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Use of pure carbon specimen holders for analytical electron microscopy of thin sections
Ultramicroscopy 2 (1976) 105-107 © North-Holland Publishing Company
SHORT NOTE USE OF PURE CARBON SPECIMEN HOLDERS FOR ANALYTICAL ELECTRON MICROSCOPY OF THIN SECTIONS
Bj6rn LILJESVAN and Godfried M. ROOMANS * Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, S-113 45 Stockholm, Sweden
Received 21 December 1976
The use of a simple specimen holder, made of pure carbon, for analytical electron microscopy of sections of biological material is reported. Interference of the specimen holder with the X-ray spectrum from the specimen is minimized. The contribution of the holder to the continuum spectrum is consistent with theoretical predictions.
1. Introduction
imen holder for electron microprobe analysis of sections of biological material.
It has been pointed out, that in X-ray microanalysis, the surroundings of the analysed part of the specimen can make an important contribution to the measured spectrum [1]. Especially when thin sections of biological material are being examined, the mass surrounding the analysed area, e.g. that of the specimen holder, is several orders of magnitude greater than that of the analysed area in the specimen. The contribution of the surrounding mass to the spectrum should be minimized for two reasons. First, in qualitative analysis, elemental peaks, especially of metals, may be introduced into the spectrum by the specimen holder. This makes it difficult to establish the presence of small quantities of these elements in the specimen. Second, if, in quantitative analysis, the continuum method is used [2], the contribution of the surrounding mass to the continuum spectrum has to be minimized to ensure accurate calculation. The contribution of the specimen holder to the measured spectrum can be greatly diminished by using a specimen holder consisting of material of low molecular weight, such as carbon [3]. Also beryllium specimen holders have been used for this purpose. In this paper, we report the use of a simple pure carbon spec-
2. Materials
Spectroscopically pure carbon (Pure Carbon DS13) plates were obtained from Pure Carbon Company, St. Mary's, Pennsylvania, USA. The carbon has to be kept sealed, as it may absorb contamination from the air, but contaminated plates can be regenerated by heating in vacuum. For use in a JEOL 100 C electron microscope, a specimen holder was made of two carbon plates in each of which a conically shaped hole was filed. In the top plate (fig. 1), two slits were filed to avoid blocking of the electron beam and absorption of the emerging X-rays. The grid was kept between the carbon plates and inserted in a JEOL side-entry goniometer specimen holder. Similar constructions can, however, be made for other transmission microscopes.
3. Electron microprobe analysis Energy-dispersive X-ray analysis of the carbon plates at various accelerating voltages did not show any obvious peaks. In comparison to a conventional metal specimen holder, the background is greatly reduced by the use of a carbon specimen holder. This
Present address: Department of Chemical Cytology, University of Nijmegen, Nijmegen, The Netherlands. 105
8. Lil]esvan et aL / Use o/pure carbon specimen homers
106
mm ...................
26
. . . . . . . . . . . . . . . . . . . . . . . ,.h 1000 o
~3
5.s
500
..4,
t!I
~,.......... 12 ....... ->' 0
i
• ~r
=; ; O.e
2
/-
6 8 ENERGY (keY)
10
Fig. 2. C o n t i n u u m i n t e n s i t y curve f i t t e d to the e x p e r i m e n t a l data. Closed circles: data used f o r t h e f i t t i n g p r o c e d u r e , open circles: o t h e r e x p e r i m e n t a l data.
ing voltage of 10 kV, was fitted to a curve of the form expressed in eq. (1):
N(E)=IeeEzlaE° -~- E
Fig. 1. (a) Upper plate of the carbon specimen holder, used in combination with a JEOL side-entry goniometer specimen holder. The holder can be tilted up to about 50° . Slit A prevents blocking of the electron beam by the specimen holder. The detector is located facing slit.B. (b) Photograph of the carbon specimen holder mounted in the JEOL specimen holder. has also been demonstrated by Mizuhira [3]. Recently, Fiori et al. [4] described a method for background prediction in energy.dispersive analysis. This method was applied to investigate, whether the obtained continuum spectra could indeed be completely attributed to the carbon specimen holder. The spectrum of the carbon plate, measured at an accelerat-
+b
(Eo E)21
(i)
in which N(E) is the number of counts at energy E, E 0 is the accelerating voltage, and Z the atomic number of the target. The absorption factor fE (absorption of X-rays of energy E in the target) was calculated as described by Fiori et al. [4]. The detector efficiency PE is determined by the thickness of the beryllium window, the silicon dead layer and the active silicon layer in the detector. The two fitting factors a and b could be determined by measuringN(E) at two separate energies, and solving the resulting two equations with two unknowns. Alternatively, the solutions of these equations were considered as initial values for a curve fitting procedure carried out with a digital computer. If carbon is used as a target, and energies above 3 keV are used for the curve fitting procedure, this is relatively simple, as both fE and PE are about constant in the energy region of 3 - 1 0 keV. In the case of low X-ray intensities, the curve fitting method may improve the accuracy of the determination of the fitting factors. The experimental data below about 2 keV can be used to adjust the estimated thickness of the beryllium window and the silicon dead layer to optimize the fit between the experimental and the calculated curve. Fig. 2 shows the curve fitted to the experimental data, taking a = 36.15 and b = - 0 . 0 1 in eq. (1). The
B. Lil/esvan et al. / Use of pure carbon specimen homers
curve fits the experimental data well. A small, though not significant, deviation occurs at the energies of the Si K lines, which may indicate a slight contamination of the column. At the upper edge of the spectrum, it can be seen, that no appreciable charging has occurred.
4. Discussion The only contribution, thus, made by the carbon specimen holder to the obtained spectrum, is a contribution to the continuum part of the spectrum. Furthermore, this contribution is consistent with theoretical predictions of the shape of the continuum generated by pure carbon, and can completely be accounted for. Compared to the baryllium specimen holder, the carbon specimen holder has the advantage, that it is made of a non-toxic material. In our experience [5] conditions for quantitative
107
microprobe analysis of thin sections of biological material are greatly improved by this simple device.
Acknowledgements The study has been made possible thanks to a grant to Dr. B.A. Afzelius from the Wallenberg Stiftelse and from the Swedish Natural Science Research Council.
References [I] P. Galle, J. MicroscopicBiol. Cell 22 (1975) 315. [2] T.A. Hall, H. Clarke Anderson and T. Appleton, J. Microsc 99 (1973) 177. [3] V. Mizuhira, Acta Histochem. Cytochem. 9 (1976) 69. [4] C.E. Fiori, R.L. Myklebust and K.F.J. Heinrich, Anal. Chem. 48 (1976) 172. [5] G.M. Roomans and L.A. Sev~.us,J. Cell. Sei. 21 (1976) 119.
SHORT NOTE USE OF PURE CARBON SPECIMEN HOLDERS FOR ANALYTICAL ELECTRON MICROSCOPY OF THIN SECTIONS
Bj6rn LILJESVAN and Godfried M. ROOMANS * Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, S-113 45 Stockholm, Sweden
Received 21 December 1976
The use of a simple specimen holder, made of pure carbon, for analytical electron microscopy of sections of biological material is reported. Interference of the specimen holder with the X-ray spectrum from the specimen is minimized. The contribution of the holder to the continuum spectrum is consistent with theoretical predictions.
1. Introduction
imen holder for electron microprobe analysis of sections of biological material.
It has been pointed out, that in X-ray microanalysis, the surroundings of the analysed part of the specimen can make an important contribution to the measured spectrum [1]. Especially when thin sections of biological material are being examined, the mass surrounding the analysed area, e.g. that of the specimen holder, is several orders of magnitude greater than that of the analysed area in the specimen. The contribution of the surrounding mass to the spectrum should be minimized for two reasons. First, in qualitative analysis, elemental peaks, especially of metals, may be introduced into the spectrum by the specimen holder. This makes it difficult to establish the presence of small quantities of these elements in the specimen. Second, if, in quantitative analysis, the continuum method is used [2], the contribution of the surrounding mass to the continuum spectrum has to be minimized to ensure accurate calculation. The contribution of the specimen holder to the measured spectrum can be greatly diminished by using a specimen holder consisting of material of low molecular weight, such as carbon [3]. Also beryllium specimen holders have been used for this purpose. In this paper, we report the use of a simple pure carbon spec-
2. Materials
Spectroscopically pure carbon (Pure Carbon DS13) plates were obtained from Pure Carbon Company, St. Mary's, Pennsylvania, USA. The carbon has to be kept sealed, as it may absorb contamination from the air, but contaminated plates can be regenerated by heating in vacuum. For use in a JEOL 100 C electron microscope, a specimen holder was made of two carbon plates in each of which a conically shaped hole was filed. In the top plate (fig. 1), two slits were filed to avoid blocking of the electron beam and absorption of the emerging X-rays. The grid was kept between the carbon plates and inserted in a JEOL side-entry goniometer specimen holder. Similar constructions can, however, be made for other transmission microscopes.
3. Electron microprobe analysis Energy-dispersive X-ray analysis of the carbon plates at various accelerating voltages did not show any obvious peaks. In comparison to a conventional metal specimen holder, the background is greatly reduced by the use of a carbon specimen holder. This
Present address: Department of Chemical Cytology, University of Nijmegen, Nijmegen, The Netherlands. 105
8. Lil]esvan et aL / Use o/pure carbon specimen homers
106
mm ...................
26
. . . . . . . . . . . . . . . . . . . . . . . ,.h 1000 o
~3
5.s
500
..4,
t!I
~,.......... 12 ....... ->' 0
i
• ~r
=; ; O.e
2
/-
6 8 ENERGY (keY)
10
Fig. 2. C o n t i n u u m i n t e n s i t y curve f i t t e d to the e x p e r i m e n t a l data. Closed circles: data used f o r t h e f i t t i n g p r o c e d u r e , open circles: o t h e r e x p e r i m e n t a l data.
ing voltage of 10 kV, was fitted to a curve of the form expressed in eq. (1):
N(E)=IeeEzlaE° -~- E
Fig. 1. (a) Upper plate of the carbon specimen holder, used in combination with a JEOL side-entry goniometer specimen holder. The holder can be tilted up to about 50° . Slit A prevents blocking of the electron beam by the specimen holder. The detector is located facing slit.B. (b) Photograph of the carbon specimen holder mounted in the JEOL specimen holder. has also been demonstrated by Mizuhira [3]. Recently, Fiori et al. [4] described a method for background prediction in energy.dispersive analysis. This method was applied to investigate, whether the obtained continuum spectra could indeed be completely attributed to the carbon specimen holder. The spectrum of the carbon plate, measured at an accelerat-
+b
(Eo E)21
(i)
in which N(E) is the number of counts at energy E, E 0 is the accelerating voltage, and Z the atomic number of the target. The absorption factor fE (absorption of X-rays of energy E in the target) was calculated as described by Fiori et al. [4]. The detector efficiency PE is determined by the thickness of the beryllium window, the silicon dead layer and the active silicon layer in the detector. The two fitting factors a and b could be determined by measuringN(E) at two separate energies, and solving the resulting two equations with two unknowns. Alternatively, the solutions of these equations were considered as initial values for a curve fitting procedure carried out with a digital computer. If carbon is used as a target, and energies above 3 keV are used for the curve fitting procedure, this is relatively simple, as both fE and PE are about constant in the energy region of 3 - 1 0 keV. In the case of low X-ray intensities, the curve fitting method may improve the accuracy of the determination of the fitting factors. The experimental data below about 2 keV can be used to adjust the estimated thickness of the beryllium window and the silicon dead layer to optimize the fit between the experimental and the calculated curve. Fig. 2 shows the curve fitted to the experimental data, taking a = 36.15 and b = - 0 . 0 1 in eq. (1). The
B. Lil/esvan et al. / Use of pure carbon specimen homers
curve fits the experimental data well. A small, though not significant, deviation occurs at the energies of the Si K lines, which may indicate a slight contamination of the column. At the upper edge of the spectrum, it can be seen, that no appreciable charging has occurred.
4. Discussion The only contribution, thus, made by the carbon specimen holder to the obtained spectrum, is a contribution to the continuum part of the spectrum. Furthermore, this contribution is consistent with theoretical predictions of the shape of the continuum generated by pure carbon, and can completely be accounted for. Compared to the baryllium specimen holder, the carbon specimen holder has the advantage, that it is made of a non-toxic material. In our experience [5] conditions for quantitative
107
microprobe analysis of thin sections of biological material are greatly improved by this simple device.
Acknowledgements The study has been made possible thanks to a grant to Dr. B.A. Afzelius from the Wallenberg Stiftelse and from the Swedish Natural Science Research Council.
References [I] P. Galle, J. MicroscopicBiol. Cell 22 (1975) 315. [2] T.A. Hall, H. Clarke Anderson and T. Appleton, J. Microsc 99 (1973) 177. [3] V. Mizuhira, Acta Histochem. Cytochem. 9 (1976) 69. [4] C.E. Fiori, R.L. Myklebust and K.F.J. Heinrich, Anal. Chem. 48 (1976) 172. [5] G.M. Roomans and L.A. Sev~.us,J. Cell. Sei. 21 (1976) 119.