by JAKOB R~LI and PER R. FLOOD, Institute of Anatomy, University of Bergen, 5000 Bergen, Norway Paper accepted 4 November 1977

Transcription

1 CJ Journal of Microscopy, Vol. 112, Pt 3, April 1978, pp Paper accepted 4 November 1977 A simple method for the determination of thickness and grain size of deposited films as used on non-conductive specimens for scanning electron microscopy by JAKOB R~LI and PER R. FLOOD, Institute of Anatomy, University of Bergen, 5000 Bergen, Norway SUMMARY Latex spheres in linear aggregates allow coating materials to accumulate only along their free surface. Transverse to the axis of the aggregate the diameter of a sphere will increase corresponding to the uncoated diameter plus twice the film thickness. Parallel to the axis of the aggregate the diameter of terminal spheres will increase with one film thickness, whereas the diameter of 'enclosed' spheres remain constant. By mounting uniform latex spheres on standard electron microscope-grids and comparing the transverse to the parallel diameter of linearly aggregated spheres in the transmission electron microscope, the film thickness produced by various coating procedures has been determined. The grain size and crack formations in the films may be examined simultaneously. INTRODUCTION In scanning electron microscopy of non-conductive biological tissues it is usual to deposit a thin metal film onto the specimens to improve their electrical conductivity and secondary electron emission. The thickness, grain size and tendency to crack formation of the metal film may, however, obscure delicate specimen details and reduce the resolution obtained on the micrographs (e.g. Echlin & Hyde, 1972). We are making some efforts to optimize our coating procedure and use a simple and reliable method to check the thickness, grain size and crack formation of the metal films. This method is based on conventional transmission electron microscopy of latex sphere test specimens. DESCRIPTION OF METHOD We find it convenient to use DOW latex spheres with a nominal diameter of pm pm (mean It: standard deviation, given by the manufacturer). The commercial solution is diluted with distilled water to faint opalescence, sonicated for 1 min, and spread onto formwar and carbon coated copper grids. These specimens are mounted together with ordinary scanning specimens in an Edwards 12EG coating unit and coated with various thicknesses of argon sputtered or thermally evaporated metals or alloys under standardized conditions. During thermal evaporation the specimens are mounted on a rotating table (30 rpm) with two evaporation sources 30" and 60" above the plane of the table. ~ /78~ / The Royal Microscopical Society 359

2 Jakob Rali and Per R. Flood As latex spheres are said to shrink slightly under certain preparative conditions (Agar & Chescoe, 1974), we have measured the diameter of our uncoated spheres after vacuum treatments similar to those used during the coating procedures. Using a Philips EM 300 electron microscope with a magnification calibrated to x 51,000 by the use of a diffraction grating of lineslinch we find the diameter of thirty unaggregated spheres to be pm pm (mean f SD). However, the aggregated spheres are compressed slightly parallel to the axis of the aggregates. For linearly aggregated spheres the transverse to parallel diameter ratio (D,/d,) of thirty spheres lodged between two other spheres (so-called enclosed spheres) is found to be 1.03 t 0.01 (mean _+ SD) whereas that of thirty spheres contacted by only one other sphere (so-called terminal spheres) is 1.02 k 0.01 (mean k SD). Due to the variable size of the spheres (SD= k 6 nm) we find a film thickness determination based on a comparison of diameters of uncoated and coated spheres too unreliable for use on films for scanning electron microscopy. However, as the coating material cannot accumulate at contact points between aggregated spheres, we may use their constant spherical (or rather slightly ovoid) form as a basis for film thickness determination to the f 2 nm level. Our method is based on the fact that the sphere-diameter transverse to the axis of linearly aggregated spheres, increases corresponding to their uncoated diameter plus twice the film thickness, whereas the diameter parallel to the axis of the aggregate remains constant for spheres lodged between two other spheres (enclosed spheres) and increases with one film thickness for terminal spheres. As the borders between aggregated spheres are quite distinct after coating, the film thickness may be determined by calculating the transverse to the parallel diameter ratio (D,/d,) of the coated spheres and comparing it to the comparable ratio of uncoated spheres (D,,/dJ. For enclosed spheres the film thickness (in nm) will be and for terminal spheres where 227 is the observed mean diameter of the uncoated spheres in nm, D,, and dce the diameter of coated enclosed spheres, transverse, respectively parallel to the axis of the aggregate, and Due and due the comparable diameters of uncoated spheres (found to give the ratio of 1.03). Dct, dct, D,t and d,t refer to similar measurements on terminal spheres (Fig. la).* Film thicknesses derived from various diameter ratios of enclosed and terminal spheres are given in Fig. l(b). As is evident from these curves a mean diameter ratio of enclosed spheres after coating of 1.05 (1.03 for terminal spheres) corresponds to a film thickness of about 2 nm (Fig. 2a). This is in good agreement with the film thickness du-ectly visualized along the edge of such spheres (Fig. 2b). D' iameter ratios of enclosed spheres of ( for terminal spheres), corresponding to a film thickness between 7 and 65 nm, may be fairly accurately determined (Figs. 3-5). Films with greater diameter ratios are difficult to examine due to poor electron penetrability (Fig. 6). The grain size and crack formations in the evaporated films may be examined in the micrographs of the supporting film in the vicinity of the latex sphere aggregate. The diameter of enclosed spheres parallel to the axis of the aggregates may serve as an internal standard (0.227 pm) for satisfactory magnification calibration. * See note added in proof. 360

3 Film thickness meawement Coated I Uncoated The designation of various parameters on coated and un- Fig. l(a). coated, enclosed and terminal latex spheres. For further explanation see text s E E L n- J Diameter ratio of coated spheres ( Dc Idc) size 0,227pm Fig. l(b). The relation between diameter ratio of coated enclosed and terminal spheres and film thickness. For further explanation see text. DISCUSSION The film thickness may probably be determined with an accuracy of about 2 nm when the enclosed sphere-diameter ratio method is used. However, to obtain this accuracy it is necessary to search for a perfectly straight aggregate where the borders of an enclosed sphere to its neighbours are close to parallel. In such cases the diameter ratio may be determined with an accuracy of about? 1%, corresponding to about _+ 1 nm. The variable diameter ratio of the uncoated spheres (SD = 0.01) is responsible for the remaining inaccuracy. Attempts to reduce the uncertainty by using smaller latex spheres have failed due to a much more variable particle size in the available suspensions of such particles. The reason why aggregated uncoated spheres appear to be slightly compressed (3%) parallel to the axis of the aggregate is unknown. A possible explanation may be the surface tension of water as it dries down around the spheres. To minimize the effect of any radial asymmetry in the magnifying power of the microscope, we recommend that aggregates of distinct radial orientation are included when the diameter ratio is to be established. Unfortunately, the perfectly straight aggregates are rare and one has to use slightly curved aggregates where the exact parallel diameter of an enclosed sphere may be difficult to determine (cf. Fig. 5). In these cases the terminal sphere-diameter 36 1

4 362 Figs. 2-6

5 Film thickness measurement ratio method is to be preferred. However, with this method the variable diameter ratio of uncoated spheres (SD = k 0.01) imposes a similar k 2 nm inaccuracy on the film thickness calculation. From these considerations it should be evident that the percent inaccuracy of our diameter ratio methods may be very large for thin films (k 5074 for 4 nm films) and relatively small for thick films (f loo() for 20 nm films). However, for the thin films (between 1 and 8 nm thick) it is possible to observe the film directly in the transmission electron microscope as a distinct layer at the edge of the spheres (cf. Fig. 2b). The accuracy of measurements on such layers may probably be better than k 1 nm. Besides the need of access to a transmission electron microscope the present method has certain advantages to other methods used for film thickness measurements (Holland, 1958; Chopra, 1969; Echlin & Hyde, It is direct, i.e. not hampered with the uncertainties of measuring a parameter more or less loosely related to the film thickness (e.g. the amount of source material and distance between specimen and evaporation source (Russ & Kabaya, 1970)). It is fairly accurate: except for very thin films as mentioned above, it is probably as accurate as the methods used hitherto (cf. Echlin & Hyde, 1972, Fig. 12). It is non-destructive, i.e. the specimens remain intact after analysis and may be used for comparative SEM studies. It is inexpensive, at least for transmission electron microscopists; expensive instruments like a quartz crystal thin film monitor or a multibeaminterferometer are not necessary. In addition there is usually no need to calibrate the microscope magnification or to follow rigid schemes to reproduce exactly the same magnification on several micrographs or specimens. A disadvantage may be that the film thickness is determined in vertically oriented films and that this thickness may differ from that of films of other orientations. When thermal evaporation sources are used, one at 45" above the plane of the rotating specimen table, or two at 30" and 60" above the table, as in this study, the film thickness will be the same in horizontally and vertically oriented films. In any case, the Fig. 2(a). Latex spheres coated by argon sputtered gold-palladium. Diameter ratio of enclosed sphere: 1.05, of terminal spheres, 1.03; calculated film thickness: 2.3 nm; grain size: 2-6 nm; cracks: absent. x 127,000. Fig. 2(b). Same specimen as Fig. 2(a), but underexposed to reveal the film thickness (43 nm) at the edge of a sphere. x 127,000. Fig. 3. Latex spheres coated by thermally evaporated gold. Diameter ratio of enclosed sphere: 1.12, of terminal spheres, 1.06; calculated film thickness: 10 nm; grain size: nm; cracks: absent. x 127,000. Fig. 4. Latex spheres coated by argon sputtered gold. Diameter ratio of enclosed sphere: 1.14, of terminal spheres, 1.07; calculated film thickness: 12 nm; grain size: nm; cracks: absent. x 127,000. Fig. 5. Latex spheres coated by argon sputtered gold-palladium. Diameter ratio of enclosed sphere: 1.37, of terminal sphere, 1.18; calculated film thickness: 38 nm; grain size: 5-20 nm; cracks: present. x 127,000. Fig. 6. Latex spheres coated by argon sputtered gold-palladium. Diameter ratio of enclosed sphere: 1.76, of terminal spheres, 1.37; calculated film thickness : 83 nm; grain size: obscured; cracks: present. x 127,

6 Jakob R0li and Per R. Flood form of biological samples is often so complex that the film thickness in one direction may be as informative as that of another. References Agar, A.W. & Chescoe, D. (1974) Checking the performance of the electron microscope. In: Practical Methods in Electron Microscopy (Ed. by A. M. Glauert), Vol. 2, pp North-Holland, Elsevier, Amsterdam. Chopra, K.L. (1969) Thin Film Phenomena. McGraw-Hill, New York. Echlin, P. & Hyde, P.J.W. (1972) The rationale and mode of application of thin film to nonconducting materials. In : Scanning Electron Microscopy (Ed. by 0. Johari), pp IIT Res. Inst., Chicago. Holland, L. (1958) Vacuum Deposition of Thin Films. Chapman & Hall, London. Russ, J.C. & Kabaya, A. (1970) Preparation of samples for scanning electron microscopy. In: Proc. 28th Annual Meeting E.M.S.A. Houston, Texas (Ed. by J. C. Arceneaux), pp NOTE ADDED IN PROOF We recently became aware of an article by Kern & Kern (1950) who found that uncoated latex spheres appeared to be about 9% smaller than spheres shadowed by a thin conductive layer of deposited metal. We have been unable to demonstrate size differences above 1% on our spheres under similar preparative conditions, and accordingly consider the results of Kern & Kern (1950) do not conflict with the use of our mean uncoated sphere diameter in the formulae for film thickness determination. Reference Kern, S.F. & Kern, R.A. (1950) The apparent size of objects as observed in the electron microscope. J. appl. Phys. 21,