J . Mol. Biol . (1966) 15, 365-371

LEITERS TO THE EDITOR

An Analogue for Negative Staining

Th e images obtained in the electron micro scope from negatively sta ined biologi calparticles are produced by the electron-dense st ain which envelops the low-densityparticles. It is the purpose of the present paper to show that t hese images can besimulated by X-ray radiography of appropriate models embedded in an X-rayabsorbing material. The ver isimilitude of the analogue depends not only on how wellthe model corresponds to thc object observed in the electron microscope, but also onthe correspondence of the distribution and relative X-ray opacit y of the medium inwhich the model is embedded to the distribution and elect ron opacity of the negativest a in used to produce the electron microscope image. Thi s X-ray radiographic methodhas previously been employed by C. F. T. Mattern (personal communicat ion ), whoradiographed wooden models, designed to represent tobacco mosaic virus, whichwere embedded in sand; the results were not published since the model was incorrect.K cllenb erger & Boy de la Tour (1964) have simulated the images of negativelystained particles by ph ot ography in visible light of t ransparent models immersed ina partially opaque liquid of matching refractive index ; bu t this method cannot beused to represent a layer of stai n of non -uniform thickness. More recently , Almeida ,Waterson & Fletcher (1965) have used direct X-ray rad iographs of rubber modelsprinted as negati ves to simulate negati ve staining. H owever , the radiograph of a modelwill correspond to t he negative of its negatively stained image only under veryrestricti ve conditions for the distribution and opacity of t he embedd ing medium.

The developm ent of t he X -ray radiographic analogue for negati ve staining de­scribed here was sti mulated by Klu g & Finch's analysis (1965) of electron micrographsof human wart virus particles. They hav e established t ha t t he protein shell of t hisvirus has the symmetry of t he T = 7d icosahedral surface la ttice and is composed of72 morphological uni t s. Some of the electron microscope images result from predomi ­nant negative staining of one side of the particle (t he side closer to t he supporting sub­st rate). The arrangement of morphological units observed in these "one-sided "ima ges corresponds to that of the 5- and 6-co-ordinated lattice points of theT = 7d icosahedral surface lattice. The majority of the images are produced byst aining on both sides of the particle, and these "two-sided" images appear confusedat first sight when compared to well-resolved one-sided images. However, whenparticles which are contrasted on both sides are in certain fav ourable orientations,distinctive images ar e produ ced which can be accounted for by superp osit ion ofdetail from the two sides of t he shell composed of 72 morphological units. It is demon­st rate d in the present pap er t hat some of the characterist ic one- and two- sidedimages observed by Klug & Finch (1965) can be qui te realisti cally reproduced byanalogue negati ve staining of a model t he design and morphology of which corre­sponds to tha t of human wart virus (Plates I and II) . Thi s ana logue method has alsobeen applied (Plate III) to represent the characte rist ic two- sided electron microscopeimages of turnip yellow mosaic virus which have been obtained by Finch & Klug(1966).

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The first requirement for a realistic analogue of negatively stained virus particleimages is a reasonably representational model of the outside of the protein coat.Klug & Finch (1965) have measured the following dimensions which characterize thesize and shape of the morphological units of human wart virus. The diameter of a6-co-ordinated unit at the "radius of mean contrast" is about 100 loA. The maximumradial extent of undeformed particles is about 280 A and the radius of mean contrastis about 220 A. The difference of 60 A between these two radii corresponds to the meandepth of the grooves between morphological units. The morphological units have ahole or depression of about 20 A diameter along their axis and their over-all length isestimated to be about 120 A. According to the theory of icosahedral virus particleconstruction (Caspar & Klug, 1962), the 6-co-ordinated morphological units arehexamers and the 5-co-ordinated units pentamers of the protein structure unitswhich form the capsid. The structure units of human wart virus should be roughlyellipsoidal in shape with a length of about 120 A and a diameter of about 30 A.Since the matter appears to be more concentrated along the lines connecting neigh­bouring morphological units than between them, it is presumed that the centers ofthe structure units lie near these lattice lines. A model (Plate I(c)) for human wartvirus was constructed at a scale of about 1 em = 100 A, to reproduce the featuresdescribed by Klug & Finch (1965). The morphological units were machined from piecesof polyethylene rod with a taper angle of 27° along half their length to represent thebonding surfaces. A small hole was drilled along their axes and the outer ends wererounded off. The diameter of the 5-co-ordinated units was made 5/6ths that of a6-co-ordinated unit to represent the difference between pentamers and hexamers.The morphological units were fixed together with rubber cement. Since these unitshad the appropriate taper angle for a T = 7 icosahedral shell they could be assembledwithout the need of a core or template. The flexibility of the rubber cement allowedthe units to arrange themselves as regularly as possible as the assembly proceeded.The rubber cement was built up as bridges between neighbouring units to representthe concentration of matter in this direction; but any more detailed representationin the substructure of the morphological units is not warranted by the resolution ofthe electron micrographs. The pattern of the T = 7d lattice was imposed in theassembly, although these units could equally well have been assembled in the T = 7licosahedral lattice which is the design of rabbit papilloma virus (Finch & Klug, 1965).In order to facilitate photography of one-sided images, the model was built in twohalves divided by a plane perpendicular to a strict 3-fold axis of the icosahedralshell. A radiograph of one-half of this model, as viewed from the outside nearly in thedirection of a 3-fold axis, is shown in Plate I(b). For reference, Plate I(a) shows adrawing of the arrangement of the 5- and 6-co-ordinated lattice points of the T =

7d icosahedral lattice on the surface of a sphere as seen in projection exactly along a3-fold axis.

The ratio of the X-ray absorption coefficient of an appropriate analogue negativestain to that of the model should be the same as the ratio of the electron scatteringpower of the actual negative stain to that of the virus particle. The relative contrastin the analogue method can be controlled by varying the mass or the electron densityof the analogue stain and also by varying the voltage of the X-ray tube. No attemptwas made to calculate the best analogue combination of stain density and X-rayvoltage for the radiographs shown here: rather, these parameters were varied empiri­cally until radiographs with a contrast comparable to that of the electron micro-

(a) (b) (c)

(d) (e)

PLATE 1. (a) Drawing of the arrangement of the 5- and 6-co-ordinated lattice points of theT = 7d icosahedral lattice as viewed exactly along a 3-fold axis. The three 5-fold pointsnearest the central 3-fold axis are emphasized.

(b) X-Ray radiograph of the top half of the model in Plate I(c), taken in a direction close to a3-fold axis.

(c) Model of human wart virus in nearly the same orientation as (a) and (b) (after Klug &Finch, 1965).

(d) Center: electron microscope image of a human wart virus particle (the top image in PlateVII of Klug & Finch, 1965). Top and bottom: analogue one-sided images. These are X-ray radio­graphs of half of the model shown in Plate I(c) with its outer surface covered, respectively, by apaste of calcium and barium sulphate and a dry powder ofthe same materials. The periphery of theanalogue image at the top has been darkened a little so that it merges better with the background.

(e) Duplicate print of Plate I(d) with the three 5-co·ordinated units and the three 6-co­ordinated units closest to the near-central 3-fold axis marked by crosses and dots, respectively.The orientation in the electron microscope image is somewhat farther from a 3-fold axis thanin the analogue images (cf. Plate I(a».

(a) (b) (e)

PLATE II. (a) Shadowgraph of a Geodestix model of the '1' = 7d icosahedral surface latticeviewed exactly down a 3-fold axis (Klug & Finch, 1965, Plate XI(i)). The orientation is identi­cal with the drawing in Plate I(a).

(b) Three different representations of a two-sided image of the structure of human wart virus.All three photographs correspond to the same orientation. Left: shadowgraph corresponding to anorientation about 2° off a 3·fold axis (cf. Plate II(a)). Top right: radiograph of the model inPlate I(c). Bottom right: analogue two-sided image obtained by radiographing the same modelwith both its top and bottom surfaces covered with the analogue stain.

(c) Top: electron microscope image of human wart virus showing a characteristic 3-foldterraced appearance (Klug & Finch, 1965, Plate XI (g) ). Bottom: duplicate of Plate II(b), bottomright.

(d)

(I)

(f)(e)

(a)

(g) (h)

(b) (i) (m)

...~ ..• .:• • ~ l,

.:..... \t .... . ,.• •

(e) (j) (k) (n)

PLATE III. (a), (b) and (c) Photographs of a cork model of turnip yellow mosaic virus takendown 2-, 3- and 5-fold axes, respectively.

(d) The cork model embedded in plaster of Paris and mounted for support in a hole in fiber­board.

(e), (f), (g) and (h) Analogue two-sided negative stained images taken close to 2-fold directions.(e) and (f): radiographs of (d) taken in very slightly different orientations close to the same 2-foldaxis. (g) and (h): radiographs of the same model (d) along two other 2-fold axes.

(i) Analogue image viewed down 3-fold axis.(j) and (k) Analogue images taken close to the same 5-fold axis of the model.(I), (m) and (n) Electron micrographs of negatively stained particles of turnip yellow mosaic

virus (Finch & Klug, 1966) showing 2., 3- and 5-fold views, respectively.The seven analogue images are not all reproduced at exactly the same scale. The small black

dots and lines correspond to patches of barium stain used to mark the orientation of the plaster.encased model.

LETTERS TO THE EDITOR 367

graphs were obtained. The radiographs shown in Plates I and II were obtained withthe model supported just above a sheet of X-ray film at a distance of about five feetfrom a copper target X-ray diffraction tube. The direct radiographs were obtained ata voltage of 20 to 25 kv with no filtration. The negative stain was a mixture of calciumand barium sulfate in various proportions and was applied either in the form of acompacted powder or a wet paste to the model supported on an indented sheet of thinplastic. The negatively stained radiographs were obtained at a voltage of 45 to 50 kvwith a cobalt filter. At this voltage the model itself is almost transparent, whereasthe stain is relatively opaque.

Calcium sulfate alone has sufficient X-ray opacity compared to the polyethyleneof the model to act as an adequate analogue negative stain. However, mixing in asmall proportion of barium sulfate in dry calcium sulfate produced a granular appear­ance similar to that observed in the electron microscope. The disadvantage of usingdry powder as the analogue stain is that it is difficult to support the powder as auniform thin layer in intimate contact with the model. On the other hand, calciumsulphate can easily be applied as a wet paste, but this sets rapidly to plaster of Paris,and it is difficult to remove the plaster or change its thickness and distribution onceit has set. A mixture of calcium and barium sulphate with water produces a thick pastewhich can easily be applied and manipulated, since it sets only very slowly and can beremoved from the model by washing. The disadvantage of this paste is its highabsorption even at the maximum X-ray voltage available from a standard diffractionunit (about 50 kv). Only very thin layers of this stain could be used and the radio­graphic images obtained had higher contrast than the electron micrographs they wereintended to represent.

Two examples of the effect of varying the texture, thickness and opacity of theanalogue stain on the radiographic images are compared in Plate l(d) and (e) with atypical one-sided electron microscope image of a human wart virus particle (the topimage in Plate VII of Klug & Finch, 1965). The model and the virus particle areviewed nearly in the direction of a 3-fold axis and the orientation of these imagesis close to that of the drawing of the T = 7d surface lattice in Plate l(a). The centersof the three 6-co-ordinated and the three 5-co-ordinated morphological unitsnearest the central 3-fold axis of the radiographic and electron microscope imagesare marked in Plate l(e). The analogue image below the virus particle image in Platel(d) and (e) was obtained from one-half of the model with its outside embedded in arelatively thick layer of dry calcium sulphate powder mixed with a small amount ofbarium sulphate. Even though this stain was applied in what appeared to be a fairlyuniform layer, it is evident that on the lower right of the particle there is a slightlygreater thickness which is sufficient to obscure this part of the structure. The textureof the image is somewhat coarser than that of the corresponding electron micrographand less detail is resolved, particularly near the periphery of the model. This loss ofdetail is not due to any limitation in resolution in recording the radiograph, but ratherto the fact that the indentations produced by the model in the layer of stain arerelatively small compared with its thickness. The sharper analogue image at the topof Plate l(d) and (e) was obtained by staining with a dense paste of calcium and bariumsulphate which filled only the interstices between the morphological units on one-halfof the model. Since the stain just outlines the morphological units, this radiographicimage is, in fact, similar to the actual external appearance of the model (Plate l(c)).Although the contrast in this analogue image is greater than that of the electron

368 D. L. D. CASPAR

micrograph, there is a very close correspondence in the disposition and appearance ofthe morphological units in both images. A variety of analogue images can be producedfrom the same model by using different staining techniques, but the most realisticimages are obtained with stain density and distribution between those of the twoexamples illustrated in Plate I.

There are some small differences between the virus particle image and the analogueimages in Plate I that are not related to differences in the staining. The virus particleillustrated appears to be slightly flattened, since its diameter is somewhat greater thanthat inferred for undeformed particles. Moreover, the relative separation of themorphological units near the periphery is slightly greater than that of correspondingunits in the radiographs of the spherical model. The extent of the flattening of thisparticle appears to be comparable to the 25 to 35% reduction in thickness whichFinch & Klug (1965) have measured stereoscopically for similar images of bothhuman and rabbit papilloma virus. The slight flattening of the particle has notsignificantly disordered the packing of the morphological units, since this arrangementcorresponds very closely to that of the regular model.

The two-sided images of human wart and rabbit papilloma virus particles in certainfavorable orientations show striking morphological features described by Klug &Finch (1965) as "terraces" and "eyes". These authors have illustrated the pattern ofthese features by shadowgraphs of a Geodestix model of the T = 7 icosahedralsurface lattice, two of which are shown in Plate II. The shadowgraph and radiographsof the model in Plate lI(b) all correspond very nearly to the same view, namelyabout 2° off a 3-fold axis. All three images illustrate the same pattern althoughtheir actual appearances are quite different. The analogue negative stain used toobtain this two-sided image of the model had the same composition as that of theone-sided analogue image at the top of Plate I(c), and was applied in a similar wayexcept that the thin layer of stain covered the model fairly uniformly on both thetop and bottom surfaces and was slightly thicker near the periphery. This two-sidedanalogue image is compared in Plate lI(c) with a characteristic two-sided image of ahuman wart virus particle (Plate XI(g) of Klug & Finch, 1965) which is in almostexactly the same orientation.

It is evident from both the shadowgraphs of the surface lattice models and theradiographs of the model stained uniformly on both sides that changes in the angle ofview by even a fraction of a degree produce significant changes in the pattern resultingfrom superposition of detail from the two sides. Since the angle of view of the modelfor these radiographs was not precisely controlled, it was fortunate that among thesmall number of analogue two-sided images obtained one should be in so nearly thesame orientation as one of the published electron micrographs. The analogue imageis sharper than the electron micrograph and there is some irregular variation in thecontrast near the periphery of the model resulting from small non-uniformity in thethickness of the stain, but on the whole there is a very close correspondence betweenthe detail that can be resolved in the electron microscope and in the analogue image. Inparticular, the pronounced eye, together with the other two weaker eyes in the centralregion of the electron microscope image, and the two terraces of small apparentmorphological units which can be seen surrounding or embracing them, are faithfullyreproduced in the analogue image. In both the virus particle and analogue images theapparent morphological units near the periphery which originate from morphologicalunits close to the equatorial plane ofthe particle are almost completely obscured by the

LETTERS TO THE EDITOR 369

negative stain. The correspondence between these images indicates that there isrelatively little, if any, flattening of this particular virus particle, which is supportedin a relatively thick layer of negative stain. The verisimilitude of these radiographsto the electron micrographs of human wart virus attests to the accuracy of Klug &Finch's analysis (1965) of the structure of its protein coat, since the model used wasconstructed to conform as closely as possible to their description of the virus particlestructure.

The electron micrographs of turnip yellow mosaic virus obtained by Finch & Klug(1966) provide the first instance of the resolution of the protein structure units in theimages of an icosahedral virus capsid, and their observations are consistent with thetheory of icosahedral particle construction (Caspar & Klug, 1962). A model of thesurface structure of turnip yellow mosaic virus, as inferred from the electron micro.graphs, was constructed at a scale of about 1 cm = 30 A, using corks to representthe projecting ends of structure units (Plate III(a), (b) and (c)). The corks do not, ofcourse, accurately represent the exact shape of the outside ends of the protein mole.cules, which is not, as yet, precisely determined. The ratio of diameter to length ofthese corks and their packing arrangement in the model do, however, closelyapproximate to the observed surface features of turnip yellow mosaic virus. The 180corks representing the 180 structure units were assembled into 12 pentamers and20 hexamers, which were then glued to the facets of a cardboard truncated icosahedronto produce the appropriate T = 3 surface lattice arrangement.

The best preservation of the structure of negatively stained turnip yellow mosaicvirus particles is observed when they are embedded in a layer of stain over holes in thecarbon film (Finch & Klug, 1966); characteristic two-sided images of these embeddedparticles are obtained when they are viewed along 2-, 3-, or 5-fold axes. Theinferred stain distribution was represented by coating the model in plaster ofParis supported in a hole in a piece of fiberboard (Plate III(d)). In order to permitradiography at oblique angles to the supporting board, the layer of the analoguestain at the periphery of the model was thinned down more than might be expectedin the electron microscope specimens. The board was held in a universal clamp aboutsix feet from a tungsten target therapeutic X-ray set. (This X-ray set was kindlymade available by the Department of Radiotherapeutics, University of Cambridge.)Because of the greater actual thickness of this model (10 cm) compared to that usedin Plates I and II (5,6 cm), voltages in the range 80 to 100 kv were required to obtainoptimum contrast. The orientation of the model was adjusted to obtain views in thedirection of the symmetry axes and the orientation was followed by direct exposureof Polaroid film until the desired setting was obtained. To facilitate orientation of themodel, the crevices at the inside of some of the hexamers and pentamers were linedwith a small amount of barium sulphate which shows up as dark narrow lines in theradiographs.

Typical analogue images viewed close to 2-, 3-, and 5-fold axes of the modelare shown in Plate III, reproduced alongside electron micrographs of the virus.The close correspondence in the appearance of the analogue and virus images showsthat the model approximates to the virus particle morphology very well and that theapproximately correct distribution of analogue stain has been applied. The deductionof the virus morphology from the electron micrographs is described by Finch &Klug (1966), in whose paper will also be found some further discussion of thecorrespondence between analogue and electron microscope images for this virus.

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370 D. L. D. CASPAR

There are, however, a number of features illustrated by these analogue imageswhich are ofgeneral interest for the interpretation of electron micrographs. The imagesin Plate III(e), (f), (g) and (h), were obtained by viewing the plaster-encased modelalong three different 2-fold axes. (The differences in orientation of the model can berecognized from the positions of the barium stain.) Since the arrangement of the struc­ture units is very nearly centrosymmetrical, the superposition of detail from the twosides viewed exactly along a 2-fold axis produces images which are similar to thosewhich would have been obtained by negative staining of half the model. The twoimages in Plate HI(e) and (f) illustrate the effect of very small changes in orientation.The blurring of detail in the image is largely the result of the projection of detail in themodel at various levels. However, irregularities in the model itself and in the distribu­tion of the stain, as well as slight mis-setting of the orientation, all contribute to theblurring and imperfect superposition of detail. The high resolution of the radiographsis indicated by the sharpness of the images of the barium, but the relatively poorresolution of structural detail in the negatively stained image of the model is largelya consequence of the way in which the stain produces the image. The relative detailwhich may be resolved in electron micrographs of negatively stained virus particleseven with optimum electron optics is not likely to be better than that of these ana­logues. In these 2-fold images, the eight morphological units (four hexamers andfour pentamers) which lie in the equatorial plane are almost completely obscured bythe stain. The near-peripheral units appear not only unconnected to their neighbours,but also detached from the body of the particle. This appearance results because thebases of the morphological units are contrasted by a thicker layer of stain than theirouter ends.

The two-sided images viewed down the 3-fold axis (Plate III(i)) and 5-foldaxis (Plate IH(j) and (k)) are different from the corresponding external appearanceof the model, since in neither case does all the detail from the two sides coincide.In the 3-fold view the image of the central hexamer from the two sides exactlysuperimposes, but the surrounding six apparent morphological units are each super.imposed images of a hexamer and pentamer from the two sides. Moreover, the peri­pheral18 morphological units which are singly contrasted by a relatively thick layerof stain are almost completely obscured. In the 5-fold view the central pentamers onthe two sides are rotated 36° relative to each other, thus their images cannot coincide.The two images in Plate IH(j) and (k) were obtained with the line of view within about1° of the 5-fold axis, but the slightly different superposition of the two pentamersgives the impression of a square array of four small units in (j), whereas a hexagonalappearance is observed at the centre of (k). The patterns of apparent morphologicalunits surrounding the centre are almost indistinguishable in the two images. Theperipheral ring of ten units is the superposition of the images of hexamers and penta­mers above and below the equatorial plane. The intermediate star-shaped patternresults from the interposition of the images of hexamers from the two sides. Analogueimages of the model obtained in orientations other than those illustrated appear moreconfused because of the more complex superposition pattern, and the orientation ofthe model can be recognized from the image itself only by comparison with a galleryof shadowgraphs (Finch & KIug, 1966) of a skeletal model of the surface lattice in aseries of different orientations.

It is evident from these radiographs that the electron microscope images of negativelystained particles can be reproduced realistically by analogue staining of representa-

LETTERS TO THE EDITOR 371

tional models. However, this method cannot conveniently be applied until the designof the particle to be represented has been established, since a considerable amount ofwork would be required to construct and test a number of very different models.The patterns observed in the electron micrographs can be interpreted by comparingthem with simpler models such as those constructed from Geodestix components orpoppet beads, which illustrate the physically plausible symmetry arid packingrelations, although they do not reproduce the actual detailed morphology. The ana­logue method described here could be applied in a more quantitative way by matchingthe relative X-ray opacity of the stain and model to the electron opacity of theactual stain and virus particle measured by photometry of electron micrographs.The detailed distribution of negative stain could be investigated in this way. It isclear from the observations described here that a negatively stained image of aparticle is not simply the negative of its positive image, and that the clearest resolu­tion of surface detail is obtained when the negative stain is in the form of a thin layerwhich closely follows the surface contours of the particle.

Drs A. Klug and J. T. Finch have provided invaluable advice and assistance in variousaspects of this work and I am indebted to them for the electron micrographs and shadow­graphs reproduced here. Dr Finch, Mr K. Harvey and Mr F. Clow have assisted with thephotography of the radiographs and models. Part of this investigation was carried out atthe Medical Research Council Laboratory of Molecular Biology, Cambridge, England, andthe hospitality of Dr M. F. Perutz and the members of the staff is gratefully acknowledged.This work has been supported by Public Health Research Grant CA-04696 from theNational Cancer Institute.

Children's Cancer Research FoundationBoston 15, Massachusetts, U.S.A.

Received 20 September 1965, and in revised form 16 October 1965

D. L. D. CASPAR

REFERENCES

Almeida, J. D., Waterson, A. P. & Fletcher, E. W. L. (1965). Nature, 206, 1125.Caspar, D. L. D. & Klug, A. (1962). Cold Spr, Harb, Symp. Quant. Biol. 27, 1.Finch, J. T. & Klug, A. (1965). J. Mol. BioI. 13, 1.Finch, J. T. & Klug, A. (1966). J. Mol. Biol. 15, 344.Kellenberger, E. & Boy de Ill. Tour, E. (1964). J. Ultrastructure Res. II, 545.Klug, A. & Finch, J. T. (1965). J. Mol. Biol. II, 403.