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Micro-PIXE analysis: importance of biological sample preparation techniques
ARTICLE IN PRESS
Radiation Physics and Chemistry 71 (2004) 785–786
Micro-PIXE analysis: importance of biological sample preparation techniques Dobros"awa Budka*, J. Mesjasz-Przyby"owicz, W.J. Przyby"owicz1 Materials Research Group, iThemba LABS, P.O. Box 722, Somerset West 7129, South Africa
1. Introduction Particle induced X-ray emission (PIXE) has been used successfully in microanalysis of the distribution and concentration of elements in different biological samples. One of the techniques used to prepare samples for PIXE measurements is high-pressure freezing followed by freeze-substitution. Various organic solvents, fixatives, stains and resins are utilised during the freezesubstitution protocols. However, the choice of a suitable freeze-substitution method is crucial for biological material preparation. Methods preserving ultrastructure may lead to elemental losses in the biological material investigated. The aim of this study was to compare the preservation of tissue structure, Ni distribution and content in Berkheya coddii leaves prepared for X-ray microanalysis according to two different freeze-substitution protocols. B. coddii, an endemic plant species characterised by high amount of Ni in its tissues (especially in the leaves), is classified as a hyperaccumulator of this metal (Mesjasz-Przyby"owicz et al., 2001).
2. Material and methods In the first procedure (I) leaf samples were cryofixed in an EM PACT high-pressure freezer (Leica) then immersed in dry acetone at 80 C in a Leica EM AFS automatic freeze-substitution system and processed according to the chemical formula described by Vander Willigen et al. (2003). In the second procedure (II) *Corresponding author. Fax: +27-218433543. E-mail address: [email protected] (D. Budka). 1 On leave from the Faculty of Physics and Nuclear Techniques, University of Mining and Metallurgy, Krako´w, Poland.
freeze-substitution was carried out in anhydrous acetone for up to 6 days at a temperature starting from 90 C, rising progressively to 20 C and eventually reaching 20 C. Specimens obtained following procedure (I) and (II) were next infiltrated with Spurr’s resin at increasing concentrations and polymerised at 60 C for 48 h. Samples were sectioned with glass knives in a Reichert Ultracut S ultramicrotome. PIXE measurements of Ni in freeze-substituted samples were made using the nuclear microprobe at iThemba LABS.
3. Results and discussion The amount of Ni found in samples freeze-substituted using pure acetone (procedure II) was on average 43 times higher than in the samples freeze-substituted in acetone with fixatives and stains (procedure I). The elemental map of Ni (Fig. 1) in samples freezesubstituted according to procedure (I) indicated that most of this metal has been washed out from the samples during processing. It is well known that glutaraldehyde and osmium tetroxide used in procedure (I) alter or destroy the permeability of cell membranes. Although these standard fixatives are routine in the electron microscope laboratory, they are invalid for Ni analysis in B. coddii leaves. The extent of the loss of a given element depends on the degree to which the element is bound to macromolecules within the cell (Warley, 1997). Thus it is possible that the most prevalent form of Ni in B. coddii leaves is an ionic form. It is interesting to note that Ni is clearly visible in epidermal layers (Fig. 1), which is in disagreement with the previously established distribution of this element in B. coddii leaves, where Ni is mostly concentrated in mesophyll cells (MesjaszPrzyby"owicz and Przyby"owicz, 2003). Therefore, this method cannot be used to prepare B. coddii leaf samples
0969-806X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2004.04.091
ARTICLE IN PRESS 786
D. Budka et al. / Radiation Physics and Chemistry 71 (2004) 785–786
Fig. 1. Micro-PIXE quantitative maps of Ni distribution in B. coddi leaf sample freeze-substituted according to the procedure I, left (dry acetone with glutaraldehyde, osmium tetroxide, uranyl acetate and tannic acid) and procedure II, right (dry acetone for up to 6 days at a starting temperature of 90 C, rising progressively to 20 C and eventually reaching 20 C). Concentrations are reported in mg g 1 d.w.
for X-ray microanalysis despite good preservation of the tissue structure. Probably, the distribution of Ni has changed due to its movement towards the epidermal layer or it is more strongly bound to epidermal rather than mesophyll cells. Pure acetone has been used for freeze-substitution for the purpose of preserving diffusible ions in biological material (Edelmann, 1990) as well as for preserving the ultrastructure of the leaves (Mendgen et al., 1991). The present study shows that the use of acetone without any fixatives or stains does not influence the distribution of Ni in B. coddii leaves because the highest concentration of Ni was found in the mesophyll layer (Fig. 1). Structural preservation of samples freeze-substituted in pure acetone is satisfactory at spatial resolution required for tissue analysis.
References Edelmann, L., 1990. Freeze-substitution and the preservation of diffusible ions. J. Microsc. 161, 217–228.
Mendgen, K., Welter, K., Scheffold, F., Knauf-Beiter, G., 1991. High pressure freezing of rust infected plant leaves. In: Mendgen, K., Lesemann, D.E. (Eds.), Elecron Microscopy of Plant Pathogens. Springer, Berlin, Heidelberg, pp. 31–40. Mesjasz-Przyby"owicz, J., Przyby"owicz, W.J., 2003. Nickel distribution in Berkheya coddii leaves by micro-PIXE and SEM/EDS. Proc. Microsc. Soc. South Afr. 33, p. 68. Mesjasz-Przyby"owicz, J., Przyby"owicz, W.J., Pineda, C.A., 2001. Nuclear microprobe studies of elemental distribution in apical leaves of the Ni hyperaccumulator Berkheya coddii. S. Afr. J. Sci. 97, 591–593. Vander Willigen, C., Pammenter, N.M., Jaffer, M.A., Mundree, S.G., Farrant, J.M., 2003. An ultrastructural study using anhydrous fixation of Eragrostis nindensis, a resurrection grass with both desiccationtolerant and sensitive tissues. Funct. Plant Biol. 30 (3), 281–290. Warley, A., 1997. . X-ray microanalysis for biologists. Practical Methods in Electron Microscopy, Vol. 16. Portland Press Ltd., UK.
Radiation Physics and Chemistry 71 (2004) 785–786
Micro-PIXE analysis: importance of biological sample preparation techniques Dobros"awa Budka*, J. Mesjasz-Przyby"owicz, W.J. Przyby"owicz1 Materials Research Group, iThemba LABS, P.O. Box 722, Somerset West 7129, South Africa
1. Introduction Particle induced X-ray emission (PIXE) has been used successfully in microanalysis of the distribution and concentration of elements in different biological samples. One of the techniques used to prepare samples for PIXE measurements is high-pressure freezing followed by freeze-substitution. Various organic solvents, fixatives, stains and resins are utilised during the freezesubstitution protocols. However, the choice of a suitable freeze-substitution method is crucial for biological material preparation. Methods preserving ultrastructure may lead to elemental losses in the biological material investigated. The aim of this study was to compare the preservation of tissue structure, Ni distribution and content in Berkheya coddii leaves prepared for X-ray microanalysis according to two different freeze-substitution protocols. B. coddii, an endemic plant species characterised by high amount of Ni in its tissues (especially in the leaves), is classified as a hyperaccumulator of this metal (Mesjasz-Przyby"owicz et al., 2001).
2. Material and methods In the first procedure (I) leaf samples were cryofixed in an EM PACT high-pressure freezer (Leica) then immersed in dry acetone at 80 C in a Leica EM AFS automatic freeze-substitution system and processed according to the chemical formula described by Vander Willigen et al. (2003). In the second procedure (II) *Corresponding author. Fax: +27-218433543. E-mail address: [email protected] (D. Budka). 1 On leave from the Faculty of Physics and Nuclear Techniques, University of Mining and Metallurgy, Krako´w, Poland.
freeze-substitution was carried out in anhydrous acetone for up to 6 days at a temperature starting from 90 C, rising progressively to 20 C and eventually reaching 20 C. Specimens obtained following procedure (I) and (II) were next infiltrated with Spurr’s resin at increasing concentrations and polymerised at 60 C for 48 h. Samples were sectioned with glass knives in a Reichert Ultracut S ultramicrotome. PIXE measurements of Ni in freeze-substituted samples were made using the nuclear microprobe at iThemba LABS.
3. Results and discussion The amount of Ni found in samples freeze-substituted using pure acetone (procedure II) was on average 43 times higher than in the samples freeze-substituted in acetone with fixatives and stains (procedure I). The elemental map of Ni (Fig. 1) in samples freezesubstituted according to procedure (I) indicated that most of this metal has been washed out from the samples during processing. It is well known that glutaraldehyde and osmium tetroxide used in procedure (I) alter or destroy the permeability of cell membranes. Although these standard fixatives are routine in the electron microscope laboratory, they are invalid for Ni analysis in B. coddii leaves. The extent of the loss of a given element depends on the degree to which the element is bound to macromolecules within the cell (Warley, 1997). Thus it is possible that the most prevalent form of Ni in B. coddii leaves is an ionic form. It is interesting to note that Ni is clearly visible in epidermal layers (Fig. 1), which is in disagreement with the previously established distribution of this element in B. coddii leaves, where Ni is mostly concentrated in mesophyll cells (MesjaszPrzyby"owicz and Przyby"owicz, 2003). Therefore, this method cannot be used to prepare B. coddii leaf samples
0969-806X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2004.04.091
ARTICLE IN PRESS 786
D. Budka et al. / Radiation Physics and Chemistry 71 (2004) 785–786
Fig. 1. Micro-PIXE quantitative maps of Ni distribution in B. coddi leaf sample freeze-substituted according to the procedure I, left (dry acetone with glutaraldehyde, osmium tetroxide, uranyl acetate and tannic acid) and procedure II, right (dry acetone for up to 6 days at a starting temperature of 90 C, rising progressively to 20 C and eventually reaching 20 C). Concentrations are reported in mg g 1 d.w.
for X-ray microanalysis despite good preservation of the tissue structure. Probably, the distribution of Ni has changed due to its movement towards the epidermal layer or it is more strongly bound to epidermal rather than mesophyll cells. Pure acetone has been used for freeze-substitution for the purpose of preserving diffusible ions in biological material (Edelmann, 1990) as well as for preserving the ultrastructure of the leaves (Mendgen et al., 1991). The present study shows that the use of acetone without any fixatives or stains does not influence the distribution of Ni in B. coddii leaves because the highest concentration of Ni was found in the mesophyll layer (Fig. 1). Structural preservation of samples freeze-substituted in pure acetone is satisfactory at spatial resolution required for tissue analysis.
References Edelmann, L., 1990. Freeze-substitution and the preservation of diffusible ions. J. Microsc. 161, 217–228.
Mendgen, K., Welter, K., Scheffold, F., Knauf-Beiter, G., 1991. High pressure freezing of rust infected plant leaves. In: Mendgen, K., Lesemann, D.E. (Eds.), Elecron Microscopy of Plant Pathogens. Springer, Berlin, Heidelberg, pp. 31–40. Mesjasz-Przyby"owicz, J., Przyby"owicz, W.J., 2003. Nickel distribution in Berkheya coddii leaves by micro-PIXE and SEM/EDS. Proc. Microsc. Soc. South Afr. 33, p. 68. Mesjasz-Przyby"owicz, J., Przyby"owicz, W.J., Pineda, C.A., 2001. Nuclear microprobe studies of elemental distribution in apical leaves of the Ni hyperaccumulator Berkheya coddii. S. Afr. J. Sci. 97, 591–593. Vander Willigen, C., Pammenter, N.M., Jaffer, M.A., Mundree, S.G., Farrant, J.M., 2003. An ultrastructural study using anhydrous fixation of Eragrostis nindensis, a resurrection grass with both desiccationtolerant and sensitive tissues. Funct. Plant Biol. 30 (3), 281–290. Warley, A., 1997. . X-ray microanalysis for biologists. Practical Methods in Electron Microscopy, Vol. 16. Portland Press Ltd., UK.