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A technique for obtaining a surface replica and a negatively stained image from the same biological object
Micron, 1974,5~51-55 with I plate
51
A technique for obtaining a surface replica and a negatively stained image from the same biological object G. J. HILLS, K. ROBERTS and M. G U R N E Y - S M I T H John Innes Institute, Colney Lane, Norwich N O R 7OF, England, U.K.
Manuscript received March 21, 1974
A new technique is described that allows precisely the same area of a biological specimen to be viewed in the electron microscope both by shadowing and by negative staining. Both techniques increase contrast; however, with shadowing the upper surface of the specimen is shown in relief while negative staining produces a two-dimensional image. With the combined technique a direct correlation can be achieved between the structure of the surface and of the interior o~ an object. Une nouvelle nffthode est dgcrite oh le m~rne emplacement d'un gchantillon biologique peut gtre observd en microscopie dlectronique simultan~ment par ombrage et par coloration nggative. Les deux techniques amgliorent le contraste. Gependant l'ombrage permet l'observation de la pattie supgrieure de la surface de l'~chantillon tandis que la coloration rdgative produit une image en deux dimensions. En combinant les deux techniques une corrglation directe peut ~tre ~tablie entre la structure de la surface et celle de l'intgrieur d'un objet. Ein neues Verfahren wird beschrieben, daft es m6glich macht, den gleichen Bereich einer biologischen Probe sowohl durch Schritgbediimpfung als anch durch Negativfarbung zu untersuchen. Beide Verfahren erh6hen den Kontrast, aber die Schritgbediimpfung zeigt das Oberfliichenreli~ der Probe, wighrend Negativfiirbung ein zweidimensionales Bild erzeugt. Mit diesem I(ombinationsverfahren ist es m6glich, die Zusammenhiinge zwischen Oberfl&henstrUktur und Innenstruktur zu bestimmen.
INTRODUCTION There are severe limitations to the retrieval of three-dimensional information fi'om biological objects in the electron microscope. Shadowing provides us with a surface view only, while negative staining, although projecting information from different depths in the specimen, only gives us a final recording in two dimensions. Clearly, more information could be obtained if these two techniques could be combined. This short paper describes our development of such a technique together with some applications. The technique provides a means of viewing precisely the same specimen area whose contrast is increased both by shadowing and by negative staining. Essentially the technique involves recording the normal negative stained image and then removing the stain followed by surface shadowing. A comparison of the optical transform (Klug and Berger, 1964) of a straight-forward replica with a replica made after the elution of a negative stain from a specimen that had been examined in the microscope, revealed that essentially the same structure is present and to the same resolution. From this we may conclude that the process of negative staining and subsequent examination in the electron microscope did not destroy the quarternary structure over about 4.0nm in the specimens that we examined. If directional shadowing is performed, detail in line with the direction of shadow is suppressed compared with detail normal to the direction of shadow. To obtain uniform information all shadowing was performed while the specimen was being rotated at about 200 r.p.m., the angle of shadow being about 15°.
52
H11,1.S, ROBERTS AND (;URNEY-SMITH M A T E R I A L S AND M E T H O D S
A suspension of the objects to be studied is mixed and dried down with a negative stain onto a map reference electron microscope grid (Micron Index II grids are very suitable) covered with an evaporated carbon film stripped from mica. The negative stain used here was a 5% aqueous solution of ammonium molybdate. The grid is inserted into the electron microscope and areas of interest photographed, noting the grid reference of that area. The grid is removed from the electron microscope and the negative stain eluted by the use of Webb's (1972) method. It has been found that the majority of negative stains normally used are removed by washing for about 20rain. Care should be taken to choose a non-reactive negative stain for the particular subject being studied as positive attachment of heavy metals may alter the surface structure o f the object. The grid is dried after complete stain elution and is then rotary shadowed at an angle of about 15 ° to the grid surface. The grid is reinserted into the electron micro.scope and areas, relocated by their grid references, are recorded. By comparison of the map references of the areas recorded on the plates from both methods of contrasting, matched pairs of plates can be sorted. R E S U L T S AND D I S C U S S I O N The above technique has been used to elucidate the structure of the cell wall of the alga Chlamydomonas reinhardi (Roberts, Gurney-Smith and Hills, 1972; Hills, GurneySmith and Roberts, 1973). Fourier transforms of micrographs of the same area of cell wall using either negative staining (Figure 1) or shadowing (Figure 2) can be directly ,compared. The relationship of parameters and angles of the lattices of the surface of the cell wall and the cell wall in depth as seen by negative staining can be determined and the Fourier transform which corresponds to the top wall and external ceil wall surface upwards selected. This technique readily gives this type of information which is necessary for structural analysis, and we believe that this is the most simple and sometimes the only way to obtain this information. If a non-reactive negative stain cannot be found for the material being studied the technique can still be used. Figures 3 and 4 illustrate an aggregate of catalase crystals together with their Fourier transforms. The Fourier transforms are from identical areas of the micrographs and demonstrate that only one of the periodicities seen in the negative stained image is due to surface structure. At least three different types of :structure are seen by shadowing catalase crystals but the Fourier transforms of negative stained crystals are similar irrespective of the surface structure. In the Fourier transform of the shadowed preparation, spots that correspond to the shadowed preparation are heavily reinforced leaving no doubt as to which spots are being generated or enhanced by surface shadowing. REFERENCES HILLS, G. J., C-URNEY-SMITH,M. and ROBERTS,K., 1973. Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardi. I I. Electron microscopy and optical diffraction analysis. 07. Ultrastruct. Res., 43:179-192. KLUG, A. and BEROER, J. E., 1964. An optical method for the analysis of periodicities in electron micrographs and some observations on the mechanism of negative staining. .7. molec. Biol., 10: 565-569.
A SURFACE REPLICA AND NEGATIVELY STAINED IMAGE
53
ROBERTS,K., GURNEY-SMITH, M. and HILLS, G. J., 1972. Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardi. I. Ultrastructure and preliminary chemical analysis. J. Ultrastruct. Res., ~ : 599-613. WEBB, M. J. W., 1973. A method for the rapid removal of sugars and salts from virus preparations on electron microscope grids. J. Microscopy, 98 : 109-111.
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t l I I.I .~. ROBERTS AN1) ( ;U RNEY-."Z,MITtt
F I G U R E S 1-4
Surface replicas and negatively stained images of the wall of Chlamydomonas reinhardi and of catalase crystals together with their Fourier trans]orms Fig,~res 1 and 2. Both figures are of the same region of the cell wall of Chlamydomonas reinhardi. However, in Figure 1, the specimen was negatively stained whereas in Figure 2 the stain was eluted and the specimen shadowed on a rotating stage. From the optical diffraction pattern shown in Figure I (insert) the apparently single wall can be identified as consisting of two walls folded back to back. The optical diffraction pattern shown in Figure 2 (insert) was obtained from the same area used to provide the diffraction pattern in Figure 1. In Figure 2 only the top surface of the upper wall is shown in relief and an exact relationship can be established between the angles and diameters of the two patterns. Two spots are ringed on each diffraction pattern that are common to both techniques, x 40,000.
Figures 3 and 4. Figure 3 is a micrograph ofcatalase crystals negatively stained with ammonium molybdate. In Figure 4 the stain has been eluted and the specimen shadowed with goldpalladium (60/40w/w). The two inserts show the optical diffraction pattern obtained following the two types of treatment. In Figure 4 some of the crystals have retained some electron density suggesting that the stain has not been completely eluted but the optical diffraction pattern is only from the surface structure of the crystals.
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51
A technique for obtaining a surface replica and a negatively stained image from the same biological object G. J. HILLS, K. ROBERTS and M. G U R N E Y - S M I T H John Innes Institute, Colney Lane, Norwich N O R 7OF, England, U.K.
Manuscript received March 21, 1974
A new technique is described that allows precisely the same area of a biological specimen to be viewed in the electron microscope both by shadowing and by negative staining. Both techniques increase contrast; however, with shadowing the upper surface of the specimen is shown in relief while negative staining produces a two-dimensional image. With the combined technique a direct correlation can be achieved between the structure of the surface and of the interior o~ an object. Une nouvelle nffthode est dgcrite oh le m~rne emplacement d'un gchantillon biologique peut gtre observd en microscopie dlectronique simultan~ment par ombrage et par coloration nggative. Les deux techniques amgliorent le contraste. Gependant l'ombrage permet l'observation de la pattie supgrieure de la surface de l'~chantillon tandis que la coloration rdgative produit une image en deux dimensions. En combinant les deux techniques une corrglation directe peut ~tre ~tablie entre la structure de la surface et celle de l'intgrieur d'un objet. Ein neues Verfahren wird beschrieben, daft es m6glich macht, den gleichen Bereich einer biologischen Probe sowohl durch Schritgbediimpfung als anch durch Negativfarbung zu untersuchen. Beide Verfahren erh6hen den Kontrast, aber die Schritgbediimpfung zeigt das Oberfliichenreli~ der Probe, wighrend Negativfiirbung ein zweidimensionales Bild erzeugt. Mit diesem I(ombinationsverfahren ist es m6glich, die Zusammenhiinge zwischen Oberfl&henstrUktur und Innenstruktur zu bestimmen.
INTRODUCTION There are severe limitations to the retrieval of three-dimensional information fi'om biological objects in the electron microscope. Shadowing provides us with a surface view only, while negative staining, although projecting information from different depths in the specimen, only gives us a final recording in two dimensions. Clearly, more information could be obtained if these two techniques could be combined. This short paper describes our development of such a technique together with some applications. The technique provides a means of viewing precisely the same specimen area whose contrast is increased both by shadowing and by negative staining. Essentially the technique involves recording the normal negative stained image and then removing the stain followed by surface shadowing. A comparison of the optical transform (Klug and Berger, 1964) of a straight-forward replica with a replica made after the elution of a negative stain from a specimen that had been examined in the microscope, revealed that essentially the same structure is present and to the same resolution. From this we may conclude that the process of negative staining and subsequent examination in the electron microscope did not destroy the quarternary structure over about 4.0nm in the specimens that we examined. If directional shadowing is performed, detail in line with the direction of shadow is suppressed compared with detail normal to the direction of shadow. To obtain uniform information all shadowing was performed while the specimen was being rotated at about 200 r.p.m., the angle of shadow being about 15°.
52
H11,1.S, ROBERTS AND (;URNEY-SMITH M A T E R I A L S AND M E T H O D S
A suspension of the objects to be studied is mixed and dried down with a negative stain onto a map reference electron microscope grid (Micron Index II grids are very suitable) covered with an evaporated carbon film stripped from mica. The negative stain used here was a 5% aqueous solution of ammonium molybdate. The grid is inserted into the electron microscope and areas of interest photographed, noting the grid reference of that area. The grid is removed from the electron microscope and the negative stain eluted by the use of Webb's (1972) method. It has been found that the majority of negative stains normally used are removed by washing for about 20rain. Care should be taken to choose a non-reactive negative stain for the particular subject being studied as positive attachment of heavy metals may alter the surface structure o f the object. The grid is dried after complete stain elution and is then rotary shadowed at an angle of about 15 ° to the grid surface. The grid is reinserted into the electron micro.scope and areas, relocated by their grid references, are recorded. By comparison of the map references of the areas recorded on the plates from both methods of contrasting, matched pairs of plates can be sorted. R E S U L T S AND D I S C U S S I O N The above technique has been used to elucidate the structure of the cell wall of the alga Chlamydomonas reinhardi (Roberts, Gurney-Smith and Hills, 1972; Hills, GurneySmith and Roberts, 1973). Fourier transforms of micrographs of the same area of cell wall using either negative staining (Figure 1) or shadowing (Figure 2) can be directly ,compared. The relationship of parameters and angles of the lattices of the surface of the cell wall and the cell wall in depth as seen by negative staining can be determined and the Fourier transform which corresponds to the top wall and external ceil wall surface upwards selected. This technique readily gives this type of information which is necessary for structural analysis, and we believe that this is the most simple and sometimes the only way to obtain this information. If a non-reactive negative stain cannot be found for the material being studied the technique can still be used. Figures 3 and 4 illustrate an aggregate of catalase crystals together with their Fourier transforms. The Fourier transforms are from identical areas of the micrographs and demonstrate that only one of the periodicities seen in the negative stained image is due to surface structure. At least three different types of :structure are seen by shadowing catalase crystals but the Fourier transforms of negative stained crystals are similar irrespective of the surface structure. In the Fourier transform of the shadowed preparation, spots that correspond to the shadowed preparation are heavily reinforced leaving no doubt as to which spots are being generated or enhanced by surface shadowing. REFERENCES HILLS, G. J., C-URNEY-SMITH,M. and ROBERTS,K., 1973. Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardi. I I. Electron microscopy and optical diffraction analysis. 07. Ultrastruct. Res., 43:179-192. KLUG, A. and BEROER, J. E., 1964. An optical method for the analysis of periodicities in electron micrographs and some observations on the mechanism of negative staining. .7. molec. Biol., 10: 565-569.
A SURFACE REPLICA AND NEGATIVELY STAINED IMAGE
53
ROBERTS,K., GURNEY-SMITH, M. and HILLS, G. J., 1972. Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardi. I. Ultrastructure and preliminary chemical analysis. J. Ultrastruct. Res., ~ : 599-613. WEBB, M. J. W., 1973. A method for the rapid removal of sugars and salts from virus preparations on electron microscope grids. J. Microscopy, 98 : 109-111.
~,i
t l I I.I .~. ROBERTS AN1) ( ;U RNEY-."Z,MITtt
F I G U R E S 1-4
Surface replicas and negatively stained images of the wall of Chlamydomonas reinhardi and of catalase crystals together with their Fourier trans]orms Fig,~res 1 and 2. Both figures are of the same region of the cell wall of Chlamydomonas reinhardi. However, in Figure 1, the specimen was negatively stained whereas in Figure 2 the stain was eluted and the specimen shadowed on a rotating stage. From the optical diffraction pattern shown in Figure I (insert) the apparently single wall can be identified as consisting of two walls folded back to back. The optical diffraction pattern shown in Figure 2 (insert) was obtained from the same area used to provide the diffraction pattern in Figure 1. In Figure 2 only the top surface of the upper wall is shown in relief and an exact relationship can be established between the angles and diameters of the two patterns. Two spots are ringed on each diffraction pattern that are common to both techniques, x 40,000.
Figures 3 and 4. Figure 3 is a micrograph ofcatalase crystals negatively stained with ammonium molybdate. In Figure 4 the stain has been eluted and the specimen shadowed with goldpalladium (60/40w/w). The two inserts show the optical diffraction pattern obtained following the two types of treatment. In Figure 4 some of the crystals have retained some electron density suggesting that the stain has not been completely eluted but the optical diffraction pattern is only from the surface structure of the crystals.
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