Cell Tiss. Res. 166, 255-263 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Silicon in Rat Liver Organelles: Electron Probe Microanalysis* Charles W. Mehard and Benjamin E. Volcani** Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California, USA
Summary. Electron probe microanalysis of unfixed freeze-substituted rat liver tissue embedded in Spurr's low viscosity epoxy resin demonstrated the occurrence of Si as well as P, S, and C1 in the nucleus, nucleolus, mitochondria, and rough endoplasmic reticulum. Chemical analysis confirmed that the Si in the organelles did not originate from instrumental contaminants. This suggests that Si may be involved in the biochemistry of these subcellular organelles. Key words: Silicon - X-ray microanalysis - Rat liver - Cell organelles - Freeze substitution.
Introduction
The biological significance of silicon (Si) is by now well established. It is required for shell formation (Richter, 1906; Lewin, 1955) and DNA synthesis in diatoms (Darley and Volcani, 1969; Sullivan and Volcani, 1973). It is needed for normal growth in certain plants (Takahashi, 1968; Chen and Lewin, 1969) and is essential for bone formation and other developmental processes in higher animals (Carlisle, 1974; Schwartz, 1974). Studies of Si metabolism, however, have been seriously hampered by a variety of difficulties, among them (1) the short half life of a~Si which limits its use as a tracer; (2) its easy extractability in aqueous Send offprint requests to. Dr. Charles W. Mehard or Dr. B.E. Volcani, Scripps Institution of Oceanography A-002, La Jolla, CA 92093, U.S.A. * Supported by Grant GM-08229-12-13 from the National Institutes of Health, USPHS. ** We are grateful to the Kevex Corporation and Mr. Glenn W. Meyer, Sales Engineer, for the use of the Kevex X-ray spectrometer; we wish to thank as well the Perkin-Elmer Corporation and their Western Branch Manager, Mr. Michael E. Mullen and Senior Microscopist, Mr. Minoru Shinorhara, for use of and assistance with the Hitachi HU 12A transmission electron microscope. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and the X-ray photographs, and Darlene Lum for technical assistance in the laboratory.
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media during organelle isolation and sample preparation (Mehard et al., 1974a; Mehard and Volcani, 1975a, b); and (3) its biological ubiquity which requires that it be followed at the subcellular level and, in addition, raises the question of contamination. A very promising tool for in situ Si studies is offered by electron probe microanalysis, since it allows very minute amounts (10- t8 g) of specific elements to be identified by their characteristic X-ray emissions. Thus the presence of Si has been detected in cells of animals (Schafer and Chandler, 1970; Weavers, 1973; Mehard and Volcani, 1974c, 1975b), plants (Kaufman et al., 1972; Hayward and Parry, 1973; Soni and Parry, 1973), and diatoms (Mehard and Volcani, 1974c, 1975b). To prevent the loss of Si during sample preparation, a method was developed for attaining maximum subcellular retention and stabilization of Si without unacceptable damage to the ultrastructure (Mehard and Volcani, 1975b). It involves freeze substitution, infiltration of the tissues with glycerol to reduce ice damage, and embedment in Spurr's low viscocity epoxy resin. Si X-ray emission signals from samples of mitochondria so prepared were shown to be intrinsic and not an artifact of contamination. This paper reports a more detailed electron probe microanalysis study on freeze-substituted rat liver cells to determine the subcellular localization sites of Si and other elements. Materials and Methods Animals. Sprague-Dawley rats (400-500 g) obtained from a commercial producer, were fed Purina Chow and water ad libitum. Organelle Isolation. Nuclei were isolated by isopycnic sucrose gradients (Mehard and Packer, 1974a). Mitochondria (Mehard et al., 1971) and microsomes (Mehard and Packer, 1974a) were isolated in 0.33 M sucrose media as previously reported. Electron Microscopy. Unfixed tissues were prepared by freeze substitution as follows: Thin (1 mm) tissue pieces were glycerinated for 40 min in 30% glycerol-0.33 M sucrose medium before being frozen at liquid N2 temperature in isopentane, and dehydrated at - 8 0 ~ in anhydrous diethylether which was changed three times during the 10-day dehydration period. Samples were brought to room temperature, equilibrated over night with vinyl cyclohexene dioxide (a component of Spurr's low viscosity embedding medium) and then equilibrated in Spurr's low viscosity embedding medium-E (Spurr, 1969) for three days prior to polymerization at 70~ C for 24 h. Thin sections were cut onto water with a diamond knife on an LKB Ultratome, mounted on copper grids coated with collodion and carbon, post stained with uranyl acetate (Watson, 1958) and analyzed by the X-ray microprobe. To obtain sufficient contrast for micrographs, the thin sections were also post stained with lead citrate (Reynolds, 1963) and examined with a Siemens Elmiskop I at 80 kV. Electron Probe X-ray Microanalysis. Thin sections (0.1 gm) of the samples were viewed with a Hitachi HU-12A transmission electron microscope (TEM) (equipped with 2 liquid nitrogen cold traps and a vacuum pump oil devoid of silicon) and assayed with the Kevex X-ray energy dispersive microanalysis attachment 5,000 A X-ray spectrometer equipped with the Kevex 6,000 data processor and Kevex 5370 monochrome video display (Kevex Corporation, Burlingame, California). The electron beam (0.3 lam in diameter) operating at 25 kV with a beam current of 25 gamp, was used as a static probe. Protein Determination. Protein was determined by the biuret method (Gornall etal., 1949) with crystalline bovine serum albumin (Sigma, St. Louis, Mo.) as the standard. Protein in the nuclear fractions was determined spectrophotometrically (Groves et al., 1968).
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Fig. la-L Electron micrographs and X-ray spectrograms of rat liver organelles (a, b) nucleus, nucleolus (x 9,800); (c, d) mitochondria (x 70,000); (e, f) rough endoplasmic reticulum (x 60,000). Nucleus is from non-glycerinated tissue; mitochondria and endoplasmic reticulum from 30% glycerol treated tissue Chemical Assay of Si and P. Si was determined colorimetrically according to Heinen (1960); to eliminate the contribution of phosphate the procedure of Jankowiak and LeVier (1971) was used. Phosphate was determined in the same samples according to Lindeman (1958).
Results Ultrastructural Preservation. T h e freeze s u b s t i t u t i o n m e t h o d a d e q u a t e l y preserves cellular u l t r a s t r u c t u r e for electron p r o b e microanalysis, b u t the organelles, espe-
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cially the mitochondria and endoplasmic reticulum, are often damaged. Some regions of the cell escape damage and more consistent preservation of these organelles is obtained by infiltrating the tissue with glycerol. However, in some preparations this affects the stability of the nuclei which show a tendency to disintegrate. An intact nucleus with well defined nucleolus can be seen (Fig. 1a) as well as relatively well preserved mitochondria and rough endoplasmic reticulum (Fig. 1c, e). Golgi elements are not easily distinguished because some vesiculation of the endoplasmic reticulum makes differentiation between the two structures difficult, particularly if vesiculation occurs in a region of the cell where ice crystals are prevalent. However, rough endoplasmic reticulum is well defined, with the uranium stained ribosomes quite prominent on the diffusely stained membrane. Unlike the well-defined double lamellar stained structures observed in conventionally fixed preparations, in the unfixed freeze substituted preparations membranes of all the organelles appear as diffuse electron transparent boundaries with remnants of electron opaque regions. The double membrane envelope of the nucleus is seen as a single diffuse line. As was previously found (Mehard and Volcani, 1975 b) mitochondrial membranes of the freeze substituted preparations show the characteristic cristae, the dark staining matrix region, and the outer limiting membrane region which appear as a light band surrounded by a gray diffuse membrane. The plasmalemma is observed as a diffuse gray line. X-ray Microanalysis. Though infiltration with glycerol prior to freezing provides better retention of cellular Si, some inter- and intracellular translocation during tissue preparation cannot be ruled out. Electron probe X-ray microanalysis of the in situ organelles shows that the nucleus (Fig. 1b, left) contains Si, P, S, and some A1. The U signal of the uranium surface-stain, which is prominent, masks the K signal, since characteristic X-rays for both occur at the same energy region; the Cu signal comes from the support grid. The nucleolus (Fig. 1b, right) contains Si, S, and C1 but the P signal is greatly reduced as compared to that in the nucleus, and the A1 peak is absent. When counts of several anaylses, corrected for background emissions (Table 1) are compared with the spectra, the Si signals are similar for nucleus and nucleolus, but those for S, C1, and especially P, are higher in the nucleus, a confirmation of the differences observable in Fig. 1b. The high standard deviations indicate that the counts vary considerably from one region to the next and suggest differential localization and/or variations in distribution within the organelle. Mitochondria (Fig. 1d) also show peaks for Si, P, S, and C1 though, as Table 1 shows more clearly, with the exception of S they are much lower than those of the nucleus. The rough endoplasmic reticulum (Fig. 1 f) contains Si, P and S, and low C1 (Table 1). The AI peak may represent an artifact of instrumentation or, more probably, actual accumulation in situ since it is not observed in all the organelles or in the embedment. Data in Table 1 show that compared to the mitochondria, the rough endoplasmic reticulum contains more Si, similar S and P and less C1; it differs from the nucleus in containing less S, P and C1.
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Table l. Electron probe analysis of in situ liver cell organelles prepared by freeze-substitution
Organelle
Counts (P-b)s - (P-b)E Si
P
S
C1
Nucleus (5) Nucleolus (4) Mitochondria(6) Endoplasmic Reticulum(6)
49.6+ 12.3 48.8_+ 8.7 26.2_+ 6.9 65.0_+31.0
199.0_+ 15.9 65.0_+ 3.7 23.7_+ 5.2 22.2-+ 4.5
85.8_+34.5 40.2_+ 15.3 34.2-+ 6.3 39.0_+ 6.3
131.0+ 6.0 100.5+23.2 28.5_+ 6.2 18.5-+ 6.2
Embedment (4)
37.0+26.0
5.0_+ 5.0
32.0_+ 7.0
17.0-+ 12.0
Organelles were assayed in the Hitachi HU-12 A transmission electron microscope. Probe size: 0.3 gm in diameter, Current: 35 p.amp, accelerating voltage: 25 kV, Counting time: 200 sec. X-ray counts were determined with the Kevex energy dispersive spectrometer. The number of counts under the elemental peak was integrated over the counting interval (P) using a window of 7 channels each of 10 ev/channel. The background counts (b) of the sample(s) were integrated over the same interval and subtracted, (P-b)s. The value of embedment (E) counts above background (P-b)E was subtracted to give the reported values, +standard deviation of the mean. Numbers in parentheses are number of examinations.
Electron probe microanalysis of the embedment (Table 1) shows that the Si, P, S and C1 counts in the epoxy resin adjacent to the tissue are usually less than half those in the organelles, with the exception of the Si in the mitochondria. Counts of these elements in thin sections of the epoxy resin alone were much lower than those of the embedment or absent entirely, and the carbon coated collodion gave no counts. Hence the embedment counts could have come either from diffusible constituents of the cell which translocated during embedment or, from scattered electrons arising from the copper grid and/or the biological material which had been surface stained with uranium. In analyzing the tissue samples, it was found that the Cu signal was usually strongest when the electron beam was closest to the edge of the copper grid; Fig. 1 b, d, f shows a sequential decline in the signal which corresponds to the distance of the sample from the Cu support grid, the nucleolus being closest and the rough endoplasmic reticulum farthest. Such scattering was corrected for by subtracting the signal minus background obtained from the resin adjacent to the embedded tissue. The mean counts of four assays of the embedment (Table l) were subtracted from the peak-minus-background (P-b) of the sample. Chemical Analysis. To confirm that the Si X-ray signal was not an instrumental artifact (Sutfin et al., 1971; Thurston and Russ, 1971), cell organelles were isolated and analyzed for Si by the silicomolybdate colorimetric method, specifically designed for determining Si in the presence of the P levels found in most animal tissues (Jankowiak and LeVier, 1971). Phosphorus was also analyzed since it was shown to manifest some biochemical similarities to Si in bacteria (Heinen, 1962). The highest concentrations of Si and P occur in the nucleus, microsomes, and mitochondria, in that order, and are strikingly higher in the nucleus than in the other two (Table 2). When the Si:P ratios of the three organelles are compared with those calculated from the amounts (counts) in
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Table 2. Silicon and phosphorus content of isolated rat liver organelles
Cell fractions
Si"
pb
Si/P
(gg/mg protein) Nuclei Mitochondria Microsomes
3.00 c 0.44 0.48
165.4 8.8 13.6
0.018 0.050 0.035
a Jankowiak and LeVier (1971) method of Si assay. b Lindeman (1958) method of P assay. Heinen (1960) method of Si assay.
Table 1 (0.25, 1.11, and 2.93, respectively), a marked difference is apparent. This difference may reflect the loss of Si during organelles isolation in aqueous medium (Mehard et al., 1974b). Discussion
The present study confirms our previous conclusions (Mehard and Volcani, 1975 b) that, despite some ice damage, the freeze substitution method adequately maintains subcellular compartmentation and reduces Si loss from in situ or isolated organelles, and that electron probe microanalysis offers the best method of detecting in situ Si at the subcellular level. The ice damage by incurred by the in situ mitochondria and endoplasmic reticulum has also been observed in plant tissues following similar treatment (L/iuchli etal., 1970; Hereward and Northcote, 1972; Spurr, 1972; Pallaghy, 1973). Reduction of damage by infiltrating the tissue with glycerol, a cryoprotectant, undoubtedly alters the cell physiology. Maintenance of cell-organelle ultrastructure is comparable to that of the glutaraldehyde fixed, freeze dried rat liver tissue of Weavers (1973) prepared by cryo-ultramicrotomy, though no ribosomes were seen on the cisternae of the endoplasmic reticulum with his method. Si was retained in the mitochondria, nucleus, and rough endoplasmic reticulum during tissue preparation. The occurrence of Si in organelles has been reported from studies of 3asi uptake in situ in diatoms (Mehard et al., 1974b) and rat tissues (Mehard and Volcani, 1975a) and by electron probe X-ray microanalysis of other biological material (Schafer and Chandler, 1970; Weavers, 1973; Dempsey et al., 1973), though the question of contamination could not be ignored. However, the finding of our previous study that the presence of Si organelles is not the result of contamination is substantiated in this study both by the use of an electron probe instrument free of Si and by chemical analysis of the organelles. Si-content of a tissue or cell depends on the nutritional status of the animal at the time of sampling, and therefore variations of more than 100% can occur in the content of this freely permeant element (LeVier, 1975; Mehard and Volcani, 1975a). The finding that the nucleus contains more Si than do the
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261
mitochondria or rough endoplasmic reticulum is in agreement with the report of LeVier (1975) for rat liver. It is difficult to assess the absolute amount of Si in organelles, since some of the element is lost during isolation (Mehard et al., 1974b). When 6 a G e w a s used as a tracer of Si, the mitochondria incurred no loss of label while loss from nuclei and microsomes was only 6% and 11% respectively (Mehard and Volcani, 1975b). Thus, analysis of elements (Si) in situ with the electron probe X-ray microanalysis technique gives a more accurate assessment of the elemental composition than does the chemical analysis of isolated organelles. This is substantiated by the fact that Si:P ratios, as shown by counts in in situ organelles (Table 1) are higher than those in isolated organelles (Table 2). The detection of S, C1, and P demonstrates that the preparation method provides good retention, since these elements are to be expected in organelles of any tissue: the S signal indicates sulfur protein (Jessen etal., 1974); the C1 is present in situ as a component of body fluids. However, Van Steveninck et al. (1974) demonstrated with the EMMA-4 (AEI) that Spurr's epoxy resin contains C1. The embedment counts in the present study are much lower than those reported by Van Steveninck which may be due to differences in X-ray generation, efficiency of collection, and/or the embedment material. The C1 counts from the organelles are sufficiently above the embedment to be considered a cellular constituent, and are similar to those reported from other microprobe studies (L~iuchli et al., 1970; Pallaghy, 1973; Dempsey et al., 1973). The presence of P, another element of biological importance, is also to be expected in detectable amounts; i.e., in the nucleus as a component of DNA and RNA, and in the mitochondria and endoplasmic reticulum as a component of phospholipids, nucleotides, and/or polyphosphates. The AI X-ray signal is probably from in situ accumulated A1 and though it could be an artifact of instrumentation it is not observed in all the organelles or in the embedment. Where A1 has been reported in the X-ray spectra of other tissues it has either been ignored or considered an artifact (Sutfin et al., 1971 ; Dempsey et al., 1973). Not enough is known at present about the biological role of Si to explain its occurrence in these organelles. It has been hypothesized that the mitochondria may participate in calcification (Lehninger, 1970), and it is known that the mitochondria can accumulate calcium and phosphate, both of which are involved in mineralization (Becker et al., 1974; Borle 1973; Chen et al., 1974). It has been shown that 3 i S i is taken up by mitochondria of diatoms and of rat liver, spleen and kidney (Mehard et al., 1974a; Mehard and Volcani, 1975a). The mitochondrion may therefore play some part in the silicification of the diatom cell walls and, since Si is required for bone formation (Carlisle, 1974; Schwartz, 1974) this organelle may be involved in a possible S i - C a relationship as well. References Becker, G.L., Chen, C.H., Greenawalt, J.W., Lehninger, A.L.: Calcium phosphate granules in the hepatopancreas of the blue carb Callinectes sapidus. J. Cell Biol. 61, 316-326 (1974) Borle, A.B. : Calcium metabolism at the cellular level. Fed. Proc. 32, 1944-1950 (1973) Carlisle, E.M.: Silicon as an essential element. Fed. Proc. 33, 1758-1766 (1974)
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Chen, C.H., Greenawalt, J.W., Lehninger, A.L. : Biochemical and ultrastructural aspects of Ca 2+ transport by mitochondria of the hepatopancreas of the blue crab Callinectes sapidus. J. Cell Biol. 61, 301-315 (1974) Chen, C.H., Lewin, J. : Silicon as a nutrient element for Equisetum arvense. Canad. J. Bot. 47, 125-131 (1969) Darley, W.M., Volcani, B.E.: Role of silicon in diatom metabolism. A silicon requirement for deoxyribonucleic acid synthesis in the diatom Cylindrotheca fusiformis. Reimann and Lewin. Exp. Cell Res. 58, 334-342 (1969) Dempsey, E.W., Agate, F.J. Jr., Lee, M., Purkerson, M.L.: Analysis of submicroscopic structures by their emitted X-rays. J. Histochem. Cytochem. 21, 580-586 (1973) Gornall, A.G., Bardawill, C.J., David, M.M. : Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751-766 (1949) Groves, W.E., Davis, F.C., Sells, B.: Spectrophotometric determination of microgram quantities of protein without nucleic acid interference. Analyt. Biochem. 22, 195-210 (1968) Hayward, D.M., Parry, D.W. : Electron probe microanalysis studies of silica distribution in barley (Hordeum sativum L). Ann. Bot. 37, 579 591 (1973) Heinen, W. : Silicium-Stoffwechsel bei Mikroorganismen. I. Aufnahme von Silicium durch Bakterien. Arch. Mikrobiol. 37, 199-210 (1960) Heinen, W. : Siliciumstoffwechsel bei Mikroorganismen. II. Beziehungen zwischen dem Silicat- und Phosphat-Stoffwechsel bei Bakterien. Arch. Mikrobiol. 41, 229-246 (1962) Hereward, F.V., Northcote, D.H. : A simple freeze substitution method for the study of ultrastructure of plant tissue. Exp. Cell Res. 70, 73-80 (1972) Jankowiak, M.E., LeVier, R.R. : Elimination of phosphorus interference in the colorimetric determination of silicon in biological material. Analyt. Biochem. 44, 462472 (1971) Jessen, H., Peters, P.D., Hall, T.A. : Sulphur in different types of keratohyaline granules: A quantitative assay by X-ray microanalysis. J. Cell Sci. 15, 359-377(1974) Kaufman, P.B., Soni, S.L., LaCroix, J.D., Rosen, J.J., Bigelow, W.C. : Electron probe microanalysis of silicon in the epidermis of rice (Oryza sativa L.) internodes. Planta (Berl.) 104, 10-17 (1972) L~iuchli, A., Spurr, A.R., Wittkopp, R.W.: Electron probe analysis of freeze substituted, epoxy resin embedded tissue for ion transport studies in plants. Planta (Berl.) 95, 341-350 (1970) Lehninger, A.L. : Mitochondria and calcium ion transport. Biochem. J. 119, 129-135 (1970) LeVier, R. : Distribution of silicon in the adult rat and rhesus monkey. Bioinorg. Chem. 4, 109-115 (1975) Lewin, J.C. : Silicon metabolism in diatoms. III. Respiration and silicon uptake in Navicula pelliculosa. J. gen. Physiol. 39, 1-10 (1955) Lindeman, W. : Observations on the behavior of phosphate compounds in Chlorella at the transition from dark to light. Proc. Sec. Int. Conf. UN on the Peaceful Uses of Atomic Energy 24, 8-15 (1958) Mehard, C.W., Packer, L.: Induction of cytochrome P-450 and P-448 in the outer membrane of mouse liver nuclei by phenobarbital and benzo[~]pyrene. Bioenergetics 6, 151-160 (1974a) Mehard, C.W., Packer, L., Abraham, S.: Activity and ultrastructure of mitochondria from mouse mammary gland and mammary adenocarcinoma. Cancer Res. 31, 2148-2160 (1971) Mehard, C.W., Sullivan, C.W., Azam, F., Volcani, B.E.: Role of silicon in diatom metabolism. IV. Subcellular localization of silicon and germanium in Nitzschia alba and Cylindrotheca fusiformis. Physiol. Plant. 30, 265-272 (1974b) Mehard, C.W., Volcani, B.E.: Electron probe X-ray microanalysis of subcellular silicon in rat tissues and the diatom, Cylindrotheeafusiformis. J. Cell Biol. 63, 220 (1974c) Mehard, C.W., Volcani, B.E.: Similarity in uptake and retention of trace amounts of 31Si and 68Ge by rat tissues and cell organelles. Bioinorg. Chem. 5, 107-124 (1975a) Mehard, C.W., Volcani, B.E.: Evaluation of silicon and germanium retention in rat tissues and diatoms during cell and organelle preparation for electron probe microanalysis. J. Histochem. Cytochem. 23, 348-358 (1975b) Pallaghy, C.K.: Electron probe microanalysis of potassium and chloride in freeze substituted leaf sections of Zea mays. Aust. J. biol. Sci. 26, 1015-1034 (1973) Reynolds, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963)
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Richter, O. : Zur Physiologic der Diatomeen (I. Mitteilung). Sitzber. Kais. Akad. Wiss. Wien, math.-nat. K1. 115, 27-119 (1906) Schafer, P.W., Chandler, J.A.: Electron probe X-ray microanalysis of a normal centriole. Science 170, 1204-1205 (1970) Schwartz, K.: Recent dietary trace element research, exemplified by tin, fluorine, and silicon. Fed. Proc. 33, 1748-1757 (1974) Soni, S.L., Parry, D.W.: Electron probe microanalysis of silicon deposition in the inflorescence bracts of the rice plant (Oryza sativa). Amer. J. Bot. 60, 111-116 (1973) Spurr, A.R. : A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) Spurr, A.R.: Freeze substitution additives for sodium and calcium retention in cells studied by X-ray analytical electron microscopy. Bot. Gaz. 133, 263-270 (1972) Sullivan, C.W., Volcani, B.E.: Role of silicon in diatom metabolism. III. The effects of silicic acid on DNA polymerase, TMP kinase and DNA synthesis in Cylindrotheeafusiformis. Biochim. biophys. Acta (Amst.) 308, 212-219 (1973) Sutfin, L.V., Holtrop, M.E., Ogilvie, R.E. : Microanalysis of individual rnitochondrial granules with diameters less than 1000 angstroms. Science 174, 947-949 (1971) Takahashi, E.: Silica as a nutrient to the rice plant. Japan Agric. Res. Quart. 3, 1-4 (1968) Thurston, E.L., Russ, J.C.: Scanning and transmission electron microscopy and microanalysis of structured granules in Fischerella ambigua. In: Proc. 4th Ann. Scanning EM Symposium, p. 511 516. Chicago, Ill., ITT Res., Inst. 1971 Van Steveninck, R.F.M., Van Steveninck, M.E., Hall, T.A., Peters, P.D. : A chlorine-free embedding medium for use in X-ray analytical electron microscope localization of chlorine in biological tissues. Histochem. 38, 173-180 (1974) Watson, M.L.: Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, 475-478 (1958) Weavers, B.A.: Combined transmission electron microscopy and X-ray microanalysis of ultrathin frozen dried sections-an investigation to determine the normal elemental compositon of mammalian tissue. J. Microscopy 97, 331-341 (1973)
Received September 16, 1975
Cell and Tissue Research 9 by Springer-Verlag 1976
Silicon in Rat Liver Organelles: Electron Probe Microanalysis* Charles W. Mehard and Benjamin E. Volcani** Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California, USA
Summary. Electron probe microanalysis of unfixed freeze-substituted rat liver tissue embedded in Spurr's low viscosity epoxy resin demonstrated the occurrence of Si as well as P, S, and C1 in the nucleus, nucleolus, mitochondria, and rough endoplasmic reticulum. Chemical analysis confirmed that the Si in the organelles did not originate from instrumental contaminants. This suggests that Si may be involved in the biochemistry of these subcellular organelles. Key words: Silicon - X-ray microanalysis - Rat liver - Cell organelles - Freeze substitution.
Introduction
The biological significance of silicon (Si) is by now well established. It is required for shell formation (Richter, 1906; Lewin, 1955) and DNA synthesis in diatoms (Darley and Volcani, 1969; Sullivan and Volcani, 1973). It is needed for normal growth in certain plants (Takahashi, 1968; Chen and Lewin, 1969) and is essential for bone formation and other developmental processes in higher animals (Carlisle, 1974; Schwartz, 1974). Studies of Si metabolism, however, have been seriously hampered by a variety of difficulties, among them (1) the short half life of a~Si which limits its use as a tracer; (2) its easy extractability in aqueous Send offprint requests to. Dr. Charles W. Mehard or Dr. B.E. Volcani, Scripps Institution of Oceanography A-002, La Jolla, CA 92093, U.S.A. * Supported by Grant GM-08229-12-13 from the National Institutes of Health, USPHS. ** We are grateful to the Kevex Corporation and Mr. Glenn W. Meyer, Sales Engineer, for the use of the Kevex X-ray spectrometer; we wish to thank as well the Perkin-Elmer Corporation and their Western Branch Manager, Mr. Michael E. Mullen and Senior Microscopist, Mr. Minoru Shinorhara, for use of and assistance with the Hitachi HU 12A transmission electron microscope. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and the X-ray photographs, and Darlene Lum for technical assistance in the laboratory.
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media during organelle isolation and sample preparation (Mehard et al., 1974a; Mehard and Volcani, 1975a, b); and (3) its biological ubiquity which requires that it be followed at the subcellular level and, in addition, raises the question of contamination. A very promising tool for in situ Si studies is offered by electron probe microanalysis, since it allows very minute amounts (10- t8 g) of specific elements to be identified by their characteristic X-ray emissions. Thus the presence of Si has been detected in cells of animals (Schafer and Chandler, 1970; Weavers, 1973; Mehard and Volcani, 1974c, 1975b), plants (Kaufman et al., 1972; Hayward and Parry, 1973; Soni and Parry, 1973), and diatoms (Mehard and Volcani, 1974c, 1975b). To prevent the loss of Si during sample preparation, a method was developed for attaining maximum subcellular retention and stabilization of Si without unacceptable damage to the ultrastructure (Mehard and Volcani, 1975b). It involves freeze substitution, infiltration of the tissues with glycerol to reduce ice damage, and embedment in Spurr's low viscocity epoxy resin. Si X-ray emission signals from samples of mitochondria so prepared were shown to be intrinsic and not an artifact of contamination. This paper reports a more detailed electron probe microanalysis study on freeze-substituted rat liver cells to determine the subcellular localization sites of Si and other elements. Materials and Methods Animals. Sprague-Dawley rats (400-500 g) obtained from a commercial producer, were fed Purina Chow and water ad libitum. Organelle Isolation. Nuclei were isolated by isopycnic sucrose gradients (Mehard and Packer, 1974a). Mitochondria (Mehard et al., 1971) and microsomes (Mehard and Packer, 1974a) were isolated in 0.33 M sucrose media as previously reported. Electron Microscopy. Unfixed tissues were prepared by freeze substitution as follows: Thin (1 mm) tissue pieces were glycerinated for 40 min in 30% glycerol-0.33 M sucrose medium before being frozen at liquid N2 temperature in isopentane, and dehydrated at - 8 0 ~ in anhydrous diethylether which was changed three times during the 10-day dehydration period. Samples were brought to room temperature, equilibrated over night with vinyl cyclohexene dioxide (a component of Spurr's low viscosity embedding medium) and then equilibrated in Spurr's low viscosity embedding medium-E (Spurr, 1969) for three days prior to polymerization at 70~ C for 24 h. Thin sections were cut onto water with a diamond knife on an LKB Ultratome, mounted on copper grids coated with collodion and carbon, post stained with uranyl acetate (Watson, 1958) and analyzed by the X-ray microprobe. To obtain sufficient contrast for micrographs, the thin sections were also post stained with lead citrate (Reynolds, 1963) and examined with a Siemens Elmiskop I at 80 kV. Electron Probe X-ray Microanalysis. Thin sections (0.1 gm) of the samples were viewed with a Hitachi HU-12A transmission electron microscope (TEM) (equipped with 2 liquid nitrogen cold traps and a vacuum pump oil devoid of silicon) and assayed with the Kevex X-ray energy dispersive microanalysis attachment 5,000 A X-ray spectrometer equipped with the Kevex 6,000 data processor and Kevex 5370 monochrome video display (Kevex Corporation, Burlingame, California). The electron beam (0.3 lam in diameter) operating at 25 kV with a beam current of 25 gamp, was used as a static probe. Protein Determination. Protein was determined by the biuret method (Gornall etal., 1949) with crystalline bovine serum albumin (Sigma, St. Louis, Mo.) as the standard. Protein in the nuclear fractions was determined spectrophotometrically (Groves et al., 1968).
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Fig. la-L Electron micrographs and X-ray spectrograms of rat liver organelles (a, b) nucleus, nucleolus (x 9,800); (c, d) mitochondria (x 70,000); (e, f) rough endoplasmic reticulum (x 60,000). Nucleus is from non-glycerinated tissue; mitochondria and endoplasmic reticulum from 30% glycerol treated tissue Chemical Assay of Si and P. Si was determined colorimetrically according to Heinen (1960); to eliminate the contribution of phosphate the procedure of Jankowiak and LeVier (1971) was used. Phosphate was determined in the same samples according to Lindeman (1958).
Results Ultrastructural Preservation. T h e freeze s u b s t i t u t i o n m e t h o d a d e q u a t e l y preserves cellular u l t r a s t r u c t u r e for electron p r o b e microanalysis, b u t the organelles, espe-
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cially the mitochondria and endoplasmic reticulum, are often damaged. Some regions of the cell escape damage and more consistent preservation of these organelles is obtained by infiltrating the tissue with glycerol. However, in some preparations this affects the stability of the nuclei which show a tendency to disintegrate. An intact nucleus with well defined nucleolus can be seen (Fig. 1a) as well as relatively well preserved mitochondria and rough endoplasmic reticulum (Fig. 1c, e). Golgi elements are not easily distinguished because some vesiculation of the endoplasmic reticulum makes differentiation between the two structures difficult, particularly if vesiculation occurs in a region of the cell where ice crystals are prevalent. However, rough endoplasmic reticulum is well defined, with the uranium stained ribosomes quite prominent on the diffusely stained membrane. Unlike the well-defined double lamellar stained structures observed in conventionally fixed preparations, in the unfixed freeze substituted preparations membranes of all the organelles appear as diffuse electron transparent boundaries with remnants of electron opaque regions. The double membrane envelope of the nucleus is seen as a single diffuse line. As was previously found (Mehard and Volcani, 1975 b) mitochondrial membranes of the freeze substituted preparations show the characteristic cristae, the dark staining matrix region, and the outer limiting membrane region which appear as a light band surrounded by a gray diffuse membrane. The plasmalemma is observed as a diffuse gray line. X-ray Microanalysis. Though infiltration with glycerol prior to freezing provides better retention of cellular Si, some inter- and intracellular translocation during tissue preparation cannot be ruled out. Electron probe X-ray microanalysis of the in situ organelles shows that the nucleus (Fig. 1b, left) contains Si, P, S, and some A1. The U signal of the uranium surface-stain, which is prominent, masks the K signal, since characteristic X-rays for both occur at the same energy region; the Cu signal comes from the support grid. The nucleolus (Fig. 1b, right) contains Si, S, and C1 but the P signal is greatly reduced as compared to that in the nucleus, and the A1 peak is absent. When counts of several anaylses, corrected for background emissions (Table 1) are compared with the spectra, the Si signals are similar for nucleus and nucleolus, but those for S, C1, and especially P, are higher in the nucleus, a confirmation of the differences observable in Fig. 1b. The high standard deviations indicate that the counts vary considerably from one region to the next and suggest differential localization and/or variations in distribution within the organelle. Mitochondria (Fig. 1d) also show peaks for Si, P, S, and C1 though, as Table 1 shows more clearly, with the exception of S they are much lower than those of the nucleus. The rough endoplasmic reticulum (Fig. 1 f) contains Si, P and S, and low C1 (Table 1). The AI peak may represent an artifact of instrumentation or, more probably, actual accumulation in situ since it is not observed in all the organelles or in the embedment. Data in Table 1 show that compared to the mitochondria, the rough endoplasmic reticulum contains more Si, similar S and P and less C1; it differs from the nucleus in containing less S, P and C1.
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Table l. Electron probe analysis of in situ liver cell organelles prepared by freeze-substitution
Organelle
Counts (P-b)s - (P-b)E Si
P
S
C1
Nucleus (5) Nucleolus (4) Mitochondria(6) Endoplasmic Reticulum(6)
49.6+ 12.3 48.8_+ 8.7 26.2_+ 6.9 65.0_+31.0
199.0_+ 15.9 65.0_+ 3.7 23.7_+ 5.2 22.2-+ 4.5
85.8_+34.5 40.2_+ 15.3 34.2-+ 6.3 39.0_+ 6.3
131.0+ 6.0 100.5+23.2 28.5_+ 6.2 18.5-+ 6.2
Embedment (4)
37.0+26.0
5.0_+ 5.0
32.0_+ 7.0
17.0-+ 12.0
Organelles were assayed in the Hitachi HU-12 A transmission electron microscope. Probe size: 0.3 gm in diameter, Current: 35 p.amp, accelerating voltage: 25 kV, Counting time: 200 sec. X-ray counts were determined with the Kevex energy dispersive spectrometer. The number of counts under the elemental peak was integrated over the counting interval (P) using a window of 7 channels each of 10 ev/channel. The background counts (b) of the sample(s) were integrated over the same interval and subtracted, (P-b)s. The value of embedment (E) counts above background (P-b)E was subtracted to give the reported values, +standard deviation of the mean. Numbers in parentheses are number of examinations.
Electron probe microanalysis of the embedment (Table 1) shows that the Si, P, S and C1 counts in the epoxy resin adjacent to the tissue are usually less than half those in the organelles, with the exception of the Si in the mitochondria. Counts of these elements in thin sections of the epoxy resin alone were much lower than those of the embedment or absent entirely, and the carbon coated collodion gave no counts. Hence the embedment counts could have come either from diffusible constituents of the cell which translocated during embedment or, from scattered electrons arising from the copper grid and/or the biological material which had been surface stained with uranium. In analyzing the tissue samples, it was found that the Cu signal was usually strongest when the electron beam was closest to the edge of the copper grid; Fig. 1 b, d, f shows a sequential decline in the signal which corresponds to the distance of the sample from the Cu support grid, the nucleolus being closest and the rough endoplasmic reticulum farthest. Such scattering was corrected for by subtracting the signal minus background obtained from the resin adjacent to the embedded tissue. The mean counts of four assays of the embedment (Table l) were subtracted from the peak-minus-background (P-b) of the sample. Chemical Analysis. To confirm that the Si X-ray signal was not an instrumental artifact (Sutfin et al., 1971; Thurston and Russ, 1971), cell organelles were isolated and analyzed for Si by the silicomolybdate colorimetric method, specifically designed for determining Si in the presence of the P levels found in most animal tissues (Jankowiak and LeVier, 1971). Phosphorus was also analyzed since it was shown to manifest some biochemical similarities to Si in bacteria (Heinen, 1962). The highest concentrations of Si and P occur in the nucleus, microsomes, and mitochondria, in that order, and are strikingly higher in the nucleus than in the other two (Table 2). When the Si:P ratios of the three organelles are compared with those calculated from the amounts (counts) in
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Table 2. Silicon and phosphorus content of isolated rat liver organelles
Cell fractions
Si"
pb
Si/P
(gg/mg protein) Nuclei Mitochondria Microsomes
3.00 c 0.44 0.48
165.4 8.8 13.6
0.018 0.050 0.035
a Jankowiak and LeVier (1971) method of Si assay. b Lindeman (1958) method of P assay. Heinen (1960) method of Si assay.
Table 1 (0.25, 1.11, and 2.93, respectively), a marked difference is apparent. This difference may reflect the loss of Si during organelles isolation in aqueous medium (Mehard et al., 1974b). Discussion
The present study confirms our previous conclusions (Mehard and Volcani, 1975 b) that, despite some ice damage, the freeze substitution method adequately maintains subcellular compartmentation and reduces Si loss from in situ or isolated organelles, and that electron probe microanalysis offers the best method of detecting in situ Si at the subcellular level. The ice damage by incurred by the in situ mitochondria and endoplasmic reticulum has also been observed in plant tissues following similar treatment (L/iuchli etal., 1970; Hereward and Northcote, 1972; Spurr, 1972; Pallaghy, 1973). Reduction of damage by infiltrating the tissue with glycerol, a cryoprotectant, undoubtedly alters the cell physiology. Maintenance of cell-organelle ultrastructure is comparable to that of the glutaraldehyde fixed, freeze dried rat liver tissue of Weavers (1973) prepared by cryo-ultramicrotomy, though no ribosomes were seen on the cisternae of the endoplasmic reticulum with his method. Si was retained in the mitochondria, nucleus, and rough endoplasmic reticulum during tissue preparation. The occurrence of Si in organelles has been reported from studies of 3asi uptake in situ in diatoms (Mehard et al., 1974b) and rat tissues (Mehard and Volcani, 1975a) and by electron probe X-ray microanalysis of other biological material (Schafer and Chandler, 1970; Weavers, 1973; Dempsey et al., 1973), though the question of contamination could not be ignored. However, the finding of our previous study that the presence of Si organelles is not the result of contamination is substantiated in this study both by the use of an electron probe instrument free of Si and by chemical analysis of the organelles. Si-content of a tissue or cell depends on the nutritional status of the animal at the time of sampling, and therefore variations of more than 100% can occur in the content of this freely permeant element (LeVier, 1975; Mehard and Volcani, 1975a). The finding that the nucleus contains more Si than do the
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mitochondria or rough endoplasmic reticulum is in agreement with the report of LeVier (1975) for rat liver. It is difficult to assess the absolute amount of Si in organelles, since some of the element is lost during isolation (Mehard et al., 1974b). When 6 a G e w a s used as a tracer of Si, the mitochondria incurred no loss of label while loss from nuclei and microsomes was only 6% and 11% respectively (Mehard and Volcani, 1975b). Thus, analysis of elements (Si) in situ with the electron probe X-ray microanalysis technique gives a more accurate assessment of the elemental composition than does the chemical analysis of isolated organelles. This is substantiated by the fact that Si:P ratios, as shown by counts in in situ organelles (Table 1) are higher than those in isolated organelles (Table 2). The detection of S, C1, and P demonstrates that the preparation method provides good retention, since these elements are to be expected in organelles of any tissue: the S signal indicates sulfur protein (Jessen etal., 1974); the C1 is present in situ as a component of body fluids. However, Van Steveninck et al. (1974) demonstrated with the EMMA-4 (AEI) that Spurr's epoxy resin contains C1. The embedment counts in the present study are much lower than those reported by Van Steveninck which may be due to differences in X-ray generation, efficiency of collection, and/or the embedment material. The C1 counts from the organelles are sufficiently above the embedment to be considered a cellular constituent, and are similar to those reported from other microprobe studies (L~iuchli et al., 1970; Pallaghy, 1973; Dempsey et al., 1973). The presence of P, another element of biological importance, is also to be expected in detectable amounts; i.e., in the nucleus as a component of DNA and RNA, and in the mitochondria and endoplasmic reticulum as a component of phospholipids, nucleotides, and/or polyphosphates. The AI X-ray signal is probably from in situ accumulated A1 and though it could be an artifact of instrumentation it is not observed in all the organelles or in the embedment. Where A1 has been reported in the X-ray spectra of other tissues it has either been ignored or considered an artifact (Sutfin et al., 1971 ; Dempsey et al., 1973). Not enough is known at present about the biological role of Si to explain its occurrence in these organelles. It has been hypothesized that the mitochondria may participate in calcification (Lehninger, 1970), and it is known that the mitochondria can accumulate calcium and phosphate, both of which are involved in mineralization (Becker et al., 1974; Borle 1973; Chen et al., 1974). It has been shown that 3 i S i is taken up by mitochondria of diatoms and of rat liver, spleen and kidney (Mehard et al., 1974a; Mehard and Volcani, 1975a). The mitochondrion may therefore play some part in the silicification of the diatom cell walls and, since Si is required for bone formation (Carlisle, 1974; Schwartz, 1974) this organelle may be involved in a possible S i - C a relationship as well. References Becker, G.L., Chen, C.H., Greenawalt, J.W., Lehninger, A.L.: Calcium phosphate granules in the hepatopancreas of the blue carb Callinectes sapidus. J. Cell Biol. 61, 316-326 (1974) Borle, A.B. : Calcium metabolism at the cellular level. Fed. Proc. 32, 1944-1950 (1973) Carlisle, E.M.: Silicon as an essential element. Fed. Proc. 33, 1758-1766 (1974)
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Chen, C.H., Greenawalt, J.W., Lehninger, A.L. : Biochemical and ultrastructural aspects of Ca 2+ transport by mitochondria of the hepatopancreas of the blue crab Callinectes sapidus. J. Cell Biol. 61, 301-315 (1974) Chen, C.H., Lewin, J. : Silicon as a nutrient element for Equisetum arvense. Canad. J. Bot. 47, 125-131 (1969) Darley, W.M., Volcani, B.E.: Role of silicon in diatom metabolism. A silicon requirement for deoxyribonucleic acid synthesis in the diatom Cylindrotheca fusiformis. Reimann and Lewin. Exp. Cell Res. 58, 334-342 (1969) Dempsey, E.W., Agate, F.J. Jr., Lee, M., Purkerson, M.L.: Analysis of submicroscopic structures by their emitted X-rays. J. Histochem. Cytochem. 21, 580-586 (1973) Gornall, A.G., Bardawill, C.J., David, M.M. : Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751-766 (1949) Groves, W.E., Davis, F.C., Sells, B.: Spectrophotometric determination of microgram quantities of protein without nucleic acid interference. Analyt. Biochem. 22, 195-210 (1968) Hayward, D.M., Parry, D.W. : Electron probe microanalysis studies of silica distribution in barley (Hordeum sativum L). Ann. Bot. 37, 579 591 (1973) Heinen, W. : Silicium-Stoffwechsel bei Mikroorganismen. I. Aufnahme von Silicium durch Bakterien. Arch. Mikrobiol. 37, 199-210 (1960) Heinen, W. : Siliciumstoffwechsel bei Mikroorganismen. II. Beziehungen zwischen dem Silicat- und Phosphat-Stoffwechsel bei Bakterien. Arch. Mikrobiol. 41, 229-246 (1962) Hereward, F.V., Northcote, D.H. : A simple freeze substitution method for the study of ultrastructure of plant tissue. Exp. Cell Res. 70, 73-80 (1972) Jankowiak, M.E., LeVier, R.R. : Elimination of phosphorus interference in the colorimetric determination of silicon in biological material. Analyt. Biochem. 44, 462472 (1971) Jessen, H., Peters, P.D., Hall, T.A. : Sulphur in different types of keratohyaline granules: A quantitative assay by X-ray microanalysis. J. Cell Sci. 15, 359-377(1974) Kaufman, P.B., Soni, S.L., LaCroix, J.D., Rosen, J.J., Bigelow, W.C. : Electron probe microanalysis of silicon in the epidermis of rice (Oryza sativa L.) internodes. Planta (Berl.) 104, 10-17 (1972) L~iuchli, A., Spurr, A.R., Wittkopp, R.W.: Electron probe analysis of freeze substituted, epoxy resin embedded tissue for ion transport studies in plants. Planta (Berl.) 95, 341-350 (1970) Lehninger, A.L. : Mitochondria and calcium ion transport. Biochem. J. 119, 129-135 (1970) LeVier, R. : Distribution of silicon in the adult rat and rhesus monkey. Bioinorg. Chem. 4, 109-115 (1975) Lewin, J.C. : Silicon metabolism in diatoms. III. Respiration and silicon uptake in Navicula pelliculosa. J. gen. Physiol. 39, 1-10 (1955) Lindeman, W. : Observations on the behavior of phosphate compounds in Chlorella at the transition from dark to light. Proc. Sec. Int. Conf. UN on the Peaceful Uses of Atomic Energy 24, 8-15 (1958) Mehard, C.W., Packer, L.: Induction of cytochrome P-450 and P-448 in the outer membrane of mouse liver nuclei by phenobarbital and benzo[~]pyrene. Bioenergetics 6, 151-160 (1974a) Mehard, C.W., Packer, L., Abraham, S.: Activity and ultrastructure of mitochondria from mouse mammary gland and mammary adenocarcinoma. Cancer Res. 31, 2148-2160 (1971) Mehard, C.W., Sullivan, C.W., Azam, F., Volcani, B.E.: Role of silicon in diatom metabolism. IV. Subcellular localization of silicon and germanium in Nitzschia alba and Cylindrotheca fusiformis. Physiol. Plant. 30, 265-272 (1974b) Mehard, C.W., Volcani, B.E.: Electron probe X-ray microanalysis of subcellular silicon in rat tissues and the diatom, Cylindrotheeafusiformis. J. Cell Biol. 63, 220 (1974c) Mehard, C.W., Volcani, B.E.: Similarity in uptake and retention of trace amounts of 31Si and 68Ge by rat tissues and cell organelles. Bioinorg. Chem. 5, 107-124 (1975a) Mehard, C.W., Volcani, B.E.: Evaluation of silicon and germanium retention in rat tissues and diatoms during cell and organelle preparation for electron probe microanalysis. J. Histochem. Cytochem. 23, 348-358 (1975b) Pallaghy, C.K.: Electron probe microanalysis of potassium and chloride in freeze substituted leaf sections of Zea mays. Aust. J. biol. Sci. 26, 1015-1034 (1973) Reynolds, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963)
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Richter, O. : Zur Physiologic der Diatomeen (I. Mitteilung). Sitzber. Kais. Akad. Wiss. Wien, math.-nat. K1. 115, 27-119 (1906) Schafer, P.W., Chandler, J.A.: Electron probe X-ray microanalysis of a normal centriole. Science 170, 1204-1205 (1970) Schwartz, K.: Recent dietary trace element research, exemplified by tin, fluorine, and silicon. Fed. Proc. 33, 1748-1757 (1974) Soni, S.L., Parry, D.W.: Electron probe microanalysis of silicon deposition in the inflorescence bracts of the rice plant (Oryza sativa). Amer. J. Bot. 60, 111-116 (1973) Spurr, A.R. : A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) Spurr, A.R.: Freeze substitution additives for sodium and calcium retention in cells studied by X-ray analytical electron microscopy. Bot. Gaz. 133, 263-270 (1972) Sullivan, C.W., Volcani, B.E.: Role of silicon in diatom metabolism. III. The effects of silicic acid on DNA polymerase, TMP kinase and DNA synthesis in Cylindrotheeafusiformis. Biochim. biophys. Acta (Amst.) 308, 212-219 (1973) Sutfin, L.V., Holtrop, M.E., Ogilvie, R.E. : Microanalysis of individual rnitochondrial granules with diameters less than 1000 angstroms. Science 174, 947-949 (1971) Takahashi, E.: Silica as a nutrient to the rice plant. Japan Agric. Res. Quart. 3, 1-4 (1968) Thurston, E.L., Russ, J.C.: Scanning and transmission electron microscopy and microanalysis of structured granules in Fischerella ambigua. In: Proc. 4th Ann. Scanning EM Symposium, p. 511 516. Chicago, Ill., ITT Res., Inst. 1971 Van Steveninck, R.F.M., Van Steveninck, M.E., Hall, T.A., Peters, P.D. : A chlorine-free embedding medium for use in X-ray analytical electron microscope localization of chlorine in biological tissues. Histochem. 38, 173-180 (1974) Watson, M.L.: Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, 475-478 (1958) Weavers, B.A.: Combined transmission electron microscopy and X-ray microanalysis of ultrathin frozen dried sections-an investigation to determine the normal elemental compositon of mammalian tissue. J. Microscopy 97, 331-341 (1973)
Received September 16, 1975
