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The further reduction of radiation damage in the electron microscope
Ultramicroscopy North-Holland.
SHORT
14 (1984) 357-358 Amsterdam
NOTE
THE FURTHER J.R. FRYER,
REDUCTION
C. McNEE
Chemist~v Depmtment, Received
351
1 August
OF RADIATION
DAMAGE IN THE ELECTRON
MICROSCOPE
and F.M. HOLLAND
The Unwersity of Gkqow,
Glasgow G12 SQQ, Scotland,
UK
1984
The results confirm the earlier hypothesis that radiation damage in the electron microscope proceeds via loss of peripheral atoms from specimen molecules. The importance of recombination reactions is shown by the addition of an inorganic halide (Cl or Br) to the specimen which prolongs the lifetime of carbon-encapsulated specimens to a greater extent than the encapsulation alone.
Previous papers [l-5] have shown that radiation damage to organic crystals can be reduced by sandwiching the specimen between continuous films. We have shown the reduction of damage to be independent of the nature of the film and proposed a free radical mechanism to explain the radiation damage [4,5]. The principles of this mechanism are that the incident electron beam excites the molecules in the crystal, resulting in the detachment of some peripheral atoms and leaving the molecule as a free radical or ion. Only the peripheral atoms are detached because the bulk of the molecule remains caged in the crystal lattice. The flux of peripheral atoms diffusing through the crystal are capable of recombining with the free radicals or ions, thus preventing their spontaneous degradation, or diffusing out of the crystal lattice. The spontaneous degradation is the structural damage that causes fading of Bragg reflections in the electron diffraction pattern. Encapsulation between carbon films reduced the loss of peripheral atoms thereby aiding the recombination reaction and reducing the effects of radiation damage. This reduction was found to be particularly effective for chlorinated and brominated compounds [2-51. It was thought significant that these halogens are very effective free-radical scavengers. To further justify this mechanism we have conducted a series of experiments in which a hydro0304-3991/84/$03.00 0 Elsevier Science Publishers (North-Holland Physics Publishing Division)
genated organic crystal was coated with a thin layer of radiation-sensitive inorganic halide to determine whether the halogen released by the radiation damage to the halide would combine with active species formed in the organic crystal, thereby deactivating them and preserving the organic crystal - albeit in a halogenated state. Copper phthalocyanine was prepared epitaxially on KC1 by evaporation [6], backed with carbon, floated on to water, picked up from above by a glass slide, floated again on to water thus turning the film over, picked up on electron microscope grids and a layer of carbon evaporated on top thus encapsulating the copper phthalocyanine crystalline film. This was the standard specimen, and for the halide additions aqueous ammonium chloride or ammonium bromide or iodine in ethanol (all at IM concentration) was sprayed onto half of the grid before the second carbon film was evaporated on. These preparations are labelled (1) in table 1 and the iodine as (a). Subsequent preparations of copper phthalocyanine are labelled (2) and (3) and iodine sublimed on in the form of iodine vapour is labelled (b). Only half of the organic layer was treated, so that the uncovered half acted as an internal standard. For a series of experiments copper phthalocyanine from the same preparation was used, as the radiation-limiting dose is thickness dependent [2]. One series was prepared with the halide outB.V.
358
J. R. Fryer et al. / Further reduction of radiation damage in electron microscope
Table 1 Sample CuPc(1) - carbon CuPc(l)+NH,Cl CuPc(l)+NH,Br CuPc(l)+I,(a) CuPc(2) - carbon CuPc(Z)+I,(b) CuPc(3) - carbon CuPc(3) - carbon
encapsulated - carbon encapsulated - carbon encapsulated carbon encapsulated encapsulated carbon encapsulated encapsulated encapsulated + Iz(a)
side of the encapsulating carbon although it had been shown [5] that the protection afforded by the carbon film was independent of the thickness of the film. The time taken for the first-order reflections in the diffraction pattern to fade was measured in the electron microscope. Radiation exposure was measured from the final screen current, as shown on the operating conditions screen of the JEOL 1200EX microscope. This final screen current was previously calibrated using a Faraday cage in the specimen position. At 120 keV, 34% of the electrons were backscattered by the final screen, and therefore the screen current only represented 66% of the electrons incident on the specimen (the comparable value at 100 keV was 68%). The intensity of the beam also affected the backscattering factor, and for a diffraction pattern the factor was 60%. This latter value was used in calculations. The microscope was operated at 120 keV and the area of specimen giving the diffraction pattern was 1.79 pm2. The results for a-copper phthalocyanine (CuPc) are shown in table 1. The results show that the dose that the specimen could withstand was enhanced by the presence of chlorine- or brominecontaining compounds within an encapsulating carbon film and diminished by an iodine-containing compound. A halide outside the film had no effect. The presence of the halide had no effect on the normal imaging of the specimen. The halide crystals could be seen in the image lying on and around the phthalocyanine crystals. Undoubtedly the system could be optimised with a more radiation-sensitive source of chlorine or bromine and a very even dispersion of this
Extinction dose (C cm-*)
Standard deviation
0.91 1.3 1.2 0.34 0.73 0.73 0.89 0.86
0.12 0.1 0.19 0.14 0.15 0.092 0.18 0.29
source over the crystal. However, the purpose of these experiments was to show that the nature of the labile atoms in and around an organic crystal affect its radiation sensitivity, and if these atoms are free-radical scavengers, such as chlorine or bromine, then the radiation sensitivity is decreased. The effect of the iodine may be that the large iodine atoms caused the lattice to be more open, thus increasing the loss of peripheral hydrogen atoms but not diffusing sufficiently rapidly itself to stabilise free radicals before they decomposed. Conclusion. The additional radiation protection given by the presence of chlorine and bromine to the carbon-encapsulated organic crystals is in accordance with the mechanism outlined above. It is also of interest chemically in that the halogen atoms behave as “hot atoms” that have been observed in reactor radiation chemistry [7]. The technique of encapsulating of chlorineor bromine-containing compound with a specimen to prolong its lifetime in the microscope may have a wide application to the electron crystallography of organic and biological crystals. References [l] S.M. Salih and V.E. Cosslett, Phil. Msg. A30 (1974) 225. [2] J.R. Fryer and F.M. Holland, Ultramicroscopy 11 (1983) 67. [3] F.M. Holland, J.R. Fryer and T. Baird, Inst. Phys. Conf. Ser. 68 (1983) 19. [4] J.R. Fryer and F.M. Holland, Proc. Roy. Sot. (London) A393 (1984) 353. [5] J.R. Fryer, Ultramicroscopy 14 (1984) 227. [6] Y. Murata, J.R. Fryer and T. Baird, J. Microscopy 108 (1976) 261. [7] J.E. Willard, Ann. Rev. Phys. Chem. 6 (1955) 141.
SHORT
14 (1984) 357-358 Amsterdam
NOTE
THE FURTHER J.R. FRYER,
REDUCTION
C. McNEE
Chemist~v Depmtment, Received
351
1 August
OF RADIATION
DAMAGE IN THE ELECTRON
MICROSCOPE
and F.M. HOLLAND
The Unwersity of Gkqow,
Glasgow G12 SQQ, Scotland,
UK
1984
The results confirm the earlier hypothesis that radiation damage in the electron microscope proceeds via loss of peripheral atoms from specimen molecules. The importance of recombination reactions is shown by the addition of an inorganic halide (Cl or Br) to the specimen which prolongs the lifetime of carbon-encapsulated specimens to a greater extent than the encapsulation alone.
Previous papers [l-5] have shown that radiation damage to organic crystals can be reduced by sandwiching the specimen between continuous films. We have shown the reduction of damage to be independent of the nature of the film and proposed a free radical mechanism to explain the radiation damage [4,5]. The principles of this mechanism are that the incident electron beam excites the molecules in the crystal, resulting in the detachment of some peripheral atoms and leaving the molecule as a free radical or ion. Only the peripheral atoms are detached because the bulk of the molecule remains caged in the crystal lattice. The flux of peripheral atoms diffusing through the crystal are capable of recombining with the free radicals or ions, thus preventing their spontaneous degradation, or diffusing out of the crystal lattice. The spontaneous degradation is the structural damage that causes fading of Bragg reflections in the electron diffraction pattern. Encapsulation between carbon films reduced the loss of peripheral atoms thereby aiding the recombination reaction and reducing the effects of radiation damage. This reduction was found to be particularly effective for chlorinated and brominated compounds [2-51. It was thought significant that these halogens are very effective free-radical scavengers. To further justify this mechanism we have conducted a series of experiments in which a hydro0304-3991/84/$03.00 0 Elsevier Science Publishers (North-Holland Physics Publishing Division)
genated organic crystal was coated with a thin layer of radiation-sensitive inorganic halide to determine whether the halogen released by the radiation damage to the halide would combine with active species formed in the organic crystal, thereby deactivating them and preserving the organic crystal - albeit in a halogenated state. Copper phthalocyanine was prepared epitaxially on KC1 by evaporation [6], backed with carbon, floated on to water, picked up from above by a glass slide, floated again on to water thus turning the film over, picked up on electron microscope grids and a layer of carbon evaporated on top thus encapsulating the copper phthalocyanine crystalline film. This was the standard specimen, and for the halide additions aqueous ammonium chloride or ammonium bromide or iodine in ethanol (all at IM concentration) was sprayed onto half of the grid before the second carbon film was evaporated on. These preparations are labelled (1) in table 1 and the iodine as (a). Subsequent preparations of copper phthalocyanine are labelled (2) and (3) and iodine sublimed on in the form of iodine vapour is labelled (b). Only half of the organic layer was treated, so that the uncovered half acted as an internal standard. For a series of experiments copper phthalocyanine from the same preparation was used, as the radiation-limiting dose is thickness dependent [2]. One series was prepared with the halide outB.V.
358
J. R. Fryer et al. / Further reduction of radiation damage in electron microscope
Table 1 Sample CuPc(1) - carbon CuPc(l)+NH,Cl CuPc(l)+NH,Br CuPc(l)+I,(a) CuPc(2) - carbon CuPc(Z)+I,(b) CuPc(3) - carbon CuPc(3) - carbon
encapsulated - carbon encapsulated - carbon encapsulated carbon encapsulated encapsulated carbon encapsulated encapsulated encapsulated + Iz(a)
side of the encapsulating carbon although it had been shown [5] that the protection afforded by the carbon film was independent of the thickness of the film. The time taken for the first-order reflections in the diffraction pattern to fade was measured in the electron microscope. Radiation exposure was measured from the final screen current, as shown on the operating conditions screen of the JEOL 1200EX microscope. This final screen current was previously calibrated using a Faraday cage in the specimen position. At 120 keV, 34% of the electrons were backscattered by the final screen, and therefore the screen current only represented 66% of the electrons incident on the specimen (the comparable value at 100 keV was 68%). The intensity of the beam also affected the backscattering factor, and for a diffraction pattern the factor was 60%. This latter value was used in calculations. The microscope was operated at 120 keV and the area of specimen giving the diffraction pattern was 1.79 pm2. The results for a-copper phthalocyanine (CuPc) are shown in table 1. The results show that the dose that the specimen could withstand was enhanced by the presence of chlorine- or brominecontaining compounds within an encapsulating carbon film and diminished by an iodine-containing compound. A halide outside the film had no effect. The presence of the halide had no effect on the normal imaging of the specimen. The halide crystals could be seen in the image lying on and around the phthalocyanine crystals. Undoubtedly the system could be optimised with a more radiation-sensitive source of chlorine or bromine and a very even dispersion of this
Extinction dose (C cm-*)
Standard deviation
0.91 1.3 1.2 0.34 0.73 0.73 0.89 0.86
0.12 0.1 0.19 0.14 0.15 0.092 0.18 0.29
source over the crystal. However, the purpose of these experiments was to show that the nature of the labile atoms in and around an organic crystal affect its radiation sensitivity, and if these atoms are free-radical scavengers, such as chlorine or bromine, then the radiation sensitivity is decreased. The effect of the iodine may be that the large iodine atoms caused the lattice to be more open, thus increasing the loss of peripheral hydrogen atoms but not diffusing sufficiently rapidly itself to stabilise free radicals before they decomposed. Conclusion. The additional radiation protection given by the presence of chlorine and bromine to the carbon-encapsulated organic crystals is in accordance with the mechanism outlined above. It is also of interest chemically in that the halogen atoms behave as “hot atoms” that have been observed in reactor radiation chemistry [7]. The technique of encapsulating of chlorineor bromine-containing compound with a specimen to prolong its lifetime in the microscope may have a wide application to the electron crystallography of organic and biological crystals. References [l] S.M. Salih and V.E. Cosslett, Phil. Msg. A30 (1974) 225. [2] J.R. Fryer and F.M. Holland, Ultramicroscopy 11 (1983) 67. [3] F.M. Holland, J.R. Fryer and T. Baird, Inst. Phys. Conf. Ser. 68 (1983) 19. [4] J.R. Fryer and F.M. Holland, Proc. Roy. Sot. (London) A393 (1984) 353. [5] J.R. Fryer, Ultramicroscopy 14 (1984) 227. [6] Y. Murata, J.R. Fryer and T. Baird, J. Microscopy 108 (1976) 261. [7] J.E. Willard, Ann. Rev. Phys. Chem. 6 (1955) 141.