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Scanning electron microscopy
Microelectromcs and Reliability
Pergamon Press 1965. Vol. 4, pp. 55-57.
Printed in Great Britain
SCANNING ELECTRON MICROSCOPY W. C. N I X O N
Engineering Laboratory, Cambridge University, Cambridge Abstract--Scanning electron microscopy is a method of microscopy that permits resolution better than that of the optical microscope (about 100 A) while examining one surface of a bulk specimen. The technique depends on electronic application to microscopy and has been developed over many years by electronic engineers rather than physicists or microscopists. The main field of application so far has been non-biological, but two biological examples are given here. The reference list includes the main papers on scanning electron microscopy written in this laboratory. INTRODUCTION
CONVENTIONAL transmission electron microscopy has reached the stage where commercially available instruments will yield routine resolution of 10 A with suitable specimens. However, suitability is sometimes hard to reproduce with thin sections that must bc used in transmission. In some cases thin sections or foils may not be prcpared at all. A resolution between that of the optical and electron microscope would bc acceptable if one polished surface of a solid specimen could be examined as in normal optical metallurgical microscopy. If this could be achieved then the specimen could be heated, cooled, strained, fractured, etc., while under obscrvation, which is not possible with a replica and only to a certain extent with thin foils. The scanning microscope produces this type of micrograph. METHOD T h e basic features of a scanning electron microscope are shown in Fig. 1 (K. C. A. Smith (1961), Encyclopedia of Microscopy, ed. G. L. Clark, Reinhold Pub. Corp., p. 241). T h e electronoptical column has the electron gun at the bottom and the electron beam is accelerated upwards by a potential of about 20 kV. Two magnetic electron lenses reduce the size of the electron source in the gun, say 50 ~, to a few hundred Angstroms at the specimen surface. With the most modern instruments using three electron lenses the probe size 55
is less than 100 A (R. F. W. Pease and W. C. Nixon, Brit. ~. Appl. Phys. (1965)). T h e resolution of the instrument is given by this electron-probe size. The electron current in such a probe is less than one ~ k and the aberrations of the present lenses prevent an increase in this current. This electron probe is scanned across the surface of the specimen by the use of beam deflecting coils within the electron-optical column. T h e scanning generator is also connected to the scanning coils of the display and photographic-recording cathode ray tubes. Magnification is achieved by reducing the current to the column coils while leaving the same current in the cathode-ray-tube coils. In this way a small raster of a few microns is scanned on the specimen while the cathode-ray-tube face contains a raster about 10 cmL T h e magnification is given by the ratio of these two rasters and can be up to 100,000 times. Several hundred lines are scanned in each frame with a frame rate of about 1 sec for visual work and up to 5 min for photography. The longer time permits integration of the signal and suppression of the "noise" due to the particulate nature of the electron beam in the main microscope column. The electron detector is now designed to collect the secondary electrons emitted from the specimen over a large solid angle by applying up to + 1 0 kV to the collector. These accelerated secondary electrons from the specimen strike a plastic scintillator and the light produced reaches a photomultiplier by passage through a perspex light pipe.
56
XV. C. N I X O N
The electrical signal from the photomuhiplier is proportional to the number of secondary electrons emitted from the surface of the specimen as the electron probe is scanned slowly across the surface to build up the picture on the long-persistancc cathode-ray-tube screen line by line. This signal modulates the brightness of the cathode-ray-tube screen and the final photograph shows the variation of collected electrons from the specimen surface. The contrast is due mainly to topographic variations as shown in the accompanying photographs. The range of contrast may be very great and so a gamma control is incorporated in the amplifier between the photomultiplier and the cathode-raytube control electrode. Contrast may thus be expanded or contracted electronically to suit the specimen. These principles have all been incorporated in the well known scanning electron-probe X-ray microanalyser with the addition of X-ray detection and therefore contrast display due to the variation of X-rays (i.e. the variation of elements) from point to point on the surface.
S C A N N I N G ELECTRON IVIICROGRAPHS
A selection of scanning electron micrographs is presented to show the main features of this type of result common to all specimens. The specimen is inclined at an angle to the electron-beam direction as shown in Fig. 1. As a result the micrographs are fore-shortened as if one were viewing the surface of the specimen at a shallow angle. The collector is set to one side and so the micrograph appears to be lit from one direction. These two features are shown in Fig. 2 where the specimen is a square grid lying on a solid surface. The square is now a rectangle and the highlights are on the edges of the grid. Once these features are recognized the unknown specimens may be interpreted correctly. The magnification varies within the micrograph depending on the direction of measurement due to this foreshortening. This is shown in the result by printing a small ellipse on the micrograph as shown in Fig. 3. The length of the major axis is written within the ellipse and the eccentricity of the ellipse is chosen to match the degree
SPECIMEN COLLECTOR PHOTOMULTIPLIER
N
,ogoo FIG. 1.
A final comment on the method is that although the micrographs are obtained by a scanning electron beam it is very difficult to see the scanning lines. The line and frame scanning rates and the electron-spot size in both the electron column and the cathode-ray tube are all adjusted so that the lines will n o t show on the subsequent micrograph. The universal acceptance of visible lines on domestic television receivers obscures the interpretation of scanning images of a higher standard.
of foreshortening so that the minor axis and in fact any line through the centre of the ellipse will also represent this length on a parallel line on the micrograph. This is a logical extension of the customary micron mark on transmission electron micrographs. CONCLUSIONS
The scanning electron microscope may be applied to selected biological specimens if care is
FIc. 2. Scanning electron micrograph of a square grid lying on a solid surface.
FxG. 3. Surface of a meal worm grub, Tenebrio molitor, silver coated. ( × 475.)
FIG. 4. As in Fig. 3 but at twenty times higher magnification showing a single bristle on the surface. ( × 9500.) More recently Thornley (1960) has been able to operate a scanning electron microscope at 1"5 kV and avoid the charging of insulating specimens. One application of biological interest is the use of frozen but not freezedried material directly in the microscope.
FIc. 5. Orlon fibre, gold-palladium coated. ( × 1800.)
facing pa~e 56
FIG. 6. Etched aluminium, specimen angle 25 ~.( "< 1800.)
FIG. 7. As in Fig. 6 but with a specimen angle of 45 ( × 1800.)
FIG. 8. Tungsten point in contact with germanium surface, before discharge of a condenser through the point of contact. ( × 600.)
FIG. 9. As in Fig. 8 hut after condenser discharge showing the ploughing up of the germanium surface ( x 60O.)
This last pair of micrographs demonstrates the use of the scanning electron microscope to observe dynamic changes in a specimen by viewing the same field during all stages of the experiment.
SCANNING ELECTRON MICROSCOPY taken in interpreting the result. T h e first commercial i n s t r u m e n t has been demonstrated at the Physical Society Exhibition by the Cambridge I n s t r u m e n t Company. Wider use of these instruments should show which fields of application, including the biological, may be usefully explored b y this relatively new method of non-conventional electron microscopy.
Acknowledgments--Fig. I has been reproduced from K. C. A. Smith (1961), Encyclopedia of Microscopy, ed. G. L. Clark, Reinhold Pub. Corp., p. 241; Figs. 3-5, 8 and 9, are reproduced from K. C. A. Smith and C. W. Oatley (1955), Brit. J. appl. Phys., 6, 391-399; Figs. 2, 6 and 7 were taken originally by K. C. A. Smith at the Engineering Laboratory, Cambridge University. This review is printed from J. Roy. Micro. Soc. 83, 213-216 (1964).
REFERENCES A. BOYDE, A. D. G. STEWART(1962), A study of the etching of dental tissues with argon ion beams..7. Ultrastruct. Res., 7, 159-172.
57
T. E. EVEItHART,K. C. A. SMITH, O. C. WELLS and C. W. OATLEY(1958), Recent developments in scanning electron microscopy. Fourth Internat. Conf. Electron Mice., Berlin, pp. 269--273. Veriag, Berlin. T. E. EVm~.RT and R. F. M. THORNLEY(1960), Wideband detector for micro-microarnpere low-energy electron currents. J. sc/. Instrum., 37, 246-248. T. E. EVF.RHART,O. C. WELLSand C. W. O^TLEY(1959), Factors affecting contrast and resolution in the scanning electron microscope. J. Electronics Control 7, 97-111. D. MCMULLAN(1953), An improved scanning electron microscope for opaque specimens. Proc. Inst. Elec. Eng. (Lond.) 100, Pt. II, 245-259. C. W. OATLEYand T. E. EVImHART(1957), The examination of p-n junctions with the scanning electron microscope. J. Electronics 2, 568-570. K. C. A. SMITH and C. W. OATLEY(1955), The scanning electron microscope and its fields of application. Brit. J. appl. Phys. 6, 391-399. A. D. G. STEWARTand A. BoYvE (1962), Ion etching of dental tissues in a scanning electron microscope. Nature, Lond. 196, 81, 82. R. F. M. THORNLEY (1960), Recent developments in scanning electron microscopy. Proc. Europ. Reg. Conf. Electron Mice., Delft, I, 173-176. O. C. W,~Ns (1959), Examination of nylon spinneret holes by scanning electron microscopy..7. Electronics Control 7, 373-376.
Pergamon Press 1965. Vol. 4, pp. 55-57.
Printed in Great Britain
SCANNING ELECTRON MICROSCOPY W. C. N I X O N
Engineering Laboratory, Cambridge University, Cambridge Abstract--Scanning electron microscopy is a method of microscopy that permits resolution better than that of the optical microscope (about 100 A) while examining one surface of a bulk specimen. The technique depends on electronic application to microscopy and has been developed over many years by electronic engineers rather than physicists or microscopists. The main field of application so far has been non-biological, but two biological examples are given here. The reference list includes the main papers on scanning electron microscopy written in this laboratory. INTRODUCTION
CONVENTIONAL transmission electron microscopy has reached the stage where commercially available instruments will yield routine resolution of 10 A with suitable specimens. However, suitability is sometimes hard to reproduce with thin sections that must bc used in transmission. In some cases thin sections or foils may not be prcpared at all. A resolution between that of the optical and electron microscope would bc acceptable if one polished surface of a solid specimen could be examined as in normal optical metallurgical microscopy. If this could be achieved then the specimen could be heated, cooled, strained, fractured, etc., while under obscrvation, which is not possible with a replica and only to a certain extent with thin foils. The scanning microscope produces this type of micrograph. METHOD T h e basic features of a scanning electron microscope are shown in Fig. 1 (K. C. A. Smith (1961), Encyclopedia of Microscopy, ed. G. L. Clark, Reinhold Pub. Corp., p. 241). T h e electronoptical column has the electron gun at the bottom and the electron beam is accelerated upwards by a potential of about 20 kV. Two magnetic electron lenses reduce the size of the electron source in the gun, say 50 ~, to a few hundred Angstroms at the specimen surface. With the most modern instruments using three electron lenses the probe size 55
is less than 100 A (R. F. W. Pease and W. C. Nixon, Brit. ~. Appl. Phys. (1965)). T h e resolution of the instrument is given by this electron-probe size. The electron current in such a probe is less than one ~ k and the aberrations of the present lenses prevent an increase in this current. This electron probe is scanned across the surface of the specimen by the use of beam deflecting coils within the electron-optical column. T h e scanning generator is also connected to the scanning coils of the display and photographic-recording cathode ray tubes. Magnification is achieved by reducing the current to the column coils while leaving the same current in the cathode-ray-tube coils. In this way a small raster of a few microns is scanned on the specimen while the cathode-ray-tube face contains a raster about 10 cmL T h e magnification is given by the ratio of these two rasters and can be up to 100,000 times. Several hundred lines are scanned in each frame with a frame rate of about 1 sec for visual work and up to 5 min for photography. The longer time permits integration of the signal and suppression of the "noise" due to the particulate nature of the electron beam in the main microscope column. The electron detector is now designed to collect the secondary electrons emitted from the specimen over a large solid angle by applying up to + 1 0 kV to the collector. These accelerated secondary electrons from the specimen strike a plastic scintillator and the light produced reaches a photomultiplier by passage through a perspex light pipe.
56
XV. C. N I X O N
The electrical signal from the photomuhiplier is proportional to the number of secondary electrons emitted from the surface of the specimen as the electron probe is scanned slowly across the surface to build up the picture on the long-persistancc cathode-ray-tube screen line by line. This signal modulates the brightness of the cathode-ray-tube screen and the final photograph shows the variation of collected electrons from the specimen surface. The contrast is due mainly to topographic variations as shown in the accompanying photographs. The range of contrast may be very great and so a gamma control is incorporated in the amplifier between the photomultiplier and the cathode-raytube control electrode. Contrast may thus be expanded or contracted electronically to suit the specimen. These principles have all been incorporated in the well known scanning electron-probe X-ray microanalyser with the addition of X-ray detection and therefore contrast display due to the variation of X-rays (i.e. the variation of elements) from point to point on the surface.
S C A N N I N G ELECTRON IVIICROGRAPHS
A selection of scanning electron micrographs is presented to show the main features of this type of result common to all specimens. The specimen is inclined at an angle to the electron-beam direction as shown in Fig. 1. As a result the micrographs are fore-shortened as if one were viewing the surface of the specimen at a shallow angle. The collector is set to one side and so the micrograph appears to be lit from one direction. These two features are shown in Fig. 2 where the specimen is a square grid lying on a solid surface. The square is now a rectangle and the highlights are on the edges of the grid. Once these features are recognized the unknown specimens may be interpreted correctly. The magnification varies within the micrograph depending on the direction of measurement due to this foreshortening. This is shown in the result by printing a small ellipse on the micrograph as shown in Fig. 3. The length of the major axis is written within the ellipse and the eccentricity of the ellipse is chosen to match the degree
SPECIMEN COLLECTOR PHOTOMULTIPLIER
N
,ogoo FIG. 1.
A final comment on the method is that although the micrographs are obtained by a scanning electron beam it is very difficult to see the scanning lines. The line and frame scanning rates and the electron-spot size in both the electron column and the cathode-ray tube are all adjusted so that the lines will n o t show on the subsequent micrograph. The universal acceptance of visible lines on domestic television receivers obscures the interpretation of scanning images of a higher standard.
of foreshortening so that the minor axis and in fact any line through the centre of the ellipse will also represent this length on a parallel line on the micrograph. This is a logical extension of the customary micron mark on transmission electron micrographs. CONCLUSIONS
The scanning electron microscope may be applied to selected biological specimens if care is
FIc. 2. Scanning electron micrograph of a square grid lying on a solid surface.
FxG. 3. Surface of a meal worm grub, Tenebrio molitor, silver coated. ( × 475.)
FIG. 4. As in Fig. 3 but at twenty times higher magnification showing a single bristle on the surface. ( × 9500.) More recently Thornley (1960) has been able to operate a scanning electron microscope at 1"5 kV and avoid the charging of insulating specimens. One application of biological interest is the use of frozen but not freezedried material directly in the microscope.
FIc. 5. Orlon fibre, gold-palladium coated. ( × 1800.)
facing pa~e 56
FIG. 6. Etched aluminium, specimen angle 25 ~.( "< 1800.)
FIG. 7. As in Fig. 6 but with a specimen angle of 45 ( × 1800.)
FIG. 8. Tungsten point in contact with germanium surface, before discharge of a condenser through the point of contact. ( × 600.)
FIG. 9. As in Fig. 8 hut after condenser discharge showing the ploughing up of the germanium surface ( x 60O.)
This last pair of micrographs demonstrates the use of the scanning electron microscope to observe dynamic changes in a specimen by viewing the same field during all stages of the experiment.
SCANNING ELECTRON MICROSCOPY taken in interpreting the result. T h e first commercial i n s t r u m e n t has been demonstrated at the Physical Society Exhibition by the Cambridge I n s t r u m e n t Company. Wider use of these instruments should show which fields of application, including the biological, may be usefully explored b y this relatively new method of non-conventional electron microscopy.
Acknowledgments--Fig. I has been reproduced from K. C. A. Smith (1961), Encyclopedia of Microscopy, ed. G. L. Clark, Reinhold Pub. Corp., p. 241; Figs. 3-5, 8 and 9, are reproduced from K. C. A. Smith and C. W. Oatley (1955), Brit. J. appl. Phys., 6, 391-399; Figs. 2, 6 and 7 were taken originally by K. C. A. Smith at the Engineering Laboratory, Cambridge University. This review is printed from J. Roy. Micro. Soc. 83, 213-216 (1964).
REFERENCES A. BOYDE, A. D. G. STEWART(1962), A study of the etching of dental tissues with argon ion beams..7. Ultrastruct. Res., 7, 159-172.
57
T. E. EVEItHART,K. C. A. SMITH, O. C. WELLS and C. W. OATLEY(1958), Recent developments in scanning electron microscopy. Fourth Internat. Conf. Electron Mice., Berlin, pp. 269--273. Veriag, Berlin. T. E. EVm~.RT and R. F. M. THORNLEY(1960), Wideband detector for micro-microarnpere low-energy electron currents. J. sc/. Instrum., 37, 246-248. T. E. EVF.RHART,O. C. WELLSand C. W. O^TLEY(1959), Factors affecting contrast and resolution in the scanning electron microscope. J. Electronics Control 7, 97-111. D. MCMULLAN(1953), An improved scanning electron microscope for opaque specimens. Proc. Inst. Elec. Eng. (Lond.) 100, Pt. II, 245-259. C. W. OATLEYand T. E. EVImHART(1957), The examination of p-n junctions with the scanning electron microscope. J. Electronics 2, 568-570. K. C. A. SMITH and C. W. OATLEY(1955), The scanning electron microscope and its fields of application. Brit. J. appl. Phys. 6, 391-399. A. D. G. STEWARTand A. BoYvE (1962), Ion etching of dental tissues in a scanning electron microscope. Nature, Lond. 196, 81, 82. R. F. M. THORNLEY (1960), Recent developments in scanning electron microscopy. Proc. Europ. Reg. Conf. Electron Mice., Delft, I, 173-176. O. C. W,~Ns (1959), Examination of nylon spinneret holes by scanning electron microscopy..7. Electronics Control 7, 373-376.