532
Anal. Chem. 1980, 52, 532-536
Organic Gallium Compounds in Epoxy Resin as Standards for the Determination of Gallium in Biological Tissues with an Electron Probe Microanalyzer Kayoko Nakamura" and Hirotake Orii Department of Radiology, Tokyo Metropolitan Institute of Medical Science, Hon-komagome, Tokyo 1 13, Japan
Organic gallium compounds in epoxy resin have been prepared as the standards for quantitative electron microanalysis of biological thin sections. The qualiiies required for the standards were investigated with respect to homogeneity, solubility, and stability under the electron beam. Gallium oxinate in Spurr resin by ultrasonic treatment has been proved a suitable standard for analysis of gallium in tissue sections. Optimal conditions (accelerating voltage, specimen current, and analyzing times) have also been established for the determination of gallium. Under the recommended conditions, a linear calibration curve was obtained with standards.
Gallium is accumulated in some malignant tumors and therefore is clinically useful as a diagnostic agent (2). However, the underlying mechanisms remain obscure. Distribution of gallium in tissues and its intracellular localization explained by the X-ray image of electron probe microanalysis (EPMA) are expected t o elucidate t h e accumulation mechanism of gallium in malignant tumors. I n spite of some investigations on the analysis of gallium with E P M A (2, 3 ) , no application to biological materials has been reported. For quantitative biological application of E P M A , a standard with the following characteristics is necessary: chemically well defined, biologically equivalent in composition, and homogeneous at the level of resolution desired. Many standards have been reported ( 4 - 3 , and reviewed by Spurr (8). According t o Spurr ( 5 ) and Chandler (91, standards of organic compounds dissolved in t h e resin used for sample embedding are amenable to sectioning a t the desired thickness. Mixing of elements, including a "biological equivalent matrix", with resins makes them mimic a biological sample also in elemental compositions. For quantitative EPMA gallium in biological thin sections, we prepared organic gallium compounds embedded in epoxy resins as standards. T h e qualities required were investigated on gallium oxinate, gallium phthalocyanine chloride, and gallium tannic acid precipitate dissolved in Epon 812 or S p u r resins. Tannic acid a n d oxine are reagents to fix metals in biological samples for observation by electron microscopy (10, 11). Gallium phthalocyanine chloride is t h e only organic gallium compound available from commercial sources. This paper also describes the basic instrumental conditions of E P M A , for good reproducibility in the quantitative determination of gallium. EXPERIMENTAL Preparation of Standards. Preparation of Gallium Oxinate. Gallium oxinate complex was prepared by the modified method for the gravimetric analysis of gallium (12). Add 100 mL of 5% oxine solution (in 2 M CH,COOH) to an equal volume of 0.1 M Ga(N0J3 solution warmed (65 "C). Adjust the pH of the mixture to 5 by the addition of NH40H and 2 M CH3COONH4,and keep mixing for 30 min a t 65 "C. Filter the precipitate by microfilter (3 pm), and then dry at 110 "C. The final product was a yellow 0003-2700/80/0352-0532$01 0010
powder; 1g of the powder was found to contain 139 mg of gallium. PrecipitatLon of Gallium a n d T a n n i c Acid. Add 25 mL of 2 M CH3COONH4to 100 mL of 0.1 M Ga(NO& solution, and heat. After the addition of 100 mL of 10% tannic acid solution, collect the precipitate on filter paper No. 5 A. The final product was a white-brown powder, and 1 g of the powder contained 76 mg of gallium. Gallium Phthalocyanine Chloride, (C8H4NJ4GaC1,was purchased from Eastman Kodak Co. Ltd. One gram of the powder contains 113 mg of gallium. Preparation of the Organic Gallium Compound-Epoxy Resin Standards. Mix the finely powdered compounds and a freshly prepared resin mixture (Epon 812 or Spurr epoxy resin) in small vials by magnetic stirring. To ensure homogeneity, treat the mixture with the ultrasonic disintegrator (Ultrasonic Ltd. USD 350). After this, pour it promptly into BEEM capsules, and polymerize as usual (13, 14). Microprobe Analysis. Cut sections 200 nm thick and mount on carbon-coated nylon grids (Effa Ltd.). Electron probe microanalysis was performed with an EDAX energy-dispersive spectrometer in combination with a Hitachi scanning electron microanalyzer X-560. The microanalyzer was fitted with an EDAX spectrometer modified so that the probespecimen distance was shorter than originally designed.
RESULTS A N D D I S C U S S I O N C h a r a c t e r i s t i c s of O r g a n i c G a l l i u m C o m p o u n d s i n Embedded Resin. The dissolved compounds did not interfere noticeably with t h e polymerization process. T h i n sections containing organic compounds on nylon grids were observed by transmission electron microscopy. In addition, the gallium content was analyzed a t several points in the sections with the electron probe microanalyzer, and the peak to background ratio, P / B , for gallium was determined (Table I). Gallium oxinate solubility was about 20% (weight fraction) both in Spurr and Epon 812 resins. No attempt was made, however, to determine maximal solubility. A thin section of Spurr resin without the ultrasonic treatment was observed by t h e electron microscope t o have signs of precipitation (Figure 1). (The same symbols and magnification are used in Figures 1-10.) The P / B ratios of the section were different from point to point. On the other hand, t h e Spurr section with the ultrasonic treatment, and the Epon 812 section with and without ultrasonic treatment, appeared homogeneous and without any obvious substructure a t a magnification of X 6000 (Figures 2-4). Point analysis showed t h a t all points in t h e thin sections had almost the same P / B ratio (Table I), thus indicating t h e homogeneity of the sections. Gallium phthalocyanine chloride was less soluble in resin than oxinate; concentrations up to 1% in Spurr resin could be prepared. However, electron micrographs showed that the sections, even if treated by the ultrasonic disintegrator, proved to be heterogeneous with some particles (Figures 5-8). T h e gallium content in the particles was larger than that in other areas judging from t h e point analysis (Table I). T h e resin containing t h e compounds was brittle and hard to section. Furthermore, the section thus obtained was unstable under the electron beam. C 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, IVO. 3, MARCH 1980
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Table I. P/B Ratios for Gallium from Points of Thin Sections Containing Organic Compounds organic compound Ga-oxinate
epoxy resin Spurr
treatmenta pointb
S
u
A
0.3-1
B C D E F G
0.56
I
0.17 0.39 2.32 0.34 0.30 0.32 0.34
H Epon 812
Ga-phthalocyanine chloride
Spurr
S
J
0.40
U
K L
0.28 0.26
S
A B C D
2.71
U
Epon 812
S U
Ga-tannic acid precipitate
Spun
Epon 812
PIB ratio
u u
E F G H I J K A B C D
E F
0.25 1.37 0.33 0.41 2.38 2.51 0.46
1.38 2.89 0.80 0.49 0. 76 1.56
Figure 2. Electron micrograph of thin section of Spurr resin containing gallium oxinate treated ultrasonically
0.84 2.24 n.d.
S : Treated by magnetic stirring; U : Treated bv ultrasonic disintegrator. Points are shown in Figures 1-10, Point analyses were conducted a t an accelerating voltage of 20 kV, a specimen current of 2 nA, and an analyzing time of 100 s.
Figure 3. Electron micrograph of thin section of Epon 812 resin containing gallium oxinate treated by magnetic stirring
Figure 1. Electron micrograph of thin section of Spurr resin containing gallium oxinate treated by magnetic stirring. I n Figures 1-10, the arrows indicate the analyzed spots whose P/B ratios are shown in Table I. The magnification is X 6000
T h e tannic acid precipitate without ultrasonic treatment settled on the bottom of the capsules during the polgmerization of the epoxy resin. Larger particles were observed in
thin sections even with the ultrasonic treatment (Figures 9 and 10). T h e point analysis also indicated that the sections were heterogeneous (Table I). Epoxy resin containing tannic acid precipitate was also brittle, but the section obtained was more stable under the electron beam than those containing other organic compounds. Judging from the homogeneity, solubility, and the stability of the standards under the electron beam, it was concluded that gallium oxinate in epoxy resin treated by the ultrasonic disintegrator was the most suitable standard for electron probe microanalysis. Basic Conditions for the Determination of Gallium in Thin Sections. Analysis was performed with thin sections
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ANALYTICAL CHEMISTRY, VOL. 5 2 , NO. 3, MARCH 1980
T a b l e 11. C o m D a r i s o n of G a l l i u m Lo a n d KQ C o u n t s M e r i t ( P 2 / B )x
(I).
Peak to B a c k g r o u n d R a t i o s
La
Ka
kV
net I
PIB
( P I I B ) x 10-3
20
1847 1842
1.15
25
2.12 2.06
30
1373
1.12 1.13
(P/B), a n d
1.55
A n a l y t i c a l F i g u r e s of
____
PIB
(P’IB) x 10-3
ratio of L~/K;
836
1.16
0.97
2.21
1091 1.39
1.26
1.37 1.22
1.32
net I
1.17
1.69
Figure 4.
Electron micrograph of thin section of Epon o i L iesin containing gallium oxinate treated ultrasonically
Figure 0. Electron micrograph of thin section of Spurr resin containing gallium phthalocyanine chloride treated ultrasonically
Figure-5. Electron micrograph of thin section of Spurr resin containing gallium phthalocyanine chloride treated by magnetic stirring
Figure 7. Electron micrograph of thin section of Epon 812 resin containing gallium phthalocyanine chloride treated by magnetic stirring
of S p u r resin containing gallium oxinate mixed ultrasonically. E f f e c t of Accelerating Voltage. Net K a and Ln counts were collected a t 20-, 2 5 , and 30-kV accelerating voltage under the following conditions: 2 nA of specimen current and 100 s of
analyzing time. In addition, P / B ratio and the analytical figure of merit. ( ( P 2 / B ) X which is a measure of analytical detection sensitivity (15),were also determined (Table 11). T h e P / B ratio for L a lines was somewhat better a t 20
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
535
E
Figure 8. Electron micrograph of thin section of Epon 812 resin
Figure 10. Electron micrograph of thin section of Epon 812 resin
containing gallium phthalocyanine chloride treated ultrasonically
containing gallium tannic acid precipitate treated ultrasonically
L .^
ic
Spec m e r
Cd.rr.71
." >I
_
r;)
Figure 11. Effect of specimen current on P/B ratios. (0)P/B ratios for Ga L a and
(a)for
Ga K a ~
Table 111. Recommended Conditiorls f o r Determination of Gallium with EPMA L C&
accelerating voltage, k V specimen current, nA analyzing time, s
Figure 9. Electron micrograph of thin section of Spurr resin containing gallium tannic acid precipitate treated ultrasonically
kV, a t which the best sensitivity, as judged by the analytical figure of merit, was also obtained. Optimal P / B ratio and greater detection sensitivity for K N lines were observed a t 25 kV. The right column of Table I1 shows that the La peak had from 1.3 to 2.2 times the net counts of the KO channel. A lower accelerating voltage is recommended to maintain the resolution capabilities. Therefore, in electron probe microanalysis of thin sections containing gallium, the collection of La counts a t 20 k V was preferred. However, many Ktu lines for light elements, including sodium (1.041 keV) and magnesium (1.253 keV), common in biological materials in large amounts, are located near the gallium La lines. Therefore,
20 1.,5--2.5
-40
Ka
2 ;i 1.0-2.5 .ti0
the window for the X-ray image should he set for transmission of the K a gallium lines (9.241 keV), and microanalysis should be carried out a t 25-kV accelerating voltage. Effect o f Specimen Current. T h e effect of the specimen current, the current from specimen to ground, on P / B ratios for gallium La and K a lines was investigated (Figure 11). Specimen current was adjusted by gun bias without a change of the condenser lens current. Theoretically, the X-ray intensity generated in the specimen is directly proportional to the specimen current (16). However, large specimen current would incur damage as shown in Figure 11 (at 3 nA). Judging from the high P / B ratios, the specimen current between 1.5 and 2.5 nA for La lines or 1.0 and 2 5 nA for K Olines was found suitable. Effect o f AnalSzing Time. Under natural conditions, the concentration of gallium in biological tissues may be low and, therefore, long analyzing time would be necessary t o collect statistically significant numbers of X-ray counts. Yet, a
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
10
i
I
20
L@
60
Analyzing Ttme
83
100
(51
Figure 12. Effect of analyzing time on P/B ratios. Symbols as in Figure
Calibration Curve of Gallium in Organic Standards. A graduated series of standards was prepared by adding appropriate amounts of the gallium oxinate complex to the resin to achieve the following gallium concentrations (mM): 40,80, 120, 160, a n d 200. Figure 13 shows calibration curves of electron probe analysis for gallium (La and K a ) . Analysis was carried out under the following conditions (refer to Table 111): 2 nA of specimen current, 100 s of analyzing time, and 20 kV ( L a )or 25 kV ( K a ) of voltage. The relation between P / B ratio a n d t h e concentration of gallium held good linearity with almost t h e same slope for the K a and La lines. Finally, the suitability of our standards has been proved by the linear fit calibration curve. Application of the standards to the microanalysis of tumor cells is now in progress.
11
ACKNOWLEDGMENT We are grateful to Akira Tanaka of the Electron Microscope Laboratory of our Institute for many suggestions and a critical reading of t h e manuscript. We also appreciate t h e skillful technical assistance of Yoshio Sekiguchi and Shizuo Kuroda of our Institute, and of Mie Nakauchi, a graduate student of J a p a n Women's University. LITERATURE CITED
Figure 13. Calibration curve for gallium in gallium oxinate complexes
in epoxy resin standards. Symbols a s in Figure 11 long-term analysis would not be tolerable to t h e specimen. With increasing analyzing time, background intensity increased, as did t h e peak intensity for gallium. Figure 12 indicated an analyzing time longer t h a n 40 s for La lines or 80 s for K a lines was necessary to give the constant P / B ratios. Recommended conditions from our experimental results are summarized in Table 111.
"Gallium-67 Imaging", Hoffer, P. B.. Bekerman, C., Henken, R. E., Eds.; John Wiley & Sons: New York, 1978. Voigt, G.; Peibst, H.; Menniger, H.; Hildsch, L. Phys. Status Solidi 1976, 3 6 , 173-179. Wittry, D. B. I n "Microprobe Analysis", Andersen. C. A,, Ed.; John Wiley & Sons: New York, 1973; Chapter 4. Roomans, G. M.; Gaal, H. L. M. J , Microsc. 1977, 109, 235-240. Spurr. A. R. In "Microprobe Analysis as Applied to Cells and Tissues", Hall, T., Echiin. P., Kaufman, R., Eds.; Academic Press: London, 1974; p 213-227. Davies, T. W.; Morgan, A. J. J , Microsc. 1976, 107, 47-54. Lechene, C . In "Microprobe Analysis as Applied to Cells and Tissues", Hall, T., Echlin, P., Kaufman, R., Eds.; Academic Press: London, 1974; pp. 351-367. Spurr, A. R . J . Microsc. Bioi. Celi1975, 22, 287-302. Chandler, J. A. J . Microsc. 1976, 106, 291-302. Mizuhira, V.; Futaesaku, Y. Proc. Electron Microsc. SOC.Am. 1971, 29, 494. Mizuhira, V.; Kimura, M. Proc. Nectron Microsc. Soc. Am. 1973, 31, 402. Rollins. 0. W.; Deischer, C.K. Anal. Chem. 1954, 26, 769-770. Spurr, A. R. J . Ultrastruct. Res. 1969, 26, 31-43. Luft, J. H. J . Biophys. Biochern. Cyfol. 1961, 9 , 409. Lifshin, E.; Ciccarelli. M. F. "Proceedings of the 6th Annual Scanning Electron Microscope Symposium, IIT Research Institute", Chicago, Ill.. 1973. Russ, J. C. In "Electron Probe Microanalysis in Biology", Erasmus. D. A.; Ed.; John Wiley & Sons: New York, 1978; Chapter 2.
RECEIVED for review September 10,1979. Accepted November 21,1979. This work was supported financially by the Ministry of Education. This research was presented a t ACS/CSJ Chemical Congress, April 1-6, 1979, Honolulu, Hawaii.
