Nuclear microprobe analysis of brain tissue: Quantitative aspects and concentration variability of minor and trace elements

Nuclear microprobe analysis of brain tissue: Quantitative aspects and concentration variability of minor and trace elements

Beam Interactions with Materials & Atoms Nuclear Instruments ELSEVIER and Methods in Physics Research B 130 (1997) 444-448 Nuclear microprobe analy...

607KB Sizes 0 Downloads 48 Views

Beam Interactions with Materials & Atoms Nuclear Instruments

ELSEVIER

and Methods in Physics Research B 130 (1997) 444-448

Nuclear microprobe analysis of brain tissue: Quantitative aspects and concentration va~ability of minor and trace elements S. Van Lierde a, W. Maenhaut

a *, J. De Reuck b, R.D. Vis ’

a Insiitute for Nuclear Sciences, Uniuersiteit Gent, Proeftuinstraat 86, B-9000 Gent, Belgium ’ Kliniek uoor Neurologie, Uniuersitair Ziekenhuis, De Pintelaan 185, B-9000 Gent, Belgium ’ Faculty of Physics and Astronomy, Vrije Unioersiteit, De Boelelaan IORI, 1081 HV Amsterdam, The Netherlands

Abstract Specimen mass loss, major element loss, and alterations in minor and trace elements during nuclear microprobe ~mbardment were studied for various types of human brain tissues. The results were quite simitar to those of an earlier study on bovine liver (Van Lierde et al., 1995). Special efforts were made to obtain accurate micro-PIXE concentration data (expressed in @g/g dry weight). This involved determination of the local specimen mass thickness. From a comparison of

the micro-PIXE data with the results of macro-PIXE it was concluded that micro-PIXE data with an accuracy of better than 20% can be obtained. The variability in elemental concentrations over various types of brain tissue and within a single tissue type were examined. From a scan over the substantia nigra, it appeared that Fe exhibits a complex concentration pattern in this tissue type. 0 1997 Elsevier Science B.V.

1. Introduction In an earlier study El], we investigated some analytical-methodological aspects of the nuclear microprobe analysis of soft biological tissues. Specifically, we examined the specimen mass loss, major element loss, and alterations in minor and trace elements for bovine liver sections of various thicknesses. In the present study, we extended on that earlier work by performing similar measurements on several types of human brain tissues. Furthermore, special efforts were devoted to obtaining accurate concentration data (in p,g/g dry weight) for the

” Corresponding author. Fax: + 32-9-264-6699; email: [email protected]

minor and trace elements in tissue sections on a micrometer scale. This involved the dete~ination of the local irradiated specimen mass thickness from the measurement of the energy loss of the transmitted beam, calibration of the set-up with thin film standards, and corrections for PIXE matrix effects. The accuracy of the concentration data obtained was assessed through comparisons with different types of macro-PIXE analyses, including analyses whereby an internal standard was added. The concentration variability of minor and trace elements over the various types of brain tissue and within each single tissue type were assessed. For some tissue samples, such as the substantia nigra, scans were made along a line covering the various subregions of this tissue. Selected results of the work are presented and briefly discussed.

0168-583X/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO168-583X(97)00237-1

.S. Van tierde

et at. / Nuci. Ins@. und ~e!~. in Phys. Res. B I30 (19971444-448

2. Experimental 2.1. Sample preparation The brain tissues examined were gray matter, white matter, nucleus caudatus, putamen, and substantia nigra. They were all taken post mortem from the right cerebral hemisphere of an individual who had no neurological disorders. For gray matter, white matter, and the three subregions of the substantia nigra (i.e., the zona inserta (lateralis), the zona compacta ventralis, and the zona compacta dorsalis), subsamples for “bulk” macro-PIXE analysis were taken from the fresh brain within 24 h after death. Adjacent subsamples of gray matter, white matter, and substantia nigra (without dividing the latter in its three subregions) and samples of the nucleus caudatus and putamen were frozen by submerging in isopentane, which was cooled in liquid nitrogen, and they were subsequently stored in a freezer (below - 20°C) until cryosectioning. Sections of four different thicknesses (i.e., 10, 20, 40, and 60 pm) were prepared with a cryomicrotome (at about - 30°C) and transferred onto 1.5 p,rn thick KIMFOL polycarbonate films, which were mounted on target frames. The lateral dimension of the roughly square microtome sections was about 5 mm. The targets were kept frozen during transfer to the freeze-drier and finally freeze-dried. The mass thickness of the freeze-dried sections ranged from about 0.2 to 2 mg/cm2. These targets were used for ex~inations with macro-PIXE and with the nuclear microprobe. The subsamples for “bulk” macro-PIXE were subjected to acid digestion in a closed Teflon vessel and subsequent target preparation, as described by Duflou et al. [2]. The procedure included the addition of yttrium as internal standard element. For the subsamples of gray and white matter a powdering by brittle fracture [3] was performed prior to the acid digestion, and two sub-subsamples were then processed for each of these two tissue types. 2.2. Macro-PIXE

analyses

All macro-PIXE analyses were performed with the PIXE set-up of the University of Gent, using a 2.4 MeV proton beam. In the measurements for “bulk” macro-PIXE (i.e., of the targets obtained after the acid digestion) a 660 p,rn thick Mylar

445

absorber was placed in front of the Si(Li) detector, and the elements from K upward were determined. Details on the analytical procedures can be found in Maenhaut et al. [4]. For the gray and white brain tissue, we also subjected targets of freeze-dried cryosections to macro-PIXE. Two targets of each thickness were irradiated, the beam diameter at the target was 2 mm, and two X-ray measurements were always performed, first with a “funny” filter in front of the Si(Li), and subsequently with the 660 @rn Mylar absorber. The net peak areas for the analyte elements were obtained by means of the AXIL-84 fitting program [5] and matrix correction factors were derived as described by Maenhaut [6]. To convert the elemental data from pg/cm’ into kg/g dry weight, three different approaches were applied: (a> using the nominal specimen mass thickness, as calculated from the microtom~ cut thickness, tissue density, and mass reduction factor during freeze-drying; (b) using the experimental mass thickness, as derived from nuclear microprobe measurements (see below) on tissue sections of the same cut thickness; and (c) using the K data from the “bulk” macro-PIXE as reference concentrations (thus, using K as some kind of internal standard). 2.3. Nuclear microprobe

irradiations

The microprobe bomb~dments were done with the Amsterdam nuclear microprobe [7], using a 2.5 MeV proton beam. The beam size on the specimen was always 4 X 20 p.m*, and the beam current was typically 200 to 400 pA. The measurements were performed in a similar way as described in Ref. [l]. A Si surface barrier detector (SB2) measured the protons that were backscattered from a thin gold film downstream of the specimen; it was used to monitor the beam fluence (proton charge) and the specimen mass thickness (the latter from the position of the Au peak in the SB2 spectrum). The backscattered protons and X-rays emerging from the specimen were measured with SBl and Si(Li) detectors, respectively. A “funny” filter, consisting of a 350 p,m thick Mylar absorber with a 6% hole, was placed in front of the Si(Li). For measurements of short duration (up to 20 min), integrated (“sum”) data collection was employed. The longer measurements (from

120 to 240 mm) were always performed with eventby-event (list-mode) data collection. Most measurements were done with the beam kept stationary on the specimen (spot mode), some were made with the 4 km wide beam being swept back and forth over a 100 pm width of the specimen at a frequency of 0.6 Hz (sweep mode), and in a few cases, the beam was kept stationary, but the specimen was mechanically advanced at a rate of 80 pm/s (scan mode). The calibration of SB2 for specimen mass thickness was achieved by bombarding Kapton polyimide foils for which the mass thickness was determined by weighing, The PIXE calibration factors for the Si(Li) detector were determined from measurements on US National Institute of Standards and Technology (NIST) thin glass film standards (SRMS 1832 and 1833) and on thin Micromatter standards [S]. 2.4. nuclear microprobe data sorting and spectrum processing

The list-mode files were sorted into bins (intervals), sub-bins, and sub-sub-bins, as described in [I]. The very first sub-sub-bin, with the signals from the first min of bombardment, served as reference in examining changes with bombarding time. The SBl spectra of the various bins were used to determine the aheration in the ratio [ Xl/S, (with [Xl surface concentration and S, surface backscattering energy loss factor) for the major elements C, N and 0 as function of bombardment time [I]. The PIXE spectra from the “sum” measurements and from the sorted list files were processed in the same way as for the macro-PIXE on the freeze-dried sections. Concentrations in Fg/g dry weight were obtained by using experimental mass thicknesses, as derived from SB2. For the list-mode files, we afways used the initial mass thickness (i.e., that obtained from the very first sub-sub-bin}. 3. Resufts and discussion 3. f. ~~mpuris~n of exper~m~nta~~ydetermined and

nominal mass thicknesses fur the freeze-dried brain tissue sections The experimental mass thicknesses of the freezedried sections, as derived from the nuclear micro-

Table 1 Ratio of ex~~~~ent~ly determined to nommal mass thickness for freeze-dried sections of gray and white brain tissue a Microtome cut thickness (pm)

Gray matter mean *s

White matter

10

1.99_+0.23

20 40 60

1.54~0.1s 0.94F0.13 0.87CO.12

1.60~0.16 1.26rtO.12 0.92+0.11 0.87 f 0.02

mean +s

’ Data listed are average ratios and associated standard deviations, as derived from measurements at up to 6 different positions for one section of each cut thickness. For list-mode data acquisitions, we retained as experimental mass thickness that from the very first sub-sub-bin (time interval).

probe measurements, deviated substantially from the nominal values, especially for the two lowest microtome cut thicknesses (IO and 20 pm). In these cases, and for all tissue types, the experimental thicknesses were always larger than the nominal data (up to a factor of 2 or more for 10 pm>. Better agreement was noted for the sections of 40 and 60 pm, but the experimental data were here always lower than the nominal values. Table I shows the comparison for sections of gray and white matter. The macro-PIXE data for the sections confirmed these observations. The concentrations obtained when using the nominal mass thicknesses decreased with increasing mass thickness, but this trend vanished when replacing the nominaf thicknesses by experimental ones. Clearly, one shouid not rely on notninal thicknesses if one wants to obtain accurate concentration data. 3.2. macro-PIXE results for gray and white brain matter and for the three subregions of the substantia nigra In the “bulk” macro-PIXE analysis, only elements from K upward are determined. Fu~he~ore, Br is lost during the acid digestion. For gray and white matter, we therefore extended the results from the “bulk” analysis with concentration data obtained by macro-PIXE on the freeze-dried sections, using K as reference element (approach (c) in Section 2.2). The combined data sets are presented in Table 2. The results are quite similar to the average values (based on 12 normal brains) reported by Duflou et al. [9]. It is worth noting, though, that

S. Van Lierde et al./Nucl.

Table 2 Concentrations (in kg/g white brain matter a

Instr. and Meth. in Phys. Rex B 130 (1997) 444-448

dry weight) for 12 elements in gny

Element

Gray matter mean f s

White matter mean k s

P S Cl K Ca Mn Fe CU Zn Se Br Rb

15500+800 4600 f 200 12700+300 17200+600 38Ok30 1.24 + 0.05 250 rt 30 22+1 78+4 0.75 f 0.03 6.5* 1.4 19.1 *0.6

18800&700 3600 + 600 4500 f 400 10000*400 147520 1.09 + 0.08 167k7 8.4 f 0.5 29+1 0.39*0.14 3.65 1.8 11.6+0.4

and

a The data were obtained by “bulk” macro-PIXE (using acid digestion), except for P, S, Cl, Ca and Br. The latter elements were determined by macro-PIXE on freeze-dried sections (see text). The “bulk” macro-PIXE data are averages and standard deviations from analyses on two subsamples (with 4 targets analyzed for each digest solution); the other macro-PIXE data are from measurements on 8 freeze-dried sections (of 4 different thicknesses).

these authors presented data for only 8 of the 12 elements of Table 2. For the three subregions of the substantia nigra, only “bulk” macro-PIXE analyses were performed. Overall, the results were consistent with the average data for the whole substantia nigra reported by Duflou et al. [9]. However, our analyses indicated that there are substantial differences in the concentration data of the three subregions, with the zona compacta ventralis and the zona compacta dorsalis exhibiting higher levels than the zona inserta (lateralis). Whereas the difference was limited to 1530% for K and Rb, it was about a factor of 2 for Zn and Se, and a factor 2-3 for Cu. The greatest difference was observed for the element Fe. In the zona inserta, its concentration (on a dry weight basis) was 168 kg/g, in the zona compacta ventralis 970 pg/g, and in the zona compacta dorsalis 510 yg/g. 3.3. Alterations during nuclear microprobe bombardment: Specimen mass loss, changes of matrix, minor and trace elements The mass loss with bombardment time was fairly similar for the various brain tissue sections and it

447

was also similar to that observed in our study on bovine liver sections [l]. Typically, an exponential decrease was observed, although, occasionally, there was an increase in mass thickness during the second half of the 2- to 4-hour bombardment after the initial gradual decrease. The percentage mass loss did not seem to depend much on the section thickness and there was also little difference between the results from the “spot” and “sweep” irradiation modes (For the “scan” mode, the alterations during bombardment were not studied). On average, after 100 min of bombardment time, the specimen mass thickness was reduced to about 85% of its original value. The alterations with bombardment time for the matrix elements (i.e., C and 0) and the minor and trace elements (P, S, Cl, K, Fe, Zn) were overall similar to those observed in the study on the liver sections [l]. The C mass thickness remained essentially constant, but there was a significant loss of 0 with time. For the minor and trace elements, there was a clear tendency for increase in mass thickness with bombardment time, with typically 10 to 20% higher values after 100 min than in the first few minutes. This result is most likely due to local shrinkage of the irradiated specimen area [l]. In the gray matter sections, S exhibited a similar behavior as the other minor and trace elements, but in the sections of white matter and the other brain tissues examined, the trend line with time for S fell systematically below the trend lines of the other elements. This suggests that S is partially lost during the bombardment of these tissue types. A similar loss was noted for bovine liver sections [l]. 3.4. Comparison of micro-PIXE elemental concentration data

and macro-PIXE

Table 3 gives a comparison of micro-PIXE elemental concentration data with results from macroPIXE for gray and white matter brain tissue. The comparison is made in terms of ratios to the macroPIXE results of Table 2, and the data listed are mean ratios and associated standard deviations from micro-PIXE measurements on specimens of all 4 thicknesses (up to 18 measurements per tissue type). The mean ratios are in general fairly close to 1 and the associated standard deviations are between 10 and 20% for the minor elements P, S, Cl and K.

IX. MEDICINE

S. Van L&de

448

et al. /NW.?.

Table 3 Concentration ratio micro-PIXE/macro-PIXE gray and white brain matter a

hstr.

for 9 elements

Element

GYav matter mean * s

White matter mean &s

P s CI K Ca Fe

1.06f0.13 1.04f0.17 1.11 kO.19 1.1610.16 1.51 f0.46 0.83f0.21 1.24+ 0.39 1.15_to.27 1.21 rto.41

0.79+0.13 1.10*0.19 1.00~0.18 0.89f0.14 o.?z4+0.31 0.83 * 0.37 1.06f0.43 0.8650.17 1.21* I.03

CU ZU

Rb

und Meth. in Ph~s. Rex 3 130

a The macro-PIXE data of Table 2 were used in making Data listed are average ratios and associated standard as derived from 12 micro-PIXE measurements for gray I8 for white matter (sections of all 4 thicknesses were

in

the ratios. deviations, matter and analyzed).

When averaging the mean ratios over the 9 elements for each tissue type, the overall average ratios are 1.15 + 0.18 for gray matter and 0.95 + 0.14 for white matter. It may therefore be concluded that the approach used in this work allows one to produce micro-PIXE data with an accuracy of better than 20% for soft biological tissues. 100

-

i

ci

P....*

IS

..~..__._-..._. ___..__._.._._~

._._____ ._.II-+-

I I9971 &W&KS

3.5. Concentration variability ojf minor and trace elements in the substantia nigra Fig. 1 presents the micro-PIXE results from a 5.2 mm linear scan over a substantia nigra section. The scan was made along a line, which first covered the zona inserta (up to around pixel 30) and then continued over the border area between the zona compacta ventralis and the zona compacta dorsalis. The alterations observed for K and Fe are in qualitative agreement with the macro-PIXE results of the subregions, as reported in Section 3.2. However, Fig. 1 clearly shows that the concentration pattern for Fe is much more complex than was deduced from the macro-PIXE analyses.

S.V.L. and W.M. acknowledge support from the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek and the Instituut voor Wetenschappelijk and Technologisch Onderzoek.

References [l] S. Van Lierde, W. Maenhaut,

J. De Reuck, RD. Vis, Nucl. Instr. and Meth. B 104 (1995) 328. (21 H. Duflou, W. Maenhaut, J. De Reuck, Biol. Trace Elem. Res. 13 (1988) 1. 131 W. Maenhaut, L. De Reu, J. Vandenhaute, Nucl. Instr. and Meth. B 3 (1984) 135. [4] W. Maenhaut, J. Vandenhaute, H. Duflou, Fresenius Z. Anal.

..-.-~~~‘~~1-~-.~,~,~,~~~,“-~~,~ /

l,,lT

1

5 3

9 7

13 11

17

15

21

19

25

23

29

27

Scan position

33

31

37

35

41

39

45

43

49

47

51

(pixel)

Fig. 1. ~ncentration variability for 5 elements in a freeze-dried section of the substantia nigra (specimen cut thickness: 20 em; beam size used: 20 pm high by 4 brn wide, total scan width: 5.2

mm).

Chem. 326 (1987) 736. [5] W. Maenhaut, J. Vandenhaute, Bull. Sot. Chim. Belg. 95 (1986) 407. [6J W. Maenhaut, Scan. Microsc. 4 (1990) 43. (71 R.D. Vis, J.L.A.M. Kramer, G.H.J. Tms, F. van Langevelde, L. Mars, Nucl. instr. and Meth. B 77 (1993) 41. [Sj J.M. Heagney, J.S. Heagney, Nucl. Ins& and Meth. 167 (1997) 137. [9] H. Duflou, W. Maenhaut, J. De Reuck, Neurochem. Res. 14 (1989) 1099.