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Electron beam penetration and X-Ray excitation depth in ice
Micron and Microscopica Acta, Vol. 16, No. 1, pp. 1-4, 1955. Printed in Great Britain
0739—6260/85 83.00+0.00 ~ 1985 Pergamon Press Ltd.
ELECTRON BEAM PENETRATION AND X-RAY EXCITATION DEPTH IN ICE K. OATES and W. T. W. POTTS Department of Biological Sciences, University of Lancaster, Lancaster, LA! 4YQ, U.K. (Received 3 August 1984: revised 30 October 1984)
Abstract—X-Ray excitation depth in ice is ofpractical importance in quantitative analysis offrozen hydrated bulk specimens. An investigation is described in which ice of known thickness was probed by a range of accelerating voltages commonly used for microprobe analysis of biological material; results are displayed in graphical forms. Electron retardation in ice and theeffect of atomic number on the emission profile for elements of biological interest are discussed. Index key words: X-Ray analysis, microprobe, frozen hydrated bulk specimens, scanning electron microscope.
INTRODUCTION Theoretical considerations by Kanaya and Okagama (1972) showed that in an uncharged solid the maximum range, R, of incident electrons would depend on the accelerating voltage E, the density p, the atomic number Z and the atomic weight A, so that 819 0.0276E’~A pZ R=4 p.m for hydrogen, 10 p.m for carbon and oxygen at a density of 1, when E=20 kV. In a non-conducting solid the build up of space charge should very rapidly reduce R. Brombach (1975) estimated that R would effectively be reduced by 90°~ within less than a second when usingabeamcurrentof0.l l.tA/l.tm2.Ontheother hand a theoretical analysis by Marshall (1982), suggested that 90°~ of the X-rays for the K~,lines of Na, K and Cl would be emitted between the surface and I p.m depth in ice or frozen protein gels at 10 kV, while at 20 kV 90°~ of the X-rays would be emitted between the surface and 4 p.m depth. As the excitation depth is of immediate practical importance in bulk quantitative analysis of frozen hydrated specimens we have attempted to determine R experimentally in ice, R
‘
—
MATERIALS AND METHODS Thin layers of ice of known thickness were prepared in micro-trays. The trays were made from a Formvar film attached to 100 mesh nickel foils of 1.65 and 5 p.m thickness (Buckbee Mears, Chicago, Illinois). The inner surfaces of the trays were coated with Sb2S3 by vacuum sublimation to provide a sulphur target underlycentral range was of elements of greatest ing the to ice. the Sulphur chosen because it is interest to biologists. The thickness of the grid was determined from photographs taken at an acute angle at known magnification, Fig. 1(a). Water, to which 0.001 % Bacitracin was added to reduce surface tension, was then flooded onto the surface of the trays to fill them and quench frozen in nitrogen slush, Fig. 1(b). The frozen preparation was transferred to the airlock of the SEM under vacuum to prevent frosting of the specimen, where it was placed on a cold pedestal at 91 K and coated with aluminium of 25 nm thickness by high vacuum evaporation. The preparation was then transferred to the microscope cold stage for examination and microprobe analysis. The specimen temperature was 88 K, measured by a thin film thermocouple, the cold stage temperature was 81 K using a cold stage of our own design. A JEOL anticontaminator, modified to permit liquid nitrogen to fill a small
K ()ate~,and W I
2
~
Vs Potts
~
~
~ -,
- -~,
L~
~
_ 1l~
_
_
_
Fig. 1(a) Low angle view ofdry nickel grid used for measurement of tray depth x 2750 (b) High angle view of nickel grid with meshes filled with Le except for a few in top right x 430
Beam Penetration and Excitation Depth
tank brazed to the anticontaminator cold plate, was used in conjunction with the cold stage. The anticontaminator cold plate was placed 4 mm above the specimen microtrays. The cold plate temperature was 81 K. Stereo pairs of micrographs were prepared using Polaroid prints and inspected under the stereoscope to select areas where the grids were filled to the brim with uniform flat layers of ice. The preparation remained on the cold stage. Measurements of the column pressure were carried out using a Penning gauge. The column pressure took 2 hr to fall to 3 mPa with the cold stage and anticontaminator in operation and observations were not begun until this pressure had been reached. The column pressure continued to fall until it reached 0.7 mPa after 6 hr.
RESULTS With a beam current 2 and count of 0.4 nA, timeand of a60repeating sec, the raster, size 1 p.mpeaks for Al, S and Sb were counts of the determined at 2 kV intervals over a range of voltages using a Link systems analyser 290 and the counts for S and Sb were expressed as percentages of the counts for Al counts. The means of six such determinations of S/Al and Sb/Al ratios, at various accelerating voltages on grids of 1.65 and 5.0 p.m thickness, are shown in Fig. 2. The take off angle was 30°. The K 2 excitation voltage of S is 2.47 kV. S was just detectable in dry grids at 2.8 kV. From Fig. 2 it will be seen that S should be just detectable at a voltage of 12.8 kV under 1.6 p.m of ice and at 19.2 kV under 5.0 p.m of ice. The probe voltage was calibrated against the maximum Bremsstrahlung radiation detectable in the Link analyser which in turn was calibrated against the _____
24
-.
22
~—
20
~_-s
~
6 4
/2”
I2~
04
~
K2 peak of manganese. There was no evidence of increasing specimen thickness due to water vapour condensation during any of the measurements. However, sometimes some specimen damage occurred in the form of a blister underneath the aluminium coat. When such damage occurred it was always somewhat larger (ca 2 p.m dia) than the area being probed. It was interpreted as radiolysis of the ice producing H ions and OH radicals leading to the formation of gases which lifted the aluminium film. DISCUSSION The number of quanta of X-rays emitted by a target is a function of both the accelerating voltage and the number of excitatory electrons. Both the current and the voltage decline as the beam penetrates the ice. The rate ofdecline of the voltage can be determined from the minimum voltage at through various whichthicknesses the sulphurof ice. can be detected As some electrons scatter immediately on entering the ice the minimum path length between the ice surface and the sulphur target must be uncertain. Theoretically a few electrons deflected through 30°at the ice surface could reach the target after traversing a minimum vertical path through the ice but most electrons deflected will suffer multiple scattering and the effective thickness will be close to t Sec 30°where t is the vertical thickness of the ice. The absorption of X-rays in the ice is low and would not substantially affect the minimum voltage at which excitation would be detectable. As a minimum excitation voltage of 2.8 kV is required to produce detectable X-rays from the sulphur, the voltage must have been reduced from 19.2 kV at the surface to 2.8 kV at the base when the ice was 5.0 p.m thick. Similarly, when the ice was 1.65 p.m thick it must have declined a retardation at the surface to 2~kV at the base, ation profile at the base of the 5.0 p.m thick ice was similar to that in the thinner ice, then with a 19.2 kV beam the voltage would be 12.8 kV 1.65 p.m above the base. As the specimen is mounted at an angle of 30°to the incident beam
-.~---~—
I 6
3
12
6
20
S/At. and Sb/At, Fig. 2. Peak ratios at various voltages. 1.65 om thick ice below. 5 gm thick ice above,
the effective ice thicknesses are 1.9 p.m and 5.77 p.m, respectively, Fig. 3. Hence the 5.77 jim oficewilldecelerateelectronsfrom 19.2 to 2.8 kV and 1.9 p.m of ice from 12.8 to 2.8 kV. The retardation profiles at 19.2 and 12.8 kV will, therefore, approximate to the solid lines in
K. Oales and W. 1. W. Potts
4
absorption edge energy hut the general shape of the profiles of elements of moderate atomic weights are similar and reasonably wel! estab-
a, 2~7// 0 4
~2O
6
________________________________________ 4 8 12 6 20 kV
Fig. 3. Decline of beam voltage with path length in ice,
Fig. 2andtheretardationprofilesat5, 10, l5and 20 kV may be obtained by interpolation and by limited extrapolation, Fig. 3. The depth distribution of X-rays emitted by a low concentration of an element, in a matrix of different composition, has been the subject of several recent studies. Parobek and Brown (1978) derived an empirical formula for X-rays emitted at moderate electron energies, 6--IS kV, which fitted reasonably well with the limited number of experimental results available but the parameters had to be changed to cover the range 15 -30 kV (Brown and Robinson, 1979) and when extended to anticathodes of low atomic numbers the agreement with both experimental results and with Monte Carlo calculations of electron trajectories was very poor (Marshall, 1982). However, the general form of the depth emission profiles should always be similar with a maximum some distance below the surface as scattered electrons take longer to traverse a given increment of depth and the agreement between observation and the Parobek-—Brown equations. for elements of moderate atomic weight, is good. Using a modified Parobek--Brown equation Marshall (1982) calculated that the X—ray emissions of potassium at 14.8 kV reach a maximum at a depth of 40 jig/cm2 (~0.4p.m at p = I ) but X-rays are emitted down to 470 jig/cm2. Assuming that the protein -water matrix has a density of 0.92. maximum emission occurs at 0.43 jim and the greatest depth from which X—rays would be emitted would be 5.0 p.m. The centre of emission would be 1.30 jim. Our results suggest that at 15 kV the maximum depth of emission would be 3 p.m. The shape of the emission profiles is a complex function of atomic weights, atomic number, electron energy. E,. and
emission that lished. ofAssuming Kat(Marshall, 15 kVthat would the be 1982) profile the 0.8 mean jim for and S resembles depths 90°,,of the X-rays would be emitted from above 60”,, of of the maximum depth. This implies that 90”,, of the X-rays of S in ice would be emitted from above 0.75 jim at 10 kV and above 4 p.m at 20 kV. Thk compares well with Marshall’s estimate (1982) of I and 4 jim, respectively. The effect of atomic number on the emi.vooti pro/tie The excitation voltages are functions of the atomic number of the element concerned hut neither the maximum nor the mean depths of emission are very sensitive to the excitation voltage. Except for the light elements, such as C and 0, which emit such soft X-rays that detection presents special problems, most elements of biological interest, Na. Mg, P. K. Cl and Ca have excitationvoltageswithinabout 1.3 kVofthatof S. An increase in the minimum excitation voltage should reduce the maximum and mean excitation depths. An increase of 1 kV should reduce the maximum excitation depth by about 0.75 p.m at 5 jim depth or by 0.25 jim at 1 p.m depth. This will be offset to some extent by a reduction in the proportion of X-rays absorbed. The centres of emission will be affected in proportion. ~ ‘Ac gratefully theA.detailed comments in the preparation of this acknowledge paper ofDi, T. I-I all of the Microprohe Laboratory, Department of Zoology. Universitr of Cambridge.
REFERENCES Br~is. J
I) and Robinson, W H 979 Quantitative analysis hr ~i (pZ) curves. In; VlieroI’(’oni l,ia/rsi’.. San
Francisco Press. San Francisco, Broinhach, J. D.. 975. Electron beam \-ray microanalysis frozen biological hulk spectmen below 13/) K II - ‘[he electrical charging ofthe sample in quantitative analysts,.! ~ Biol (‘cli, 22: 233 235 Green, M and (‘osslctt. V. F.. 1961 l’he eflicienc~ al production of characteristic X-radiation in thick targets hr a ~‘ure element. Proc. P/it.’. Sm’.. 78: I 206 Kanay a. K. nid Okay ama. S.. 1972. Penet rat ton and cnerg\ los’. theory it solid targets .1 P/irs. 1) app! l’lit”... 5:43 55 Marshall. A. I.. 1952. Application of i/i (p// curves and a windowless detector to the quantitative X-ray niicroanalysis
of frozen-hydrated
hulk
biological
specimens
3m ui’.,.. 26)). Atomic number and L.Elet andtwit Brown. .1. 1)243 975. absorption corrections in microanalysis at low electron energies. .\ -Rat’ .Spei’irooieirr. 7: 26 3/).
~ Parohek, ‘~
0739—6260/85 83.00+0.00 ~ 1985 Pergamon Press Ltd.
ELECTRON BEAM PENETRATION AND X-RAY EXCITATION DEPTH IN ICE K. OATES and W. T. W. POTTS Department of Biological Sciences, University of Lancaster, Lancaster, LA! 4YQ, U.K. (Received 3 August 1984: revised 30 October 1984)
Abstract—X-Ray excitation depth in ice is ofpractical importance in quantitative analysis offrozen hydrated bulk specimens. An investigation is described in which ice of known thickness was probed by a range of accelerating voltages commonly used for microprobe analysis of biological material; results are displayed in graphical forms. Electron retardation in ice and theeffect of atomic number on the emission profile for elements of biological interest are discussed. Index key words: X-Ray analysis, microprobe, frozen hydrated bulk specimens, scanning electron microscope.
INTRODUCTION Theoretical considerations by Kanaya and Okagama (1972) showed that in an uncharged solid the maximum range, R, of incident electrons would depend on the accelerating voltage E, the density p, the atomic number Z and the atomic weight A, so that 819 0.0276E’~A pZ R=4 p.m for hydrogen, 10 p.m for carbon and oxygen at a density of 1, when E=20 kV. In a non-conducting solid the build up of space charge should very rapidly reduce R. Brombach (1975) estimated that R would effectively be reduced by 90°~ within less than a second when usingabeamcurrentof0.l l.tA/l.tm2.Ontheother hand a theoretical analysis by Marshall (1982), suggested that 90°~ of the X-rays for the K~,lines of Na, K and Cl would be emitted between the surface and I p.m depth in ice or frozen protein gels at 10 kV, while at 20 kV 90°~ of the X-rays would be emitted between the surface and 4 p.m depth. As the excitation depth is of immediate practical importance in bulk quantitative analysis of frozen hydrated specimens we have attempted to determine R experimentally in ice, R
‘
—
MATERIALS AND METHODS Thin layers of ice of known thickness were prepared in micro-trays. The trays were made from a Formvar film attached to 100 mesh nickel foils of 1.65 and 5 p.m thickness (Buckbee Mears, Chicago, Illinois). The inner surfaces of the trays were coated with Sb2S3 by vacuum sublimation to provide a sulphur target underlycentral range was of elements of greatest ing the to ice. the Sulphur chosen because it is interest to biologists. The thickness of the grid was determined from photographs taken at an acute angle at known magnification, Fig. 1(a). Water, to which 0.001 % Bacitracin was added to reduce surface tension, was then flooded onto the surface of the trays to fill them and quench frozen in nitrogen slush, Fig. 1(b). The frozen preparation was transferred to the airlock of the SEM under vacuum to prevent frosting of the specimen, where it was placed on a cold pedestal at 91 K and coated with aluminium of 25 nm thickness by high vacuum evaporation. The preparation was then transferred to the microscope cold stage for examination and microprobe analysis. The specimen temperature was 88 K, measured by a thin film thermocouple, the cold stage temperature was 81 K using a cold stage of our own design. A JEOL anticontaminator, modified to permit liquid nitrogen to fill a small
K ()ate~,and W I
2
~
Vs Potts
~
~
~ -,
- -~,
L~
~
_ 1l~
_
_
_
Fig. 1(a) Low angle view ofdry nickel grid used for measurement of tray depth x 2750 (b) High angle view of nickel grid with meshes filled with Le except for a few in top right x 430
Beam Penetration and Excitation Depth
tank brazed to the anticontaminator cold plate, was used in conjunction with the cold stage. The anticontaminator cold plate was placed 4 mm above the specimen microtrays. The cold plate temperature was 81 K. Stereo pairs of micrographs were prepared using Polaroid prints and inspected under the stereoscope to select areas where the grids were filled to the brim with uniform flat layers of ice. The preparation remained on the cold stage. Measurements of the column pressure were carried out using a Penning gauge. The column pressure took 2 hr to fall to 3 mPa with the cold stage and anticontaminator in operation and observations were not begun until this pressure had been reached. The column pressure continued to fall until it reached 0.7 mPa after 6 hr.
RESULTS With a beam current 2 and count of 0.4 nA, timeand of a60repeating sec, the raster, size 1 p.mpeaks for Al, S and Sb were counts of the determined at 2 kV intervals over a range of voltages using a Link systems analyser 290 and the counts for S and Sb were expressed as percentages of the counts for Al counts. The means of six such determinations of S/Al and Sb/Al ratios, at various accelerating voltages on grids of 1.65 and 5.0 p.m thickness, are shown in Fig. 2. The take off angle was 30°. The K 2 excitation voltage of S is 2.47 kV. S was just detectable in dry grids at 2.8 kV. From Fig. 2 it will be seen that S should be just detectable at a voltage of 12.8 kV under 1.6 p.m of ice and at 19.2 kV under 5.0 p.m of ice. The probe voltage was calibrated against the maximum Bremsstrahlung radiation detectable in the Link analyser which in turn was calibrated against the _____
24
-.
22
~—
20
~_-s
~
6 4
/2”
I2~
04
~
K2 peak of manganese. There was no evidence of increasing specimen thickness due to water vapour condensation during any of the measurements. However, sometimes some specimen damage occurred in the form of a blister underneath the aluminium coat. When such damage occurred it was always somewhat larger (ca 2 p.m dia) than the area being probed. It was interpreted as radiolysis of the ice producing H ions and OH radicals leading to the formation of gases which lifted the aluminium film. DISCUSSION The number of quanta of X-rays emitted by a target is a function of both the accelerating voltage and the number of excitatory electrons. Both the current and the voltage decline as the beam penetrates the ice. The rate ofdecline of the voltage can be determined from the minimum voltage at through various whichthicknesses the sulphurof ice. can be detected As some electrons scatter immediately on entering the ice the minimum path length between the ice surface and the sulphur target must be uncertain. Theoretically a few electrons deflected through 30°at the ice surface could reach the target after traversing a minimum vertical path through the ice but most electrons deflected will suffer multiple scattering and the effective thickness will be close to t Sec 30°where t is the vertical thickness of the ice. The absorption of X-rays in the ice is low and would not substantially affect the minimum voltage at which excitation would be detectable. As a minimum excitation voltage of 2.8 kV is required to produce detectable X-rays from the sulphur, the voltage must have been reduced from 19.2 kV at the surface to 2.8 kV at the base when the ice was 5.0 p.m thick. Similarly, when the ice was 1.65 p.m thick it must have declined a retardation at the surface to 2~kV at the base, ation profile at the base of the 5.0 p.m thick ice was similar to that in the thinner ice, then with a 19.2 kV beam the voltage would be 12.8 kV 1.65 p.m above the base. As the specimen is mounted at an angle of 30°to the incident beam
-.~---~—
I 6
3
12
6
20
S/At. and Sb/At, Fig. 2. Peak ratios at various voltages. 1.65 om thick ice below. 5 gm thick ice above,
the effective ice thicknesses are 1.9 p.m and 5.77 p.m, respectively, Fig. 3. Hence the 5.77 jim oficewilldecelerateelectronsfrom 19.2 to 2.8 kV and 1.9 p.m of ice from 12.8 to 2.8 kV. The retardation profiles at 19.2 and 12.8 kV will, therefore, approximate to the solid lines in
K. Oales and W. 1. W. Potts
4
absorption edge energy hut the general shape of the profiles of elements of moderate atomic weights are similar and reasonably wel! estab-
a, 2~7// 0 4
~2O
6
________________________________________ 4 8 12 6 20 kV
Fig. 3. Decline of beam voltage with path length in ice,
Fig. 2andtheretardationprofilesat5, 10, l5and 20 kV may be obtained by interpolation and by limited extrapolation, Fig. 3. The depth distribution of X-rays emitted by a low concentration of an element, in a matrix of different composition, has been the subject of several recent studies. Parobek and Brown (1978) derived an empirical formula for X-rays emitted at moderate electron energies, 6--IS kV, which fitted reasonably well with the limited number of experimental results available but the parameters had to be changed to cover the range 15 -30 kV (Brown and Robinson, 1979) and when extended to anticathodes of low atomic numbers the agreement with both experimental results and with Monte Carlo calculations of electron trajectories was very poor (Marshall, 1982). However, the general form of the depth emission profiles should always be similar with a maximum some distance below the surface as scattered electrons take longer to traverse a given increment of depth and the agreement between observation and the Parobek-—Brown equations. for elements of moderate atomic weight, is good. Using a modified Parobek--Brown equation Marshall (1982) calculated that the X—ray emissions of potassium at 14.8 kV reach a maximum at a depth of 40 jig/cm2 (~0.4p.m at p = I ) but X-rays are emitted down to 470 jig/cm2. Assuming that the protein -water matrix has a density of 0.92. maximum emission occurs at 0.43 jim and the greatest depth from which X—rays would be emitted would be 5.0 p.m. The centre of emission would be 1.30 jim. Our results suggest that at 15 kV the maximum depth of emission would be 3 p.m. The shape of the emission profiles is a complex function of atomic weights, atomic number, electron energy. E,. and
emission that lished. ofAssuming Kat(Marshall, 15 kVthat would the be 1982) profile the 0.8 mean jim for and S resembles depths 90°,,of the X-rays would be emitted from above 60”,, of of the maximum depth. This implies that 90”,, of the X-rays of S in ice would be emitted from above 0.75 jim at 10 kV and above 4 p.m at 20 kV. Thk compares well with Marshall’s estimate (1982) of I and 4 jim, respectively. The effect of atomic number on the emi.vooti pro/tie The excitation voltages are functions of the atomic number of the element concerned hut neither the maximum nor the mean depths of emission are very sensitive to the excitation voltage. Except for the light elements, such as C and 0, which emit such soft X-rays that detection presents special problems, most elements of biological interest, Na. Mg, P. K. Cl and Ca have excitationvoltageswithinabout 1.3 kVofthatof S. An increase in the minimum excitation voltage should reduce the maximum and mean excitation depths. An increase of 1 kV should reduce the maximum excitation depth by about 0.75 p.m at 5 jim depth or by 0.25 jim at 1 p.m depth. This will be offset to some extent by a reduction in the proportion of X-rays absorbed. The centres of emission will be affected in proportion. ~ ‘Ac gratefully theA.detailed comments in the preparation of this acknowledge paper ofDi, T. I-I all of the Microprohe Laboratory, Department of Zoology. Universitr of Cambridge.
REFERENCES Br~is. J
I) and Robinson, W H 979 Quantitative analysis hr ~i (pZ) curves. In; VlieroI’(’oni l,ia/rsi’.. San
Francisco Press. San Francisco, Broinhach, J. D.. 975. Electron beam \-ray microanalysis frozen biological hulk spectmen below 13/) K II - ‘[he electrical charging ofthe sample in quantitative analysts,.! ~ Biol (‘cli, 22: 233 235 Green, M and (‘osslctt. V. F.. 1961 l’he eflicienc~ al production of characteristic X-radiation in thick targets hr a ~‘ure element. Proc. P/it.’. Sm’.. 78: I 206 Kanay a. K. nid Okay ama. S.. 1972. Penet rat ton and cnerg\ los’. theory it solid targets .1 P/irs. 1) app! l’lit”... 5:43 55 Marshall. A. I.. 1952. Application of i/i (p// curves and a windowless detector to the quantitative X-ray niicroanalysis
of frozen-hydrated
hulk
biological
specimens
3m ui’.,.. 26)). Atomic number and L.Elet andtwit Brown. .1. 1)243 975. absorption corrections in microanalysis at low electron energies. .\ -Rat’ .Spei’irooieirr. 7: 26 3/).
~ Parohek, ‘~