- Email: [email protected]
Identification and quantitive analysis of calcium phosphate microparticles in intestinal tissue by nuclear microscopy
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 249 (2006) 665–669 www.elsevier.com/locate/nimb
Identification and quantitive analysis of calcium phosphate microparticles in intestinal tissue by nuclear microscopy Inmaculada Gomez-Morilla a,*, Vinay Thoree b, Jonathan J. Powell c, Karen J. Kirkby a, Geoffrey W. Grime d a Advanced Technology Institute, University of Surrey, Guildford GU2 7XH, UK Gastrointestinal Laboratory, Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK MRC Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, UK d Department of Physics, University of Surrey, GU2 7XH, UK b
c
Available online 8 May 2006
Abstract Microscopic particles (0.5–2 lm diameter), rich in calcium and phosphorus, are found in the lumen of the mid-distal gut of all mammals investigated, including humans, and these may play a role in immuno-surveillance and immune regulation of antigens from food and symbiotic bacteria that are contained in the gut. Whether these particles can cross in to tissue of the intestinal mucosa is unclear. If so, characterising their morphology and chemical composition is an important task in elucidating their function. The analysis of calcium phosphate in biological tissues has been approached in several ways including optical microscopy, scanning electron microscopy and, most recently in this work, with nuclear microscopy. In this paper, we describe the use of microPIXE and microRBS to locate these particles and to determine, accurately, the ratio of phosphorus to calcium using the information on sample thickness obtained from RBS to allow the PIXE ratios to be corrected. A commercial sample of hydroxy apatite was used to demonstrate accuracy and precision of the technique. Then, in a pilot study on intestinal tissue of mice, we demonstrated the presence of calcium phosphate microparticles, consistent with confocal microscopy observations, and we identified the average molar P:Ca molar ratio as 1.0. Further work will confirm the exact chemical speciation of these particles and will examine the influence of differing calcium containing diets on the formation of these microparticles. 2006 Elsevier B.V. All rights reserved. PACS: 81.70. q; 82.80. d; 87.64. t; 82.80.Yc Keywords: Calcium phosphate; Microparticles; PIXE; RBS; Gastrointestinal
1. Introduction The gastrointestinal tract has specialised features, termed intestinal lymphoid aggregates, which facilitate the uptake of luminal macromolecules. The purpose of this remains unclear although some sort of ‘immuno-surveillance’ has long been postulated. In such a manner the immune system of the gut is ‘armed’ in case of uncontrolled *
Corresponding author. Tel.: +44 1483 682214; fax: +44 (0) 1483 686091. E-mail address: [email protected] (I. Gomez-Morilla). 0168-583X/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2006.03.076
penetration of the gut tissue by foreign material. In the distal small intestine (ileum) intestinal lymphoid aggregates are especially pronounced and are referred to as ‘Peyer’s patches’ [1]. The surface cells of the Peyer’s patch differ from regular intestinal epithelial cells and are termed M-cells. M-cells are known to readily transport micronand sub-micron-sized particles from the lumen into the underlying lymphoid patch. Non-biological microparticles are widely ingested as food additives, pharmaceutical excipients or dust and soil contaminants but are also formed in situ in the gut lumen, specifically in the form of calcium phosphate (0.5–2 lm
666
I. Gomez-Morilla et al. / Nucl. Instr. and Meth. in Phys. Res. B 249 (2006) 665–669
diameter) [2]. The reason for luminal calcium phosphate formation is unclear, although it may be an inevitable result of calcium and phosphate homeostasis, since they are secreted from the mucosa into the lumen where they co-precipitate. Our group (JJP, VT et al.) has postulated that calcium phosphate particles may act as ‘Trojan horses’ carrying luminal products into immune cells of intestinal tissue, especially via the Peyer’s patch uptake route, enabling immuno-surveillance and contributing to immune regulation [2]. Two key questions with respect to this hypothesis are: (i) can discrete calcium phosphate particles be demonstrated in the intestinal mucosa, especially around the region of Peyer’s patches and (ii) can accurate quantification of calcium:phosphorus ratios be achieved to facilitate determination of the specific form of calcium phosphate (termed in the following text as CaP due to its unknown stoichiometry). This is a difficult characterisation problem, since the particles are small (typically around 1 lm diameter) and located randomly in the tissue. In addition, freshly precipitated CaP readily dissociates/re-dissolves and so may be strongly disturbed by any form of chemical processing of the tissue. Depending on the analytical technique applied, these effects can influence the measured composition and it is imperative to have a reliable technique, or combination of techniques, that enable accurate and precise analysis. The majority of characterisation studies of CaP particles in tissue have so far employed either confocal microscopy, labelling the tissue with calcein for identification of mineralised calcium, or scanning electron microscopy to locate the particles followed by EDX for quantitative analysis. Confocal microscopy is an important routine technique for visualising the localisation of particles within tissue but it is unable to provide data on particle composition, while the specificity and sensitivity of fluorescent labels (e.g. calcein) is not guaranteed without confirmation using physical analytical techniques. Moreover, the technique often requires chemical processing of the tissue which may affect the physico-chemical characteristics of the analyte. Scanning electron microscopy allows the particles to be located using the Z contrast in backscattered electrons. This is sensitive mainly to particles located close to the surface of the sample but would not distinguish CaP particles from other dense regions of the sample (e.g. dust contamination). Once the particles have been located they can be analysed using energy dispersive X-ray spectroscopy (EDX). The quantitative accuracy of determination using this technique is limited by the rapid scattering of the electrons, which means that the extent of the excitation volume within the tissue section is not well known and may not coincide with the particle. In essence, despite the development of algorithms to allow elemental quantification of particles in complex samples using EDX, it remains a semi-quantitative technique. Nuclear microscopy (NM) using a focused beam of MeV protons to excite characteristic X-rays (PIXE) could also be used to characterise the CaP particles in tissue
samples. In addition to the low detection limits of PIXE, the advantages of NM in this application include the fact that the long range and low lateral straggling of the ions means that the spatial resolution is maintained through the tissue and thicker tissue sections can be analysed, which will improve the probability of finding the particles. This also means that X-rays are excited from deep within the sample so that it is important to know the particle thickness in order to correct the PIXE yields for the effects of energy loss and X-ray absorption [3]. 2. Experimental To determine whether NM may allow identification of CaP particles, and quantification of Ca:P ratios, in the intestinal mucosa, multiple analyses of tissue sections were undertaken using gut samples from two different mice. Seven-week-old female Balb/c mice were maintained on a powdered diet of defined, normal calcium and phosphate content (0.5% Ca and molar Ca:P ratio of 1.3:1; HarlanTeklad, Madison, USA). Twenty-four days later the mice were killed and their intestinal tracts removed and immediately preserved by placing in liquid nitrogen. Peyer’s patch-containing areas of the tissue were sectioned (5 lm thickness) using a cryomicrotome and prepared for NM analysis as follows: tissue sections were mounted on a thin layer of pioloform film (Agar Scientific Ltd., UK) stretched across 5 mm diameter holes in aluminium holders [4]. A calibration standard was prepared with particles of the calcium phosphate, hydroxy apatite (HA), embedded in gelatine. The composition of HA is Ca5(PO4)3OH, which means that the P:Ca ratio (w/w) for this material is 0.46. The samples were mounted in the sample chamber of the nuclear microprobe at the University of Surrey Ion Beam Centre [5] and were scanned using a beam of 3 MeV protons from a 2 MV Tandetron accelerator focused to a diameter of 1–2 lm. The average beam current varied
Fig. 1. PIXE elemental maps showing collections of calcium phosphate particles in a section of murine gut wall. Dark regions indicate a higher concentration of the element and although each particle is mainly micronsized or smaller, these accumulate in lysosomes of phagocytic cells and so appear as larger aggregates. A typical phagocytic cell (immature dendritic cell or macrophage) is of the order of 30 lm diameter so the above aggregates are clearly within multiple cells.
I. Gomez-Morilla et al. / Nucl. Instr. and Meth. in Phys. Res. B 249 (2006) 665–669
between 100 pA and 200 pA. Induced X-rays were detected using a lithium-drifted silicon detector at a variable distance of 20–50 mm from the sample (Gresham Scientific Ltd., 80 mm2 active area) and backscattered protons were detected using a PIPS detector with active area 50 mm2 (EGG – Ortec) mounted at a scattering angle of 155 and a distance of 40 mm from the sample. Based upon parallel confocal microscopy observations, elemental maps of likely CaP-containing areas and ranging from 1 · 1 mm2 to 100 · 100 lm2, were recorded in order to locate the microparticles in the tissue and relate their position to the tissue morphology (see Fig. 1). PIXE and RBS spectra from individual particles were recorded by positioning the beam on each particle sequentially. The spectra were processed using the Dan32 software [6,7], which fits the RBS spectra to obtain the sample matrix composition and uses this as an input to GUPIX [8] to obtain accurate relative elemental concentrations corrected for absorption. 3. Results and discussion Using confocal microscopy, with fluorescent calcein staining for mineralised calcium particles, in combination with NM, we have been able to identify calcium phosphate microparticles as a normal occurrence in the Peyer’s patch-rich ileal (distal small intestinal) mucosa of mice on a regular Ca- and P-containing diet (Fig. 1). Moreover, quantification of P:Ca ratios was possible using NM. In NM, X-rays are excited from deep within the particle so it is important to know the particle thickness in order to correct the PIXE yields for the effects of energy loss and X-ray absorption. The significance of this is demonstrated in Fig. 2, which shows the P:Ca ratio which would be
calculated from the measured PIXE spectra of a hydroxy apatite particle as a function of the assumed particle thickness. Graphs are presented for two cases (each relating to a measured spectrum): a thin particle less than 0.5 lm thick and a larger particle with a diameter of around 5 lm. The calculations were carried out using GUPIX [8] assuming that the particle can be represented as a flat homogenous slab. The curves show that in order to keep the relative error on the P:Ca ratio to less than 10% it is necessary to know the particle thickness to an accuracy of better than 2 lm. This can be achieved using Rutherford backscattering (RBS) analysis simultaneously with PIXE, allowing the determination of the thickness and major element content of the particle. Fig. 3 shows typical PIXE and RBS spectra from an aggregate of CaP particles in a 5 lm thick tissue section. It can be seen that the RBS spectrum is fairly complex, and is not easy to interpret, especially in the region corresponding to the light elements in the sample, but the recoils from calcium and phosphorus are reasonably isolated and can be used to obtain a relatively unambiguous estimate of the particle thickness. The complexity of modelling PIXE and RBS yields from these small irregular particulate samples also means that it is difficult to determine absolute PIXE yields even if the sample matrix structure is accurately known. For this reason it is much easier to determine elemental ratios and for the rest of this project the measured parameter is the ratio of phosphorus to calcium concentration. The overall accuracy of this measurement was evaluated using a sample of hydroxy apatite (HA) particles with diameters ranging from sub-micrometre to clumps of several micrometres which were dispersed in gelatine to
^5
0.75
667
P
Ca
^4
Calculated P:Ca ratio (w/w)
0.70 Mn
^3
0.65
Fe
^2
0.60
Cu Zn
Data from thick particle Data from thin particle
0.55
^1 ^0 2
0.50
4
6 C
0.45
8
N O
10
12
P Ca
100
0.40 0.35 0.30 0
2
4
6
8
10
50
Assumed particle thickness (μm) Fig. 2. The calculated P:Ca ratio obtained from two experimental PIXE spectra from thin (lower line) and thick (upper line) hydroxy apatite particles plotted as a function of the assumed particle thickness. The calculations were carried out using GUPIX via the Dan32 interface. The horizontal line indicates the stoichiometric ratio calculated from the chemical formula.
0 1600
2000
2400
2800
3200
Fig. 3. PIXE and RBS spectra of a point selected from one of the particlerich aggregates shown in the PIXE maps in Fig. 1.
668
I. Gomez-Morilla et al. / Nucl. Instr. and Meth. in Phys. Res. B 249 (2006) 665–669 Table 1 Summary of P:Ca ratios (mean SD) in the standard hydroxy apatite particles (expected 0.46 w/w) and in naturally occurring mucosal calcium phosphate particles in the ileum of mice
Measured P:Ca ratio (w/w)
0.8 0.7 0.6
Sample
0.5
Standard Mice
0.4
N
0.46 (0.08) 0.78 (0.07)
8 26
N is the number of measurements. 0.3 0.2 0.1 0 0
2
4
6
8
10
12
14
RBS particle thickness (μm)
Fig. 4. The P:Ca ratio for eight hydroxy apatite particles of a range of diameters calculated using the RBS particle thickness and composition to determine the thick target PIXE correction. The horizontal line indicates the stoichiometric ratio.
form a block from which samples were prepared and analysed in exactly the same way as the tissue sections. Fig. 4 shows the measured P:Ca ratio for eight particles plotted as a function of their thickness determined from the RBS spectrum. The fact that there is no obvious dependence on particle thickness indicates that the RBS thickness measurement can be used with some confidence to correct the PIXE data (compare this with the graphs in Fig. 2). The remaining scatter of the points (relative standard deviation of 18%) is probably related to (i) inadequacies in other assumptions in the model and is an indication of the maximum limits of reliability of nuclear microscopy in these analyses and (ii) heterogeneity within the individual particles of HA. Nonetheless, the mean P:Ca ratio (0.46 w/w) is close to the expected value of 0.46. Fig. 5 and Table 1 summarise the results of the determination of P:Ca ratios for the CaP particles in the mice. A figure of 0.78 ± 0.07(w/w) was found for P:Ca ratios in Peyer’s patch microparticles of the ileal mucosa. These 1
0.8 P:Ca (w/w)
Hydroxy apatite Normal Ca and P diet
P:Ca ratio as w/w
results are preliminary but suggest a molar P:Ca ratio close to 1:1. Point analyses were performed also outside the particles in the surrounding tissue in order to determine the levels of calcium and phosphorus present naturally in the tissue. These were found to be two orders of magnitude less than those in the particles and the contribution of this to the error in the calculated ratios in the particles was considered not to be significant. Further studies will investigate this further including their chemical speciation and findings in other parts of the intestine, as well as the effects of different calcium-containing diets on these parameters. 4. Conclusion The results of this preliminary study confirm that nuclear microscopy can be a valuable tool for characterising the chemical composition of mineralised (i.e. calcium) particles embedded in tissue. The long penetration of MeV protons allows the entire thickness of the sample to be measured, which simplifies the localisation of the particles, while the use of PIXE and RBS simultaneously allows the PIXE data to be corrected for the varying thickness of the sample, enhancing the accuracy of the technique. Work is in progress with further mice and human samples. Acknowledgements The authors are grateful to the UK Engineering and Physical Sciences Research Council for supporting access to the Surrey Ion Beam Centre through a pump-priming grant and to the Sir Halley Stewart Trust for the support of Vinay Thoree. The authors also wish to thank Mr. Adrian Cansell and Mr. Mark Browton from IBC for their valuable help in the technical implementation of the measurements.
0.6
References
0.4
0.2
0
HA
Control
Mouse 1
Mouse 2
Gut Mucosal
Fig. 5. Measured P:Ca ratios (w/w) for calcium phosphate (CaP) particles either embedded in gelatine (hydroxy apatite (HA) control) or naturally present in the gut mucosa of two different mice. The triangles and circles in mouse 1 indicate measurements made on two different tissue sections.
[1] T.T. MacDonald, G. Monteleone, Immunity, inflammation, and allergy in the gut, Science 307 (2005) 1920. [2] M.C.E. Lomer, R.P.H. Thompson, J.J. Powell, Fine and ultrafine particles of the diet: influence on the mucosal immune response and association with Crohn’s disease, Proc. Nutr. Soc. 61 (1) (2002) 123. [3] E.F. Garman, G.W. Grime, Elemental analysis of proteins by microPIXE, Prog. Biophys. Mol. Bio. 89 (2005) 173. [4] D. Yates et al., Characterisation of thin support films for microPIXE analysis of metals in biological materials, in: 10th International
I. Gomez-Morilla et al. / Nucl. Instr. and Meth. in Phys. Res. B 249 (2006) 665–669 Conference on Particle Induced X-Ray Emission and its Analytical Applications, http://pixe2004.ijs.si/proceedings/, p. 946.1. [5] A. Simon et al., The new Surrey ion beam analysis facility, Nucl. Instr. and Meth. B 219–220 (2004) 405. [6] G.W. Grime, The Q factor method: quantitative microPIXE analysis using RBS normalisation, Nucl. Instr. and Meth. B 109–110 (1996) 170.
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[7] G.W. Grime, DAN32: recent developments in the Windows interface to GUPIX, in: 10th International Conference on Particle Induced X-Ray Emission and its Analytical Applications, http://pixe2004.ijs.si/ proceedings/, p. 827.1. [8] J.L. Campbell et al., The Guelph PIXE software package III: Alternative proton database, Nucl. Instr. and Meth. B 170 (2000) 193.
Nuclear Instruments and Methods in Physics Research B 249 (2006) 665–669 www.elsevier.com/locate/nimb
Identification and quantitive analysis of calcium phosphate microparticles in intestinal tissue by nuclear microscopy Inmaculada Gomez-Morilla a,*, Vinay Thoree b, Jonathan J. Powell c, Karen J. Kirkby a, Geoffrey W. Grime d a Advanced Technology Institute, University of Surrey, Guildford GU2 7XH, UK Gastrointestinal Laboratory, Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK MRC Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, UK d Department of Physics, University of Surrey, GU2 7XH, UK b
c
Available online 8 May 2006
Abstract Microscopic particles (0.5–2 lm diameter), rich in calcium and phosphorus, are found in the lumen of the mid-distal gut of all mammals investigated, including humans, and these may play a role in immuno-surveillance and immune regulation of antigens from food and symbiotic bacteria that are contained in the gut. Whether these particles can cross in to tissue of the intestinal mucosa is unclear. If so, characterising their morphology and chemical composition is an important task in elucidating their function. The analysis of calcium phosphate in biological tissues has been approached in several ways including optical microscopy, scanning electron microscopy and, most recently in this work, with nuclear microscopy. In this paper, we describe the use of microPIXE and microRBS to locate these particles and to determine, accurately, the ratio of phosphorus to calcium using the information on sample thickness obtained from RBS to allow the PIXE ratios to be corrected. A commercial sample of hydroxy apatite was used to demonstrate accuracy and precision of the technique. Then, in a pilot study on intestinal tissue of mice, we demonstrated the presence of calcium phosphate microparticles, consistent with confocal microscopy observations, and we identified the average molar P:Ca molar ratio as 1.0. Further work will confirm the exact chemical speciation of these particles and will examine the influence of differing calcium containing diets on the formation of these microparticles. 2006 Elsevier B.V. All rights reserved. PACS: 81.70. q; 82.80. d; 87.64. t; 82.80.Yc Keywords: Calcium phosphate; Microparticles; PIXE; RBS; Gastrointestinal
1. Introduction The gastrointestinal tract has specialised features, termed intestinal lymphoid aggregates, which facilitate the uptake of luminal macromolecules. The purpose of this remains unclear although some sort of ‘immuno-surveillance’ has long been postulated. In such a manner the immune system of the gut is ‘armed’ in case of uncontrolled *
Corresponding author. Tel.: +44 1483 682214; fax: +44 (0) 1483 686091. E-mail address: [email protected] (I. Gomez-Morilla). 0168-583X/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2006.03.076
penetration of the gut tissue by foreign material. In the distal small intestine (ileum) intestinal lymphoid aggregates are especially pronounced and are referred to as ‘Peyer’s patches’ [1]. The surface cells of the Peyer’s patch differ from regular intestinal epithelial cells and are termed M-cells. M-cells are known to readily transport micronand sub-micron-sized particles from the lumen into the underlying lymphoid patch. Non-biological microparticles are widely ingested as food additives, pharmaceutical excipients or dust and soil contaminants but are also formed in situ in the gut lumen, specifically in the form of calcium phosphate (0.5–2 lm
666
I. Gomez-Morilla et al. / Nucl. Instr. and Meth. in Phys. Res. B 249 (2006) 665–669
diameter) [2]. The reason for luminal calcium phosphate formation is unclear, although it may be an inevitable result of calcium and phosphate homeostasis, since they are secreted from the mucosa into the lumen where they co-precipitate. Our group (JJP, VT et al.) has postulated that calcium phosphate particles may act as ‘Trojan horses’ carrying luminal products into immune cells of intestinal tissue, especially via the Peyer’s patch uptake route, enabling immuno-surveillance and contributing to immune regulation [2]. Two key questions with respect to this hypothesis are: (i) can discrete calcium phosphate particles be demonstrated in the intestinal mucosa, especially around the region of Peyer’s patches and (ii) can accurate quantification of calcium:phosphorus ratios be achieved to facilitate determination of the specific form of calcium phosphate (termed in the following text as CaP due to its unknown stoichiometry). This is a difficult characterisation problem, since the particles are small (typically around 1 lm diameter) and located randomly in the tissue. In addition, freshly precipitated CaP readily dissociates/re-dissolves and so may be strongly disturbed by any form of chemical processing of the tissue. Depending on the analytical technique applied, these effects can influence the measured composition and it is imperative to have a reliable technique, or combination of techniques, that enable accurate and precise analysis. The majority of characterisation studies of CaP particles in tissue have so far employed either confocal microscopy, labelling the tissue with calcein for identification of mineralised calcium, or scanning electron microscopy to locate the particles followed by EDX for quantitative analysis. Confocal microscopy is an important routine technique for visualising the localisation of particles within tissue but it is unable to provide data on particle composition, while the specificity and sensitivity of fluorescent labels (e.g. calcein) is not guaranteed without confirmation using physical analytical techniques. Moreover, the technique often requires chemical processing of the tissue which may affect the physico-chemical characteristics of the analyte. Scanning electron microscopy allows the particles to be located using the Z contrast in backscattered electrons. This is sensitive mainly to particles located close to the surface of the sample but would not distinguish CaP particles from other dense regions of the sample (e.g. dust contamination). Once the particles have been located they can be analysed using energy dispersive X-ray spectroscopy (EDX). The quantitative accuracy of determination using this technique is limited by the rapid scattering of the electrons, which means that the extent of the excitation volume within the tissue section is not well known and may not coincide with the particle. In essence, despite the development of algorithms to allow elemental quantification of particles in complex samples using EDX, it remains a semi-quantitative technique. Nuclear microscopy (NM) using a focused beam of MeV protons to excite characteristic X-rays (PIXE) could also be used to characterise the CaP particles in tissue
samples. In addition to the low detection limits of PIXE, the advantages of NM in this application include the fact that the long range and low lateral straggling of the ions means that the spatial resolution is maintained through the tissue and thicker tissue sections can be analysed, which will improve the probability of finding the particles. This also means that X-rays are excited from deep within the sample so that it is important to know the particle thickness in order to correct the PIXE yields for the effects of energy loss and X-ray absorption [3]. 2. Experimental To determine whether NM may allow identification of CaP particles, and quantification of Ca:P ratios, in the intestinal mucosa, multiple analyses of tissue sections were undertaken using gut samples from two different mice. Seven-week-old female Balb/c mice were maintained on a powdered diet of defined, normal calcium and phosphate content (0.5% Ca and molar Ca:P ratio of 1.3:1; HarlanTeklad, Madison, USA). Twenty-four days later the mice were killed and their intestinal tracts removed and immediately preserved by placing in liquid nitrogen. Peyer’s patch-containing areas of the tissue were sectioned (5 lm thickness) using a cryomicrotome and prepared for NM analysis as follows: tissue sections were mounted on a thin layer of pioloform film (Agar Scientific Ltd., UK) stretched across 5 mm diameter holes in aluminium holders [4]. A calibration standard was prepared with particles of the calcium phosphate, hydroxy apatite (HA), embedded in gelatine. The composition of HA is Ca5(PO4)3OH, which means that the P:Ca ratio (w/w) for this material is 0.46. The samples were mounted in the sample chamber of the nuclear microprobe at the University of Surrey Ion Beam Centre [5] and were scanned using a beam of 3 MeV protons from a 2 MV Tandetron accelerator focused to a diameter of 1–2 lm. The average beam current varied
Fig. 1. PIXE elemental maps showing collections of calcium phosphate particles in a section of murine gut wall. Dark regions indicate a higher concentration of the element and although each particle is mainly micronsized or smaller, these accumulate in lysosomes of phagocytic cells and so appear as larger aggregates. A typical phagocytic cell (immature dendritic cell or macrophage) is of the order of 30 lm diameter so the above aggregates are clearly within multiple cells.
I. Gomez-Morilla et al. / Nucl. Instr. and Meth. in Phys. Res. B 249 (2006) 665–669
between 100 pA and 200 pA. Induced X-rays were detected using a lithium-drifted silicon detector at a variable distance of 20–50 mm from the sample (Gresham Scientific Ltd., 80 mm2 active area) and backscattered protons were detected using a PIPS detector with active area 50 mm2 (EGG – Ortec) mounted at a scattering angle of 155 and a distance of 40 mm from the sample. Based upon parallel confocal microscopy observations, elemental maps of likely CaP-containing areas and ranging from 1 · 1 mm2 to 100 · 100 lm2, were recorded in order to locate the microparticles in the tissue and relate their position to the tissue morphology (see Fig. 1). PIXE and RBS spectra from individual particles were recorded by positioning the beam on each particle sequentially. The spectra were processed using the Dan32 software [6,7], which fits the RBS spectra to obtain the sample matrix composition and uses this as an input to GUPIX [8] to obtain accurate relative elemental concentrations corrected for absorption. 3. Results and discussion Using confocal microscopy, with fluorescent calcein staining for mineralised calcium particles, in combination with NM, we have been able to identify calcium phosphate microparticles as a normal occurrence in the Peyer’s patch-rich ileal (distal small intestinal) mucosa of mice on a regular Ca- and P-containing diet (Fig. 1). Moreover, quantification of P:Ca ratios was possible using NM. In NM, X-rays are excited from deep within the particle so it is important to know the particle thickness in order to correct the PIXE yields for the effects of energy loss and X-ray absorption. The significance of this is demonstrated in Fig. 2, which shows the P:Ca ratio which would be
calculated from the measured PIXE spectra of a hydroxy apatite particle as a function of the assumed particle thickness. Graphs are presented for two cases (each relating to a measured spectrum): a thin particle less than 0.5 lm thick and a larger particle with a diameter of around 5 lm. The calculations were carried out using GUPIX [8] assuming that the particle can be represented as a flat homogenous slab. The curves show that in order to keep the relative error on the P:Ca ratio to less than 10% it is necessary to know the particle thickness to an accuracy of better than 2 lm. This can be achieved using Rutherford backscattering (RBS) analysis simultaneously with PIXE, allowing the determination of the thickness and major element content of the particle. Fig. 3 shows typical PIXE and RBS spectra from an aggregate of CaP particles in a 5 lm thick tissue section. It can be seen that the RBS spectrum is fairly complex, and is not easy to interpret, especially in the region corresponding to the light elements in the sample, but the recoils from calcium and phosphorus are reasonably isolated and can be used to obtain a relatively unambiguous estimate of the particle thickness. The complexity of modelling PIXE and RBS yields from these small irregular particulate samples also means that it is difficult to determine absolute PIXE yields even if the sample matrix structure is accurately known. For this reason it is much easier to determine elemental ratios and for the rest of this project the measured parameter is the ratio of phosphorus to calcium concentration. The overall accuracy of this measurement was evaluated using a sample of hydroxy apatite (HA) particles with diameters ranging from sub-micrometre to clumps of several micrometres which were dispersed in gelatine to
^5
0.75
667
P
Ca
^4
Calculated P:Ca ratio (w/w)
0.70 Mn
^3
0.65
Fe
^2
0.60
Cu Zn
Data from thick particle Data from thin particle
0.55
^1 ^0 2
0.50
4
6 C
0.45
8
N O
10
12
P Ca
100
0.40 0.35 0.30 0
2
4
6
8
10
50
Assumed particle thickness (μm) Fig. 2. The calculated P:Ca ratio obtained from two experimental PIXE spectra from thin (lower line) and thick (upper line) hydroxy apatite particles plotted as a function of the assumed particle thickness. The calculations were carried out using GUPIX via the Dan32 interface. The horizontal line indicates the stoichiometric ratio calculated from the chemical formula.
0 1600
2000
2400
2800
3200
Fig. 3. PIXE and RBS spectra of a point selected from one of the particlerich aggregates shown in the PIXE maps in Fig. 1.
668
I. Gomez-Morilla et al. / Nucl. Instr. and Meth. in Phys. Res. B 249 (2006) 665–669 Table 1 Summary of P:Ca ratios (mean SD) in the standard hydroxy apatite particles (expected 0.46 w/w) and in naturally occurring mucosal calcium phosphate particles in the ileum of mice
Measured P:Ca ratio (w/w)
0.8 0.7 0.6
Sample
0.5
Standard Mice
0.4
N
0.46 (0.08) 0.78 (0.07)
8 26
N is the number of measurements. 0.3 0.2 0.1 0 0
2
4
6
8
10
12
14
RBS particle thickness (μm)
Fig. 4. The P:Ca ratio for eight hydroxy apatite particles of a range of diameters calculated using the RBS particle thickness and composition to determine the thick target PIXE correction. The horizontal line indicates the stoichiometric ratio.
form a block from which samples were prepared and analysed in exactly the same way as the tissue sections. Fig. 4 shows the measured P:Ca ratio for eight particles plotted as a function of their thickness determined from the RBS spectrum. The fact that there is no obvious dependence on particle thickness indicates that the RBS thickness measurement can be used with some confidence to correct the PIXE data (compare this with the graphs in Fig. 2). The remaining scatter of the points (relative standard deviation of 18%) is probably related to (i) inadequacies in other assumptions in the model and is an indication of the maximum limits of reliability of nuclear microscopy in these analyses and (ii) heterogeneity within the individual particles of HA. Nonetheless, the mean P:Ca ratio (0.46 w/w) is close to the expected value of 0.46. Fig. 5 and Table 1 summarise the results of the determination of P:Ca ratios for the CaP particles in the mice. A figure of 0.78 ± 0.07(w/w) was found for P:Ca ratios in Peyer’s patch microparticles of the ileal mucosa. These 1
0.8 P:Ca (w/w)
Hydroxy apatite Normal Ca and P diet
P:Ca ratio as w/w
results are preliminary but suggest a molar P:Ca ratio close to 1:1. Point analyses were performed also outside the particles in the surrounding tissue in order to determine the levels of calcium and phosphorus present naturally in the tissue. These were found to be two orders of magnitude less than those in the particles and the contribution of this to the error in the calculated ratios in the particles was considered not to be significant. Further studies will investigate this further including their chemical speciation and findings in other parts of the intestine, as well as the effects of different calcium-containing diets on these parameters. 4. Conclusion The results of this preliminary study confirm that nuclear microscopy can be a valuable tool for characterising the chemical composition of mineralised (i.e. calcium) particles embedded in tissue. The long penetration of MeV protons allows the entire thickness of the sample to be measured, which simplifies the localisation of the particles, while the use of PIXE and RBS simultaneously allows the PIXE data to be corrected for the varying thickness of the sample, enhancing the accuracy of the technique. Work is in progress with further mice and human samples. Acknowledgements The authors are grateful to the UK Engineering and Physical Sciences Research Council for supporting access to the Surrey Ion Beam Centre through a pump-priming grant and to the Sir Halley Stewart Trust for the support of Vinay Thoree. The authors also wish to thank Mr. Adrian Cansell and Mr. Mark Browton from IBC for their valuable help in the technical implementation of the measurements.
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References
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0.2
0
HA
Control
Mouse 1
Mouse 2
Gut Mucosal
Fig. 5. Measured P:Ca ratios (w/w) for calcium phosphate (CaP) particles either embedded in gelatine (hydroxy apatite (HA) control) or naturally present in the gut mucosa of two different mice. The triangles and circles in mouse 1 indicate measurements made on two different tissue sections.
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