JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 18:424-428 (1991)

Biological Applications of Electron Probe Microanalysis in France CARMEN QUINTANA AND GHISLAIN NICAISE U 194 INSERM, 75013 Paris, and Centre de Biologie Cellulaire CNRS, 94205 Iury-sur-Seine (C.Q.1, and Cytologie Experimentale (URA CNRS 6511 and Centre Commun de Micrascopie Appliquee, Uniuersite de Nice-Sophia Antipolis, 06034 Nice (G.N.1, France

KE Y WORDS

X-ray microanalysis, Biology, French science

ABSTRACT The contribution of French science to the field of biological X-ray microanalysis is reviewed. The main analytical microscopy centers are listed, and their methods and main results are summarized.

INTRODUCTION Electron probe microanalysis (EPMA) or X-ray microanalysis (XRMA) began its existence in France in 1951, with the work of R. Castaing (Castaing, 1951, 1960). During the next decades other French investigators such as Philibert (1963, 1965, 1970). Henoc (Henoc et al., 1963), Maurice (Henoc and Maurice, 1976), Tixier (Philibert and Tixier, 19751, contributed to develop EPMA a s a very effective method for quantitative analysis in physics and geology. One of the first authors who applied EPMA in the biomedical field was Galle (1964). However, in a n extensive review of “the application of X-Ray microanalysis in TEM to the study of ultrathin biological specimens” published 14 years later (Chandler, 1978), only 9 of the 272 quoted articles were written by French investigators. Ten years later, in 1988, 11 review articles were published in Ultramicroscopy and Journal of Electron Microscopy Technique on the application of X-ray analytical microscopy to biological problems. Only one of them was written by a French author (Roinel, 1988) and in the 10 others only 5 of the 1,057 bibliographical references gathered by the authors of these articles were of French origin. After being the first to develop the technique and to use i t in biology, French authors had obviously little bibliographical impact in biological EPMA. Was their research weak, too provincial, or badly advertised? The present survey will not answer directly these questions but will try to call the attention of the reader to the French literature in the field. FRENCH ANALYTICAL CENTERS AND INSTRUMENTS The Saclay Center of the Commissariat d’Energie Atomique (CEA) has been the pioneer in the quantitative analysis of biological fluids since 1969. As already mentioned in the Introduction, the work done in this laboratory is well known beyond French boundaries, Another center of XRMA established in France at the beginning of the 1970s is the Service Commun de Microanalyse Appliquee a la Biologie (CNRS-INSERM) of Creteil, where a British-French symposium took place in 1975. This center from the start mainly specialized in the qualitative analysis of mineralaccu-

0 1991 WILEY-LISS, INC.

mulations and toxic elements both in experimental and clinical pathology (Galle group) and in physiopathology of invertebrates (Martoja group). In 1978, the Centre de Microscopie Electronique Appliquee a la Biologie e t la Geologie of the Claude Bernard University (Lyon-Villeurbanne) acquired in analytical electron microscope. A group started working on quantitation of calcium in soft tissues with this equipment, adopting Hall’s method of quantitative analysis of thin sections (Amsellem et al., 1983). The instruments used by these centers are the Castaing microprobe (MS-46) and the SEM microanalyser Camebax (from Cameca instruments) mainly built to be equipped with X-ray wavelength dispersive spectrometers (WDS) and only secondarily with energy dispersive spectrometers (EDS). The Camebax instruments of the Creteil and Villeurbanne centers are fitted with a transmission electron microscope (TEM) accessory of 4 nm resolution which permits thin section analysis with a minimum detectable mass (MDM) of 7.10-18 g in the usual working conditions, i.e., acceleration voltage 50 kV, probe current 140 nA for a 500 nm spot, collection time 100 seconds (Quintana, 1980). An alternative instrumental choice was made in other countries with standard high resolution TEM, only slightly modified to accept EDS, which proved to be very effective in quantitation (Shuman and Somlyo, 1976) and led to early breakthroughs, particularly in muscle cell biology (Somlyo et al., 1977). Few transmission analytical microscopes of this type are currently used in France by biologists. One of the first (Philips EM 400 + Tracor 2000 EDS system) was acquired by the University of ,Marseille43 Jer6me in 1981. A more recent and more sophisticated version (Philips CM12 + Tracor 5400 EDS system) was installed in 1987 in the Centre Commun de Microscopie Appliquee of Nice University. Another analytical microscope of the last generation was installed in 1989 in Unit 314 of the French medical research council

Received May 14, 1990; accepted in revised form August 3, 1990. Address reprint requests to Dr. C . Quintana, U 194 INSERM, 91 Bd. de I’HBpita1 75013 Paris, France.

BIOLOGICAL EPMA IN FRANCE

(INSERM) in Reims (Philips CM30 system).

+ Edax 9900 EDS

SAMPLE PREPARATION The XRMA of mineral bioaccumulations (natural or induced) or accumulations of toxic elements, which are generally present at high concentration within stable compounds, can be achieved on conventional electron microscopy preparations, even fixed with Os04 or stained with uranyl and lead (Galle, 1975). However, the use of unstained sections was advised by Martoja et al. (1979) and other authors regularly used specimens in which staining with heavy metals was omitted, except for postfixation with OsO, (Hernandez-Nicaise et al., 1982; Nicaise and Hernandez-Nicaise, 1980; Nicaise et al., 1982). It has also been reported that the elimination of osmium postfixation leads to a decrease of MDM in the Camebax analyser from 9.10-ls to 7.10-Is g (Quintana, 1980). Since the 1970s biologists have been trying to apply EPMA to the study of physiological processes, and particularly to the study of diffusible elements such as Na, K, C1, as well as partially diffusible elements such as Mg, Ca, etc. Various cytochemical (precipitative) methods have been used but they cannot provide quantitative physiological data. For this purpose, a new way of preparing the samples is required to preserve the concentration and distribution of these elements as close as possible to the in vivo state. Cryomethods are compulsory, though they demand more complex and often more expensive instruments and experimental requirements than the conventional methods. The analysis of biological fluids was made on frozen and freeze-dried droplets (Morel and Roinel, 1969; Roinel, 1975). For XRMA of tissue sections, cryomethods such as cryofixation and cryosubstitution followed by conventional resin embedding have been used by Zylberberg et al. (19851, Blaineau et al. (19871, Beaulaton et al. (19861, and Nicaise et al. (1989). Dehydration at progressively lower temperatures (PLT) and cryoembedding in HM20 or K4M Lowicryl resins has been used by Quintana et al. (1987). We are now testing in the CNRS laboratory of Ivry XRMA on dry cut sections after rapid cryofixation, freeze-substitution, and cryoembedding in the new Lowicryl resins, K l l M and HM23 (Quintana, 1991).Although it is generally considered abroad that cryosections of freshfrozen tissue are the most reliable way to preserve a physiological distribution of the diffusible ions, very few French publications were done with this technique, it is only used for a semiquantitative estimation in Meyran et al. (1986) and as a control of the freezesubstitution technique in Blaineau et al. (1987). EXPLOITATION OF XRMA DATA The study of the variation in different populations (cells or cellular organelles) of a certain quantitative character (XRMA data) involves the use of statistical concepts. All the classical statistical methods of data treatment are based on probabilistic hypotheses. In this respect it is worth noting the contribution of French authors (Ancey et al., 1975, 1977; Trebbia, 1989) to the establishment of better criteria in the de-

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termination of minimal detectable concentrations, to the control of optimization of measurements, and to the automatization of quantitative analysis. Other methods of data processing have recently been developed, called multivariate statistical analysis of data (MSA). These methods are essentially applied in cases where there are more than two variables; they represent a set of descriptive methods which use no starting point hypothesis, study individuals and characters of a population globally, and present the results graphically. They are fully widespread, especially the correspondence factorial analysis, developed by Benzecri (1969). These methods have been utilised in the processing of EPMA data by Ballan-Dufranqais et al. (1985) and Quintana and Ollacarizqueta (1989).

RESULTS A quantitative EPMA method for the study of biological fluids and isolated cells has been carried out in the Saclay Center (Morel and Roinel, 1969; Roinel, 1975,1981, 1988; Roinel and Desportes, 1979). The applications of this method in renal physiology are numerous (Morel et al., 1981; Roinel and Rouflignac, 1988; Rouffignac et al., 1984). The main achievements in the Creteil Center were published in the field of experimental and human pathology and invertebrate physiopathology, including the following. 1. Studies of pathological mineral accumulations in human tissues (Berry et al., 1976; Galle, 1964). 2. Studies on the toxicity of different metals: gold (Galle, 1974; Stuve and Galle, 1970), chrome (Berry et al., 19781,uranium (Galle, 19741,indium (Galle, 19811, and principally aluminum (Cournot-Witmer et al., 1986; Duckett et al., 1985; Galle, 1981,1986;Galle and Giudicelli, 1982; Galle et al., 1980, 1983, 1984; Plachot et al., 1984; Watrin and Galle, 1986). The main results on aluminum are related to its presence in different organs of hemodialysed patients: brain, kidney, liver, bone, parathyroid gland. 3. Studies of the role of lysosomes in mineral elements concentration in different cell types (Galle, 1974, 1983; Galle and Berry, 1980). 4. Studies of the role of spherocrystals in natural mineral bioaccumulations in insects (Ballan-Dufranqais, 1970, 1972; Martoja and Ballan-Dufrancais, 1984). 5. Studies of the role of spherocrystals and of lysosomes in bioaccumulations induced by heavy metals, Hg, Cd, Ag (Ballan-Dufrancais et al., 1979; Jeantet et al., 1974, 1977,1980, 1985; Lauberjat et al., 1989; Martoja et al., 1988a) or U (Chassard-Bouchaud, 1982).The incidence of these mechanisms was considered at the ecological level (Martoja et al., 197513) and particularly in pollution studies (Coulon et al., 1987; Martoja et al., 1984, 1986, 198813). Temporary associations of biologists with physicists or mathematicians have led to semiquantitative XRMA (Ballan-Dufranqais et al., 1980) and the applications of the MSA at the EPMA data (Ballan-Dufranqais et al., 1985). Information on the molecular form of the stored Hg was deduced from semiquantitative XRMA (Ballan-Dufrancais et al.,

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1980). The multivariate statistical methods showed a correlation between the occurrence of some elements and cell types as epithelial cells and hemocytes (Ballan-Dufranqais et al., 1985). 6. Cytochemical applications: XRMA has been used to validate cytochemical methods (Ballan-Dufrangais and Martoja, 1971; Berry et al., 1978,1982;Chastellier and Ryter, 1981; Martoja et al., 1975; Mentre and Halpern, 1988). 7. Studies of biomineralization: structure and composition of the quail egg shell (Quintana and Sandoz, 1978; Quintana et al., 1980), and mineral phase in teleost elasmoid scale (Zylberberg et al., 1985). The main results obtained in Lyon and Nice centers concern the study of subcellular calcium stores. 1. Calcium-containing membrane-bound granules in the glio-interstitial cells of annelids (Nicaise and Bilbaut, 1975) and molluscs (Blaineau et al., 1987; Keicher and Nicaise, 1988; Nicaise e t al., 1984). 2. Calcium storage in tooth soft tissue (Magloire and Joffre, 1979; Magloire and Nicaise, 1977). 3. Calcium binding sites in the mitochondria (Nicaise and Hernandez-Nicaise, 1980; Hernandez-Nicaise et al., 1982) and under the plasma membrane (Nicaise et al., 1982) of the giant smooth muscle cell, discovered in a planctonic invertebrate. 4. Calcium-t,ransporting epithelium in a terrestrial crustacean (Meyran et al., 1984, 1986). 5 . Calcium in programmed cell death of insect muscles (Beaulaton, 1986, 1988; Beaulaton et al., 1986) or postmortem evolution of mammalian muscle (Vignon et al., 1989). 6. Effect of OsO, postfixation on the calcium content of cardiac tissue (Blaineau et al., 1988). 7. Calcium distribution in the sea urchin egg (Gillot et al., 1989).

Since 1984 this group has been making use of cryofixation and cryosubstitution in the presence of oxalic acid which permits in situ immobilization of calcium (Beaulaton e t al., 1986; Blaineau e t al., 1987; Gillot et al., 1989; Nicaise et al., 1984, 1989) or barium (Amsellem et al., 1988). Since 1985 and within the international cooperation program between CNRS, INSERM (France), and CSIC (Spain), one of us (C.Q.) has been able to work with transmission analytical microscopes fitted with a n EDS system (Phillips EM 420 + Edax 9100 a t CIB, Madrid, and Hitachi H800 + Kevex 8000 at Barcelona University). This study was focused on the composition of animal cell nuclei, with the following results. 1. The chromatin can be distinguished from the nucleolus by different SIP values. 2. The metals, Al, Fe, Cu, and Zn regularly occur in cell nuclei (Quintana e t al., 1987). 3. A nucleolar component is richer in sulphur-containing proteins and A1 and Zn metals (Quintana, 1991; Quintana and Ollacarizqueta, 1989).

CONCLUSION If we keep in mind the international trends in biological XRMA, not reviewed in this short survey, it is possible to suggest that the poor bibliographic impact of French science in the field (save a few exceptions) was probably due to a lack of physiological and quantitative studies. With the recent acquisitions of instruments and the development of cryotechniques, particularly in the Reims INSERM unit and in Nice University, we should expect a rapid development of biological applications of XRMA in physiopathology that will update French research in a few years. At the same time, it can be expected that XRMA will become less a specialized field and hopefully will become more integrated i n general studies involving other cell and molecular biology techniques.

ACKNOWLEDGMENTS We would like to thank Mrs. Isabelle Angelchic for typing this manuscript.

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Biological applications of electron probe microanalysis in France.

The contribution of French science to the field of biological X-ray microanalysis is reviewed. The main analytical microscopy centers are listed, and ...
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