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Electron Microscopy of Negatively Stained and Unstained Fibrinogen (Scanning Transmission Electron Microscopy) LEONARD F

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Electron Microscopy of Negatively Stained and Unstained Fibrinogen (Scanning Transmission Electron Microscopy) LEONARD F Proc. Natl. Acad. Sci. USA Vol. 77, No. 6, pp. 3139-3143, June 1980 Biochemistry Electron microscopy of negatively stained and unstained fibrinogen (scanning transmission electron microscopy) LEONARD F. ESTIS* AND RUDY H. HASCHEMEYER Department of Biochemistry, Cornell University Medical College, 1300 York Avenue, New York, New York 10021 Communicated by Alton Meister, December 26,1979 ABSTRACT Electron microscopic images of negatively are always met: (i) The great majority of images of "apparent stained fibrinogen are predominantly asymmetric rods 450 A particles" in any field are of fibrinogen molecules. (Ui) The in length and about 60 A in width. The molecules appear to have considerable flexibility, and mass distribution along the major number of identifiable fibrinogen molecules is defined by fi- axis is not uniquely distinguished despite apparent beading in brinogen concentration and application time and not by the some particles. Scanning transmission electron microscopy of buffer, pH, or staining conditions. (Mii) Large-field views of unstained fibrinogen again demonstrates that a majority of fibrinogen contain predominantly well-separated molecules molecules are rodlike. The results differ from those obtained with clearly defined morphology and dimensions. by negative staining in that a substantial fraction of images are Conditions trinodular with striking resemblance to those obtained by C. required to achieve these goals are primarily E. Hall and H. S. Slayter IJ Biophys. Bioehem. Cytol. (1959) 5, related to attachment of fibrinogen to the substrate film. Once 11-161 using the mica replictecaique. The above results were this problem was solved, images of the unstained molecule were obtained on glow-discharged carbon substrate films by a simple also obtained by high-resolution scanning transmission electron low-concentration, lon&-attachment-time modification of microscopy (STEM). standjard deposition methods that is diffusion controlled and The data presented here may be combined with published depnds on concentration and time but is independent of pH, buffer, and other staining conditions. Evidence is presented gat work to present an overall interpretation of the general mor- standard attachment procedures result in artifactual images. phology of the fibrinogen molecule that is free of ambigui- Any model of fibrinogen in solution consequently must en- ties. compass properties that permit its visualization as an asym- metric rod by electron microscopy as first suggested by Hall and MATERIALS AND METHODS Slayter 20 years ago. The fibrinogen used in this work was a kind gift of Birger Electron microscopic investigation of the size and shape of the Blomback. For the neutral pH studies, bovine or human fraction fibrinogen molecule and fibrin clot substructure has led to a I-4 (>95% clottable), lyophilized or frozen in 0.1 M Tris.HCl multitude of images and conflicting interpretations (1-10). pH 7.4, was dialyzed against 0.02 M sodium phosphate buffer. Consistently well-defined large-field images have to date re- For the low pH studies, the fibrinogen was dialyzed against 0.01 sulted primarily from the shadowed images obtained by the M acetic acid. Covalent crosslinking of fibrinogen was ac- mica replica technique. This work was originally done by Hall complished by adding 10% glutaraldehyde to a solution of bo- and Stayter in 1959 (2) and was later reproduced by Gorman vine fibrinogen at 1.5 mg/ml (0.2 M sodium phosphate buffer, et al. (11), and essentially identical results are readily obtained pH 7.0), to give a final glutaraldehyde concentration of 2%. The in our own laboratory. From the Hall and Slayter work, the solution was allowed to react at room temperature for 90 min rodlike trinodular structure of fibrinogen was proposed. and was then extensively dialyzed at 40C against 0.02 M sodium Shadowed replicas of freeze-etched fibrinogen recently ob- phosphate buffer, pH. 7.0. Despite long dialysis and numerous tained by Bachmann et al. (10) also indicate that the molecules buffer changes, negative staining of the glutaraldehyde-fixed have a rodlike structure with length similar to that obtained by protein often resulted in "glutaraldehyde-induced background" Hall and Slayter but with a greater width indicative of more images as if the fixative had not been removed. extended protein domains. Fibrinogen was negatively stained by first diluting the pro- Electron microscopic studies involving the high-resolution tein to a concentration of about 1-2 ,g/ml in either the 0.02 technique of negative staining have resulted either in over- M sodium phosphate buffer or 0.01 M acetic acid. A copper grid crowded images wherein one cannot see isolated molecules or with attached thin carbon film (100-150 A), which had been in small viewing fields where it is difficult to be certain if a subjected to a low-vacuum (t10-1 torr; 1 torr = 1.333 X 102 "particle" is fibrinogen or the result of electron microscopic Pa) glow discharge at about 5000 V for 2 min, was floated on artifacts. These studies have been interpreted in terms of a the surface of the fibrinogen solution. The grid was then number of models for the fibrinogen molecule, ranging from transferred to the surface of the staining solution, either 2% a much more elongated multibeaded molecule (4, 7) to an iso- sodium phosphotungstate (NaPTA) or 0.5% uranyl acetate. The tropic pentagonal dodecahedron (5). We present evidence here grid was removed after 1 to several minutes and excess stain was that conventional negative staining methods, which are so removed by touching the edge with a piece of filter paper. In successful in imaging well-behaved macromolecules, fail to a typical time series experiment, four grids were floated on four provide negatively stained fields of fibrinogen that fulfill rea- separate fibrinogen solutions at identical concentration and sonable prerequisites for meaningful interpretation. were removed after various times to permit variation in the time Our major purpose here is to define precise conditions for the allowed for fibrinogen attachment to the carbon film-e.g., 5, negative staining of fibrinogen wherein the following criteria 10, 15, and 20 min. The publication costs of this article were defrayed in part by page Abbreviations: STEM, scanning transmission electron microscopy; charge payment. This article must therefore be hereby marked "ad- NaPTA, sodium phosphotungstate. vertisemenz" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: Waksman Institute of Microbiology, Rutgers Uni- this fact. versity, P.O. Box 759, Piscataway, NJ 08854. 3139 Downloaded by guest on September 28, 2021 3140 Biochemistry: Estis and Haschemeyer Proc. Natl. Acad. Sci. USA 77 (1980) C. Ni\ $ *r-J ?t mw~~~~~ 2ak FIG. 1. Fibrinogen negatively stained with NaPTA. (Upper) Human. (X225,000.) (Lower) Bovine. (X440,000.) Electron microscopy was performed on a JEOL 100-B mi- using procedures to minimize beam damage to the specimens. croscope equipped with an anticontamination device. The An area near to that to be photographed was visualized at high operating voltage was 80 kV and a 20-um objective aperture magnification (X100,000) to correct astigmatism. The same was used. The typical electron optical magnification employed nearby area was then brought into near focus at the photo- for photography was X50,000. Photography was performed by graphing magnification of X50,000. The desired area was then 4 $ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~is i 4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1r<. ;..Ad<*4'ta FIG. 2. Bovine fibrinogen negatively stained with uranyl acetate. (X225,000.) Downloaded by guest on September 28, 2021 W.**>4 Biochemistry: Estis and Haschemeyer Proc. Nati. Acad. Sci. USA 77 (1980) 3141 4 qrich FIG. 3. Bovine fibrinogen negatively stained with NaPTA after varying time of protein attachment: 1 min (Left); 5 min (Center); 10 min (Right). (X235,000.) brought into position, the electron beam was deflected, and, Fig. 1 Upper shows the characteristic reproducible images after a short period of time to permit stage stabilization, the obtained when a grid is floated for 10 min on a dilute solution beam was recentered and the noncontaminated area was of human fibrinogen (-1-Ig/ml) in 0.02 M phosphate buffer quickly photographed. (pH 7.0), followed by floatation on 2% NaPTA (pH 8.0) for 2 Areas chosen for photography were typically near the edge min. A similar result is obtained with bovine fibrinogen (Fig. of a smooth wavelike depression in the carbon film. These de- 1 Lower). The predominant images in the field of view are seen pressions, or channels, presumably formed as the carbon film to be rodlike with dimensions of approximately 450 A in length was attached by sweeping the grid smoothly upward from and 60 A in width. Some of the images appear to be bent or below the water surface, show a gradation in density of negative S-shaped and, in some, apparent beading is evident. Sometimes stain. The spreading of negative stain on the glow-discharged images appear to have a different staining intensity in the two grids presents a final smooth surface with very light stain over halves defined by the center of the rod out to each end along most of the grid to thick stain in the center of the channels. the long axis. Results obtained with highly purified human and Fibrinogen is poorly contrasted in areas of very light stain bovine fibrinogen show no distinguishable differences. compared to other proteins, whereas near the center of the Other images in the field of view, which are generally channel the stain is too thick. Areas near the periphery of the spherical in appearance with a wide range of diameters (10-200 channels provide excellent contrast and were selected for most A), are often seen in negatively stained preparations of other of the negatively stained photographs. proteins and in protein-free controls. Their origin appears to Magnification calibration utilized catalase crystals (Worth- be due to minor contamination of the buffers used.
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