rays have already been taken [32-34]: see also the review [6]. PEM has a different set of advanlages and disadvantages from SEM and the microprobe methods, so it is likely that ail of these instruments will be used in combination to study biological specimens. The current resolution of PEM is 10 nm with a possibility of eventually reaching 2 nm. TEM will remain the highest resolution technique but there are many cases where this high resolution is not realized, or where contrast factors, information content, or specimen damage become more important. In the final analysis, Mother Nature provides a range of challenges far greater then the capabilities of any one technqiue, and all of these methods are needed ili gi ~rt,au-spcctrum approach to so~xang structure-function problems in cell bioloov I
I
.
1
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Acknovdedgements We are pleased to acknowledge the many contributions of our colleagues Drs. G. Bruce Birreii
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O.H. Griffith, G.F. Rempfer / Photoelectron imaging and photoelectron labeling
and Karen K. Hedberg, and Douglas L. Habliston, Walter P. Skoczylas and Loreene Evans. This work was supported by PHS grant CA 11695 from the National Cancer Institute and a grant from the M.J. Murdock Charitable Trust.
References [1] P. Echlin, Ed., Analysis of Organic and Biological Surfaces (Wiley, New York, 1984) pp. 1-646. [2] D.L. Taylor, A.S. Wag,goner, R.F. Murphy, F. Lanni and R.R. Birge, Eds., Applications of Fluorescence in the Biomedical Sciences (Liss, New York, 1986) pp. 1-639. [3] R.A. Schwarzer, Microsc. Acta 84 (1981) 51. [4] O.H. Griffith, K.K. Nadakavukaren and P.C. Jost, in: Scanning Electron Microscopy/1982, Ed. O. Johari (SEM, AMF O'Hare, IL, 1984) p. 633. [5] O.H. Griffith and G.F. Rempfer, Ann. Rev. Biophys. Biophys. Chem. 14 (1985) 113. [6] O.H. Griffith and G.F. Rempfer, in: Advances in Optical and Electron Microscopy, Vol. 10, Eds. R. Barer and V.E. Cosslett (Academic Press, London, 1987). [7] G.F. Rempfer and M.S. Mauck, in: Proc. 43rd Annual EMSA Meeting, Louisville, KY, 1985, Ed. G.W. Bailey (San Francisco Press, San Francisco, CA, 1985) p. 132. [8] G.F. Rempfer, K.K. Nadakavukaren and O.H. Griffith, Ultramicroscopy 5 (1980) 437. [9] W.A. Houle, W. Engel, F. Willig, G.F. Rempfer and O.H. Griffith, Ultramicroscopy 7 (1982) 371. [10] G.F. Rempfer, K.K. Nadakavukaren and O.H. Gfiffith, Ultramicre~copy 5 (1980) 449. [11] M. Bode, G. Pfefferkom, K. Schur and L. Wegmann, J. Microscopy 95 (1972) 323. [12] O.H. Griffith, G.F. Rempfer and K.K. Nadakavukaren, in: Electron Microscopy, Proc. 10th Intern. Congr. on Electron Microscopy, Hamburg, 1982, pp. 59-68. [13] P. Bell, Jr., in: Scanning Electron Microscopy/1981, Voi. II, Ed. O. Johari (SEM, AMF O'Hare. iL, 1981) p. 130. [14] L.B. Chen, I.C. Summerhayes, K.K. Nadakavukaren, T.J. Lampidis, S.D. Bemal and E.L. Shepherd, in: Cancer
Cells, Vol. 1, The Transformed Phenotype (Cold Spring Harbor Laboratory, 1984) pp. 75-86. [151 K.K. Nadakavukaren and O.H. Griffith, Ultramicroscopy 17 (1985) 31. [16] O.H. Griffith, G.F. Rempfer and G.H. Lesch, in: Scanning Electron Microscopy/1981, Vol. II, Ed. O. Johafi (SEM, AMF O'Hare, IL, 1981) p. 123. [171 G.F. Rempfer, J. Appl. Phys. 57 (1985) 2385. [181 G.B. Birrell, D.L. Habliston, K.K. Nadakavukaren and O.H. Gdffith, Proc. Natl. Acad. Sci. USA 82 (1985) 109. [191 A.H. Coons, H.J. Creech and R.N. Jones, Proc. Scc. Expti. Biol. Med. 47 (1941) 200. [2o] R.S. Molday, in: Techniques in Immunocytochemistry, Vol. 2, Eds. G.R. Bullock and P. Petrusz (Academic Press, London, 1983) pp. 117-154. [211 J. De Mey, in" lmmunocytochemistry: Practical Applications in Pathology and Biology, Eds. J.M. Polak and S. Van Noorden (Wright, Bristol, 1983) p. 82. [221 J. Roth, in: Techniques in Immunocytochemistry, Vol. 2, Eds. G.R. Bullock and P. Petrusz (Academic Press, London, 1983) pp. 217-284. [231 G. Danscher and J.O.R. Norgaard, J. Histochem. Cytochem. 31 (1983) 1394. 1241 O.H. Griffith, D.L. Hoimbo, D.L. Habliston and K.K. Nadakavukaren, Ultramicroscopy 6 (1981) 149. [251 G.B. Birrell, S.M. Rose and O.H. Griffith, Ultramicroscopy 12 (1983) 213. [26] G.B. BirreU, D.L. Habliston, K.K. Hedberg and O.H. Griffith, J. Histochem. Cytochem. 34 (1986) 339. [271 P.M. Blumberg, CRC Critical Rev. Toxicology 8 (1980) 153. I281 R.O. Hynes, So.. Am. 254 (1986) 42. [29] K.M. Yamada, in: Cell Interactions and Development, Ed. K.M. Yamada (Wiley, New York, 1983) pp. 231-249. [301 E. Ruoslahti, M. Pierschbacher, E.G. Hayman and E. Engvall, Trends Biochem. Sci. 7 (1982) 188. [311 D.L. Habliston, G.B. Birrell, K.K. Hedberg and O.H. Griffith, European J. Cell Biology 41 (1986) 222. [32] J. Cazaux, Rev. Physique Appl. 8 (1973) 371. [33] J. Cazaux, in: Scanning Electron Microscopy/1984, Vol. III, Ed. O. Johari (SEM, AMF O'Hare, IL, 1984) p. 1193. [341 G. Beamson, H.Q. Porter and D,W. Turner, Nature 290 (1981) 556.