Silicate particles engulfed in human alveolar macrophages: An analytical electron microscopy study

Mikrochim. Acta 114/115, 285-291 (1994)

Mikrochimica Acta 9 Springer-Verlag 1994 Printed in Austria

Silicate Particles Engulfed in Human Alveolar Macrophages: An Analytical Electron Microscopy Study Marco Diociaiuti*, Mario Falchi, and Luigi Paoletti Department of Ultrastructures, Istituto Superiore di Sanith, Viale Regina Elena 299, 1-00161 Roma, Italy

Abstract. The chemical composition and crystalline structure of silicate particles

engulfed in human alveolar macrophages were investigated by transmission analytical electron microscopy. A crystalline ZrSiO4 and an amorphous Si2A10 4 particle were identified. Small crystalline Fe microparticles (2-4 nm), which are likely ferritin molecules, were found concentrated mainly at the amorphous particle-tissue interface. Key words: analytical electron microscopy, structural analysis, silicates, particles.

Silicate particles are present in atmospheric aerosol and can be easily found engulfed in human alveolar macrophages. These compounds show different toxicities and while the activity of silica particles is well known, the activity of other silicates is not [1]. Until recently, the alterations induced on the particle surface by macrophage action have not been investigated. In particular, it is not clear if the surface atomic arrangement of the silicate particles is modified after its interaction with these cells which are involved in chemical digestion. In the narrow interface of molecular dimension (1 nm) which exists between this material and tissue, important primary reactions, such as corrosion and dissolution, protein adsorption and denaturation and catalytic effects can take place [2, 3]. These initiating reactions can subsequently cause secondary reactions due to corrosion products, denatured proteins or compounds produced in the catalyzed reactions. In order to characterize the primary interaction zone existing around silicate particles engulfed in human alveolar macrophages, we have used in a high voltage (300 keV) transmission electron microscope (TEM), imaging and selected area electron diffraction (SAED), energy dispersive X-ray (EDX) spectroscopy and electron energy loss spectroscopy (EELS) elemental microanalysis.

* To whom correspondence should be addressed

286

M. Diociaiuti et al.

Experimental Alveolar macrophages were obtained from bronchoalveolar lavage fluids (BALF) performed on a pottery worker exposed to silicate particles. After centrifugation, cell pellet was fixed by the conventional glutheraldehyde-osmium technique for TEM. Sample was then dehydrated in alcohol and embedded in Agar 100. Sections were cut by diamond knife with thickness ranging between 90 and 50 nm, mounted on 400 mesh copper grids and observed in a TEM, without addition of contrasting media. The instrument used in our measurements was a TEM Philips EM430 equipped with a LaB 6 gun, operating at a primary beam energy of 300 keV and equipped with an EDX spectrometer for the elemental microanalysis. EDX spectroscopy is based on the analysis of the energy spectrum of X-ray emitted under the electron bombardment. An energy-dispersive solid-state Si(Li) spectrometer, cooled by liquid nitrogen, is connected to the TEM column and a multi-channel-amplifier (MCA) enables discrimination of the signal as a function of the X-ray photon energy. Standardless semiquantitative elemental analysis is performed according to the Cliff-Lorimer ratio method [4] by evaluating the characteristic emission intensities. The high energy beam allows us to consider valid the "thin film criterion", that is, to consider negligible absorption and fluorescence of X-rays during their passage out of the specimen. For this reason, no matrix correction is applied and elemental concentrations are obtained as follows:

Na -

K~BI~pb, Ibpa

(1)

where N a, ND are the numbers of atoms, p,, Pb the atomic weights, I,, I b the integral under the peaks after the background subtraction and K~b the Cliff-Lorimer factors [4] experimentally determined. In this type of spectroscopy, the accuracy is ultimately limited by the counting statistics because no difficulties in the background subtraction [5] exist. Assuming Gaussian statistics, a typical CliffLorimer factor error of about 370 and counts in the order of 10.000, we can estimate an error of about

107o. TEM have also allowed us to study the "long-range" crystalline order by SAED technique [6]. Diffraction patterns can be used, after a camera length calibration performed on Au crystalline standards, to identify the atomic arrangement and, in the case of crystals, to measure the lattice parameters. The error associated to the lattice parameter measurement can be estimated to be about

37o. EELS spectra, including low-loss (0-100 eV) and high-loss (100-1200 eV) spectra, were acquired by a serial magnetic sector energy analyzer (Gatan 607) connected to the TEM. Counting times of about 0.2 s per channel were used in the acquisition of EELS spectra for elemental microanalysis. EELS measurements were performed with primary electrons of 300 keV corresponding to an electron wavelength of 1.97 • 10 -~2 m, an acquisition angle of 18.2 mrad and over areas of about 100 nm in diameter. EELS quantitative microanalysis is performed according to the Egerton ratio method [7]. The background under the edges was modelled by interpolating the spectrum in an energy window of F --- 100 eV before the edge by a function: B(E) = AE -r,

(2)

where A and r are coefficients calculated by means of a least-squares fitting. The correlation coefficients between the experimental points and the power law of Eq. (2) was used as test of fit. This coefficient was always close to 1 (0.99) in the energy loss regions before the edge jump. After the subtraction of the background, spectra were integrated in an energy window A = 50 eV above the edge onsets and the atomic ratio Nx/Ny was determined from the appropriate partial integrated cross-sections a x and ay [8] according to: N x _ Sx(Cq A)o'y(~, A)

Ny

Sy(~,A)O'x(~, A)'

(3)

Silicate Particles Engulfed in Human Alveolar Macrophages

287

where Nx and Ny are the numbers of x and y atoms, ax and try are the values of the integrals under the x and y edges, ct is the acquisition angle. Error sources in EELS microanalysis have been extensively discussed [7, 9]. Statistical limitations, approximate calculations of the appropriate cross-sections and uncertainties in the background subtraction are the principal sources. In our case, the edges studied are the O-K, A1-K and Si-K located at 532, 1560 and 1839 eV, respectively. All edges are of the same type (K edges) and consequentely errors from calculation of the cross-sections are reduced. Moreover, K edges do not overlap and the analysis is not affected by troublesome tail edge subtractions. The main error source is the uncertainty in the background subtraction. Considering the energy windows used in our analysis (F = 100 and A = 50 eV) and the statistics, the accuracy in our EELS analysis can be estimated to be about 10700.

Fig. 1. TEM micrographs of the two studied particles (a, c) together with relative SAED patterns (b, d). Magnification in the images and camera length in the diffraction pattern were 193000 X and 1357 mm, respectively

288

M. Diociaiuti et al.

Results and Discussion

Results relative to two silicate particles are reported: an amorphous SixAlyOz particle of about 300 nm diameter and a SixZryO z crystalline particle of the same size. Figure 1 shows images relative to both particles together with diffraction patterns recorded from a selected area of about 400 nm diameter which includes the whole particle. The diffraction pattern (Fig. 1 b) relative to the particle of Fig. 1 a shows the superposition of three well-defined Debye rings superimposed over broadened peaks. The broadened peaks can be attributed to the amorphous particle core and the weak rings to the dark microparticles that can be observed around

9

i

,

I

]

I

J ....

I

II I

~

i ,

I II

A 1 Kc,~

]

I

I

~I

t

r~L.,

I I I I

I

.....

.

il ll,l

1

9

E. 0~ laENT

a

4, E10

6,00

5

~ 4 4 K E V

.

1

i 1 il i

8,00

i~. ~0

t~Z!eV.."oh A

SiK~ I

EEDAX

b

I I

Zr+.::

I ~ I N T

5 .

4 4 K E V

!E~eV,'"oh

A

EDF~X

Fig. 2. (a, b) EDXS spectra relative to the two particles of Fig. 1. Microanalytical results are summarized in Table 1

Silicate Particles Engulfed in Human Alveolar Macrophages

289

the particle core (Fig. 1 a). These microparticles, which are about 2-4 nm in diameter, are present outside the particle and, in high concentration, at the particle-tissue interface. Debye rings indicate that microparticles are characterized by "long-range" order. According to the Scherrer relation [10], the small size of those microparticles causes a broadening of the Debye peaks. Measurements of the Debye rings leads to an estimation of interplanar spacing of 0.207 ___0.006, 0.148 __+0.004 and 0.083 __+ 0.003 nm. These values are in agreement with values relative to the interplanar spacing of FeOOH crystals [11] growing in the inner core of tbrritin proteins. Ferritin proteins have been found around silicate particles engulfed in alveolar macrophages [12] and asbestos fibres [13] or self-organized in siderosomes 1-14]. The diffraction pattern of Fig. 1 d, relative to the particle of Fig. 1 c, clearly shows well-defined diffraction spots, suggesting that this particle is composed mainly of a single crystal. The diffraction pattern is confused by spots due to the occurrence in the particle of misoriented crystalline domains. Figure 2 shows EDX spectra relative to both particles. The amorphous particle is composed mainly of Si, A1; Fe traces are present. The crystalline particle contains Si, Zr; Fe and Ca traces are present. The spatial resolution of this technique does not allow discrimination between different particle zones. Figure 3 shows EELS spectra relative to both particles recorded from a zone of about 400 nm in diameter which includes the whole particle. Both spectra are characterized by strong O-K edges. The spectrum relative to the amorphous particle (Fig. 3 a) shows A1-K and Si-K edges together with weak Fe-L2, 3 edges. The spectrum relative to the crystalline particle is characterized by Si-K edge and weak F e - L 2 , 3 , C a - L 2 , 3 edges. Zr-L2, a and -M4, 5 a r e located outside the energy loss window chosen. Table 1 summarizes all EXD and EELS element percentages. All values are expressed in ~o atomic ratio and normalized to Si = 2, for both EDX and EELS microanalysis.

0

a

AI Si

20x

r

>._ I-CO Z Ld I-Z

0

1

1000

2000

ENERGY LOSS ( e V )

Fig. 3. (a, b) EELS spectra relative to the two particles of Fig. 1. Microanalytical results are summarized in Table 1

290

M. Diociaiuti et al.

Table 1

Element Part. 1 (amorph.)

EDX (At. %)

Si A1 O Fe

Part. 2 (cryst.)

2 • 0.2

0.96 • 0.1

EELS (At. %) 2 _+ 0.2 1.18 _ 0.1 4.52 + 0.4

0.08 + 0.01

Si Zr O A1 Fe Ca

2 • 0.2 1.59 • 0.2

2 • 0.2 9.18 • 0.9

0.04 • 0.01 0.06 • 0.01 0.02 • 0.01

EDXS (column 1) and EELS (column 2) microanalytical results relative to the chemical composition of amorphous (part. 1) and crystalline (part. 2) particles. All values are expressed in ~o atomic ratio and normalized to Si -~ 2~o, for both EDX and EELS microanalysis.

a

r-

L.

I

I

1200

>-

800 ,.

b

Z W HZ

"....,. I

I

600

1000 ENERGY LOSS (eV)

Fig. 4. Fe-L2,3 edges relative to the amorphous particle of Fig. 1 a. In a spectrum a has been acquired in a zone centred on the particle-tissue interface while spectrum b in a zone centred on the particle core. b shows the spectrum difference c = a - b

E E L S m i c r o a n a l y s i s is c a p a b l e of h i g h s p a t i a l r e s o l u t i o n a n d in Fig. 4 a, Fe-L2, 3 edges a c q u i r e d in a z o n e of 100 n m in d i a m e t e r c e n t r e d o n the a m o r p h o u s particle c o r e (curve b in Fig. 4 a) a n d o n the a m o r p h o u s paticle-tissue interface (curve a in Fig. 4 a), are r e p o r t e d . F i g u r e 4 b s h o w s the s p e c t r u m difference (curve e in Fig. 4 b is o b t a i n e d as a - b in Fig. 4 a). This result is in a g r e e m e n t with the results o b t a i n e d b y S A E D a n d clearly

Silicate Particles Engulfed in Human Alveolar Macrophages

291

shows that, in the amorphous particle, Fe atoms are concentrated at the particletissue interface.

Conclusions The combined application of standard transmission electron microscopy and spectroscopic techniques, such as EDXS and EELS, allowed us to study t h e chemical composition of two silicate particles engulfed in h u m a n alveolar rnacrophages. An amorphous Si2A10 4 and a crystalline ZrSiO4 particle, were found. Structural data relative to "long-range" order (SAED) clearly show that the ZrSiO 4 particle is crystalline while the Si2A10 4 is amorphous. As far as the particle-tissue interface is concerned, all data suggest that macrophage action leads to the accumulation in this zone of ferritin molecules which contain F e O O H cores, as an early stage of the formation of "ferruginous bodies" around engulfed particles, as described in the literature [12, 13, 14]. It is important to note that the dimension of these particles is in the range of 2 - 4 nm while 7 n m is the diameter generally reported in the literature [13]. However, Iancu et al. [14] found particles in the cytosol with diameters in the range of 2 - 4 nm. These particles were related to ferritin by Mossbauer spectroscopy [15] and interpretated as being subunits or degraded ferritin. Further investigation is in progress to test this hypothesis.

Acknowledgment. This study has been supported by the CNR-ENEL Project "Interactions of energy

systems with human health and environment" Rome, Italy.

References [1] J. Bignon Health Related Effects of Phyllosilicates, NATO ASI Series, Springer, Berlin Heidelberg New York Tokyo, 1990, p. 21. [2] J. E. Gallagher, G. George, A. R. Brody, Am. Rev. Respir. Dis. 1987, 135, 1345. [3] R. P. Nolan, A. M. Langer, J. S. Harington, G. Oster, I. J. Selikoff,Environ. Res. 1981, 26, 503. [4] G. Cliff, G. W. Lorimer, J. Microsc. 1975, 103, 203. [5] A. D. Roming, J. I. Goldstein, Met. Trans. 1980, llA, 1151. [6] R. D. Heidenreich, Fundamentals of Transmission Electron Microscopy, Wiley, New York, 1964, p. 174. [7] R.F. Egerton, Electron Energy Loss Spectroscopy in the Electron Microscope, Plenum, New York, 1986. [8] R. F. Egerton, Ultramicroscopy 1979, 4, 169. [9] C. Colliex, Advances in Optical and Electron Microscopy, Academic Press, London, 1984. [10] P. Scherrer, G6ttinger Nachr. 1918, 2, 98. [11] F. D. Pooley, Environ. Res. 1972, 5, 369. [12] J. P. Berry, P. Henoc, P. Galle, R. Pariente, Am. J. Path. 1976, 83, 427. [13] C. Janguillame, J. P. Berry, C. Colliex, P. Galle, M. Tence, P. Trebbia, J. Physique 1984, 45, C2-577. [14] T. C. Iancu, Electron Microsc. Rev. 1992, 5, 209. [15] E. R. Bauminger, T. C. Iancu, G. Link, A. Pinson, C. Hershko, Hyperfine Interactions 1987, 33, 249.