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Quantitative microautoradiography by X-ray emission micro-analysis
1395
Notes Geochimica
et Cosmochlmlca
Acta
Quantitative
1977. Vol. 41. pp. 1395 to 1397. Pergamon
Press.
microautoradiography
Printed
in Great
Britain
by X-ray emission micro-analysis
JOHN R. HOLLOWAY Division of Geochemistry, Department of Chemistry, Arizona State University, Tempe, AZ 85281, U.S.A. and MICHAEL J. DRAKE Department of Planetary Sciences and Lunar and Planetary Laboratory, University of Arizona. Tucson, AZ 85721, U.S.A. (Received 24 January 1977; accepted in revised form 3 Maq’ 1977)
Abstract-Silver concentration in developed photographic emulsions exposed by p-emission may be measured on spots with a minimum diameter of about 10pm using an electron microprobe or a scanning electron microscope. Silver concentration is directly proportional to the integrated flux of P-particles bombarding the emulsion. Thus analysis for silver provides a method for quantitative microautoradiography which is comparable in accuracy and precision to other available methods and which can be performed on readily available equipment.
INTRODUCTION THE TECHNIQUEof autoradiography
has been used extensively in the last three decades to provide a map of the distribution of beta (/$-particle emitting radiogenic nuclides in biological systems. Exposure of photographic emulsions to particles has also been utilized in the field of nuclear physics as a means of determining particle flux. Descriptions of the technique as applied to biologic problems have been published by numerous authors e.g. FISCHERand WERNER(1970), LCJTTGE(1972) and ROGERS (1973). ROGERS(1973) provides a comprehensive analysis of the interaction of p-particles with emulsions. Autoradiography has also been applied to experimental measurement of trace element partition coefficients in petrologic systems by MYSENand SEITZ(1975). Since then, it has been used extensively to measure CO, solubility in silicate melts (MYSENet al., 1976), to determine the amount of liquid formed in partial melting experiments (MYSENand KUSHIRO.1977). and to measure diffusion coeff&ients in silicate melts (HO~MANN,1975). Autoradiography has thus become a powerful tool for a wide variety of applications in experimental geochemistry and petrology. The method involves the following steps: (1) introduction of the radiogenic nuclide into the sample; (2) distribution of the nuclide between two or more phases in the sample during an experimental process; (3) preparation of a planar surface on the sample; (4) exposure of a photographic emulsion to the planar sample; (5) development of the emulsion; and (6) qualitative, semiquantitative, or quantitative estimation of the density of nuclear-particle tracks in the developed emulsion. In this paper we present a new technique for performing the sixth step in the autoradiographic procedure given above-the quantitative determination of nuclear particle track density. Existing techniques include visual grain counting, visual track counting, photometric measurements in reflected light, optical density measurements, image analysis by television scanning systems and grain counting with the scanning electron microscope (ROGERS,1973). In the technique described here, silver concentration in a developed emulsion is measured using an electron microprobe or an analytical scanning electron microscope. We show that silver concentration is directly proportional to the number of /?-particles entering the emulsion and that the spatial resolution for quantitative analysis is better than IOpm.
6.C.A. 4119-N
EXPERIMENTS
AND RESULTS
The technique relies on the measurement of characteristic L, silver X-rays generated by bombarding the developed emulsion with electrons. The area of the emulsion bombarded must remain constant from sample to sample and with time. The analytical methods used are those described in standard texts on X-ray emission microanalysis (e.g. BIRKS, 1971). The exposed emulsion is coated with carbon prior to analysis. For quantitative analyses, “background” must be subtracted from “peak” counts. “Background” refers to areas of the emulsion which are not exposed to the radiogenic sample. The analyses reported here were performed at the University of Arizona on an Applied Research Laboratories Scanning Electron Microprobe Quantometer equipped with a Tracer-Northern TN 1310 automation system. The operating conditions were: PET crystal spectrometer, 90nA beam current, 5 nA sample current on the emulsion, and 15 kV accelerating potential. We used a 5 nA sample current because larger currents caused extensive damage to the emulsions. However. the emulsion may suffer considerable damage without noticeable loss of Ag L, X-ray intensity. The electron beam diameter was estimated either by measuring the area of the emulsion where a change in visual appearance occurred, or by measuring the size of the cathodolumenescence spot on an anorthite specimen. All emulsions used in this study were the Ilford K-5 type with a 25 pm emulsion on a 2.5 x 3.75 cm glass plate. All exposures were made using a calcite (C&O,) standard containing 14C (specific activity 7.74 x 10m4mCi/mg). The preparation of the calcite standard is similar to the description in MYSEN and SEITZ (1975). The emulsions were developed using Kodak D-19 developer. Development time was 10 min followed by 5 min in a stop bath, 15 min in a fixer solution, and a 30min rinse in water. We designed our experiments to determine: (1) the minimum practical electron beam diameter which could be used; (2) analytical reproducibility; and (3) the relation between p-particle density and measured silver concentration. The minimum electron beam diameter was determined on an emulsion exposed for 30min. Five 50 set counts were made for a series of beam diameters from 1OOpm to less than 5 pm. A single spot was used for each diameter tested. For diameters between 1OOpm and about 10pm. no systematic change in count rate was observed from one
50s~ count to the next. A large decrease in count rate w;t~ ohserved using a 2 5 ,trn beam diameter. We then switched to 5 set counting intervals and made between IO and 40 repeated readings using beams of approximately 20 + 2 pm, 10 + 2 nm and 3 + 2 pm diameter. No change in count rate was observed with time for the 20 and IO pm beams, but a sharp decrease occurred using the 3 pm beam. Analytical reproducibility was tested by repeated readings on the same spot and by analyzing several closely spaced spots. For beam diameters greater than about IO pm. the scatter of repeated readings was not significantly greater than that expected from counting statistics alone. Consequently the precision of silver analysis of emulsions is expected to be comparable to that achieved on polished minerals and alloys. The correlation between silver concentration and b-particle density was tested by measuring emulsions exposed to the calcite standard for periods of 5-940 min. To correct for variations due to slight differences in development procedure from one emulsion to another. IO and 30 min expo-
sures were made on each emulsion in addition to other exposures of varying duration. Silver concentrations ohtaincd on different emulsions were then normalized using these exposures as internal monitors, For each exposure time. seven measurements of 50sec duration were made using a 5Opm diameter beam. Backgrounds were determined from seven measurements at the Ag L, peak position on areas of emulsion which were not exposed directly to the B-emitting isotope. These measurements were always higher than would be obtained by offsetting the crystal spectrometer from the Ag L, peak position. For b-particle densities below emulsion saturation values, the number of tracks per unit area of emulsion has been shown to be directly proportional to exposure time for a sample of constant p activity (ROGERS,1973, p. 166). Thus any relationship between measured silver concentration and track density can be examined by comparing silver concentration to exposure time. The comparison is made in Fig. 1, which shows our results plotted on two scales. Examination of the full scale plot shows that large deviations from
I I
I
I
100 IO
I
I
I
t
I
EXPOSURE TIME
1
1
I
.O 1000 130
500 50 (MINUTES)
Fig. I. Background subtracted silver L, count rate measured on exposed emulsions vs exposure time. Note double scales, dashed line refers to normal scale (left ordinate), solid line to expanded scale (right ordinate). Error bars represent two standard deviations based on seven repeated measurements. One wt o/0Ag is approximately equivalent to 300 counts/set. Note that there are three separate points for the 10 and 30 min exposure times, and that the error bar is offset from the points.
1397
Notes linearity occur for exposure times greater than a few tens of minutes. From the expanded scale it can be seen that a linear relation exists between silver concentration and exposure time for periods of 0 to about 30 min. The linear region is pr~umably limited by coincidence of p-particles on silver halide grains and consequently will vary with the grain size of the emulsion and also with the development procedure (ROGERS, 1973). The linear region (530min exposure) in Fig. I corresponds to track densities of about I 8 y IO’ tracks/cm’ as reported by MYSEN and SEITZ (1975) for i“C. This is about the same range as is useful for visual track counting and hence the detection limits reported by MYSEN and SEITZ(1975, Table 6) will also be appropriate for the silver analysis technique. The relative standard deviation (la) for repeated, background subtracted, readings in the linear region of Fig. I range from 4 to 10%. The precision reported by MY.SE> and SEITZ (1975) and MYSEN et al. (1976) using visual counting techniques spans the same range, but in most analyses is reported as better than 5% (lo). However, the precision reported by Mysen and co-workers refers to peak counts only. Inclusion of variations in background counts would result in somewhat larger standard deviations. Visual track counting is somewhat faster than silver analysis. hut requires considerable operator skill. Silver analysis can be done by anyone familiar with microprobe techniques and makes the P-track technique much less operator dependent. CONCLUSIONS Analysis of silver by X-ray emission microanalysis offers a precise means of obtaining quantitative information from microautoradiographs. The linear response over a &fold range of exposure intervals provides ample flexibility in isotope concentration versus exposure time. Areas of 10 pm diameter or less may be measured. suthcient for many element partitioning experiments. Although we have emphasired the application of electron probe microanalysis to
NOTE
ADDED
autoradiography in this letter, the technique should be generally applicable to the analysis of any developed silver halide emulsion. Acknowfrtiyrmmts-This research was supported by the Earth Sciences Section, National Science Foundation, Grant EAR75-21619. John R. Holloway thanks Dr. BJORN MYSENof the Geophysical Laboratory, Carnegie Institute of Washington for the many hours of instruction in autoradiographic techniques. Michael J. Drake thanks Mr. GILBERTMCLAUGHLINfor assistance with emulsion exposure and development techniques. The scanning electron microprobe is ably maintained by Mr. THOMASTESKA.We thank B. J. Wool for his criticism of the manuscript. REFERENCES BIRKSL. S. (1971) Electron Probe Micraurta~~ysis, 190 pp. Wiley. FISHERH. A. and WERNERG. (1971) ~u~o~~iogr~~~y, 199 pp. De Gruyter. HOFMANNA. W. (1975) Diffusion of calcium and strontium in a basalt melt. Carnegie Inst. Wash. Yea&k. 74, 183-189. LIITTAGE U. (1972) Microautoradiography and Electron Probe Analysis, 242 pp. Springer. MYSENB. O., EGGLERD. H.. SEITZM. G. and HOLLOWAY J. R. (1976). Carbon dioxide in silicate melts and crystals. Part I. Solubility measurements. Am. J. Sci. 276, 455479. MYSEN B. 0. and KUSBIROI. (1977) Compositional variations of coexisting phases with degree of melting of peridotite in the upper mantle. Am. Mineru[ogist. To be published. MYSENB. 0. and SEITZM. G. (1975) Trace element partitioning determined by beta track mapping: an experimental study using carbon and samarium as examples. J. Geophys. Res. SO, 2627-2635. ROGERSA. W. (1973) Techniques of Autoradiography, 2nd edition, 372 pp. Elsevier.
IN PROOF
In a paper in preparation (instrumental techniques for beta-track mapping. Carnegie Inst. Wash. Yearbook 76), T. M. BENJAMIN,N. T. ARNT and J. R. HOLLOWAYshow that a correction can be applied to measured silver contents above the saturation limit (equivalent to exposure times greater than 30min in Fig. I). Use of the correction allows a much wider range in track density in a given exposure which greatly simplifies partition coefficient determinations.
Notes Geochimica
et Cosmochlmlca
Acta
Quantitative
1977. Vol. 41. pp. 1395 to 1397. Pergamon
Press.
microautoradiography
Printed
in Great
Britain
by X-ray emission micro-analysis
JOHN R. HOLLOWAY Division of Geochemistry, Department of Chemistry, Arizona State University, Tempe, AZ 85281, U.S.A. and MICHAEL J. DRAKE Department of Planetary Sciences and Lunar and Planetary Laboratory, University of Arizona. Tucson, AZ 85721, U.S.A. (Received 24 January 1977; accepted in revised form 3 Maq’ 1977)
Abstract-Silver concentration in developed photographic emulsions exposed by p-emission may be measured on spots with a minimum diameter of about 10pm using an electron microprobe or a scanning electron microscope. Silver concentration is directly proportional to the integrated flux of P-particles bombarding the emulsion. Thus analysis for silver provides a method for quantitative microautoradiography which is comparable in accuracy and precision to other available methods and which can be performed on readily available equipment.
INTRODUCTION THE TECHNIQUEof autoradiography
has been used extensively in the last three decades to provide a map of the distribution of beta (/$-particle emitting radiogenic nuclides in biological systems. Exposure of photographic emulsions to particles has also been utilized in the field of nuclear physics as a means of determining particle flux. Descriptions of the technique as applied to biologic problems have been published by numerous authors e.g. FISCHERand WERNER(1970), LCJTTGE(1972) and ROGERS (1973). ROGERS(1973) provides a comprehensive analysis of the interaction of p-particles with emulsions. Autoradiography has also been applied to experimental measurement of trace element partition coefficients in petrologic systems by MYSENand SEITZ(1975). Since then, it has been used extensively to measure CO, solubility in silicate melts (MYSENet al., 1976), to determine the amount of liquid formed in partial melting experiments (MYSENand KUSHIRO.1977). and to measure diffusion coeff&ients in silicate melts (HO~MANN,1975). Autoradiography has thus become a powerful tool for a wide variety of applications in experimental geochemistry and petrology. The method involves the following steps: (1) introduction of the radiogenic nuclide into the sample; (2) distribution of the nuclide between two or more phases in the sample during an experimental process; (3) preparation of a planar surface on the sample; (4) exposure of a photographic emulsion to the planar sample; (5) development of the emulsion; and (6) qualitative, semiquantitative, or quantitative estimation of the density of nuclear-particle tracks in the developed emulsion. In this paper we present a new technique for performing the sixth step in the autoradiographic procedure given above-the quantitative determination of nuclear particle track density. Existing techniques include visual grain counting, visual track counting, photometric measurements in reflected light, optical density measurements, image analysis by television scanning systems and grain counting with the scanning electron microscope (ROGERS,1973). In the technique described here, silver concentration in a developed emulsion is measured using an electron microprobe or an analytical scanning electron microscope. We show that silver concentration is directly proportional to the number of /?-particles entering the emulsion and that the spatial resolution for quantitative analysis is better than IOpm.
6.C.A. 4119-N
EXPERIMENTS
AND RESULTS
The technique relies on the measurement of characteristic L, silver X-rays generated by bombarding the developed emulsion with electrons. The area of the emulsion bombarded must remain constant from sample to sample and with time. The analytical methods used are those described in standard texts on X-ray emission microanalysis (e.g. BIRKS, 1971). The exposed emulsion is coated with carbon prior to analysis. For quantitative analyses, “background” must be subtracted from “peak” counts. “Background” refers to areas of the emulsion which are not exposed to the radiogenic sample. The analyses reported here were performed at the University of Arizona on an Applied Research Laboratories Scanning Electron Microprobe Quantometer equipped with a Tracer-Northern TN 1310 automation system. The operating conditions were: PET crystal spectrometer, 90nA beam current, 5 nA sample current on the emulsion, and 15 kV accelerating potential. We used a 5 nA sample current because larger currents caused extensive damage to the emulsions. However. the emulsion may suffer considerable damage without noticeable loss of Ag L, X-ray intensity. The electron beam diameter was estimated either by measuring the area of the emulsion where a change in visual appearance occurred, or by measuring the size of the cathodolumenescence spot on an anorthite specimen. All emulsions used in this study were the Ilford K-5 type with a 25 pm emulsion on a 2.5 x 3.75 cm glass plate. All exposures were made using a calcite (C&O,) standard containing 14C (specific activity 7.74 x 10m4mCi/mg). The preparation of the calcite standard is similar to the description in MYSEN and SEITZ (1975). The emulsions were developed using Kodak D-19 developer. Development time was 10 min followed by 5 min in a stop bath, 15 min in a fixer solution, and a 30min rinse in water. We designed our experiments to determine: (1) the minimum practical electron beam diameter which could be used; (2) analytical reproducibility; and (3) the relation between p-particle density and measured silver concentration. The minimum electron beam diameter was determined on an emulsion exposed for 30min. Five 50 set counts were made for a series of beam diameters from 1OOpm to less than 5 pm. A single spot was used for each diameter tested. For diameters between 1OOpm and about 10pm. no systematic change in count rate was observed from one
50s~ count to the next. A large decrease in count rate w;t~ ohserved using a 2 5 ,trn beam diameter. We then switched to 5 set counting intervals and made between IO and 40 repeated readings using beams of approximately 20 + 2 pm, 10 + 2 nm and 3 + 2 pm diameter. No change in count rate was observed with time for the 20 and IO pm beams, but a sharp decrease occurred using the 3 pm beam. Analytical reproducibility was tested by repeated readings on the same spot and by analyzing several closely spaced spots. For beam diameters greater than about IO pm. the scatter of repeated readings was not significantly greater than that expected from counting statistics alone. Consequently the precision of silver analysis of emulsions is expected to be comparable to that achieved on polished minerals and alloys. The correlation between silver concentration and b-particle density was tested by measuring emulsions exposed to the calcite standard for periods of 5-940 min. To correct for variations due to slight differences in development procedure from one emulsion to another. IO and 30 min expo-
sures were made on each emulsion in addition to other exposures of varying duration. Silver concentrations ohtaincd on different emulsions were then normalized using these exposures as internal monitors, For each exposure time. seven measurements of 50sec duration were made using a 5Opm diameter beam. Backgrounds were determined from seven measurements at the Ag L, peak position on areas of emulsion which were not exposed directly to the B-emitting isotope. These measurements were always higher than would be obtained by offsetting the crystal spectrometer from the Ag L, peak position. For b-particle densities below emulsion saturation values, the number of tracks per unit area of emulsion has been shown to be directly proportional to exposure time for a sample of constant p activity (ROGERS,1973, p. 166). Thus any relationship between measured silver concentration and track density can be examined by comparing silver concentration to exposure time. The comparison is made in Fig. 1, which shows our results plotted on two scales. Examination of the full scale plot shows that large deviations from
I I
I
I
100 IO
I
I
I
t
I
EXPOSURE TIME
1
1
I
.O 1000 130
500 50 (MINUTES)
Fig. I. Background subtracted silver L, count rate measured on exposed emulsions vs exposure time. Note double scales, dashed line refers to normal scale (left ordinate), solid line to expanded scale (right ordinate). Error bars represent two standard deviations based on seven repeated measurements. One wt o/0Ag is approximately equivalent to 300 counts/set. Note that there are three separate points for the 10 and 30 min exposure times, and that the error bar is offset from the points.
1397
Notes linearity occur for exposure times greater than a few tens of minutes. From the expanded scale it can be seen that a linear relation exists between silver concentration and exposure time for periods of 0 to about 30 min. The linear region is pr~umably limited by coincidence of p-particles on silver halide grains and consequently will vary with the grain size of the emulsion and also with the development procedure (ROGERS, 1973). The linear region (530min exposure) in Fig. I corresponds to track densities of about I 8 y IO’ tracks/cm’ as reported by MYSEN and SEITZ (1975) for i“C. This is about the same range as is useful for visual track counting and hence the detection limits reported by MYSEN and SEITZ(1975, Table 6) will also be appropriate for the silver analysis technique. The relative standard deviation (la) for repeated, background subtracted, readings in the linear region of Fig. I range from 4 to 10%. The precision reported by MY.SE> and SEITZ (1975) and MYSEN et al. (1976) using visual counting techniques spans the same range, but in most analyses is reported as better than 5% (lo). However, the precision reported by Mysen and co-workers refers to peak counts only. Inclusion of variations in background counts would result in somewhat larger standard deviations. Visual track counting is somewhat faster than silver analysis. hut requires considerable operator skill. Silver analysis can be done by anyone familiar with microprobe techniques and makes the P-track technique much less operator dependent. CONCLUSIONS Analysis of silver by X-ray emission microanalysis offers a precise means of obtaining quantitative information from microautoradiographs. The linear response over a &fold range of exposure intervals provides ample flexibility in isotope concentration versus exposure time. Areas of 10 pm diameter or less may be measured. suthcient for many element partitioning experiments. Although we have emphasired the application of electron probe microanalysis to
NOTE
ADDED
autoradiography in this letter, the technique should be generally applicable to the analysis of any developed silver halide emulsion. Acknowfrtiyrmmts-This research was supported by the Earth Sciences Section, National Science Foundation, Grant EAR75-21619. John R. Holloway thanks Dr. BJORN MYSENof the Geophysical Laboratory, Carnegie Institute of Washington for the many hours of instruction in autoradiographic techniques. Michael J. Drake thanks Mr. GILBERTMCLAUGHLINfor assistance with emulsion exposure and development techniques. The scanning electron microprobe is ably maintained by Mr. THOMASTESKA.We thank B. J. Wool for his criticism of the manuscript. REFERENCES BIRKSL. S. (1971) Electron Probe Micraurta~~ysis, 190 pp. Wiley. FISHERH. A. and WERNERG. (1971) ~u~o~~iogr~~~y, 199 pp. De Gruyter. HOFMANNA. W. (1975) Diffusion of calcium and strontium in a basalt melt. Carnegie Inst. Wash. Yea&k. 74, 183-189. LIITTAGE U. (1972) Microautoradiography and Electron Probe Analysis, 242 pp. Springer. MYSENB. O., EGGLERD. H.. SEITZM. G. and HOLLOWAY J. R. (1976). Carbon dioxide in silicate melts and crystals. Part I. Solubility measurements. Am. J. Sci. 276, 455479. MYSEN B. 0. and KUSBIROI. (1977) Compositional variations of coexisting phases with degree of melting of peridotite in the upper mantle. Am. Mineru[ogist. To be published. MYSENB. 0. and SEITZM. G. (1975) Trace element partitioning determined by beta track mapping: an experimental study using carbon and samarium as examples. J. Geophys. Res. SO, 2627-2635. ROGERSA. W. (1973) Techniques of Autoradiography, 2nd edition, 372 pp. Elsevier.
IN PROOF
In a paper in preparation (instrumental techniques for beta-track mapping. Carnegie Inst. Wash. Yearbook 76), T. M. BENJAMIN,N. T. ARNT and J. R. HOLLOWAYshow that a correction can be applied to measured silver contents above the saturation limit (equivalent to exposure times greater than 30min in Fig. I). Use of the correction allows a much wider range in track density in a given exposure which greatly simplifies partition coefficient determinations.