JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 18:176-182 (1991)

The Effect of Aluminium Coating on Elemental Standards in X-Ray Microanalysis DIANE M. HOPKINS, ALAN D. JACKSON, AND KENNETH OATES Institute of Environmental and Biological Sciences, Division of Biology, Lancaster University, Lancaster LA1 4YQ, England (D.M.H., K.O.), and Gatty Marine Laboratories, University of St. Andrews, Fife (A.D.J.),Scotland

KEY WORDS

Quantitative X-ray microanalysis, Coating parameters, Frozen hydrated specimens

ABSTRACT The standardisation of frozen hydrated bulk biological specimens using gelatin standards is described. The relationship between corrected elemental X-ray counts and ionic concentration was found to be linear, and minimum detectable limits for each element are stated. Variations in uncorrected standard curves were found to be due to changes in aluminium coating thickness. There was a n inverse relationship between coating thickness and elemental X-ray counts. The factors causing this are discussed. To avoid errors arising from inconsistent aluminium thickness, experimental material should only be compared with standards of similar aluminium net counts. This can be achieved most easily by mounting and analysing specimen and standard together. MATERIALS AND METHODS Standard curves X-ray microanalysis (X-rma) is a useful tool for The preparation of gelatin standards has been studying biological material, a s i t allows the analysis described in detail by Wyness et al. (19871, and will of individual cells within normal (Potts and Oates, only be briefly described here. All glassware to be used 1983) and pathological (Wyness et al., 1987; Zeyen, in the preparation of standards was acid washed for 1982) tissue samples. Much work has been carried out 24 hours in dilute nitric acid in order to remove on thin sections of biological material, fixed (Marshall, insoluble cations from the surface of the glass. Since 19831, frozen hydrated (Saubermann, 1988; Yousuf e t gelatin has been shown to contain detectable amounts al., 1978), and freeze-dried (von Zglinicki and Bimmler, of chlorine (Wyness et al., 19871, it was dialysed in 1987). Analysis of frozen hydrated bulk material, how- distilled, deionised water, which was constantly ever, is less popular, probably due to initial problems in stirred, with 10 2-hourly changes of water. All spatial resolution and difficulty in quantitation. Prep- standards were made up in 20% gelatin using the aration of frozen hydrated bulk specimens is easier appropriate electrolyte solutions, and frozen until (Gupta and Hall, 1981; Wyness et al., 1987), and it is likely that the material will more closely resemble the required, to avoid evaporative losses which would alter in vivo state of the tissue, as the electrolytes in super- standard concentrations. Standard solutions were prepared using the followficial cells will be instantly immobilized in situ (Gupta ing analytically pure salts (AnalaR): sodium chloride, and Hall, 1981; Staff et al., 1985). As mathematical chloride, calcium hydroxide, ammonium correction procedures developed for thin sections are potassium chloride, magnesium acetate, potassium hydrogen cardifficult to apply to bulk material (Wyness et al., 1987), bonate, sodium acetate, and ammonium dihydrogen orquantitation must be carried out using a standard thophosphate, and were acidified with nitric acid in which closely resembles the biological tissue of interest order to remove calcium precipitates. In each concen(Roomans, 1980). tration series, only one of the electrolytes was varied, It is necessary to coat all specimens for X-rma with a conductive coating, in order to dissipate space charge the other being maintained a t approximately physiolevels: potassium, 140 mM; sodium, 30 mM; calwhich would distort the volume of analysis (Marshall, logical cium, 5 mM; magnesium, 3 mM; phosphorous, 120 mM; 1980; Wyness et al., 1987; Yakowitz and Goldstein, chloride, 50 mM. The calcium standard, however, was 1977). Aluminium is becoming the choice metal for evaporative coating due to its low toxicity and mass absorption co-efficient (Yakowitz and Goldstein, 1977). It is also available in a high purity foil which allows accurate and even coating of the specimen. This study Received April 11, 1990; accepted in revised form August 24, 1990. has examined the effects of aluminium coating Address reprint requests to Diane M. Hopkins, Institute of Environmental and thickness on the elemental counts in gelatine stan- Biological Sciences, Division of Biology, Lancaster University, Bailrigg. Landards. caster, LA1 4YQ, England. INTRODUCTION

C 1991 WILEY-LISS. INC

ALUMINIUM COATING IN X-rma

made without potassium (the adjacent element in the X-ray spectrum), as the KP energy of potassium overlaps with the K a energy of calcium, and would have caused elevated calcium counts. It is possible to "strip" the KP potassium peak, but this would also remove some of the K a calcium peak, again reducing the accuracy of results. The concentrations of the variable elements in each series were as follows:

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standard was used for each analysis, but the aluminium thickness increased. The standard was analysed in the same way as described above, and was not exposed to air a t any time. The data from four replications were pooled. The livetime was noted for all analyses a t both 0.5 nA and 1 nA.

Relationship between aluminium coating thickness and weight evaporated 1. Potassium 12,l0,50,100,200l mM The specimen holder of the cryochamber of the mi2. Sodium [15,20,50,100,200] mM croscope was replaced by a crystal oscillator. The dis3. Calcium 15,10,20,50,100,200] mM tance of the crystal from the tungsten evaporating fil4. Magnesium [5,10,20,30,50,100,2001 mM ament corresponded to the central point of a n angled 5. Phosphorous [2,10,20,50,100,200] mM stub (60 mm). The crystal was coated by evaporation 6. Chloride [2,10,20,50,100,200] mM with aluminium foil strips of similar weights to those used in the previous paragraph. The coating thickness Cryofixation of standards for X - r m a . Standards obtained from different weights of aluminium was were mounted onto aluminium stubs milled to a n angle noted over a number of evaporations. Readings were of 30", with Leit C conductive carbon cement (Agar also taken after heating the filament in the absence of Scientific). The mounted specimens were rapidly frozen aluminium, in order to determine the degree of drift in by contact with a mirror-finished copper block cooled to the crystal caused by the evaporation process. Drift - 195°C in liquid nitrogen, and transferred, without was found to be negligible. exposure to the air, to the cryochamber (Hexland CT RESULTS 1000, Oxford Instruments) of a JEOL JSM 840A scanStandard curves ning electron microscope (SEM). All corrected standard curves for the elements invesX-rma. The standards were coated under high vacuum with aluminium foil vaporised on a tungsten fil- tigated showed a linear relationship between elemenament. They were analysed using a Kevex energy dis- tal counts and concentration, with net counts increaspersive detector and Link Systems 860 series I1 500 ing a s concentration increased. Sodium, phosphorous, analyser (Link Analytical Ltd.). The following param- chloride, and potassium are shown in Figure 1,as these eters for data acquisition were used: accelerating volt- elements were also used in the second part of the inage = 15 kV; probe current = 0.6 nA; working dis- vestigation. The minimum detectable limits detertance = 18.5 mm; analysis area = 90 km2; preset mined from these calibration curves were as follows: integral = 80,000 A1 counts; and take-off angle = 45". sodium, 50 mM Kg-' water; magnesium, 20 mM Kg-' The net counts of the variable element in each concen- water; phosphorous, 2 mM Kg-' water; chloride, 20 tration series were corrected to take into account vari- mM Kg-' water; potassium, 20 mM Kg-' water; and ations in the constant elements caused by changes in calcium, 5 mM Kg -'water. When uncorrected standard curves were plotted, the the aluminium coating thickness a s follows: relationship between elemental counts and concentra1. The mean number of counts for each of the con- tions was not linear. The variations in the standard stant elements phosphorous, potassium, and chlorine curves corresponded reciprocally to variations in net within a series was calculated. (Sodium, magnesium, aluminium counts. Phosphorous is shown in Figure 2, and calcium were not used in these corrections, a s they but the relationship was true for all the elements inwere present in the standard at concentrations lower vestigated. than their minimum detectable limits, and would not, Effects of aluminium coating thickness therefore, give reliable count rates.) As the weight of aluminium evaporated increased, 2. The net counts for phosphorous, potassium, and chlorine in each analysis were calculated as a percent- the net elemental X-ray counts of all other elements age of the series mean. The average of these (y) was decreased. The absolute decrease in elemental counts used in the following correction equation: xly x 100. became greater a s the atomic number of the element increased. This was true for both 0.5 nA (Table 1)and Where x is the net integral for the variable element. 1nA (Table 2) as shown by the gradients for each of the Effects of aluminium coating thickness elements (taken from graphs of absolute elemental A standard solution was prepared by the method de- counts against aluminium counts): scribed above, but the elements investigated were 1. 1 nA: sodium = -0.16; phosphorus = -0.49; fewer, and were kept a t constant concentrations throughout the investigation, as follows: sodium, 100 chloride = -0.73; potassium = -1.19 2. 0.5 nA: sodium = -0.17; phosphorus = -0.50; mM; phosphorous, 100 mM; chloride, 100 mM; potassium, 200 mM. The parameters for data acquisition chloride = -0.77; potassium = -1.21. were as above, except that specimens were analysed at 0.5 nA and 1 nA with a n analysis area of 1,600 km2. The percentage decrease in elemental counts with inThe standard was sequentially coated by evaporation creasing aluminium coating thickness, however, was with weighed strips of aluminium foil, so that the same similar for all elements (Table 3), as shown by the gra-

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Fig. 1. A standard curve for sodium; B: standard curve for phosphorus; C: standard curve for chloride; D: standard curve for potassium.

200

ALUMINIUM COATING IN X-rma

Aluminium Curve 86000

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179

ness and weight of aluminium evaporated. There was a direct linear relationship between the weight of aluminium evaporated and the thickness of deposit on the specimen (Table 4). The relationship between aluminium weight and net aluminium counts, however, was not linear (Fig. 4). Aluminium counts increased with the weight of aluminium evaporated, but the increase was only linear in the lower count range, tailing off a t around 55,000 net aluminium counts.

DISCUSSION To quantify electrolyte levels in bulk frozen hydrated biological material, standards must be used which match closely the biological material of interest (Roomans, 1980). Any standard must be able to fulfill several criteria: 1)be of known composition (Gupta et al., 1977) and as close to biological tissue as possible (Heinrich, 1982; Roomans, 1980; Wyness et al., 1987); 2) be stable under the electron beam (Yakowitz and 42000 Goldstein, 1977); and 3) conform to the requirements of 40000 I 1 I 1 I X-rma, having a flat surface and being able to with0 40 80 120 160 200 stand high vacuums (Ingram et al., 1972). Gelatin has Phosphorobs Concentration (mMol/Kg water] been previously shown to fulfill these criteria (Wyness et al., 1987). For reliable quantitation to occur, the relationship Uncorrected Phosphorous Curve between concentration and X-ray counts of a given element has to be established. It is also necessary to de7000termine the minimum detectable limits of the electrolytes of interest. The uncorrected X-ray data for all 0000elements investigated had a non-linear relationship to concentration, which was found to be due to variations n in aluminium coating thickness (Fig. 2). This will be 2 30003 discussed below. All corrected X-ray data, however, 0 showed a linear relationship between elemental counts 0 4000and concentration (Fig. 1,A-D). 3 For accurate X-rma to be carried out, it is necessary 2 3000for specimens to be coated with a conductive material 0 in order to avoid specimen charging (Marshall, 1980; a 0 Marshall and Condron, 1985a). In uncoated specimens, I 2000n the number of backscattered electrons produced increases as the analysis proceeds as a result of space charge (Marshall and Condron, 1985b). Backscattered electrons can cause excitation of X-rays from areas outside the probe spot and can also penetrate the detector ' 0 0 O 40 80 o 1200 160 200 L window, registering as background counts (bremss(Statham, 1980). The background radiation Phosphorous Concentration (mMol/Kg water) trahlung) is highest at the low energy end of the spectrum, so Fig. 2. Comparison of the uncorrected phosphorous standard curve coating is beneficial in the detection of the lighter elements, such as sodium (Wyness et al., 1987). In this with aluminium counts. study aluminium was the conductive coating chosen; it is of low toxicity and has a low mass absorption coefficient (Yakowitz and Goldstein, 1977). Carbon coating dients taken from graphs of percentage decrease in el- was not used, as it was found by Oates (1984) to cause emental counts against aluminium counts: sodium = the loss of sodium, phosphorous, and potassium due to -0.0047; phosphorus = -0.0045; chloride = -0.0044; thermal heating of standards during evaporation. and potassium = -0.0044. The percentage decrease in When using aluminium as a conductive coating, Marsodium counts was slightly greater than those of the shall (1980) has found that frozen specimens coated other elements, but not significantly so (data were com- external to the SEM did not yield a consistent aluminium coating, black oxides of aluminium sometimes bepared using Student's t test). At both 0.5 nA and 1nA, the livetime for each anal- ing formed. These deposits probably reduce elemental ysis decreased as the aluminium coating thickness in- X-ray counts by increasing absorption. In the present work, coating was carried out in the SEM airlock under creased (Fig. 3). torr). The Relationship between aluminium coating thick- the high vacuum of the microscope (5 x

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D.M. HOPKINS ET AL. TABLE I . Relationship between elemental and aluminium netcounts in a gelatine standard (0.5 nAl

Nrt elemental X-rav counts (2SE) A1 Na 3,685.5 i 176.1 2,549.2 i 202.5 1,412.1 i 138.1 1,049.7 i108.6 851.4 2 71.7 562.5 2 95.9

39.893.1 i 202.0 48,009.6 2 260.8 53,191.3 t 318.5 55,481.3 i 286.1 57,049.1 f 239.2 58.024.8 -t 157.0

P

c1

K

11,277.4 ? 543.3 7,849.0 i510.4 4,574.0 t 358.9 3,694.5 i 332.2 2,679.9 i 203.9 2,225.6 i 208.4

17,561.8 i 865.2 12,161.4 t 709.4 7,442.7 t 578.7 5,750.0 2 535.2 4,571.1 i 409.8 3,716.8 i 351.5

27,317.1 2 1,350.9 18,782.9 t 1,231.3 11,304.5 2 857.4 8,556.6 767.9 7,837.3 f 876.8 5,256.2 t 475.6

TABLE 2. Relationship between elemental and aluminium netcounts in Net elemental X-ray counts ( i S E ) A1 Na 3,408.0 i 268.4 2,151.8 i 169.1 1,222.9 i132.4 1.043.0 i 95.9 800.3 i102.2 651.1 i 51.7

39,536.9 2 260.3 47,425.8 2 377.8 52,393.8 i 278.6 54.514.8 -t 187.5 56;189.6 2 227.8 57,448.1 2 187.9

c1

K

10,884.5 +- 739.3 6,877.7 t 485.5 4,113.1 i311.9 3,399.5 i 243.7 2,488.5 t 268.0 2,309.1 i 184.7

16,729.1 i- 1,174.4 10,919.5 t 767.7 6,919.1 t 463.9 5,841.9 i447.38 4,480.8 i 388.1 3,863.9 i 251.3

26,662.2 t 1,800.8 16,674.4 i 1,224.2 10,446.4 2 737.4 8,511.9 i 617.1 6,489.9 2 549.8 5,540.7 i 382.1

400

Reduction of elemental counts expressed as 2 of initial count

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48,009.6 53,191.3 55,481.3 57,049.1 58.024.8

gelatine standard (I nA)

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TABLE 3 . Reduction in elemental X-ray counts with increasing aluminium coating thickness Aluminium counts

Q

*

Na

P

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100 69.2 38.3 28.5 23.1 15.3

100 69.6 40.6 32.8 23.8 19.7

100 69.3 42.4 32.8 26.0 21.2

100 68.8 41.4 31.3 28.7 19.2

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TABLE 4 . Relationship of aluminium weight to coating thickness Mass (mg)

Thickness (nm)

2.7 4.3 5.7 7.0 8.8

24.5 f 1.1 39.9 i 0.4 51.6 2 1.3 66.5 i 1.0 82.4 i0.5

SEM includes three cold surfaces a t around -180°C onto which vapours condense. Bright aluminium coatings were consistently produced. The thickness of aluminium deposited is directly proportional to the weight evaporated (Table 41,and not to the area of aluminium used, a s has been suggested previously (Wyness et al., 1987). The X-ray counts for aluminium, however, are not directly proportional to weight evaporated (Fig. 4). This is due to the pulse pile-up rejection circuit within the analyser. The aluminium counts tail off (Fig. 4)as the count rate reaches its maximal point and many of the aluminium counts are simply rejected (Statham, 1981). In order to reduce rejection of elemental counts, coating thickness should not exceed 50 nm (around 5.3 mg). When choosing the coating thickness, time available should also be considered, as increasing the thickness will reduce analysis time (Fig. 3). As the aluminium coating thickness increased, the counts for all other elements decreased (Tables 1, 2);

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0

30000

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60000

aluminium counts Fig. 3. The relationship between aluminium counts and livetime a t two probe currents.

this occurs for several reasons. 1)The analyzer was set to a preset integral of 80,000 gross aluminium counts (aluminium counts plus background); this gives a total spectrum count of around 300,000, a figure considered to ensure statistically significant differences between elemental peaks and background (Statham, 1980). As the aluminium thickness increased the preset integral will be reached more quickly (Fig. 3 gives a n indication of this, although the real time of a count is the combination of dead time and livetime) and the analyser will therefore record fewer elemental counts. 2) As a result of the pulse pile-up rejection circuit, the number of re-

181

ALUMINIUM COATING IN X-rma $3000

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jected counts, and therefore dead time, will increase as the total number of counts originating from an area increases, thus more elemental counts will be rejected as aluminium increases. 3) The aluminium layer affects attenuation of the electron beam into the specimen, and absorption of X-rays produced (Kotrba, 1979), both of which will increase as the conductive coating thickness increases. Attenuation causes a decrease of current into the specimen and increased scattering of electrons within the conductive layer, thus reducing the electrons available to produce X-rays from the elements in the standard. It has been argued that these factors are of greater importance to the lighter elements, such as sodium (Kerrick et al., 1973). In the present investigations, however, the relative decrease in counts was similar for all elements (Table 31, indicating that the lighter elements are not affected to a greater extent by attenuation of the incident electron beam and absorption of characteristic X-rays. This contradicts the work carried out by Kerrick et al. (19731, but it should be pointed out that the coating of choice in their work was carbon, which has a higher mass ab-

sorption coefficient than aluminium: in the case of sodium, carbon has a mass absorption of 1,534, compared with 1,021 for aluminium (Heinrich, 1966), and will therefore absorb more sodium X-rays than a similar thickness of aluminium. That all elements were affected equally, in relative terms, by increasing aluminium thickness indicates that the thickness of aluminium used in the present investigation did not reach a critically limiting point in terms of attenuation and absorption. The absolute decrease became greater as the mass of the element increased (Tables 1, 2). This second phenomenon is again probably due t o rejection of overlapping pulses reaching the analyzer: heavier elements produce more X-rays, and will therefore have a greater number rejected than lighter elements. In conclusion, it is necessary to ensure that the standard and unknown being analysed have the same aluminium coating in order to ensure accuracy of quantitation. The best way to ensure a comparable coating on both standard and specimen is to coat and analyse them together. When mounting the standard and spec-

182

D.M. HOPKINS ET AL.

imen together, care must be taken to avoid evaporative losses from the standard, and to ensure that the biological material is frozen quickly enough to avoid translocation of the electrolytes of interest. Where this is not possible, specimens should be mounted separately, and compared with standards of a similar aluminium coating thickness.

ACKNOWLEDGMENTS This work was carried out while D.M.H. was supported by the North West Cancer Research Fund, grant No. CR55, and A.D.J. was in receipt of a n SERC Case studentship (8651045x1. We would like to thank Dr. Henry Huddart,for his valuable advice during the preparation of this paper. REFERENCES Gupta, B.L., and Hall, T.A. (1981) The X-ray microanalysis of frozen hydrated sections in scanning electron microscopy: an evaluation. Tissue Cell, 13:623-643. Gupta, B.L., Hall, T.A., and Moreton, R.B. (1977) Electron probe Xray microanalysis. In: Transport of Ions and Water in Animals. B.L. Gupta and R.B. Moreton, eds. Academic Press, London, New York, San Francisco, pp 83-143. Heinrich, K.F.J. (1966) in Reed, S.J.B. (1975) Electron Microprobe Analysis, p. 257, Cambridge University Press, London. Heinrich, K.F.J. (1982) The accuracy of quantitation in X-ray microanalysis, particularly of biological standards. SEM I:281-287. Ingram, F.D., Ingram, M.J., and Hogben, C.A.M. (1972) Quantitative electron probe analysis of soft biological tissue for electrolytes. J . Histochem. Cytochem., 20:716-722. Kerrick, D.M., Eminhizer, L.B., and Villaume, J.F. (1973) The role of carbon film thickness in electron microprobe analysis. Am Minerologist, 58:920-925. Kotrba, Z. (1979)The influence of conductive coatings on the accuracy of X-ray microanalysis. Microscopia Acta., 82:59-68.

Marshall, A.T. (1980) Quantitative X-ray microanalysis of frozen hydrated bulk biological specimens. SEM., II:335-348. Marshall, A.T. (1983) X-ray microanalysis of copper and sulphur-containing granules in the fat body cells of Homopteran insects. Tissue Cell, 15:311-315. Marshall, A.T., and Condron, R.J. (1985a) X-ray microanalytical resolution in frozen hydrated biological bulk samples. J. Microsc., 140: 109-118. Marshall, A.T., and Condron, R.J. (198513) Normalization of light element X-ray intensities for surface topography effects in frozen hydrated biological bulk samples. J. Microsc., 140:99-108. Oates, K. (1984) PhD thesis, University of Lancaster. Potts, W.T.W., and Oates, K. (1983) The ionic concentrations in the mitochondria rich or chloride cell of Fundulus heteroclitus. J . Exp. Zoo., 227:349-359. Roomans, G.M. (1980) Problems in quantitative X-ray microanalysis in biological specimens. SEM., II:309-320. Saubermann, A.J. (1988) Quantitative electron microprobe analysis of cryosections. Scanning Microsc., 2:2207-2218. Staff, W.G., Middleton, J.P.S., Morris, J.A., and Oates, K. (1985) Low temperature and conventional scanning electron microscopy of human urothelium. J . Urol., 57:lO-19. Statham, P. (1980) Problems of qualitative and quantitative analysis with X-ray energy spectrometry. J . Microsc. Spectrosc. Electron., 5:47-61. Statham, P. (1981) X-ray microanalysis with Si(Li) detectors. J . Microsc., 123:1-23. van Zglinicki, T., and Bimmler, M. (1987) The intracellular distribution of ions in rat liver and heart muscle. J . Microsc., 146:77-85. Wyness, L.E., Morris, J.A., Oates, K., Staff, W.G., and Huddart, H. (1987) Quantitative X-ray microanalysis of bulk hydrated specimens: a method using gelatine standards. J . Pathol., 153:61-69. Yakowitz, H., and Goldstein, J.I. (1977) Practical aspects of X-ray Microanalysis. In: Practical Scanning Electron Microscopy. J.I. Goldstein and H. Yakowitz, eds. Plenum Press, New York, pp. 422433. Yousuf, AS., Wisby, A,, and Gray, J.C. (1978) Electron probe analysis of cryosections of epiphyseal cartilage. Metab. Bone Dis. Rel. Res., 1:97-103.. Zeyen, R.J. (1982) Applications of in situ microanalysis in underlying disease: X-ray microanalysis. Ann. Rev. Phytopathol., 20:119-142.