Anal. Chem. 2007, 79, 6074-6080
Quantitative Imaging of Selenium, Copper, and Zinc in Thin Sections of Biological Tissues (Slugs-Genus Arion) Measured by Laser Ablation Inductively Coupled Plasma Mass Spectrometry J. S. Becker,*,† A. Matusch,‡ C. Depboylu,‡ J. Dobrowolska,†,§ and M. V. Zoriy†
Central Division of Analytical Chemistry, Research Centre Juelich, D-52425 Ju¨lich, Germany, and Experimental Neurology, Department of Neurology, Philipps-University of Marburg, Marburg D-35033, Germany
Quantitative imaging analysis of endogenous an exogenous elements throughout entire organisms is required for studies of bioavailability, transport processes, distribution, contamination and to monitor environmental risks using indicator organisms. An imaging mass spectrometric technique using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) was developed to analyze selenium and metal distributions in longitudinal sections (thickness, 100 µm) of entire slugs (genus arion). Slugs were fed with either a placebo or solutions containing 1000 µg mL-1 Se. Samples (raster area, 25 mm × 45 mm) were scanned together with synthetic matrixmatched standards with a focused beam of a Nd:YAG laser (wavelength, 266 nm; diameter of laser crater, 50 µm; laser power density, 3 × 109 W cm-2) in a large laser ablation chamber. The ablated material was transported with argon as carrier gas to the ICP ion source at a double focusing sector field ICPMS. Ion intensities of selenium (78Se+, 82Se+) were measured together with 13C+, 63Cu+, and 64Zn+ within the entire tissue section. The regression coefficient of the calibration curve was 0.998. Inhomogeneous distributions for Se but also for C, Cu, and Zn were found. Selenium was enriched in the kidney (150 µg g-1 in Se-treated animals versus 15 µg g-1 in the placebo-treated animal, respectively) and in the digestive gland (200 µg g-1 versus 25 µg g-1). Highest Se concentrations were detected in the gut of Se-treated slugs (250 µg g-1), and additional Se occurred in the skin of these animals. Cu was enriched in the heart and the mucous ventral skin. Interestingly, in addition to the localization in the digestive gland, Zn was detected only in the dorsal skin but not the ventral skin. The developed analytical technique allows the quantitative imaging of selenium together with selected metals in thin sections of biological * To whom correspondence should be addressed. Telephone: 0049 2461 612698. Fax: 0049 2461 612560. E-mail:
[email protected]. † Research Centre Juelich. ‡ Philipps-University of Marburg. § Present address: Faculty of Chemistry, Jagiellonian University, PL-30060 Krakau.
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tissue with limits of detection at the submicrogram per gram range. Over the past years, the development and the application of imaging mass spectrometric techniques such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS),1-4 secondary ion mass spectrometry (SIMS), or laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) to determine quantitative distribution (imaging or mapping) of organic compounds, metals, nonmetals, and metalloids in thin sections of biological tissue is rapidly growing.5-9 An increasing interest in protein, peptide, and small-molecule analysis by imaging mass spectrometry using MALDI-MS and SIMS to study biological surfaces or in tissues was demonstrated by several papers published recently.10-16 The quantitative imaging of elements by mass spectrometry in thin tissue slices or on biological surfaces and gels is also a challenging task in analytical chemistry and (1) Todd, P. J.; Schaaf, T. G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrom. 2001, 36, 355-368. (2) Chaurand, P.; Cornett, D. S.; Caprioli, R. M. Curr. Opin. Biotechnol. 2006, 17, 431-436. (3) Heeren, R. M. A.; Mc.Donnell, L. A.; Amstalden, E.; Luxambourgh, S. L.; Altelaar, A. F. M.; Piersma, S. R. Appl. Surf. Sci. 2006, 252, 6827-6835. (4) McDonnel, L. A.; Piersma. S. R.; Maarten Altelaar, A. F.; Mize, T. H.; Luxembourg, S. L.; Verhaert, P. D. E. M.; van Minnen, J.; Heeren, R. M. A. J. Mass Spectrom. 2005, 40, 160-168. (5) Brunelle, A.; Touboul, D.; Lapre´vote, O. J. Mass Spectrom. 2005, 40, 985999. (6) Touboul, D.; Halgand, F.; Brunelle, A.; Kersting, R.; Tallarek, E.; Hagenhoff, B.; Laprevote, O. Anal. Chem. 2004, 76, 1550-1559. (7) Becker, J. S. Inorganic Mass Spectrometry: Principles and Applications, Wiley, 2007. (8) Becker, J. S.; Zoriy, M.; Dehnhardt, M.; Pickhardt, C.; Zilles, K. J. Anal. At. Spectrom. 2005, 20, 912-917. (9) Becker, J. S.; Zory, M.; Becker, J. Su.; Dobrowolska, J.; Matusch, A. J. Anal. At. Spectrom. 2005, 22, 736-744. (10) Groseclose, M. R.; Andersson, M.; Hardesty, W. M.; Caprioli, R. M. J. Mass Spectrom. 2007, 42, 254-262. (11) Altelaar, A. F. M.; Luxembourg, S. L,; McDonnell, L. A.; Piersma, S. R.; Heeren, R. M. A. Nature Protocols 2007, 2, 1185-1196. (12) Li, Y,; Shrestha, B.; Vertes, A. Anal. Chem. 2007, 79, 523-532. (13) McMahon, G.; Glassner, B. J.; Lechene, C. P. Appl. Surf. Sci. 2006, 252, 6895-6906. (14) Sjo ¨vall, P.; Johansson, B.; Lausmaa, J. Appl. Surf. Sci. 2006, 252, 69666974. (15) Norris, J. L.; Cornett, D. S.; Mobley, J. A.; Andersson, M.; Seeley, E. H.; Chaurand, P.; Caprioli, R. M. Int. J. Mass Spectrom. 2007, 260, 212-221. (16) Wu, L.; Lu, X.; Kulp, K. S.; Knize, M. G.; Berman, E. S. F.; Nelson, E. J.; Felton, J. S.; Wu, K. J. J. Int. J. Mass Spectrom. 2007, 260, 137-145. 10.1021/ac0700528 CCC: $37.00
© 2007 American Chemical Society Published on Web 07/11/2007
relevant in different areas of biological and medical research.8,17-20 From investigations using several element-specific staining techniques or imaging mass spectrometry, distinct distribution patterns of essential and toxic elements in biological tissues are known. This was demonstrated, for example, for Cu, Zn, P, and Pb in the human brain within regions like the hippocampus7 or the insula analyzed by LA-ICPMS in our laboratory.21 For imaging of elements on biological surfaces or on tissue sections, SIMS6,22,23 and LA-ICPMS are the most common sensitive surface analytical techniques.24-26 SIMS can directly produce images of element distribution22 and of organic compounds1 in tissue with a lateral resolution in the low-micrometer and submicrometer range using liquid metal primary ion sources or cluster beams to produce secondary ions of the biological sample.6 The main drawbacks of SIMS are huge matrix effects and high formation rate of polyatomic ions, which do not allow an easy quantification of mass spectrometric measurements. Due to less matrix effects and significantly lower formation rate of polyatomic ions, the quantification of LA-ICPMS is relatively simple if suited matrix-matched standard reference materials are available. The limits of detection in LA-ICPMS (in the ng g-1 range) are in general lower than in SIMS.27 LA-ICPMS,28-30 which uses a focused laser beam for evaporation of solid sample and where the ablated material is transported in the inductively coupled plasma of an ICPMS using Ar as carrier gas, is a powerful element analytical technique for determining the element composition of trace elements in quite different materials.31,32 LA-ICPMS is increasingly employed for characterization of biological and environmental samples.25 Because there is a lack of suitable matrix-matched standard reference materials in LA-ICPMS, different calibration strategies were developed: the application of homemade laboratory standards21,33 or solutionbased calibration,7,27 which was used, for example, for the determination of phosphorus in protein spots in gels separated by two-dimensional gel electrophoresis or for imaging of human (17) Becker, J. S.; Zoriy, M.; Becker, J. Su.; Pickhardt, C.; Damoc, E.; Juhacz, G.; Palkovits; Przybylski, M. Anal. Chem. 2005, 77, 5851-5860. (18) Hutchinson, R. W.; Cox, A. G.; McLeod, C. W.; Marshall, P. S.; Harper, A.; Dawson, E. L.; Howlett, D. R. Anal. Biochem. 2005, 346, 225-233. (19) Zoriy, M.; Dehnhardt, M.; Reifenberger, G.; Zilles, K.; Becker, J. S. Int. J. Mass Spectrom. 2006, 257, 27-33. (20) Lobinski, R.; Moulin, C.; Ortega, R. Biochimie 2006, 88, 1591-1604. (21) Dobrowolska, J.; Dehnhardt, M.; Matusch, A.; Zoriy, M.; Koscielniak, P.; Zilles, K.; Becker, J. S. Talanta. Submitted. (22) Morrison, G. H.; Gay, I.; Chandra, S. Scan. Microsc. 1994, 8 (Suppl), 359370. (23) Chandra, S. Appl. Surf. Sci. 2003, 203-204, 679-683. (24) Feldmann, J.; Kindness, A.; Ek, P. J. Anal. At. Spectrom. 2002, 17, 813818. (25) Kindness, A.; Sekaran, N.; Feldmann, J. Clin. Chem. 2003, 49, 1916-1923. (26) Ghazi, A. M.; Wataha, J. C.; O’Dell, N. L.; Singh, B. B.; Simmons, R.; Shuttleworth, S. J. Anal. At. Spectrom. 2002, 17, 1295-1299. (27) Becker, J. S. Spectrochim. Acta, B 2002, 57, 1805-1820. (28) Durrant, S. F. J. Anal. At. Spectrom. 1999, 14, 1385-1403. (29) Becker, J. S.; Dietze, H.-J. Int. J. Mass Spectrom. 2003, 228, 127-150. (30) Becker, J. S.; Pickhardt, C.; Dietze, H.-J. Int. J. Mass Spectrom. 2000, 203, 283-297. (31) Gu ¨ nther, D.; Jackson, S. E.; Longerich, H. P. Spectrochim. Acta, B 1999, 54, 381-409. (32) Pickhardt, C.; Brenner, I. B.; Becker, J. S.; Dietze, H. J. Fresenius’ J. Anal. Chem. 2000, 368, 79-87. (33) Zoriy, M.; Matusch, A.; Spruss, T.; Becker, J. S. Int. J. Mass Spectrom. 2007, 260, 102-106.
brain tissues or tumor regions in brain.34-36 By imaging mass spectrometry using LA-ICPMS it was possible to distinguish between several layered structures in human brain tissue21,36 or to characterize small brain tumors,8,19 as described in previous studies. Selenium is one of the most investigated beneficial elements, a crucial nutrient for higher organisms (minimum requirement: 1 µg/kg per day in rats),37 and exerts important functions in processes such as antioxidant defense, DNA synthesis, or reproduction. Several studies demonstrated that selenium supplementation can reduce the risk of cancer and further diseases;38 seleniumrich yeast is an often investigated selenium-containing nutrition supplement. Conversely, in higher concentrations, almost all selenium species are toxic, the middle lethal dose (LD 50) of selenite p.e. ranging at 11 mg of Se/kg in chicken,39 4.5 mg of Se/kg in rabbits,40 and 22.5 mg of Se/kg in worms. In vivo, selenite reacts with thiol groups such as in glutathione and can be completely reduced under formation of radical anions. This leads to hemolysis and vacuolar damage of liver cells.41 The selenium speciation analyses of selenium-enriched supplements (in extracts of Se-yeast and Se-methylselenocysteine) by size exclusion, anionexchange, and reversed-phase ion-pair HPLC-ICPMS and ESIMS-MS were described by Infante et al.42 Selenodrug metabolites were characterized using MALDI-q-TOF-MS and LA-ICPMS in McLeod’s working group.43 In spite of the significance of selenium in life sciences, there are only a few studies on selenium distribution in biological tissues.44 The aim of the present work was to develop a microanalytical technique using LA-ICPMS for a quantitative determination of selenium at a concentration level down to submicrogram per gram in thin sections of biological tissues. Such an analytical technique would be of great importance to get new information on the lateral and in-depth distribution of selenium for future studies in life sciences. EXPERIMENTAL LA-ICPMS Instrumentation. A double-focusing sector-field ICPMS (ICP-SFMS, Element, Thermo Fisher Scientific, Bremen, Germany) coupled to the laser ablation system CETAC 200 (CETAC Technologies, Omaha, NE) was used for imaging of selenium, copper, zinc, and carbon in thin biological tissue sections (thickness, 100 µm). The experimental arrangement of LA-ICPMS (34) Becker, J. S.; Boulyga, S. F.; Becker, J. Su.; Pickhardt, C.; Damoc, E.; Przybylski, M. Int. J. Mass Spectrom. 2003, 228, 985-997. (35) Becker, J. S.; Boulyga, S. F.; Becker, J. Su.; Dobrowolska, J.; Dehnhardt, M.; Matusch, A. Phys. Stat. Sol. (C) 2007, 4, 1775-1784. (36) Becker, J. S.; Zoriy, M. V.; Pickhardt, C.; Palomero-Gallagher, N.; Zilles, K. Anal. Chem. 2005, 77, 3208-3216. (37) Schwarz, K.; Foltz, C. M. J. Am. Chem. Soc. 1957, 79, 3292-3293. (38) Rayman, M. P. Proc. Nutr. Soc. 2002, 61, 203. (39) Wang, W.; Kang, S.; Wang, Z.; Qu, Z.; Liu, M.; Li, X. Zhongguo Shouyi Xuebao 1996, 16, 387-389. (40) Berschneider, F.; Hess, M.; Neuffer, K.; Willer, S. Arch. Exp. Veterina ¨ rmed. 1976, 30, 525-531. (41) Spallholz, J. E. Free Radical Biol. Med. 1994, 17, 45-64. (42) Infante, H. G.; O’Connor, G.; Rayman, M.; Wahlen, R.; Entwisle, J.; Norris, P.; Hearn, R.; Catterick, T. J. Anal. At. Spectrom. 2004, 19, 1524-1528. (43) Dickson, H.; Bunch, J.; Stokes, S.; Mead, R.; McLeod, C. W. 17th International Mass Spectrometry Conference, Prague, 2006; ThP-143. (44) Becker, J. S.; Becker, J. Su.; Zoriy, M.; Dobrowolska, J.; Matusch, A. Eur. J. Mass Spectrom. 2007, 13, 1-6.
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Table 1. Optimized Operating Conditions of Developed Imaging LA-ICPMS Procedure for Determination of Lateral Distribution of Se, C, Cu, and Zn in Thin Cross Sections of Slugs laser ablation system wavelength of Nd-YAG laser, nm laser power density, W cm-2 laser scan speed, µm s-1 number of lines per analyzed sample repetition frequency, Hz laser beam diameter, µm inductively coupled plasma mass spectrometer RF power, W cooling gas flow rate, L‚min-1 auxiliary gas flow rate, L‚min-1 nebulilzer gas flow rate, L‚min-1 extraction lens potential, V sampler cone skimmer cone mass resolution, m/∆m mass window, % runs passes scanning mode analysis time, h
CETAC, LSX-200 266 3 × 109 40 150 20 50 Element 1 1200 18 0.65 1.2 2000 nickel, 1.1-mm orifice diameter nickel, 0.9-mm orifice diameter 300 10 12000-14000 1 peak hopping 5-6
Table 2. Characteristics of the Slugs Used for LA-ICPMS Analysisa treatment
[Se], µg mL-1
[Na], mM
weight of slug, g
Na2SeO3/NaNO3
1000
185
6.1
Na2SeO3
1000
25
8.8
H2O aLiquid
6.4
destiny hypoactive, frosted at 60 h death at 60 h, emesis inconspicuous, frosted at 60 h
supply, 2 mL × 2 per day.
with cooled laser ablation chamber is shown elsewhere.45 The laser ablation of thin biological sections was performed with a frequency quadrupled Nd:YAG laser (wavelength, 266 nm; repetition frequency, 20 Hz; spot diameter, 50 µm; laser power density, 3 × 109 W cm-2) in the ablation chamber. Samples and matrixmatched calibration standards were arranged in a commercial large laser ablation chamber (CETAC Technologies) together in order to allow an imaging analysis under the same experimental conditions. The ablated material was transported by argon as a carrier gas into the ICP. The ions formed in the ICP were extracted in the sector field mass spectrometer and separated according to their mass-to-charge ratios. The ICP torch was shielded with a grounded platinum electrode (GuardElectrode, Thermo Electron Corp.). The experimental parameters of ICPMS were optimized with respect to the maximal ion intensity of 78Se+ using a 10 µg L-1 selenium solution introduced by an ultrasonic nebulizer (USN, CETAC Technologies Inc.) as described elsewhere.35 Maximal ion intensity was observed at a carrier gas flow rate of 1 L min-1. Thin tissue sections of slugs were investigated (45) Zoriy, M.; Kayser, M.; Izmer, A.; Pickhardt, C.; Becker, J. S. Int. J. Mass Spectrom. 2005, 242, 297-302.
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Figure 1. 1. Calibration curve of selenium from matrix-matched standards of slug homogenates with added concentrations in the range from 1 to 1000 mg L-1 measured by LA-ICPMS.
with respect to the element distribution of Se, Zn, Cu, and C. The background intensity of the analytes of interest was determined directly by LA-ICP-SFMS in a “blank” slug tissue homogenate (preparation of homogeneous synthetic laboratory standard is described below). The optimized experimental parameters for LAICP-SFMS measurements of thin slug tissues are summarized in Table 1. Thin biological tissue sections of slug were analyzed together with the synthetic laboratory standards under the same experimental conditions by LA-ICPMS (rastering line by line). The region of slug tissues analyzed by LA-ICPMS using a large laser ablation chamber (CETAC Technologies Inc.) varied between 25 × 35 mm2 and 35 × 45 mm2. Samples and Sample Preparation. Slugs (genus arion) naturally occurring at the area of Research Centre Juelich were collected and kept in separate air-permeable transparent boxes (4 slugs/box) at 25-30 °C room temperature onto lettuce leafs. Slugs stayed without treatment during 24 h for accommodation. Than every 12 h solutions containing either water or 1000 mg L-1 selenium as selenite (Na2SeO3; corresponding to 25 mM Na+) alone or in combination with 160 mM NaNO3 (≡ 10 mg mL-1 NO3-) were applied. The former selenite solution contained 3.33 mg mL-1 Na2SeO3‚5H2O (Sigma-Aldrich; Seelze, Germany). The latter (Na2SeO3/NaNO3) solution resulted from 1:10 dilution followed by neutralization with NaOH of a 10 mg mL-1 ICP standard solution of SeO2 in 10% HNO3 (NIST, Gaithersburg, MD). Further details about the sample preparation procedure are presented in Table 2. All the solutions contained 0.05% sodium dodecyl sulfate (Sigma-Aldrich; Seelze, Germany) to allow better moistening of surfaces. Two hundred microliters of liquid was dropped directly onto the mouth of slugs and 1800 µL onto the leafs. The slugs treated with selenite alone showed the highest decrease in lettuce consumption (from 1 leaf per night to
