Evaluation of Several Types of Replicas Preparation of Substrates f o r High Resolution Electron Microscopy J. J. COMER AND F. A. HAMM’ General Aniline and Film Corp., Easton, P a . These investigations were begun primarily for two reasons: to compare one-step silica and two-step polystyrene-silica replicas from the standpoint of resolution and accuracy of reproduction of the original surface, and to prepare substrates exhibiting a minimum in their inherent structure, so that the resolution of particulate material resting on these substrates would not be seriously impaired. The one-step silica replica is shown to be superior for studying the topography of relatively soft organic surfaces. Correlation between the crystal size, which accounts for undesirable background in the final replica, and the thickness of an intermediate layer of evaporated sodium chloride is demonstrated. The inherent structure in substrates of silica and silicon monoxide with a thickness dependence is demonstrated. For critical work, the need for more than one type of replica is shown. For resolution of structure at the 50 A. level, the effects of shadowing metal and inherent structure in the substrate arc found to impose serious limitations.
F
OR several years in this laboratory replica techniques have been applied to a few related research problems having to
do with photographic film base. hlore recently interest was focused on the attempted resolution of single macromolecules of highly polymeric materials. In general, these efforts were concentrated on the use of shadowed silicon monoxide and silicon dioxide (silica) replicas. In the more recent phase of this investigation replicas of mica were used as supporting membranes for the polymeric subject matter. Consequently, these replicas of mica are here referred to as substrates. In all this work the authors were constantly confronted with the problem of evaluating the background structure inherent in the shadowed silicon monoxide and silicon dioxide specimens. This is somewhat of an enigma to all electron microscopists who are concerned with high resolution studies. This paper was written in an effort to crystallize some experience in this regard. It is dangerous to illustrate by means of a relatively few micrographs information that has been periodically recorded during about 3 pears. This is especially true in this study, because the operating conditions of the electron microscope could readily introduce changes in the image which might erroneously be interpreted as being inherent in the specimen. For this reason the micrographs are for the most part illustrated in groups representing specimens prepared and photographed under carefully controlled conditions. The subject of this paper is divided into threeparts: 1. To compare the quality of replication of one-step silica replicas and two-step polystyrene-silica replicas (9) of the surface of gelatin cast on a cellulose ester photographic film base. The replicas are evaluated on the basis of a comparison with the gelatin film itself. As a result of a separate study not disclosed in this paper it was found that the gelatin film could be shadonTed with chromium and isolated from the cellulose ester base without danger of changing the original surface. As the same specific area of the gelatin film could not be shadowed for examination in the electron microscope and then be replicated, or vice versa, it was necessary to prepare replicas of areas immediately adjoining the 1
Present address, Burroughs Adding Machine Co., Paoli, Pa.
portion of gelatin film selected for esamination. Tests indicated, furthermore, that a given gelatin film could be treated in such a manner as to cause a known, uniform rough or smooth type of structure throughout the treated region. Shadowed replicas of such gelatin films 1-iei-e compared with the shadowed original sample. 2. To evaluate the effect on the resolution of a silica replica prepared by the use of a hydrophilic layer of sodium chloride ( 17 ) evaporated onto the gelatin film before deposition of the silica. Any st’ructure that’ may have been introduced by the sodium chloride \vas evaluated by comparing replicas prepared in this manner with t,he shadowed original gelatin film or with shadowed one-step silica replicas of this film. 3. To demonstrate how substrates of silicon dioxide and silicon nionoxide may be prepared with a minimum of structure, so that they map be used for high resolution studies. In extending the application of the one-step silica replica technique, silica substrates were prepared by evaporation of silica onto freshly cleaved mica and subsequent se aration under water. A comparison wari made of both silica a n 8 silicon monoxide substrates of different, thicknesses and shadowed differently with platinum-palladium. The results are interesting in view of a recent paper by Williams and Backus ( 1 6 ) . Throughout the paper the term “replicas” refers to silica replicas of either the one-step or two-step (polystyrene-silica j type prepared according to established methods. The films discussed in this paper, unless otherwise stated, are thin gelatin films, approximately 1 micron thick, cast on cellulose ester photographic film bases from which they can be separated for examination in the electron microscope. The silica and silicon monoxide substrates are actually replicas of the smooth surface of freshly cleaved mica. Formvar substrates were prepared in the usual way by casting a thin film of the material on the surface of water. These specimens are referred to as substrates because they are later used to support particular objects such as bacteria, pigments, and polymers.
Table I. Material Evaporated
Conditions for Evaporation
Source
Filament
.-
o_.
lament
Current (20 Volts), Amperes
45
Time? Seconds
1.?I
Time a t maximum current. b Sold by Johns-Manville as Hyflo-Super-Cel. National Research Corp., Cambridge, Mass. d A. D. Mackay, S e w York, N. Y.
a
SURFACES EXAMINED
The surfaces used in this comparative study of replicas are classified as fine structure and rough structure. These structures were deliberately prepared by treating the gelatin films in a special manner, All treated films were checked for uniformity as follows: Four or five areas of each of several 3 X 4 inch sections of the film were chosen at widely separated points,chromeshadowed and, upon separation of the gelatin from the cellulose ester base, examined in the electron microscope. On the basis of the similarity of all the test areas it was possihle to make significant comparison of the shadowed gelatin films, the onestep silica replicas, the two-step silica replica, and replicas pre1006
V O L U M E 2 4 , NO. 6, J U N E 1 9 5 2 pared with the layer of sodium chloride. The micrographs shown represent typical surfaces observed on many preparations of each typc of replica. PREP4RATION OF SPECIMENS
Condtions for the evaporation of silica, silicon monoxide, etc.. are listed in Table I. The platinum-palladium alloy was prepared by wrapping 4.9 mg. of I-mil platinum foil around 1.2 mg. of 2-mil palladium foil. This pornbination was flattened into a thin strip and wrap ed around the wire of a conical basket or a V-shaped tungsten &ament. Best results were observed when the metal strip was wrapped around a wire of an ordinary conical basket, using a thfferent position each time. When 30-mil tungsten wire is used, a t least five complete evaporations may be carried out before the filament breaks. Usually only two heating cycles are possible with a V-shaped filament. The filament is heated very briefly with 40 amperes (20 volts) of current in order to fuse the alloy. As some metal is lost with occasional sputtering, the specimen should be positioned after the alloy has been fused and cooled. It is possible, however, to fuse the alloy and complete the shadoning in one operation. Based on the density of platinum, if a 10-cm. target distance and 10" angle of incidence are used, complete evaporation of all the metal would give a film thickness of about 3 A. Recent experience suggests that it is best to evaporate chromium from a tungsten boat rather than from a basket; the calculated amounts of chromium may be weighed and placed in the boat, but these small amounts are not always retained by a conical basket. Calculations based on the use of a boat show that less than one half as much metal is necessary than when a basket is used. At a target distance of 7 cm. and an angle of incidence of 20°, 1 mg. of chromium when completely evaporated should give a film about 16 A. thick using 40 amperes for 5 to 10 seconds. In general, the complete evaporation of calculated amounts of material is more satisfactory than a partial evaporation of a large piece of metal using a time-amperage basis in conjunction with a basket. Although there is no intent t o prescribe specific pressure limits for the preparation of replica specimens, the conditions described In Table I1 (see also Table I ) have been used with good results
Table 11. Pressure Limits Material Silicon dioxide Silicon monoxide Chromium Gold Nickel Platinum-palladium aiio3 Calibrated Philips gage values. hIaterials can at higher pressures.
Preasure,a Micron 0 . 0 4 t o 0.08 0.04 t o 0.08
0.08 t o 0 . 1 5
in all cases be evaporated
It has been pointed out (9,16,17) that it is impossible to calculate precisely the thickness of evaporated films. However, calculations were made and certain thicknesses have been established as desirable goals. Experience has shown that the roughness of the original surface partly determines the thickness of d i c a required to make a coherent replica. The equation 0.75 rn tan w t =
4w2d
113s been used in these calculations (15). Arbitrary values of film thickness, t, viere used in calculating quantities, m, of materials to be evaporated. CY is the inclination of the target to the perpendicular rays emanating from the apex of the tungsten basket, d is the density of material, and r is the distance of the center of the target from the top of the tungsten basket.
Chromium granules for evaporation may be readily prepared from larger fragments. Sodium chloride may be pelletized or granules from a source of rock salt may be conveniently weighed. Silicon dioxide may be weighed in the form of quartz fibers, diatomaceous earth, Ludox ( I ) , Celite (Johns-Manville, New York 16,
1007
S.Y.), etc. Powdered silicon monoxide may be easily handled on aluminum foils. All these materials may be completely evaporated in preparing the replica, so that the calculated thickness is at least a good approximation. The thickness of platinum-palladium alloys was more difficult to ascertain, because usually not all Of the alloy is evaporated during the shadowing cycle. In the preparation of the shadowed original gelatin films, chromium was evaporated directly onto the surface of the film a t an angle of incidence of 20"; the thickness of the chromium was about 16 A. and the thickness of the gelatin film was approximately 1 micron. The specimen was scored in the usual manner and immersed in a solvent for the cellulose ester base. As the sections of the shadowed gelatin film came free from the base, they were transferred to a second dish of solvent. After 10 t o 15 minutes they were removed to a third dish of solvent for 5 to 10 minutes before being picked up on the specimen screens. One-step silica replicas of the gelatin films were prepared by evaporating silica at normal incidence directly onto the surface to be studied. Some of the silica re licas were then shadowed on the air side with chromium at an angre of incidence of 20" before being removed from the gelatin; others were shadowed on the side that had been in contact with the original surface. The first type may be considered to be a positive replica. The method of separating the silica from the gelatin was the same as for the shadowed gelatin films, with the addition of a second step in which the gelatin was separated from the silica by digestion in an enzyme (Rhozyme). In the preparation of the two-step (polystyrene-silica) replica it was necessary to prepare a negative replica of the gelatin film by casting polystyrene onto the surface from a 1% solution in benzene. The thickness of the negative replica was increased to the desired degree by adding 5 to 10% polystyrene solution after the initial coating had dried. When dry, the negative replicas were easily stripped from the film. Previous tests on gelatin films with very smooth surfaces showed that the polystyrene solution caused no change in the gelatin surface that could be detected in the electron microscope. Silica replicas were prepared from the negative replica in the established manner (9); these silica replicas were then chrome-shadowed on the side that had been in contact with the polystyrene. The resulting specimen is a positive replica. The third type of replica was prepared by evaporating a layer of sodium chloride at normal incidence directly onto the surface of the gelatin film. Using a tungsten boat it is ssible to eliminate spattering of the salt by adding water and t g n drying the salt by passing a very small current through the boat a t atmospheric pressure. This was followed by a layer of silica of the usual thickness (see later discussion) deposited on the sodium chloride also a t normal incidence. The minimum amount of the salt which could be used to allow separation of the silica from the film was determined by gradually decreasing the thickness of the evaporated salt on successive replicas until the silica could no longer be teased from the surface upon immersion in water. A minimum amount of sodium chloride is necessary to preclude undesirable background structure. To separate the silica it is first necessary to immerse just one end of the preparation carefully a t a slight angle, with the silica side up, in a dish of disiilled water. In doing so, the squares of silica were left floating on the surface. They were transferred to a second dish of water by means of a microscope cover glass. The glass, held by tweezers, was brought up under the floating silica, so that some of the water was transferred with the replica. The silica squares were washed again in a third dish of water and then picked up on specimen screens. All traces of sodium chloride are eliminated. Subsequent electron microgra hs will illustrate the effect of different amounts of sodium cgloride on the background structure. Silicon dioxide and silicon monoxide substrates were made by evaporating the materials directly onto the surface of freshly cleaved mica. Mica was used instead of glass because of the ease with which thin substrates of silicon dioxide could be separated from its surface. These replicas were shadowed on the air side at angles of incidence of 10' with approximately 3 A. of the platinum-palladium alloy. After being scored with a sharp blade, the shadowed replicas were separated from the mica by careful immersion in a distilled water. It is imperative to immerse only one end of the mica in water at a low angle and allow the water to diffuse between the mica and evaporated film before continuing the lowering. If properly done, the substrate will float free upon the surface of the water. It may be then readily picked u p on 0.125inch specimen screens. For substrates up to 100 A. thick the alternative method of picking up the entire unscored substrate on large sections of screen may be used. From these the 0.125-inch screens may be punched. Thicker substrates usually tend to separate from the screen in the punch. Using the procedure described first, very little difficulty wm encountered in removing films as thin aa 40 A. If all the squares
ANALYTICAL CHEMISTRY
1008 of substrate do not float after the first immersion, repeated immersions in water will often separate the remaining sections. Silicon monoxide substrates could be separated from mica only when the relative humidity was less than 40%, but silicon dioxide substrates from 100 t o 200 A. could be separated even when the relative humidity was over SOY0. If i t becomes necessary, the substrate can be removed by coating it withalayer of polystyrene (from solution), which when stripped from the mica removes the silicon dioxide or silicon monoxide substrate with it. Tnen the substrate can be separated from the polystyrene by immersions in a solvent. Both types of substrate are more readily separated under water as their thickness is increased.
. The substrates were prepared with carefully weighed amounts of silicon monoxide and silicon dioxide calculated to give^ the desired thickness. Although no absolute measurements of film thicknesses have been made, relative measurements were made using an electron microscope photometer, which accurately measures the electron beam intensity in the final focused image. The illuminating conditions of the RCA EMU electron microscope are controlled so as to give a specific beam current density and constant aperture of illumination on thespecimen. Photometer readings for the various thicknesses of silicon monoxide and silicon dioxide are then taken. Readings for both types of specimen indicate that the electron transmissivity of a specimen 200 A. thick is one half that of a specimen 100 A. thick. Similarly, equal thicknesses (calculated) of both types of specimen exhibit the same electron transmissivity within the limits of error attributable t o the photometer. According to a thorough study by Hass (7), these films of silicon monoxide should he very stable toward air oxidation t o the dioxide because they are thin and formed by rapid deposition in a very good vacuum. A very slow partial conversion to the dioxide over a period of weeks might be expected. The purity of the supply of silicon monoxide is not accurately known. However, the supply must have been very pure, because it sublimed at a fast rate (20-second heating cycle) a t about 1100‘ C. Silicon dioxide would require a temperature approximately twice as great. The higher vapor pressure of the silicon monoxide might be considered an advantage. INSTRUMENT ADJUSTMMTS
gelatin _Film (3Sz0O0X) ExhibitFigure 1. Unshadowed . -
h
...
I
...
Figure 2.
Gelatin Film of Figure 1 (55,OOOX) after Shadowing with Chromium Nms cavities and smooth ‘‘plsfeaus”
The relative virtues of thin silica and silicon monoxide replicas and substrates, especially from the standpoint of inherent struoture, required a critical evaluation of the perfomulnee of the electron microscope and the shaxpness of focus. For example, increasing the apparent size of fine structures in underfocusing the image is a common error. These factom have been discussed by Hillier (IO). Serious effort was maintained during this investigation to keep the performance of the electron microscope a t an optimum, so that the comparisons of the various replicas would not be affected hy image quality. After all the factors limiting the resolving power of the instrument have been eliminated, or a t least evaluated, it was realized that astigmatism waa the most important factor limiting reaolution of structures h e r than 50 A. This condition was eliminated before taking the high magnification micrographs illustrated in thia paper. The objective pole piece (6) was lapped and polished until the image at 20,000 diameters (instrument) was essentially free of astigmatism. No compensatingohjeetivepolepiece spacer was used. Because of the importance of instrument adjustments, the pertinent information is summarized in Tahle 111. The column alignment and illuminating conditions were standardized for the micrographs shown herein in so far as i t was possible. This presupposes that the differences in structure ohmwed on the micrographs of the various thicknesses of silicon monoxide and silicon dioxide replicas are not due t o electron-optical effects. This may be the subject of some controversy. Experiments, other than those described in this paper, designed to demonstrate that replica structure differences are not due to the thickness factor as claimed herein, would he difficult to carry out. Although the use of limiting apertures is still the subject of contmversy, experience suggests that high quality images are possible if the contamination that forms around the periphery of the aperture is removed periodically. The low contrsllt in the specimens described herein made theuse of thisaperture desirable. Heating the platinum aperture in a Bunsen burner flame a p pmently hums off the prohibitive contamination. Similarly, the tarnish on the faces and the bore of the objective pole piece should he removed by polishing with a powder of particles 1 micron or less at least once a month, preferably more often (6).
V O L U M E 24, NO, 6, J U N E 1 9 5 2
1009
Table 111. Electron Miemsoope Adjustments (EMU-2A)= %If-biased eleotron gun Beam ourrent (saturated). 200 to 250 microamperes Besm ourrent density at specimen. IO-s ampere per sq. om. during photographic expo*ure Condenser aperture. 20 mils. Condenser lens current of about 21 ma. resulting in an illumination with an angular aperture of about
- .--
A , Y ? n - g.V d a n
Objectiveaperture. Z-mif platinum periodically ignited o v e r ~Bunsen burner to rninimiee contamination Angular aperture of rays leaving objective lens, 1.3X 10-2 radian.
mediate viewing assemblyObjective focusing circuit. modified to give threefold sensitivity over standard circuit Projector lens control, low gain waeer with high projeotor tap "sed to photograph images at less than 12,000 X. This is to minimize pm-cushion distortion Micrographs were tbken at 7000, 12,000, and 20,000 X. Some of the data were estimated from information received from Hillier
(if).
DISCUSSION O F ELECTRON MICROGRAPHS
The quality of the various specimens illustrated was evaluated from two points of viea: The replicas are primarily considerrd with regpeot to their faithfulness of reproduction and resolution,
Figure 3. Shadowed Gelatin Film (125,OOOx) Exhibiting Relatively Smooth Topography Fine s t r ~ c t u r e
Figure 4. Chrome-Shadowed One-Step Silica Replica of Gelatin Film Exhibiting Fine Structure (125,OOOX) Compare with Fixmre 3
vary from 50 t o 100 A. in depth and from 100 to 700A. in width at tho widest points. These two shadowed specimens of the original gelatin film are
ANALYTICAL CHEMISTRY
1010 atandnrda on which, the amlit,v of the reolioas was
Figure 7.
Gelatin Film of Fiwure 6 (100,OOOX) Compze with Fiwrea 2 and 5
been distorted, probably during removal of the polystyrene from the film. Examination of a large number of these replicas suggests that this type of two-step replica is limited to a resolution of about 150 A. if the original structure is relatively rough. Finer structures permit a higher resolution. The chrome01 betarin
rum nxnsoinng nougn ~ ~ r u c r u r ~e w,uvun)
The one-step silica replica technique bas been found especially useful in studying surface structure of dyed fibers (6) and metal particles. Material adhering to the surface, such as dye crystals or metal oxides, can sometimes be transferred to the silica replica, so that the image is interpreted as though one were looking a t the original surface. Other workers (9, 4 ) have reported similar transferral of surface material when using the twc-step polystyrene-silica technique. The authors‘ experience suggests a less complete transferral Then using the latter technique. The extensive applications inthis laboratory of the one-step silica r e p lica technique to dyed cotton fibers and carbonyl iron powder have not yet been prepared for publication, However, this technique hras been found very useful. TWO-STEP SILICA REPLICAS.The polystyrene-silica replicas of the gelstin film exhibit a relatively poor reproduction of the original surface. Comparison of Figures 4 and 8, for examplc, illustrates the difference; the reproduction of the cavities has suffered. Two or more of the shallow cavities in Figure.4 are dy one in Figure 8. Figures 6 and 7 and a comparison of these with 5 demonstrate the differences in the two types of most significant change in the appearance of this ,by appears t o be in the very narrow “ridges” . . ting the cavities. These thin ridges appear to have
-
Firnure8. C h r o m e - S h a d o w d Two-step Silioa Replica of Gelatin Film Exhibiting Fine Structure (100,000 X)
V O L U M E 24, NO. 6, J U N E 1 9 5 2
1011
the silica replica from the suriace of a cellulose ester photographic film base was between 0.2 and 0.3 mg. (tungsten boat) calculated t o give an evaporated layer of sodium chloride between 13and 20 A. thick. The structure observed, due t o the sodium chloride, i8 not always uniform, but tends to be larger in area6 where the surface is rough. Furthermore, the size of the cubic structure wm found to increase with the thickness of the sodium chloride layer. Experience has shown that the minimum thickness of sodium chloride may depend upon the mmple. Gelatin, which is much more hydrouhilic than the cellulose est,%rfilm base, required a 300 A. thick layer of salt (5.4mg. in a bas [ret at 7 em.). Figure 9 illustrates the effect of this minimum am0unt of sodium chloride on a typical line structure. It should be ciimpared with Figure 4; adjoining areas oi the original sample we76' used for both types of replicas. The sodium chloride appems to widen the cavities, but the depth of the cavities is reduced. The specimens compared were shsdowcast simultaneously, the:reby precluding differences possibly to slight differences in th e angle of shadowing. This structure, obtained by exposing the' 300 A. thick layer of sodium chloride t o a normal laboratoiy atmosphere before evmoration of the silica. did not differ appreeisbly from that obtained by evaporating the silica onto a, layer of sodium ohlw ride of the *me thickness before exposinE! it to the stmosDhere. Figure 9. One-Step Silica Replica (55,OOOX) Preparedby means ofhydrophiliolayerofnodivmchloride Fins sfmcflll.~ gelatin Com~are with Figure 4
shadowed gelatin film described above (original gelatin film) has been used as the basis formaking the comparisons. HYDROPHILICLAYEROFSODIUMCHLORIDE. Theuseofanintermediate layer of sodium chloride is sometimes desirable where the replica may be most readily isolated under water. Although this technique is not new, there wa6 no critical evaluation in the literature of the effect of the sodium chloride on the resolution of fine Structure. This investigation suggests that structures finer than about 200 A. cannot properly be resolved whenasodium chloride layer is used t o prepare the replica. I n practice it was obsorved that the minimum amount of evaporated sodium chloride that would permit separation oi
Figure 11. One-Step Silica Replica Shadowed (Z5,OOOX) of Flexible Cellulose Ester Film Base
Fimre 10. One-SteD ReDliea (55,OOOX)
This was also found t o be true for sodium chloride layers varying between 100 and 600 A. The results of using the minimum amount of sodium chloride for gelatin films are again illustrated in Figure 10. By comparing this micrograph with Figures 2 and 5 it may be seen that the relatively smooth area8 are rougher when sodium chloride is used. A similar comparison is illustrated in Figures 11and 12, using 8 sodium chloride layer of only 60 A. These micrographs illustrate chromeshadowed onestep silica replicas of a cellulose ester photographic film base. Even a t low magnification the smallest amount of sodium chloride introduces its own grain. However, it was found that the structure seen
ANALYTICAL CHEMISTRY
1012 in this esse could be made less prominent by not exposing the salt layer to the atmosphere before evaporation of the silica. In order to demonstrate the importance of using the minimum amount of sodium chloride, about twice this amount (11 mg.) was used in the replication of the fine structure previously illustrated in Figures 3, 4, 9, etc. The result is shown in Figure 13. The large, cubic structure would preclude any interpretation of fine detail that might have been present in the original sample.
Figure 13. Replica Similar to Specimen Illuatrated i n Figure 9 Twice optimum amount of sodium chloride used. Note
Figure 12. Replica Similar to Specimen in Figure 11 (25,OOOX), Intermediate Layer of Sodium Chloride Used Note “grain”
The technique of evaporating the silica onto the sodium chloride layer before exposing it to the atmosphere failed to alter this structure. A rather complete study suggested that the use of a hydrophilic hyer of sodium chloride has serious limitations when attempting to resolve detail smaller than 200 A. It may be worth while always to prepare a similar replica of some smooth surface such as mica. Any inherent detail due to the salt should then be considered when interpreting the replica of some unknown stNCtUre. The importance of not exposing the hydrophilic layer of sodium chloride t o the atmesphere prior to the evs,por% tion of the silica may depend on the affinity that the object has for water. For example, gelatin, especially under desiccating conditions, is extremely hygroscopic; certainly more hygroscopic than sodium chloride. It may be for this reason that there was no significant grain growth when sodium chloride over a wide range of thicknesses was applied t o gelatin. The 60 A. layer of sodium chloride on the hydrophobic cellulose ester film base did show evidence of increasing crystal size on exposure t o atmospheric moisture. It is conceivable that for certain objects resolution of structure finer than 200 A. is permissible when acdium chloride is used as an intermediate layer. This bas not been the authom’ experience, based on a limited number of different specimens. However, the effect of the total amount of sodium chloride evaporated onto the specimen does have a definite bearing on the size of the individual cubic crystals, which may in turn impair the resolution in the micrograph. This correlation between the siae of the individual sodium chloride crystals and the total thickness of the evaporated layer haz a
w(I
A. cubes
parallel in the study of silver and aluminum reported by Hass and Scott (8). SUBSTRATES OF SILICON DIOXIDE ANDSILICON MONOXIDE.Figure 14 shows differences in structures observed for Some silioon monoxide and silicon dioxide substrates. The silicon dioxide substrate (200 A. thick) exhibits the least structure. Next would he the 40 A. silicon monoxide substrate, although the differences between this and 100 A. silicon dioxide substrate seem to be slight. There is a far greater differencein structure between 200 A. silicon monoxide and 200 A. silicon dioxide substrates. Occasionrtlly some replicated StNCtUe characteristic of the mica is found. Thia structure usually consists of small tetrahedra and O&II easily be distinguished from that BtNCtUre inherent in the substrates.
and the silica was evaporated onto them simultaneously. The sub8trate was separated from one section by immersion in water. This substrate was picked up on a smooth sheet of polystyrene with the side that had been in contact with the mica now exposed to the air. The polystyrene with adhering substrate was mounted beside the other section of mica still retaining the silicon dioxide film. The two substrates were then shadowed simultaneously with approximately 3 A. of platinum-palladium a t an angle of incidence of 10”. A comparison of four micrographs of each substrate taken a t different area6 showed no consistent differences between the air side and the contact side. Mica was also found useful in the reparation of preshadowed replicas. The s ecimen was placeBon the surface of freshly cleaved mica a n t w a s shadowed with 3 A. of palladium. A layer of silicon dioxide, 75 A. thick, was evaporated onto the metal a t normal incidence and the replica was separated from the mica in water. A minimum of background structure w&sobtained in this manner. Figure 15 shows silicon monoxide substrates 40,100, and 200 A. thick. The structure here may be similar to that observed by Picard and Duffendsck (19) and later by Sennett and Scott (15) in their studies of evaporated metal films. Here also there appear to be agglomerates separated by channels, and the micrographs suggest an increase in the siae of these agglomerates as the thickness of the substrate is increased. This change in structure with increasing thickness is further evidence that the differences observed between the various substrates described herein are
V O L U M E 24, NO. 6, J U N E 1 9 5 2
1013
n
n
c
and silicon monoxide substrate already discussed, the minimum amount of platinum-palladium yielding a percep tible shadow had been used to avoid the introduction of what might be called a shadowing artifact. Figures 18, 19, and 20 show 40 A. silicon monoxide, 200 A. silicon dioxide, and Formvar substrates supporting polyvinyl, pyrrolidone. All were shadowed with 3 A. of platinumpalladium at an angle of 10". The results again show the silicon dioxide substrate (200 A,) to exhihit the least structure. For .this reman this substrate would be expected to make visible finer detail in the specimen being examined. For practical use the 200 A. silicon dioxide substrate may be of value 8 6 a support for substances of high electron-scattering power, where the 10% of contrast due to the thickness of the substrate will not interfere with interpretation. I n using the technique of Siege1 et a!. ( 1 4 ) for the determination of molecular weights by the m e a s urement of single molecules sprayed from solution onto the substrates, one might find these films of great value. ,Thinner substrates, such as silicon monoxide about 40 A. thick, while exhibiting more inherent structure than the 200 A. silicon dioxide are still smoother than the usual collodion substrates and afford greater contra&. SUMMARY AND CONCLUSIONS
Figure 14.
Substrates (150,OOOX)
A. 40 A. silicon monoxide B. ZW A. ailicon monoxide
C. 100 A. a i l i c o n dioxide D. ZWA. silicondioxide
Gelatin films, per se andchrome-shadowed, wereused as standards for comparing one-step and twmtep polystvrene-silica reolicss from the standnoint of wcuracv of reproduction and ease of preparation. Unlike the usual standards (glass, metals) which cannot he examined in the electron microscope, these organio films permit a more
Shadowed with 3 A. platinum-pall=dium at an sngle of innidence of 10'
inherent and not due to replication of the mica. surface. Reference t o Figure 14 reveals an opposite effect for silicon dioxidenamely, a decrease in substrate structure with increasing thickness. The electron-scattering powers (transmissivity) of equal thicknesses of silicon monoxide and silicon dioxide are the same. It is conceivable that the inherent structure io silioon monoxide increases with increasing thickness, because the vapor condensates become more orystalline. Very thin and rapidly formed evaparated films of silicon monoxide are amorphous, according to Has8 (7). On the other hand, silicon dioxide films prepared similarly &re crystalline (8) in all thicknesses, regardless of speed of formation. Consequently, in this material the greater overlapping of the cryrtalline aggregates a t greater film thicknesses may account for the lesser structures simply because the resolution of the individual crystalline aggregates is no longer possible. This theory may not be completely tenable. However, the phenomena are not readily explainable without providing room for reservation. I n shadowing specimens for electron microscopy it must be remembered that structure is also introduced by evaporated metal films. If the minimum amount of the metal required for perceptible shadows is used, there is usually little danger of a serious impairment of resolution. Figure 16 shows the results of a fourfold increase in the thickness of evaporated platinumpalladium on a silicon monoxide substrate 40 A. thick. I n comparing the specimens, it is apparent that the structure visible in the heavily shadowed film is that of the metal itself. Figure 17 shows a similar case where sections of a 100 A. silicon dioxide substrate were shadowed with 3 and 12 A. of platinum-palladium, respectively. The increase in size of the structure on both thicknesses of substrate indicates that the smoothest surface is of little value if care is not used in the shadowing. . - I n comparing what is believed to be the inherent structure of the silicon dioxide
A ~.
Figure 15.
R
C
Silicon Monoxide Substrates (l50,OOOX)
Shadowed with 3 A. platinum-pallsdium at an ztngle of
.I_."_ I--.,.,I"--~ ...":Ao""-
A.
40 A. thick
B . 100 A. fhiak
C. ZOO A. thick
1014
ANALYTICAL CHEMISTRY
critical evaluation of these replica techniques. Possible effects of high vacuum and solvents appear to be nonexistent, thereby permitting this comparison. From the standpoint of ease of preparation and accuracy of reproduction the one-step silica or silicon monoxide replica. sppears ta be the best and should be used wherever possible. For the renlieation of fine structure the use of nlatinum-oslladium
or uranium is preferred to chromium for shadowing because of the difference in thickness required for a given amount of eontrast. Chromium also introduces some structure, possibly through its oxidation. Depressions of 25 A. based on shadow lengths and plrtner distances of 80 A. were resolved. This technique is particularly advantageous whenever the original sample can be dissolved away from the replica. Textile fibers, shect plastics, many organic films, and crystals (inorganic and organic) are common examples. In so far as the materials examined in this investigation are .concerned, the cast polystyrene-silica replica is less desirable, primarily because its accuracy of reproduction is more limited, Distortion of line structure, especially when dispersed throughout a coarse structure, presumably occurs during the preparation of the neeative resin renlica. This techniaue R i sdvantaeeous and sometimes necessary when replicating some metals in bulk extremely rough or sharp surfaces, materials sensitive to light oP high vacuum, or specific areas on samples that are to be periadi oally replicated. Unlike the one-step silica replies, differences ii surface elevations of less than 150 A. were not always sharp11 defined. Fine horizontal or planar structure on the order of 1M A. was resolved. Kegative polystyrene replicas prepared hj compression molding could not he prepared because of the natun of the samples under investigation; hence, cast and molded repli a s could not be compared. A hydrophilic layer of sodium chloride should be used onlj when the sample makes it imperative. The thickness of thii1 layer must be carefully controlled so as to minimize its inherenit I
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rtmrbrro and a t +.haw m o" "+.;ma narmit lj, I".l""~."I..~I" "~ ^ ramnrial nf +he &lira ~ m A"T " i " "
A
B
Figure 16. Effect of Thickness of Shadowing Metal bn Structure Observed on 40 A. Silicon Monoxide Substrates (150,OOOX) Platinum-palladium at an angle of incidence of 10" ahadowed with A . 3 A. B . 12A.
mter. In any ease, a control replica of some very smooth surface such as mica should be prepared, so that proper cognizance of the structure may be taken. Under optimum conditions for the gelatin surface used in this investigation crystals of sodium chloride of the order of 200 A. are formed; therefore, resolution of structure on the original sample is limited by this value. The nature of the condensing surface may determine the amount of sodium chloride necessary and so this limit on resolution may be variable. Substrates of silicon monoxide and silicon dioxide can be prepared by the evaporation of the materials onto the surface of
A
Figure 17. Effect of Thickness of Shadowing ,Metal on Structure Observed on 100 A. Silicon Dioxide Substrates
Figure 18. Silioon Monoxide Substrates 40 A. Tbiek Supporting Polyvinylpyrrolidone (125,OOOX)
Shadowed with platinum- alladium at art angle of incidence of 103.4. B . 1 2 A .
Shadowed with 3 A. pl,latinum-palladium at so angle of incidence of 10-
8.
1015
V O L U M E 2 4 8 NO. 6, J U N E 1 9 5 2
Figure 20. Formvar Substrate Supporting Polyvinylpyrrolidone (150,000 X)
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freshly cleaved mica. The resulting structures appear t o he those inherent in the evaporated film. This structure is not unlike that reported for films of 8ome evaporated metals of the aame order of thickness. The size of this structure on silicon monoxide substrates increased with increasing thickness of the evaporated films. Films of silicon dioxide over a range of thicknesses have also been exhaustively studied, and there is good evidence that the substrates 200 A. thick exhibit less structure than the 100 A. substrates aqd hence may he desirable for high resolution studies in casea where the loss of contrast due to the inoreased thickness of the substrate m u he tolerated. Thin (40 A,) silioou monoxide substrates, while exhibiting more S t N C ture, afford greater contrast in the final image because of their lower electron-scattering power. The relatively thin iilms of platinum-pallsdium used in shadowing may introduce their own structure a8 the thickneas is increased over the minimum required for perceptible shadows. A0 interesting observation pertaining to specimen drift, a serious factor in high magnification electron micrography, is that speoimens prepared by means of silicon dioxide Rubstrates exhibit less drift than those prewred using Formvar. The importance of thermal conductivity in this regard in not known, although in the bulk state silica has the higher thermal condu5 tivity. The conclusions concerning the relative merits of the replicas may be somewhat specific because they are based on specific samples. Nevertheless, one fact stmds out boldly, for critical evaluation of certain materials-more than one type of replica should be prepared whenever possible. The differences in structure, resulting from variations in evaporated silica thickness (see Figures 14 and 151, are considerably more dearly illustrated by the original prints. Some fine detail is last during reproduction for publication. I n fact, micrographs representing the more subtle effects of intermediate thicknesses were not shown for this reason. LITERATURE CITED
(1) Comer, J. J., and Hamm, F. A,, ANAL. CHEM.,21, 418-19 (1949). (2) Electron Microsoope Society of America, Committee on Resolution, J . Applied Phys., 17, 989-96 (1946).
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(3) N l e m . E. F.. and Savage, R. H., Ibid., 19, 6 W 6 1 (1948) (4)Gerould, C. H.,Ibid., 18, 33343 (1947). (5) Hamm, F. A., Ihid.. 21, 271-8 (1950). (6) Hamm. F. A,. and Comer. J. J., ANAL. C n m . . 20, 861-70 (1948).
(7) Hass. G., J . Am. Cwem. Soc., 33, 355-60 (1950). ( 8 ) Hass, G.. and Scott. N. W., J. phm. radium,11,394-402 (1950). (9) Heidenreich. R. D.. and Peck, Y.G., J . Applied P h g ~ .14, , 23-9
(1943). (10) Hillier. J., "Adjustment and Manipulation of Electron Microcope," presented at meeting of Electron Microscope Society
of America, Toronto, Canede, Sept. 9 to 11, 1948. (11) Hillier, James, RCA Laboratories, Princeton, N. J., private communication. (12) Pioard. R. G., and Duffendaok, U. S., J . Applied Phm.. 14,291305 (1943). (13) Sennett. R. S., and Scott, G. D., J . Optical Soe. Am.. 40,203 (1950). (14) Sieeel. B.M..Johnson. D. H.. and Mark.. H... J . Polvner 6%.. 5.. iG.'i, 111120 (issoj. (15) Strong. John. "Procedures in Experimental Physics," p. 177, New York, Prentioe-Hall, 1944. (16) Williams. R. C., and Baokus. R. C., J . Applied Phw.. 20,9&106
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(1949).
(17) Willis*ms.R. C., and Wyckoff,R. W. G.. I b d . . 17,23-33 (1946).
Recerveo for review March 14, 1950. Accepted March 21, 1952. A paper describing work ~n replicas was presented before the Electron M ~ D ~ O B C O D Society. September 9, 1048, at Toronto, Canads. A p a p e r o n ~ t Dresented before the aame moiety September 14, 1950. at Detr,
Corrections I n the paper on "Automatic Measuremcnt of Optical Rotation" [Levy, G. B., ANAL.CHEM.,23,1089 (195111the heading of Table I should be "Dependcnoe of Reaction Rate on Penicillinase Concentration." I n the papers by J. H. Mahon and R. A. Chapman on "Estimsi tion of Antioxidants in Lard and Shortening" [ANAL. CHEM.,23, 1116 (1951)], "Butylated Hydroxyanisole in Lard and Shortening" [ANAL.CHEM.,23, 1120 (1951)], and "Estimation of 2- and 3-tert-Butyl-4hydroxyanisole Isomers" [ANAL. CHEM.,24, 534 (1952)], thereagentindicated its l,l'-bipyridine should have been called Z,Z'-bipyridine.
