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The reduction of radiation damage in the electron microscope
Ultramicroscopy 11 (1983) 67-70 North-Holland Publishing Company
67
SHORT NOTE THE REDUCTION OF RADIATION DAMAGE IN THE ELECTRON MICROSCOPE J.R. FRYER and F. HOLLAND Chemistry Department, University of Glasgow, Glasgow G12 8QQ, UK Received 25 January 1983
The major factor limiting the resolution in organic and biological specimens in the electron microscope is radiation damage caused by the electron beam. This work describes the application of an encapsulation method at room temperature that reduces radiation damage by a factor of 3-12 and provides an explanation for the nature of the damage mechanism. The most stable organic crystal hitherto examined in the electron microscope is hexadecachlorocopper phthalocyanine. It has been possible to image the molecule [1,2] and examine the structural changes caused by radiation damage [2-4]. The effect of radiation is to create a random distribution of damaged areas where degradation of the crystal lattice proceeds rapidly; and since the prerequisite for this rapid degradation is an adjacent non-crystalline region, it is clear that this more open region permits diffusion of active species [3]. A further observation on this compound [4] is that crystalline copper oxide (both Cu 2° and CuO) occasionally forms in the damaged area, particularly in thick specimens. The extent of the damage area is proportional to the time elapsed since it was initiated, and since this initiation is random a series of kinetic measurements can be made relating the extent of the damaged area to the amount of copper in the copper-oxide crystal, showing that the rate-determining step for growth of damaged areas corresponds to a diffusion value o f -- 10 - 7 c m 2 S -1. T h i s value is much lower than surface diffusion rates and corresponds to bulk diffusion values in organic molecular crystals. In view of the assumptions made in this calculation, it cannot be regarded as much more than a suggestion that bulk diffusion is the rate-determin-
ing step, but it promoted the idea of covering the specimen with a continuous layer of carbon that would encapsulate active species within the crystal, providing more opportunity for recombination of species displaced by the primary radiation impact. Similar radiation protection factors to those observed here had been observed by Salih and Cosslett [5,6] using carbon, gold and aluminium layers covering the specimen, but because separate specimens were used the precision of the results was low, and an explanation was put forward based on the conductive properties of these materials. Bright field high resolution images of chlorinated copper phthalocyanine, metal-free phthalocyanine, polydiacetylene and coronene had shown that damage was initiated at random sites on the crystal and at crystal edges. The damaged area expanded rapidly from these sites in a circular manner until the whole crystal was destroyed [2]. The similarity in behaviour among these different compounds suggested that a similar damage mechanism pertained. Therefore epitaxial films of hexadeca-halocopper phthalocyanine, metal-free phthalocyanine, perylene and the paraffin n-hexatriacontane (C36H74) were prepared by deposition onto KC1, the first three by vacuum evaporation and the paraffin by crystallisation from petroleum ether solution. The crystalline layer on the KC1 was backed with a carbon layer deposited by evaporation, floated off on water and picked up on an electron microscope grid. The other side of the specimen was then half covered with evaporated carbon so that the final arrangement was that shown in fig. 1. In addition, a specimen of catalase was prepared on carbon from suspension and half
0304-3991/83/0000-0000/$03.00 © 1983 North-Holland
J.R. Fryer, F. Holland / Reduction of radiation damage in E M
68
damage was total extinction of diffraction spots determined both by observation of the final screen and sequential micrographs. Absolute values of radiation dose are often quoted as the dose De to achieve 1 / e of the original intensity of first-order diffraction spots under particular operating conditions. In the present work this was not necessary because the aim was to obtain comparative values between covered and uncovered parts of a homogeneous specimen. Thus the values for radiation dose may not accord with absolute values but are self-consistent for one specimen. In practice it was found that there was good agreement between different specimens of the same preparation and thickness, as shown by the standard deviation in results. The results are shown in table 1, and the encapsulating carbon film gave a protection factor of 6-12 for halogenated molecules and a factor of - 3 for hydrogenated. There was no significant difference in protection between aliphatic and aromatic compounds when hydrogen was the peripheral atom in the compound. An important feature was that thickness was significant and may account for the diversity of values of radiation critical doses that have been observed previously. For example, the factor between the thin uncovered specimen of chlorinated copper phthalocyanine and the thick covered specimen appears to be - 2 0 , but this neglects the role of specimen
/ _ j _ _ C o . r bon ~-..~~...:-..-:-.-.-.-.~ - . ~ " [~
[]
[]
Specimen []~
Coxbon
~Grid
bar
Fig. 1. Schematic cross-section of half-covered specimen arrangement.
covered with carbon, as the other specimens. This half covering permitted identical radiation damage measurements to be made between covered and uncovered areas of specimen on the same specimen grid. This is particularly important since it ensured that the microscope conditions for the comparative measurements were the same; the same beam diameter and all other operating conditions were maintained for measurements on the covered and uncovered parts of the same specimen. The epitaxial preparation ensured that all areas of the specimen were of the same thickness and orientation. The electron dose was measured by means of a flat copper plate in the image plane, and a backscattering factor of 30% was used. This factor has been shown to be reproducible [7]; and certainly under these experimental conditions, which involved only moving from one part of a specimen to another, there would be no change in the backscattering factor. The criterion for radiation
Table 1 Electron doses required for extinction of first-order reflections in the diffraction pattern; JEOL 100C electron microscope 100 kV with 200/~m diameter condenser aperture and 0.16/~m 2 selected area Sample
With carbon coating ( C / e r a 2 )
Without carbon coating ( C / c m 2)
Thickness (A.)
No. of measurements
Factor and standard deviation
Paraffin C36H 74
0,04 5:0.005
0.013 + 0.003
Unknown
15
3.1 + 0.4
Perylene
0,11 + 0.02 0,07 -t- 0.02
0.04 + 0.01 0.024 4:-0.01
330 70
35 42
2.6 5:0.7 3.0 +_ 1.7
Phthalocyanine
0.47 + 0.08
0.175 + 0.02
100
26
2.7 5:0.5
2.1 6.3
54 103
4 16
9.3+0.8 6.1+2.4
9
3.1 + 0.4
Chlorinated copper phthalocyanine Catalase Brominated copper phthalocyanine
19.5 + 5 38.9 + 1 4 0.05 5:0.001 10.6 + 0.2
+0.15 +0.87
0.015 + 0.002 0.92 + 0.17
Unknown 35
10
11.6 + 2
J.R. Fryer, F.. Holland / Reduction of radiation damage in E M
thickness. The effect of the encapsulating carbon diminished as the specimen thickness increased, but since dynamical scattering effects begin to be significant at 15 nm, this would reduce any advantages of using thicker specimens for high resolution studies [8]. The effect of the encapsulating layer ( - 5 rim) did not appear to affect the resolution obtainable in the specimens as shown by lattice imaging. Consideration of the protection factors indicates the nature of the radiation damage mechanism. The similarity of protection factors between metal-free phthalocyanine, perylene, catalase and paraffin show that the chemical structure of the molecular carbon framework is not important. Neither are the electronic properties of the molecule or crystal, in view of the considerable differences in electron transfer properties between paraffin (i.e. down the c axis) and metal-free phthalocyanine. Furthermore, if the carbon simply provided a conduction path, as suggested previously [5,6], then the differences between hydrogenated and halogenated compounds would not occur. The cross-sections of the atom involved also have little effect, as both chlorine and bromine have lower electron capture cross-sections than carbon, and if carbon loss predominated, then the protection factors for both of these materials would be the same. The factor that explains these results is the ability of the peripheral atoms to diffuse away from the parent molecules. The theoretical ratio of diffusion rates for H 2, C12 and Br 2 as a function of the square root of their molecular weights is H2:C12:Br2 = 9: 1.4: 1. Therefore, as the protection factor would be expected to vary inversely with the diffusion rates, the theoretical ratio would be H2:CI2:Br2 = 1 : 6 : 9 . The protection factors measured for molecules containing these peripheral atoms are 3 : 9 : 12; i.e., approximately RH : RC1 : RBr = 1 : 3 : 4. Therefore the experimental results follow the same
69
trend as the theoretical ratio based on the concept of diffusion and for the two chemically similar halogen molecules show a reasonable quantitative agreement. Thus the damage mechanism would consist of an initial excitation of the molecule by the electron beam in which all of the atoms would gain sufficient energy to break the chemical bonds. However, the atoms are held in a lattice so that the predominant reaction would be recombination into the original molecule. Peripheral atoms are less tightly held, so that a proportion can diffuse away from the parent molecule, which in turn, as a charged moiety, may spontaneously degrade. It is this degradation that is observed in the electron microscope, and hence absolute values of radiation damage for different compounds vary by orders of magnitude, depending upon the structure of the parent moiety, but the protection factor of the encapsulating film that reduces diffusion and hence aids recombination is only dependent upon the nature of the peripheral atom. The smaller the atom, then, the more easily it could diffuse, as illustrated by the differences between hydrogen and halogens in these experiments. It would be expected, then, that all parts of a molecule exposed to an electron beam would be in a constant state of fission and recombination, with permanent damage occurring when a peripheral atom diffused away and the remaining charged species decomposed. The effect of cooling the specimen would be to slow down the diffusion, but also slow down the recombination reaction, and to stabilise the charged moiety remaining in its lattice site. At very low temperatures, e.g. 4 K, the degradation would be dependent upon the sublimation of hydrogen atoms into the surrounding vacuum, but subsequent heating would result in rapid specimen degradation. Conclusion: The major factor limiting the resolution in organic and biological specimens is radiation damage caused by the electron beam. Application of the reported encapsulation method at room temperature reduces radiation damage by a factor of 3 to 12 and clarifies the damage mechanism.
70
J.R. Fryer, F. Holland / Reduction of radiation damage in E M
References [1] N. Uyeda, T. Kobayashi, E. Suito, Y. Harada and M.J. Watanabe, J. Appl. Phys. 43 (1972) 5181. [2] Y. Murata, J.R. Fryer, T. Baird and H. Murata, Acta Cryst. A33 (1977) 108. [3] W.R.K. Clark, J.N. Chapman and R.P. Ferrier, Ultramicroscopy 5 (1980) 195.
[4] J.R. Fryer, R.A. Camps and D.J. Smith, in: Proc. 10th Intern. Congr. on Electron Microscopy, Hamburg, 1982, Vol. 2, p. 449. [5] S.M. Salih and V.E. Cosslett, Phil. Ma 8. 30 (1974) 225. [6] V.E. Cosslett, J. Microscopy 113 (1978) 113. [7] W.A.P. Nicholson, J. Microscopy 121 (1980) 141, [8] M.A. O'Keefe, J.R. Fryer and D.J. Smith, Inst. Phys. Conf. Ser. 61 (1982) 337.
67
SHORT NOTE THE REDUCTION OF RADIATION DAMAGE IN THE ELECTRON MICROSCOPE J.R. FRYER and F. HOLLAND Chemistry Department, University of Glasgow, Glasgow G12 8QQ, UK Received 25 January 1983
The major factor limiting the resolution in organic and biological specimens in the electron microscope is radiation damage caused by the electron beam. This work describes the application of an encapsulation method at room temperature that reduces radiation damage by a factor of 3-12 and provides an explanation for the nature of the damage mechanism. The most stable organic crystal hitherto examined in the electron microscope is hexadecachlorocopper phthalocyanine. It has been possible to image the molecule [1,2] and examine the structural changes caused by radiation damage [2-4]. The effect of radiation is to create a random distribution of damaged areas where degradation of the crystal lattice proceeds rapidly; and since the prerequisite for this rapid degradation is an adjacent non-crystalline region, it is clear that this more open region permits diffusion of active species [3]. A further observation on this compound [4] is that crystalline copper oxide (both Cu 2° and CuO) occasionally forms in the damaged area, particularly in thick specimens. The extent of the damage area is proportional to the time elapsed since it was initiated, and since this initiation is random a series of kinetic measurements can be made relating the extent of the damaged area to the amount of copper in the copper-oxide crystal, showing that the rate-determining step for growth of damaged areas corresponds to a diffusion value o f -- 10 - 7 c m 2 S -1. T h i s value is much lower than surface diffusion rates and corresponds to bulk diffusion values in organic molecular crystals. In view of the assumptions made in this calculation, it cannot be regarded as much more than a suggestion that bulk diffusion is the rate-determin-
ing step, but it promoted the idea of covering the specimen with a continuous layer of carbon that would encapsulate active species within the crystal, providing more opportunity for recombination of species displaced by the primary radiation impact. Similar radiation protection factors to those observed here had been observed by Salih and Cosslett [5,6] using carbon, gold and aluminium layers covering the specimen, but because separate specimens were used the precision of the results was low, and an explanation was put forward based on the conductive properties of these materials. Bright field high resolution images of chlorinated copper phthalocyanine, metal-free phthalocyanine, polydiacetylene and coronene had shown that damage was initiated at random sites on the crystal and at crystal edges. The damaged area expanded rapidly from these sites in a circular manner until the whole crystal was destroyed [2]. The similarity in behaviour among these different compounds suggested that a similar damage mechanism pertained. Therefore epitaxial films of hexadeca-halocopper phthalocyanine, metal-free phthalocyanine, perylene and the paraffin n-hexatriacontane (C36H74) were prepared by deposition onto KC1, the first three by vacuum evaporation and the paraffin by crystallisation from petroleum ether solution. The crystalline layer on the KC1 was backed with a carbon layer deposited by evaporation, floated off on water and picked up on an electron microscope grid. The other side of the specimen was then half covered with evaporated carbon so that the final arrangement was that shown in fig. 1. In addition, a specimen of catalase was prepared on carbon from suspension and half
0304-3991/83/0000-0000/$03.00 © 1983 North-Holland
J.R. Fryer, F. Holland / Reduction of radiation damage in E M
68
damage was total extinction of diffraction spots determined both by observation of the final screen and sequential micrographs. Absolute values of radiation dose are often quoted as the dose De to achieve 1 / e of the original intensity of first-order diffraction spots under particular operating conditions. In the present work this was not necessary because the aim was to obtain comparative values between covered and uncovered parts of a homogeneous specimen. Thus the values for radiation dose may not accord with absolute values but are self-consistent for one specimen. In practice it was found that there was good agreement between different specimens of the same preparation and thickness, as shown by the standard deviation in results. The results are shown in table 1, and the encapsulating carbon film gave a protection factor of 6-12 for halogenated molecules and a factor of - 3 for hydrogenated. There was no significant difference in protection between aliphatic and aromatic compounds when hydrogen was the peripheral atom in the compound. An important feature was that thickness was significant and may account for the diversity of values of radiation critical doses that have been observed previously. For example, the factor between the thin uncovered specimen of chlorinated copper phthalocyanine and the thick covered specimen appears to be - 2 0 , but this neglects the role of specimen
/ _ j _ _ C o . r bon ~-..~~...:-..-:-.-.-.-.~ - . ~ " [~
[]
[]
Specimen []~
Coxbon
~Grid
bar
Fig. 1. Schematic cross-section of half-covered specimen arrangement.
covered with carbon, as the other specimens. This half covering permitted identical radiation damage measurements to be made between covered and uncovered areas of specimen on the same specimen grid. This is particularly important since it ensured that the microscope conditions for the comparative measurements were the same; the same beam diameter and all other operating conditions were maintained for measurements on the covered and uncovered parts of the same specimen. The epitaxial preparation ensured that all areas of the specimen were of the same thickness and orientation. The electron dose was measured by means of a flat copper plate in the image plane, and a backscattering factor of 30% was used. This factor has been shown to be reproducible [7]; and certainly under these experimental conditions, which involved only moving from one part of a specimen to another, there would be no change in the backscattering factor. The criterion for radiation
Table 1 Electron doses required for extinction of first-order reflections in the diffraction pattern; JEOL 100C electron microscope 100 kV with 200/~m diameter condenser aperture and 0.16/~m 2 selected area Sample
With carbon coating ( C / e r a 2 )
Without carbon coating ( C / c m 2)
Thickness (A.)
No. of measurements
Factor and standard deviation
Paraffin C36H 74
0,04 5:0.005
0.013 + 0.003
Unknown
15
3.1 + 0.4
Perylene
0,11 + 0.02 0,07 -t- 0.02
0.04 + 0.01 0.024 4:-0.01
330 70
35 42
2.6 5:0.7 3.0 +_ 1.7
Phthalocyanine
0.47 + 0.08
0.175 + 0.02
100
26
2.7 5:0.5
2.1 6.3
54 103
4 16
9.3+0.8 6.1+2.4
9
3.1 + 0.4
Chlorinated copper phthalocyanine Catalase Brominated copper phthalocyanine
19.5 + 5 38.9 + 1 4 0.05 5:0.001 10.6 + 0.2
+0.15 +0.87
0.015 + 0.002 0.92 + 0.17
Unknown 35
10
11.6 + 2
J.R. Fryer, F.. Holland / Reduction of radiation damage in E M
thickness. The effect of the encapsulating carbon diminished as the specimen thickness increased, but since dynamical scattering effects begin to be significant at 15 nm, this would reduce any advantages of using thicker specimens for high resolution studies [8]. The effect of the encapsulating layer ( - 5 rim) did not appear to affect the resolution obtainable in the specimens as shown by lattice imaging. Consideration of the protection factors indicates the nature of the radiation damage mechanism. The similarity of protection factors between metal-free phthalocyanine, perylene, catalase and paraffin show that the chemical structure of the molecular carbon framework is not important. Neither are the electronic properties of the molecule or crystal, in view of the considerable differences in electron transfer properties between paraffin (i.e. down the c axis) and metal-free phthalocyanine. Furthermore, if the carbon simply provided a conduction path, as suggested previously [5,6], then the differences between hydrogenated and halogenated compounds would not occur. The cross-sections of the atom involved also have little effect, as both chlorine and bromine have lower electron capture cross-sections than carbon, and if carbon loss predominated, then the protection factors for both of these materials would be the same. The factor that explains these results is the ability of the peripheral atoms to diffuse away from the parent molecules. The theoretical ratio of diffusion rates for H 2, C12 and Br 2 as a function of the square root of their molecular weights is H2:C12:Br2 = 9: 1.4: 1. Therefore, as the protection factor would be expected to vary inversely with the diffusion rates, the theoretical ratio would be H2:CI2:Br2 = 1 : 6 : 9 . The protection factors measured for molecules containing these peripheral atoms are 3 : 9 : 12; i.e., approximately RH : RC1 : RBr = 1 : 3 : 4. Therefore the experimental results follow the same
69
trend as the theoretical ratio based on the concept of diffusion and for the two chemically similar halogen molecules show a reasonable quantitative agreement. Thus the damage mechanism would consist of an initial excitation of the molecule by the electron beam in which all of the atoms would gain sufficient energy to break the chemical bonds. However, the atoms are held in a lattice so that the predominant reaction would be recombination into the original molecule. Peripheral atoms are less tightly held, so that a proportion can diffuse away from the parent molecule, which in turn, as a charged moiety, may spontaneously degrade. It is this degradation that is observed in the electron microscope, and hence absolute values of radiation damage for different compounds vary by orders of magnitude, depending upon the structure of the parent moiety, but the protection factor of the encapsulating film that reduces diffusion and hence aids recombination is only dependent upon the nature of the peripheral atom. The smaller the atom, then, the more easily it could diffuse, as illustrated by the differences between hydrogen and halogens in these experiments. It would be expected, then, that all parts of a molecule exposed to an electron beam would be in a constant state of fission and recombination, with permanent damage occurring when a peripheral atom diffused away and the remaining charged species decomposed. The effect of cooling the specimen would be to slow down the diffusion, but also slow down the recombination reaction, and to stabilise the charged moiety remaining in its lattice site. At very low temperatures, e.g. 4 K, the degradation would be dependent upon the sublimation of hydrogen atoms into the surrounding vacuum, but subsequent heating would result in rapid specimen degradation. Conclusion: The major factor limiting the resolution in organic and biological specimens is radiation damage caused by the electron beam. Application of the reported encapsulation method at room temperature reduces radiation damage by a factor of 3 to 12 and clarifies the damage mechanism.
70
J.R. Fryer, F. Holland / Reduction of radiation damage in E M
References [1] N. Uyeda, T. Kobayashi, E. Suito, Y. Harada and M.J. Watanabe, J. Appl. Phys. 43 (1972) 5181. [2] Y. Murata, J.R. Fryer, T. Baird and H. Murata, Acta Cryst. A33 (1977) 108. [3] W.R.K. Clark, J.N. Chapman and R.P. Ferrier, Ultramicroscopy 5 (1980) 195.
[4] J.R. Fryer, R.A. Camps and D.J. Smith, in: Proc. 10th Intern. Congr. on Electron Microscopy, Hamburg, 1982, Vol. 2, p. 449. [5] S.M. Salih and V.E. Cosslett, Phil. Ma 8. 30 (1974) 225. [6] V.E. Cosslett, J. Microscopy 113 (1978) 113. [7] W.A.P. Nicholson, J. Microscopy 121 (1980) 141, [8] M.A. O'Keefe, J.R. Fryer and D.J. Smith, Inst. Phys. Conf. Ser. 61 (1982) 337.