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Experimental investigations into sample preparation of Alzheimer tissue specimens for nuclear microprobe analysis
186
Nuclear Instruments and Methods in Physics Research B54 (1991) 186-190 North-Holland
Experimental investigations into sample preparation specimens for nuclear microprobe analysis
of Alzheimer tissue
T. Pinheiro a,1, U.A.S. Tapper b, K. Sturesson ’ and A. Brun ’ a CEC-JRC, Central Bureau for Nuclear Measurements, Steenweg op Retie, B-2440 Geel,
Belgium b Department of Nuclear Physics, Lund Institute of Science and Technology, Siilvegatan 14, S-223 62 Lund, Sweden ‘Division of Neuropathology, Department of Pathology, Lund University Hospital, S-221 85 Lund, Sweden
Nuclear microprobe analysis was applied to the study of elemental distribution in brains sections of patients with a diagnosis of Alzheimer’s disease. Stained and nonstained cryosections were studied. The work carried out shows that serious elemental losses follow the sample staining procedure. Major losses occurred in a simple rinse of the tissue section, probably reducing most of the in-vivo gradients, which shows that generally very little information can be gained from stained sections. However, in many cases stained sections are compulsory because of the requirement to recognize the area which is to be studied. All the elemental maps obtained for the neurofibrillary deposits indicate a localized concentration for Si and probably also Al, associated with the senile plaque core. Neither of these elements were found in the staining solutions used. The validity of the results is discussed as well as the possible link of Al and/or Si in the development of Alzheimer’s disease.
1. Introduction To explore whether the inorganic content of cells correlates with specific pathogenesis (e.g. Alzheimer’s disease), it is advantageous to use an analytical technique that can probe for a large range of elements with high sensitivity and in tiny sample masses. The nuclear microprobe technique as well as proton-induced X-ray emission have been recognized so far as powerful and sensitive techniques for the study of elemental composition and regional distribution in biomedical specimens [1,2]. However, the identification of cellular and subcellular structures is in most cases practically impossible to achieve without specimen pre-treatment. To identify and characterize Alzheimer’s neurofibrillary plaques, stains have to be used [3]. Therefore contamination or elemental loss will occur [4,5]. However, when sampling Alzheimer specimens, post-mortem material will be the only tissue available which means that already some of the in-vivo gradients have vanished. In this work thin sections of human brain tissue originating from a patient with Alzheimer’s disease were stained for the identification of senile plaque B-amyloid protein. Nuclear microprobe analysis was applied to the analysis of senile plaques in stained cryosections. Comparisons with nonstained cryosections were made concerning elemental concentration determination. Rat
i On leave from LNETI, Sacavtm,
Portugal.
0168-583X/91/$03.50
Physics
Department,
ENlO,
P-2685
~ 0 1991 - Elsevier
Science
Publishers
brains with known calcium and potassium gradients were investigated in order to study eventual elemental redistribution due to the staining.
2. Experimental 2.1. Specimen preparation Brain tissue of patients with Alzheimer’s disease was removed by autopsy from hippocampus and amygdala regions. The brain tissue was immediately dissected, quickly frozen in isopentane, cooled in liquid nitrogen and stored at - 70 o C in polyethylene vessels. Rat brains of a male Wistar SPF strain were used as reference samples for possible staining-induced elemental redistribution. Epileptic seizures of 60 min duration were induced in the animals. Analysis of the element levels after such an experiment shows that there is a direct link between the elemental levels and the duration of the seizure. The brains were frozen in situ, removed and stored at - 70 o C as described elsewhere
t61.
Both tissues were sectioned in a cryomicrotome at - 20 o C with a stainless-steel knife. The specimens were glued on a cork disk in embedding medium (Tissue TEK, Ames Div. Miles). Sections of 10 to 14 urn thick were obtained. The sections were mounted on a microscopy copper grid for a better orientation during proton microprobe analysis. The grid was sandwiched between a polycarbonate KimfolTM foil stretched out
B.V. (North-Holland)
187
T. Pinheiro et al. / Sample preparation of Alzheimer tissue specimens
on plastic rings and a Formvar film (0.4% in dioxane) to avoid cracks in the sections. Some of the cryosections were stained prior to freeze-drying, others were freezedried without any further treatment. All tools and containers were of polyethylene. Samples were always handled in a laminar flow bench with the exception of the staining steps which were carried out in a chemical fume cupboard. 2.2. Staining procedure Cryosections were fixed in acetone vapour and incubated in a damp chamber at room temperature, with three different antisera raised against p-amylase peptide diluted in normal swine serum (NSS, Dako X901, 20% in a phosphate buffer solution at pH 7.0). The antisera were applied separately. After tipping each antiserum, the sections were washed in a phosphate buffer solution. The antibody bound complex was visualized using a dia~no-benzene (Sigma D-5637) solution containing hydrogen peroxide. Finally the sections were washed in double-distilled water and allowed to dry in a clean bench. This procedure was a modification from that of Beer [7]. 2.3. Nuclear
microprobe
analysis
The analyses were carried out at the microprobe PIXE setup of Lund University [S]. The microprobe analyses were performed with a 2.55 MeV proton beam, generated from an electrostatic tandem accelerator (Pelletron 3UDH). The proton beam was collimated and
focussed by a magnetic quadrupole triplet. The beam size was estimated to be approximately 7 X 7 pm’. The beam current was kept below 150 pA/pm2. Determination of areal mass density was carried out using a silicon surface barrier detector at an angle of 160”, relative to the incoming beam for the detection of backscattered protons from the sample [9]. The X-rays were detected in a Si(Li) detector (active area 30 mm2) placed at an angle of 135O relative to the beam direction. The X-ray spectra were evaluated with the aid of the pea-fitting program HEX, with matrix correction capabilities, as described elsewhere [lO,ll].
3. Results and discussion Spot analysis was performed for freeze-dried and stained cryosections. For both brain specimens analyzed (human and rat brain) large concentration variations were observed for all elements detected. Cerebral cortex and substantia nigra regions of rat brains were used in this study. The human brain tissue originated from amigdala region of a patient affected with Alzheimer’s disease. In the human stained cryosections losses higher than 60% occurred for all elements with respect to the unstained adjacent cryosection. Si as well as other trace elements, i.e. Ni, Zn, Br, and Rb, were encountered in unstained cryosections of brains of patients with Alzheimer’s disease. However, in adjacent cryosections that have been stained these element concentrations were below the minimum detection limit. On the other hand, in rat-brain stained cryosections a
1O3-
I,,,,,,,1 ,,rimr
T,,IIIII,II.,IIIII(,,III,..I,II,,11I,I,,,,,r,,,,,,,,,~,,,
100
200
300
400
CHANNEL
Fig. 1. PIXE spectrum
500
600
700
800
and buffers)
used.
$
0
NUMBER
of the stain solutions
(antisera
V. MEDICINE
T. Pinheiro et al. / Sample ptep~~ation of Aizheimer tissue specimens
Aluminum
Aluminum
- mass
normalized
(4
Silicon
Silicon
- mass
normalized
Fig. 2. Elemental maps of aluminum (a) and silicon (b) from the analysis of a P-amyloid neurofibriilary deposit (central region of the scan), in a stained cryosection originating from Alzheimer’s disease affected brain tissue. The data for both elements normalized to area1 density taken from the RBS spectra are also shown. The square (lower right) shows the copper grid which was used as a support for the section.
T. Pinheiro et al. / Sample preparation
of Alzheimer
189
tissue specimens
found for Si and probably Al is circumscribed to what is thought to be the senile plaque core (fig. 2). Area1 mass normalization (carbon and oxygen peak area taken from RBS spectra) of the elemental maps is used to correct the differences in thickness of the cryosection and as a consequence apparent higher elemental concentration. The mass normalization confirms the existence of the Al and Si enriched structure (fig. 2). PIXE spectral analysis of selected pixel groups (20 to 50 pixels) corresponding to this core region (see figs. 2 and 3) showed the presence of Si in a concentration range of 150 to 600 ppm. Also an evident focal deposition of Al was detected after scanning and mass normalization. Spectra analysis of the pixels corresponding to the plaque core reveal an Al concentration slightly above the detection limit. The high detection limit for Al is due to the low system transmission for the Al X-ray energy. Therefore, Al concentrations will not be quoted. PIXE sum spectra of the entire scanning area of 64 x 64 pixels corresponding to the B-amyloid deposit area, did not reveal Si OT Al. This may indicate that both elements exist as a focal deposit rather than a homogeneous dispersion in the tissue section. However, Si was detected in the human brain tissue, as discussed above. After staining, a major percentage of the elements present is lost, as was observed in the tissue analyzed. Therefore it could be assumed that the related core silicon is more strongly bound to the amyloid neurofibrillary deposit and some still remains after staining.
general loss was not always observed. Some of the concentrations increased in relation to the correspondent region of unstained cryosections. The results clearly reveal unpredictable elemental changes, occurring during staining of biological materials, as was stressed by several authors [5,12-141. Nevertheless, staining techniques allow an histological confirmation of the pathology. Except for all the artifacts and the alteration of elemental concentrations found, the staining procedure used is rather convenient. The serum solutions employed were found to be relatively clean. Nine elements were detected in a macroPIXE analysis of the solutions used in the staining (i.e. P, S, Cl, K, Ca, Fe, Cu. Zn and Br). The analysis was carried out on a much higher solution concentration and amount than that required for the staining of a single tissue section (fig. 1). Typical neurofibrillary deposits with a core and dense senile plaques of stained cryosections were analyzed by the microprobe. Elemental maps for Al, Si and S, resulting from the scanning analysis of one senile plaque type containing a central core is presented in fig. 2. Other elements such as Cl, K, Ca, Mn, Fe and Zn were also detected. The copper peak was affected by the strong signal from the copper grid used as support and orientation for the cryosection. so any data concerning this element will be omitted. The distribution of S, Ca and K seems to follow the same pattern and is spread over a region which should correspond to the senile plaque amyloid deposit. On the other hand the focal deposit
_ CHANNEL
Fig. 3. PIXE spectrum of 21 pixels (aproximately
10 X 10 pm’)
-
NUMBER
corresponding
to the senile plaque
core region.
Total accumulated
charge of 0.19 PC. V. MEDICINE
190
T. Pinheiro et al. / Sample preparaiion
The plaque region was also found to be P-, S- and K-enriched after mass normalization. The region that corresponds to what is thought to be the plaque core, presented an enhanced concentration level for the majority of the elements detected. The analysis of dense neurofibrillary amyloid deposits (without any core) showed a more homogeneous distribution for all elements, although a hot spot for silicon was found in a central region of the amyloid deposit. However, a big variability was found for the concentration of those three elements in the different plaques analyzed, e.g. for S from 200 to 1000 ppm and for P from 150 to 800 ppm. If elemental maps are normalized to sulphur content all the focal deposits observed vanish, as was reported earlier [15]. However, it seems that the concentration variations for these elements (P, S and K) are related to a local increase in protein content due to bound antibody complex formation and contaminations resulting from staining.
4. Conclusions Although there are some signs of a possible link of aluminium and silicon deposits in the cores of senile plaques we should be aware of the unpredictable changes induced by staining procedures that could lead to artifacts and a misunderstanding of results. More investigations have to be done to confirm whether Al and/or Si and other elements as well are connected with Alzheimer’s disease neurofibrillary deposits. The results of this study indicate that stained sections will not be able to give a certain answer. Two approaches to the problem could be: (1) the staining technique proposed in ref. [15], or (2) to explore the possibility to use the scanning electron microscope for
of Alzheimer
tissue specimens
identification of the senile plaques in their location on the copper grid. Finally, the results presented in this paper are based on the sampling of only one brain, therefore the analysis of more material will be necessary as well.
References [l] R.D. Vis, in: The Proton Microprobe: Applications in the Biomedical Field (CRC Press, New York, 1986). [2] F. Watt and G.W. Grime, in: Principles and Applications of High Energy Ion Microbeams, eds. F. Watt and G.W. Grime (Adam Hilger, 1987). [3] C.L. Joachim, H. Mori and D.J. Selkoe, Nature 341 (1989) 226. [4] G.J.F. Legge and P. Mazzolini, Nucl. Instr. and Meth. 168 (1980) 563. [5] G.J.F. Legge, P. Mazzolini, A.F. Rocziniok and P.M. O’Brien, Nucl. Instr. and Meth. 197 (1982) 191. [6] V. Ponttn, R. Ratcheson, L.G. Salford and B.K. Siesjii, J. Neurochem. 21 (1973) 1127. [7] J. Beer, private communication, 1988. [8] U.A.S. Tapper, R. Hellborg, M.B. Hult, N.P.-0. Larsson, N.E.G. Lovestam, K.G. Malmqvist, J. Pallon and K. Themner, Nucl. Instr. and Meth. B49 (1990) 425. [9] K. Themner and K.G. Malmqvist, Nucl. Instr. and Meth. B15 (1986) 404. [lo] L.-E. Carlsson, K.G. Malmqvist, G.I. Johansson and R.K. Akselsson, Nucl. Instr. and Meth. 181 (1981) 179. [ll] G.I. Johansson, X-ray Spectr. 11 (1982) 194. [12] G.M. Roomans, J. Wroblewski and R. Wroblewski, Scanning Microsc. 2 (1988) 937. [13] R. Wroblewski, J. Wroblewski and G.M. Roomans, Histochemistry 81 (1984) 469. [14] K. Zierold, Scanning Electron Microsc. 3 (1982) 1205. [15] N.P.-0. Larsson, U.A.S Tapper, K. Sturesson, R. Odselius and A. Bnm, Nucl. Instr. and Meth. B49 (1990) 472.
Nuclear Instruments and Methods in Physics Research B54 (1991) 186-190 North-Holland
Experimental investigations into sample preparation specimens for nuclear microprobe analysis
of Alzheimer tissue
T. Pinheiro a,1, U.A.S. Tapper b, K. Sturesson ’ and A. Brun ’ a CEC-JRC, Central Bureau for Nuclear Measurements, Steenweg op Retie, B-2440 Geel,
Belgium b Department of Nuclear Physics, Lund Institute of Science and Technology, Siilvegatan 14, S-223 62 Lund, Sweden ‘Division of Neuropathology, Department of Pathology, Lund University Hospital, S-221 85 Lund, Sweden
Nuclear microprobe analysis was applied to the study of elemental distribution in brains sections of patients with a diagnosis of Alzheimer’s disease. Stained and nonstained cryosections were studied. The work carried out shows that serious elemental losses follow the sample staining procedure. Major losses occurred in a simple rinse of the tissue section, probably reducing most of the in-vivo gradients, which shows that generally very little information can be gained from stained sections. However, in many cases stained sections are compulsory because of the requirement to recognize the area which is to be studied. All the elemental maps obtained for the neurofibrillary deposits indicate a localized concentration for Si and probably also Al, associated with the senile plaque core. Neither of these elements were found in the staining solutions used. The validity of the results is discussed as well as the possible link of Al and/or Si in the development of Alzheimer’s disease.
1. Introduction To explore whether the inorganic content of cells correlates with specific pathogenesis (e.g. Alzheimer’s disease), it is advantageous to use an analytical technique that can probe for a large range of elements with high sensitivity and in tiny sample masses. The nuclear microprobe technique as well as proton-induced X-ray emission have been recognized so far as powerful and sensitive techniques for the study of elemental composition and regional distribution in biomedical specimens [1,2]. However, the identification of cellular and subcellular structures is in most cases practically impossible to achieve without specimen pre-treatment. To identify and characterize Alzheimer’s neurofibrillary plaques, stains have to be used [3]. Therefore contamination or elemental loss will occur [4,5]. However, when sampling Alzheimer specimens, post-mortem material will be the only tissue available which means that already some of the in-vivo gradients have vanished. In this work thin sections of human brain tissue originating from a patient with Alzheimer’s disease were stained for the identification of senile plaque B-amyloid protein. Nuclear microprobe analysis was applied to the analysis of senile plaques in stained cryosections. Comparisons with nonstained cryosections were made concerning elemental concentration determination. Rat
i On leave from LNETI, Sacavtm,
Portugal.
0168-583X/91/$03.50
Physics
Department,
ENlO,
P-2685
~ 0 1991 - Elsevier
Science
Publishers
brains with known calcium and potassium gradients were investigated in order to study eventual elemental redistribution due to the staining.
2. Experimental 2.1. Specimen preparation Brain tissue of patients with Alzheimer’s disease was removed by autopsy from hippocampus and amygdala regions. The brain tissue was immediately dissected, quickly frozen in isopentane, cooled in liquid nitrogen and stored at - 70 o C in polyethylene vessels. Rat brains of a male Wistar SPF strain were used as reference samples for possible staining-induced elemental redistribution. Epileptic seizures of 60 min duration were induced in the animals. Analysis of the element levels after such an experiment shows that there is a direct link between the elemental levels and the duration of the seizure. The brains were frozen in situ, removed and stored at - 70 o C as described elsewhere
t61.
Both tissues were sectioned in a cryomicrotome at - 20 o C with a stainless-steel knife. The specimens were glued on a cork disk in embedding medium (Tissue TEK, Ames Div. Miles). Sections of 10 to 14 urn thick were obtained. The sections were mounted on a microscopy copper grid for a better orientation during proton microprobe analysis. The grid was sandwiched between a polycarbonate KimfolTM foil stretched out
B.V. (North-Holland)
187
T. Pinheiro et al. / Sample preparation of Alzheimer tissue specimens
on plastic rings and a Formvar film (0.4% in dioxane) to avoid cracks in the sections. Some of the cryosections were stained prior to freeze-drying, others were freezedried without any further treatment. All tools and containers were of polyethylene. Samples were always handled in a laminar flow bench with the exception of the staining steps which were carried out in a chemical fume cupboard. 2.2. Staining procedure Cryosections were fixed in acetone vapour and incubated in a damp chamber at room temperature, with three different antisera raised against p-amylase peptide diluted in normal swine serum (NSS, Dako X901, 20% in a phosphate buffer solution at pH 7.0). The antisera were applied separately. After tipping each antiserum, the sections were washed in a phosphate buffer solution. The antibody bound complex was visualized using a dia~no-benzene (Sigma D-5637) solution containing hydrogen peroxide. Finally the sections were washed in double-distilled water and allowed to dry in a clean bench. This procedure was a modification from that of Beer [7]. 2.3. Nuclear
microprobe
analysis
The analyses were carried out at the microprobe PIXE setup of Lund University [S]. The microprobe analyses were performed with a 2.55 MeV proton beam, generated from an electrostatic tandem accelerator (Pelletron 3UDH). The proton beam was collimated and
focussed by a magnetic quadrupole triplet. The beam size was estimated to be approximately 7 X 7 pm’. The beam current was kept below 150 pA/pm2. Determination of areal mass density was carried out using a silicon surface barrier detector at an angle of 160”, relative to the incoming beam for the detection of backscattered protons from the sample [9]. The X-rays were detected in a Si(Li) detector (active area 30 mm2) placed at an angle of 135O relative to the beam direction. The X-ray spectra were evaluated with the aid of the pea-fitting program HEX, with matrix correction capabilities, as described elsewhere [lO,ll].
3. Results and discussion Spot analysis was performed for freeze-dried and stained cryosections. For both brain specimens analyzed (human and rat brain) large concentration variations were observed for all elements detected. Cerebral cortex and substantia nigra regions of rat brains were used in this study. The human brain tissue originated from amigdala region of a patient affected with Alzheimer’s disease. In the human stained cryosections losses higher than 60% occurred for all elements with respect to the unstained adjacent cryosection. Si as well as other trace elements, i.e. Ni, Zn, Br, and Rb, were encountered in unstained cryosections of brains of patients with Alzheimer’s disease. However, in adjacent cryosections that have been stained these element concentrations were below the minimum detection limit. On the other hand, in rat-brain stained cryosections a
1O3-
I,,,,,,,1 ,,rimr
T,,IIIII,II.,IIIII(,,III,..I,II,,11I,I,,,,,r,,,,,,,,,~,,,
100
200
300
400
CHANNEL
Fig. 1. PIXE spectrum
500
600
700
800
and buffers)
used.
$
0
NUMBER
of the stain solutions
(antisera
V. MEDICINE
T. Pinheiro et al. / Sample ptep~~ation of Aizheimer tissue specimens
Aluminum
Aluminum
- mass
normalized
(4
Silicon
Silicon
- mass
normalized
Fig. 2. Elemental maps of aluminum (a) and silicon (b) from the analysis of a P-amyloid neurofibriilary deposit (central region of the scan), in a stained cryosection originating from Alzheimer’s disease affected brain tissue. The data for both elements normalized to area1 density taken from the RBS spectra are also shown. The square (lower right) shows the copper grid which was used as a support for the section.
T. Pinheiro et al. / Sample preparation
of Alzheimer
189
tissue specimens
found for Si and probably Al is circumscribed to what is thought to be the senile plaque core (fig. 2). Area1 mass normalization (carbon and oxygen peak area taken from RBS spectra) of the elemental maps is used to correct the differences in thickness of the cryosection and as a consequence apparent higher elemental concentration. The mass normalization confirms the existence of the Al and Si enriched structure (fig. 2). PIXE spectral analysis of selected pixel groups (20 to 50 pixels) corresponding to this core region (see figs. 2 and 3) showed the presence of Si in a concentration range of 150 to 600 ppm. Also an evident focal deposition of Al was detected after scanning and mass normalization. Spectra analysis of the pixels corresponding to the plaque core reveal an Al concentration slightly above the detection limit. The high detection limit for Al is due to the low system transmission for the Al X-ray energy. Therefore, Al concentrations will not be quoted. PIXE sum spectra of the entire scanning area of 64 x 64 pixels corresponding to the B-amyloid deposit area, did not reveal Si OT Al. This may indicate that both elements exist as a focal deposit rather than a homogeneous dispersion in the tissue section. However, Si was detected in the human brain tissue, as discussed above. After staining, a major percentage of the elements present is lost, as was observed in the tissue analyzed. Therefore it could be assumed that the related core silicon is more strongly bound to the amyloid neurofibrillary deposit and some still remains after staining.
general loss was not always observed. Some of the concentrations increased in relation to the correspondent region of unstained cryosections. The results clearly reveal unpredictable elemental changes, occurring during staining of biological materials, as was stressed by several authors [5,12-141. Nevertheless, staining techniques allow an histological confirmation of the pathology. Except for all the artifacts and the alteration of elemental concentrations found, the staining procedure used is rather convenient. The serum solutions employed were found to be relatively clean. Nine elements were detected in a macroPIXE analysis of the solutions used in the staining (i.e. P, S, Cl, K, Ca, Fe, Cu. Zn and Br). The analysis was carried out on a much higher solution concentration and amount than that required for the staining of a single tissue section (fig. 1). Typical neurofibrillary deposits with a core and dense senile plaques of stained cryosections were analyzed by the microprobe. Elemental maps for Al, Si and S, resulting from the scanning analysis of one senile plaque type containing a central core is presented in fig. 2. Other elements such as Cl, K, Ca, Mn, Fe and Zn were also detected. The copper peak was affected by the strong signal from the copper grid used as support and orientation for the cryosection. so any data concerning this element will be omitted. The distribution of S, Ca and K seems to follow the same pattern and is spread over a region which should correspond to the senile plaque amyloid deposit. On the other hand the focal deposit
_ CHANNEL
Fig. 3. PIXE spectrum of 21 pixels (aproximately
10 X 10 pm’)
-
NUMBER
corresponding
to the senile plaque
core region.
Total accumulated
charge of 0.19 PC. V. MEDICINE
190
T. Pinheiro et al. / Sample preparaiion
The plaque region was also found to be P-, S- and K-enriched after mass normalization. The region that corresponds to what is thought to be the plaque core, presented an enhanced concentration level for the majority of the elements detected. The analysis of dense neurofibrillary amyloid deposits (without any core) showed a more homogeneous distribution for all elements, although a hot spot for silicon was found in a central region of the amyloid deposit. However, a big variability was found for the concentration of those three elements in the different plaques analyzed, e.g. for S from 200 to 1000 ppm and for P from 150 to 800 ppm. If elemental maps are normalized to sulphur content all the focal deposits observed vanish, as was reported earlier [15]. However, it seems that the concentration variations for these elements (P, S and K) are related to a local increase in protein content due to bound antibody complex formation and contaminations resulting from staining.
4. Conclusions Although there are some signs of a possible link of aluminium and silicon deposits in the cores of senile plaques we should be aware of the unpredictable changes induced by staining procedures that could lead to artifacts and a misunderstanding of results. More investigations have to be done to confirm whether Al and/or Si and other elements as well are connected with Alzheimer’s disease neurofibrillary deposits. The results of this study indicate that stained sections will not be able to give a certain answer. Two approaches to the problem could be: (1) the staining technique proposed in ref. [15], or (2) to explore the possibility to use the scanning electron microscope for
of Alzheimer
tissue specimens
identification of the senile plaques in their location on the copper grid. Finally, the results presented in this paper are based on the sampling of only one brain, therefore the analysis of more material will be necessary as well.
References [l] R.D. Vis, in: The Proton Microprobe: Applications in the Biomedical Field (CRC Press, New York, 1986). [2] F. Watt and G.W. Grime, in: Principles and Applications of High Energy Ion Microbeams, eds. F. Watt and G.W. Grime (Adam Hilger, 1987). [3] C.L. Joachim, H. Mori and D.J. Selkoe, Nature 341 (1989) 226. [4] G.J.F. Legge and P. Mazzolini, Nucl. Instr. and Meth. 168 (1980) 563. [5] G.J.F. Legge, P. Mazzolini, A.F. Rocziniok and P.M. O’Brien, Nucl. Instr. and Meth. 197 (1982) 191. [6] V. Ponttn, R. Ratcheson, L.G. Salford and B.K. Siesjii, J. Neurochem. 21 (1973) 1127. [7] J. Beer, private communication, 1988. [8] U.A.S. Tapper, R. Hellborg, M.B. Hult, N.P.-0. Larsson, N.E.G. Lovestam, K.G. Malmqvist, J. Pallon and K. Themner, Nucl. Instr. and Meth. B49 (1990) 425. [9] K. Themner and K.G. Malmqvist, Nucl. Instr. and Meth. B15 (1986) 404. [lo] L.-E. Carlsson, K.G. Malmqvist, G.I. Johansson and R.K. Akselsson, Nucl. Instr. and Meth. 181 (1981) 179. [ll] G.I. Johansson, X-ray Spectr. 11 (1982) 194. [12] G.M. Roomans, J. Wroblewski and R. Wroblewski, Scanning Microsc. 2 (1988) 937. [13] R. Wroblewski, J. Wroblewski and G.M. Roomans, Histochemistry 81 (1984) 469. [14] K. Zierold, Scanning Electron Microsc. 3 (1982) 1205. [15] N.P.-0. Larsson, U.A.S Tapper, K. Sturesson, R. Odselius and A. Bnm, Nucl. Instr. and Meth. B49 (1990) 472.