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Transmission electron microscopy of human saliva
Colloids and Surfaces B: Biointerfaces 9 (1997) 257–267
Transmission electron microscopy of human saliva Morten Rykke a,*, Alix Young b, Gunnar Ro¨lla b, Tove Devold c, Gro Smistad d a Department of Cariology, Faculty of Dentistry, University of Oslo, Oslo, Norway b Department of Pedodontics, Faculty of Dentistry, University of Oslo, Oslo, Norway ˚ s, Norway c Food Science, Agricultural University of Norway, A d Department of Pharmaceutics, Faculty of Science, University of Oslo, Oslo, Norway Received 21 January 1997; accepted 29 April 1997
Abstract Globular, or micelle-like, structures have been demonstrated in human parotid saliva by photon correlation spectroscopy and transmission electron microscopy (TEM, negative staining). However, preparation of the TEM specimens may lead to artefact formations. To verify the presence of micelle-like structures in human parotid saliva, the application of cryo-transmission electron microscopy may prove valuable. The aim of this study was therefore to examine human parotid saliva using cryo-TEM and to correlate these results with parallel TEM examinations of parotid saliva prepared with negative staining. Whole saliva was also examined for the presence of these structures, as they may be of importance in oral physiology. Human and bovine milk were included as the presence of casein micelles in these fluids is well established. Freshly collected parotid saliva was prepared for cryo-TEM by instantaneous vitrification in liquid nitrogen. Specimens were also prepared with negative staining (2% ammonium molybdate) from freshly collected parotid saliva, from 0.45 mm filtered parotid saliva, from glutaraldehyde fixed parotid saliva, and from clarified whole saliva. Specimens of human and bovine milk were prepared from freshly obtained milk as well as from glutaraldehyde fixed milk samples. Cryo-TEM demonstrated globular structures in parotid saliva, mostly in the size range about 100–350 nm, appearing as single structures or in clusters. Similar globular structures were observed in negatively stained samples of parotid and whole saliva, and appeared to be multi-globular. TEM of human and bovine milk showed numerous multi-globular casein micelles with a structure similar to the globular structures observed in human saliva. The study demonstrates globular structures in human parotid and whole saliva similar to the casein micelles of milk, indicating the presence of salivary micelle-like structures as an important fraction of human saliva and that the negative staining technique may be suitable for TEM examinations of ultra-structural phenomena in saliva. © 1997 Elsevier Science B.V. Keywords: Casein micelles; Electron microscopy; Human saliva; Salivary micelle-like structures; Salivary proteins
1. Introduction The surface morphology of the acquired enamel pellicle has been described as being predominantly globular or with a knotted surface structure based * Corresponding author. Tel: +47 22 85 22 87; Fax: +47 22 85 23 44; e-mail: [email protected] 0927-7765/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 09 2 7 -7 7 6 5 ( 9 7 ) 0 0 0 31 - 3
on electron microscopic examinations [1,2]. Previously, globular structures have been demonstrated in human parotid saliva by photon correlation spectroscopy (PCS ) and by transmission electron microscopy ( TEM ) of saliva specimens prepared with negative staining [3,4]. PCS analyses have indicated the presence of particles in human parotid saliva in the size range about
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100–200 nm, consistent with both the globular structures observed by TEM of negatively stained saliva samples and the globular structures observed in the acquired enamel pellicle [2,3]. Dierent structural components, including globular structures, have also been described in human saliva by Glantz and coworkers [5–7]. It has therefore been suggested that the globular surface morphology of the acquired enamel pellicle may be the result of an adsorption of salivary protein aggregates in the form of such globular structures [3]. The uptake of lipid-soluble dyes by these structures indicates a hydrophobic interior, and zeta potential measurements demonstrate a net negative surface potential [3,4,8]. The salivary globular structures, therefore, exhibit the characteristics of micelle-like structures. As biological material has poor electron scattering characteristics, it must therefore be visualised by either negative contrast or positive staining. In the negative contrast, using the negative staining technique, the stain does not directly interact with the specimen structures, rather it embeds them by occupying hydrated regions in and around the structures. Therefore, the technique has been shown to be very useful in studying macromolecular structures like lipoproteins as well as in the study of particle size distribution and particle morphology in small and dilute samples [9,10]. However, the specimen preparation may lead to artefact formations, since the adsorption of the specimens to the grids may induce conformational changes in the sample structures [11]. In addition, dehydration of the specimens may also induce conformational changes in the structures such as distortions, and filtration of the saliva samples may induce shear forces causing sample structures to rupture with a time dependent reformation of other non-naturally occurring structures [12]. Hydrophobic regions of the grids may stain unevenly, repelling the stain, thus creating the appearance of negatively stained particles [10]. Furthermore, clumping of the structures may clearly interfere with the assessment of the particle morphology or particle sizes. However, particle aggregation may under certain conditions provide additional information about the particle morphology and their physical properties [10].
The application of cryo-TEM may, therefore, provide valuable additional information when studying saliva samples. By instantaneous vitrification and cryo-TEM, these samples can be observed without the addition of any chemicals and with only minimal sample handling, a technique which may lend itself to negligible artefact formation. Instantaneous vitrification of liquid samples and cryo-TEM have been described by Dubochet et al. [13]. TEM examinations of human parotid saliva and the salivary globular structures including cryo-TEM were thus thought to be of interest as these structures may be of importance in oral physiology, e.g. in pellicle formation and bacterial agglutination. Whole saliva was therefore also examined for the presence of similar globular structures. The aim of this study was to demonstrate the micelle-like protein structures in human parotid saliva by cryo-TEM, and to compare these results with parallel TEM examinations of negatively stained samples of parotid and whole saliva. The major proteinaceous component of milk comprises the caseins which, due to their phosphorylation and amphiphilic nature, interact and form large spherical complexes involving calcium phosphates. These casein micelles have been demonstrated by TEM of milk specimens, showing spherical particles with a raspberry-like appearance, consisting of sub-units [14–18]. The amphiphilic proteins of parotid saliva exhibit structural similarities to the casein molecules of milk [14,19– 22]. The amphiphilic salivary proteins could therefore possibly form similar aggregates. Human and bovine milk were therefore included as controls.
2. Material and methods 2.1. Collection of saliva samples Parotid saliva was collected from four healthy subjects (two females and two males), aged 32–42 yr, by individually fitted appliances made of ProvilA-P impression material (Bayer Dental ). The impression material was made to fit the entire buccal vestibulum and drainage was obtained through tubes from excavations in the appliances
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at the locations of the parotid papillae. Sour stimulated parotid saliva was collected bilaterally and immediately prior to the specimen preparation by means of sour sweets (SurSild, AB Malaco, Sweden). Parotid saliva was collected at a flow rate of 0.9–2.3 ml min−1. Whole saliva was collected during paran wax stimulated chewing and clarified at 2000×g for 10 min. Samples of fixed parotid saliva were obtained by addition of glutaraldehyde to a final concentration of 2% glutaraldehyde in 3 ml samples of freshly collected saliva and stored for 24 h at 4°C before the TEM specimen preparations. 2.2. Collection of milk Bovine milk was collected from a single cow from the herd (Norwegian Red Cattle) at the Agricultural University of Norway. Fat was removed by centrifugation at 2000×g for 30 min at 30°C, followed by chilling. Samples with fixed casein micelles were obtained by addition of 4% glutaraldehyde in a simulated milk ultra-filtrate (SMUF ) buer [23], pH 6.7, to a final concentration of 2% glutaraldehyde in 3 ml samples of milk, and stored for 24 h at 4°C, prior to the TEM specimen preparations. Human milk was obtained from a healthy 38 yr old subject, 3 months post natum, and 3 ml samples were prepared as described for bovine milk. 2.3. Cryo-transmission electron microscopy Copper grids (200 mesh) covered with a perforated supporting film (average hole diameter 3–4 mm) and coated with carbon were used as specimen support. The grids were prepared as described by Fukami and Adachi [24]. The film-coated grids were made hydrophilic (negatively charged) by glow discharging for 30 s, prior to the specimen preparations. The specimen preparation was carried out in a flow of humid air. The grids (held in fine forceps) were mounted on a simple guillotinelike frame 13 cm above a 4 ml recipient of liquid ethane cooled by liquid nitrogen. 3 ml of parotid saliva was applied to the grid and excess fluid drained o immediately with a filter paper until a
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thin film remained. The grid was then plunged into the liquid ethane to instantaneously vitrify the sample, then transferred to liquid nitrogen and further to the microscope using a Gatan 626 cryotransfer system. The specimens were examined at −170°C using a Philips CM12 transmission electron microscope operating at 80 kV. Low magnification (×3.400) and binoculars, or a video screen, were used when examining the samples to minimise the exposure of the specimens to electron irradiation. Representative electron micrographs ( Kodak SO-163) were taken at magnifications of ×17.000 at 1 s exposure time and finally developed for 12 min in a Kodak Developer D-19 at full strength.
2.4. Transmission electron microscopy and negative staining Standard Formvar carbon-coated copper grids (200 mesh) were used as specimen support. The copper grids were prepared as described by Forte and Nordhausen [10]. The film-coated grids were positively ionised by glow discharging for 30 s immediately prior to specimen preparations. 5 ml of the prepared samples was immediately after sampling applied to the grids, and excess fluid drained o with a filter paper after 2 min. The specimens were then negatively stained with a 2% ammonium molybdate solution for 1 min (pH 7.8, 0.22 mm filtered). Excess stain was drained o until a thin film remained and the specimens were airdried. Specimens were prepared directly from freshly collected parotid saliva, from 0.45 mm filtered parotid saliva (MillexA-HV filters with DuraporeA membranes), from glutaraldehyde fixed parotid saliva, and from clarified whole saliva. A further three specimens were prepared from glutaraldehyde fixed human milk samples obtained by adding 100 ml of the fixed human milk to 3 ml samples of SMUF buer (pH 6.7), and three specimens were prepared from glutaraldehyde fixed bovine milk samples obtained by adding 50 ml of the fixed bovine milk to 3 ml samples of SMUF buer. Specimens were also prepared from human and bovine milk samples obtained by adding 100 ml of freshly collected human milk and
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50 ml of freshly collected bovine milk respectively to 3 ml samples of SMUF buer. The specimens were examined using a Philips CM12 transmission electron microscope operating at 80 kV. Representative electron micrographs were taken at magnifications of ×10.000 to ×28.000.
3. Results The results of the transmission electron microscopy are shown as representative micrographs presented in Figs. 1–7. The micrographs are judged to represent the main findings after a thorough scanning of all the prepared specimens. Transmission electron microscopy of negatively stained specimens revealed globular structures in human parotid saliva in the size range about 100–400 nm in diameter. The globular structures appeared predominantly as single units from 90–180 nm ( Fig. 1), or in clusters with a total size of about 500–900 nm, each cluster comprising up to about 10–15 units or more ( Fig. 2). These salivary globular structures seemed to consist of smaller sub-units in that the structures exhibited a
multi-globular structure or a ‘‘raspberry-like’’ appearance (Figs. 1 and 2). Such smaller sub-units with a size of about 40–50 nm were also observed as single units in the clusters and in the background of the micrographs (arrows in Figs. 1 and 2). Similar globular structures of about 100–350 nm in diameter appearing as single units, or in clusters, were also observed in the micrographs from the cryo-TEM (Fig. 3). Smaller units of about 50 nm are also observed in these clusters and, furthermore, small units of about 20 nm scattered all over the background. Some of the globular structures in the cryo-TEM showed signs of degradation in that a number of vacuoles were observed to be formed during the examination of the specimens. Electron micrographs of specimens prepared from glutaraldehyde fixed parotid saliva showed similar globular structures of about 190–350 nm ( Fig. 4). However, the background appeared to be more grainy, or variegated, mostly consisting of small units of 20–30 nm. Micrographs of whole saliva showed similar multi-globular structures of about 100–200 nm, appearing in clusters and surrounded by abundant small units of 30–40 nm ( Fig. 5).
Fig. 1. Transmission electron micrograph of freshly collected parotid saliva, negatively stained with 2% ammonium molybdate for 1 min. The globular structures of about 90–180 nm in diameter appear as single units. The structures have a multi-globular structure consisting of sub-units of 40–50 nm, also observed as single units in the micrograph (arrows). Bar represents 200 nm.
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Fig. 2. Transmission electron micrograph of freshly collected parotid saliva, negatively stained with 2% ammonium molybdate. The globular structures of about 100–400 nm in diameter appear in clusters of 10–15 units with a total size of 500–900 nm. The structures appear to be multi-globular, consisting of sub-units of 40–50 nm, also observed as single units in the clusters and in the background of the micrograph (arrows). Bar represents 200 nm.
Fig. 3. Cryo-transmission electron micrograph of freshly obtained human parotid saliva instantaneously vitrified in liquid nitrogen. The globular structures of 100–350 nm appear in a cluster of 10–15 units. Smaller units of about 50 nm are observed in the cluster as well as small globular structures of about 20 nm all over the background. Bar represents 200 nm.
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Fig. 4. Transmission electron micrograph of parotid saliva fixed with 2% glutaraldehyde prior to negative staining with 2% ammonium molybdate. Salivary multi-globular structures of 190–350 nm in diameter may be observed consisting of sub-units of 30–40 nm. The grainy background comprises smaller units of about 20–30 nm. Bar represents 200 nm.
Fig. 5. Transmission electron micrograph of freshly obtained whole saliva clarified at 2000×g for 10 min and negatively stained with 2% ammonium molybdate. Multi-globular salivary structures of about 100–200 nm may be seen surrounded by numerous small units of 30–40 nm. Bar represents 200 nm.
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Globular structures, i.e. the casein micelles, were observed in specimens prepared from both human (Fig. 6) and bovine milk (Fig. 7), and they appeared to be similar to the globular structures observed in the salivary samples. The casein micelles appeared partly in clusters and partly as single structures. These casein micelles also exhibited a multi-globular appearance consisting of subunits of about 20 nm, similar to the salivary globular structures. The casein micelles appeared to be smaller than the salivary structures, i.e. mostly in the size range about 80–120 nm in diameter, the largest observed to be about 140 nm. Furthermore, the casein micelles of human milk (Fig. 6) and fixed bovine milk samples were predominantly observed as single structures.
4. Discussion In this study, globular structures consistent with salivary micelle-like structures were demonstrated in human saliva by TEM. Cryo-TEM of vitrified saliva specimens and TEM of negatively stained samples showed similar globular structures in par-
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otid and whole saliva. Casein micelles were demonstrated by TEM in both human and bovine milk samples, and these structures appeared to be similar to the multi-globular structures observed in human saliva. Cryo-TEM allows parotid saliva to be examined by TEM without the addition of any chemicals or stains, and with only minimal sample handling of the freshly obtained samples, as described by Dubochet et al. [13]. The specimen preparations were performed in a flow of humid air to reduce evaporation and drying eects as described by Cyrkla et al. [12], and were then transferred to the microscope in liquid nitrogen without any devitrification. Therefore, artefact formations may be negligible when using cryo-TEM, and a method for specimen preparation providing instantaneous vitrification of the freshly collected samples. The globular structures observed by the cryoTEM appeared to be similar to the globular structures demonstrated in parotid saliva by TEM in a previous study [3], and similar to the globular structures demonstrated in negatively stained saliva specimens in the present study. The fact that fewer globular structures were observed by the
Fig. 6. Transmission electron micrograph of freshly obtained human milk negatively stained with 2% ammonium molybdate. Fat was removed by centrifugation prior to the specimen preparation. The casein micelles with a size of about 80–120 nm are similar to the salivary globular structures, i.e. multi-globular, consisting of sub-units of about 20 nm. Bar represents 200 nm.
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Fig. 7. Transmission electron micrograph of freshly obtained bovine milk fixed with 2% glutaraldehyde prior to negative staining with 2% ammonium molybdate. The multi-globular casein micelles appear predominantly as single units with a size of about 80–120 nm, and appear to be similar to the human casein micelles and the salivary micelle-like structures. Fat was removed by centrifugation prior to specimen preparation. Bar represents 200 nm.
cryo-TEM may be due to the specimen preparation. In specimens prepared with negative staining, the salivary proteins were allowed to adsorb to the grids for 2 min prior to removal of excess fluid and staining. In some cryo-electron micrographs, small vacuoles were observed inside the globular structures. These were observed to form during the examination of the specimens, indicating the onset of boiling of organic material due to excessive electron irradiation. TEM of negatively stained parotid saliva samples demonstrated globular structures of about 40–400 nm appearing in clusters, or as single units. This is consistent with the size measurements of these structures as estimated by photon correlation spectroscopy (PCS ), also indicating polydispersity of the structures [8]. This study suggests a time dependent increase of the globular structures as the mean hydrodynamic diameter increased after sampling. This observation may be caused by the fact that the structures aggregate into clusters as shown in the micrographs, also indicating a spherical morphology of the structures [10]. Similar globular structures were observed in
saliva specimens prepared from 0.45 mm filtered saliva samples and in glutaraldehyde fixed saliva as in specimens prepared from freshly obtained parotid saliva, indicating that the structures are resistant to dierent sample handling procedures. Furthermore, the globular structures did not seem to be dependent on dierent salivary flow rates as they appeared in all specimens from all the subjects. The micrographs from the glutaraldehyde fixed parotid saliva samples showed similar globular structures, however, the background appeared to be more grainy or variegated. The variegated background appeared in repeated experiments, and may represent proteinaceous material visualised in TEM due to the glutaraldehyde fixation, also shown in the cryo-TEM as the small units of about 20 nm in the background of these micrographs. Multi-globular structures surrounded by small units were also found in the micrographs of whole saliva, indicating that proteinaceous aggregates in the form of such globular, or micelle-like, structures may be an important fraction of human saliva. TEM of both human and bovine milk showed
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numerous multi-globular structures consistent with the casein micelles. The amphiphilic nature and phosphorylation of the casein molecules result in interactions involving calcium phosphates, forming spherical complexes of 100–600 nm consisting of sub-units of 20–30 nm [14–18,25–27]. These casein micelles have been extensively studied and demonstrated by TEM of milk specimens prepared with dierent techniques, all showing spherical structures with a raspberry-like appearance [14–18]. The casein micelles demonstrated in the present study appear to be similar to the casein micelles previously described. Based on the electron micrographs, size measurements of the casein micelles vary from 20 to about 140 nm. These measurements correspond to size calculations as obtained by PCS, also indicating polydispersity of the particles [25,26 ]. Smaller discrepancies in the size measurements are of little significance as the casein micelles may vary in size between individuals, and the fact that the PCS analysis may underestimate the fraction of smaller particles [25]. Also, the human casein micelles appeared to vary in size from about 30 to 200 nm in diameter and with a more irregular shape, as observed in the micrographs. The casein micelles appeared to be similar in specimens prepared from glutaraldehyde fixed samples and in specimens from freshly obtained samples. However, the casein micelles in glutaraldehyde fixed bovine milk samples appeared predominantly as single structures, and it therefore seems that the fixation of the casein micelles may prevent aggregation into clusters. It has been shown that approximately 70% of the parotid salivary proteins comprise amphiphilic proteins, of which the proline-rich proteins constitute a major component [28]. Several of these proteins have been characterised, and shown to exhibit structural similarities to the casein molecules of milk [19–22]. Due to their phosphorylation and amphiphilic nature, these salivary proteins may also interact and form similar aggregates, or globular structures. Therefore, the globular structures demonstrated in this study both by cryoTEM and TEM of negatively stained saliva samples most probably represent salivary micellelike structures. Calcium has also been shown to be
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of importance, as the addition of calcium complexing agents like EDTA and pyrophosphate causes disintegration of the structures [3]. Previously, human saliva has been examined by Glantz and coworkers [5–7]. TEM of specimens prepared from whole saliva have demonstrated dierent globular structures observed lying within a network structure. Some of these structures appear to be similar to the globular structures demonstrated in this study. The acquired enamel pellicle has also been examined by TEM, showing the presence of globular structures of 100–200 nm in diameter, consistent with the salivary structures demonstrated in the present study [2,29]. These structures may be adsorbed to the enamel surfaces as an important fraction of the acquired pellicle, thus accounting for the globular morphology, as previously reported [1,2]. The negative staining technique for TEM has been extensively described by Forte and Nordhausen [10] and Spiess et al. [9]. The biological material has to be free in suspension, available at a minimum concentration, and exhibit anity for the supporting material. Saliva is assumed to fulfil these requirements. The ammonium molybdate solution is isotonic and exhibits a pH of 7.0–7.4, which is convenient when studying biological fluids like saliva with a neutral pH. It is assumed that the stain solution is able to occupy hydrated regions in and around the structures to be studied, the degree of stain penetration into the structures being dependent on local charges and degree of hydration. The stain penetration into hydrated regions of the salivary structures was demonstrated as the structures appeared to be multi-globular. This sub-division of the structures was not observed in the cryo-TEM. The negative staining technique may, therefore, be very useful when studying salivary protein structures. Controls of the negative staining technique were performed deleting sample application to the grids. These results showed that the stain solution did not contain impurities, and that the grids did not contain artefacts in the supporting membrane, e.g. hydrophobic regions staining unevenly. All specimens were processed immediately after sample collection and examined promptly to minimise the possibilities of artefacts. Thus, pH rise due to air
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exposure is unlikely, and particles resembling calcium phosphate precipitates or distorted particles were not observed. Furthermore, haphazard clumping of the structures was not observed, rather an organised aggregation into clusters dependent on their spherical geometry [10]. Saliva was collected from four subjects, and a previous study indicates that the structures appear in parotid saliva samples collected from several individuals [8]. The milk samples were diluted in SMUF buers, as described, to obtain samples with a micellar concentration suitable for studying in the electron microscope. The casein micelles are known to be resistant to dierent sample handling techniques, including heat treatment, acidification, centrifugation and fixation with glutaraldehyde [17,18,30]. Filtration of the saliva samples was performed using 0.45 mm Millex-HVA filters which are known to adsorb only negligible amounts of proteins [31]. The glutaraldehyde solution was suspended to a 4% concentration in a SMUF buer [23] at pH 6.7 to avoid pH drops in the samples. It has been shown that glutaraldehyde fixation does not influence the pH of biological material, and that glutaraldehyde preserves the micellar structures better than formaldehyde and osmium tetroxide fixation [30]. Controls verified that the pH remained stable in the samples after glutaraldehyde addition, consistent with the known buering capacity of the saliva and the SMUF buer. Human saliva and the acquired enamel pellicle have been claimed to exhibit important functions in oral physiology, i.e. maintaining calcium supersaturation of saliva, bacterial agglutination, lubrication of oral tissues and remineralization of teeth [20,32–34]. The salivary globular structures demonstrated by TEM in this study may be of importance in these aspects.
Acknowledgment This work was supported by the Research Council of Norway, Grant No. 107573/320.
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Transmission electron microscopy of human saliva Morten Rykke a,*, Alix Young b, Gunnar Ro¨lla b, Tove Devold c, Gro Smistad d a Department of Cariology, Faculty of Dentistry, University of Oslo, Oslo, Norway b Department of Pedodontics, Faculty of Dentistry, University of Oslo, Oslo, Norway ˚ s, Norway c Food Science, Agricultural University of Norway, A d Department of Pharmaceutics, Faculty of Science, University of Oslo, Oslo, Norway Received 21 January 1997; accepted 29 April 1997
Abstract Globular, or micelle-like, structures have been demonstrated in human parotid saliva by photon correlation spectroscopy and transmission electron microscopy (TEM, negative staining). However, preparation of the TEM specimens may lead to artefact formations. To verify the presence of micelle-like structures in human parotid saliva, the application of cryo-transmission electron microscopy may prove valuable. The aim of this study was therefore to examine human parotid saliva using cryo-TEM and to correlate these results with parallel TEM examinations of parotid saliva prepared with negative staining. Whole saliva was also examined for the presence of these structures, as they may be of importance in oral physiology. Human and bovine milk were included as the presence of casein micelles in these fluids is well established. Freshly collected parotid saliva was prepared for cryo-TEM by instantaneous vitrification in liquid nitrogen. Specimens were also prepared with negative staining (2% ammonium molybdate) from freshly collected parotid saliva, from 0.45 mm filtered parotid saliva, from glutaraldehyde fixed parotid saliva, and from clarified whole saliva. Specimens of human and bovine milk were prepared from freshly obtained milk as well as from glutaraldehyde fixed milk samples. Cryo-TEM demonstrated globular structures in parotid saliva, mostly in the size range about 100–350 nm, appearing as single structures or in clusters. Similar globular structures were observed in negatively stained samples of parotid and whole saliva, and appeared to be multi-globular. TEM of human and bovine milk showed numerous multi-globular casein micelles with a structure similar to the globular structures observed in human saliva. The study demonstrates globular structures in human parotid and whole saliva similar to the casein micelles of milk, indicating the presence of salivary micelle-like structures as an important fraction of human saliva and that the negative staining technique may be suitable for TEM examinations of ultra-structural phenomena in saliva. © 1997 Elsevier Science B.V. Keywords: Casein micelles; Electron microscopy; Human saliva; Salivary micelle-like structures; Salivary proteins
1. Introduction The surface morphology of the acquired enamel pellicle has been described as being predominantly globular or with a knotted surface structure based * Corresponding author. Tel: +47 22 85 22 87; Fax: +47 22 85 23 44; e-mail: [email protected] 0927-7765/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 09 2 7 -7 7 6 5 ( 9 7 ) 0 0 0 31 - 3
on electron microscopic examinations [1,2]. Previously, globular structures have been demonstrated in human parotid saliva by photon correlation spectroscopy (PCS ) and by transmission electron microscopy ( TEM ) of saliva specimens prepared with negative staining [3,4]. PCS analyses have indicated the presence of particles in human parotid saliva in the size range about
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100–200 nm, consistent with both the globular structures observed by TEM of negatively stained saliva samples and the globular structures observed in the acquired enamel pellicle [2,3]. Dierent structural components, including globular structures, have also been described in human saliva by Glantz and coworkers [5–7]. It has therefore been suggested that the globular surface morphology of the acquired enamel pellicle may be the result of an adsorption of salivary protein aggregates in the form of such globular structures [3]. The uptake of lipid-soluble dyes by these structures indicates a hydrophobic interior, and zeta potential measurements demonstrate a net negative surface potential [3,4,8]. The salivary globular structures, therefore, exhibit the characteristics of micelle-like structures. As biological material has poor electron scattering characteristics, it must therefore be visualised by either negative contrast or positive staining. In the negative contrast, using the negative staining technique, the stain does not directly interact with the specimen structures, rather it embeds them by occupying hydrated regions in and around the structures. Therefore, the technique has been shown to be very useful in studying macromolecular structures like lipoproteins as well as in the study of particle size distribution and particle morphology in small and dilute samples [9,10]. However, the specimen preparation may lead to artefact formations, since the adsorption of the specimens to the grids may induce conformational changes in the sample structures [11]. In addition, dehydration of the specimens may also induce conformational changes in the structures such as distortions, and filtration of the saliva samples may induce shear forces causing sample structures to rupture with a time dependent reformation of other non-naturally occurring structures [12]. Hydrophobic regions of the grids may stain unevenly, repelling the stain, thus creating the appearance of negatively stained particles [10]. Furthermore, clumping of the structures may clearly interfere with the assessment of the particle morphology or particle sizes. However, particle aggregation may under certain conditions provide additional information about the particle morphology and their physical properties [10].
The application of cryo-TEM may, therefore, provide valuable additional information when studying saliva samples. By instantaneous vitrification and cryo-TEM, these samples can be observed without the addition of any chemicals and with only minimal sample handling, a technique which may lend itself to negligible artefact formation. Instantaneous vitrification of liquid samples and cryo-TEM have been described by Dubochet et al. [13]. TEM examinations of human parotid saliva and the salivary globular structures including cryo-TEM were thus thought to be of interest as these structures may be of importance in oral physiology, e.g. in pellicle formation and bacterial agglutination. Whole saliva was therefore also examined for the presence of similar globular structures. The aim of this study was to demonstrate the micelle-like protein structures in human parotid saliva by cryo-TEM, and to compare these results with parallel TEM examinations of negatively stained samples of parotid and whole saliva. The major proteinaceous component of milk comprises the caseins which, due to their phosphorylation and amphiphilic nature, interact and form large spherical complexes involving calcium phosphates. These casein micelles have been demonstrated by TEM of milk specimens, showing spherical particles with a raspberry-like appearance, consisting of sub-units [14–18]. The amphiphilic proteins of parotid saliva exhibit structural similarities to the casein molecules of milk [14,19– 22]. The amphiphilic salivary proteins could therefore possibly form similar aggregates. Human and bovine milk were therefore included as controls.
2. Material and methods 2.1. Collection of saliva samples Parotid saliva was collected from four healthy subjects (two females and two males), aged 32–42 yr, by individually fitted appliances made of ProvilA-P impression material (Bayer Dental ). The impression material was made to fit the entire buccal vestibulum and drainage was obtained through tubes from excavations in the appliances
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at the locations of the parotid papillae. Sour stimulated parotid saliva was collected bilaterally and immediately prior to the specimen preparation by means of sour sweets (SurSild, AB Malaco, Sweden). Parotid saliva was collected at a flow rate of 0.9–2.3 ml min−1. Whole saliva was collected during paran wax stimulated chewing and clarified at 2000×g for 10 min. Samples of fixed parotid saliva were obtained by addition of glutaraldehyde to a final concentration of 2% glutaraldehyde in 3 ml samples of freshly collected saliva and stored for 24 h at 4°C before the TEM specimen preparations. 2.2. Collection of milk Bovine milk was collected from a single cow from the herd (Norwegian Red Cattle) at the Agricultural University of Norway. Fat was removed by centrifugation at 2000×g for 30 min at 30°C, followed by chilling. Samples with fixed casein micelles were obtained by addition of 4% glutaraldehyde in a simulated milk ultra-filtrate (SMUF ) buer [23], pH 6.7, to a final concentration of 2% glutaraldehyde in 3 ml samples of milk, and stored for 24 h at 4°C, prior to the TEM specimen preparations. Human milk was obtained from a healthy 38 yr old subject, 3 months post natum, and 3 ml samples were prepared as described for bovine milk. 2.3. Cryo-transmission electron microscopy Copper grids (200 mesh) covered with a perforated supporting film (average hole diameter 3–4 mm) and coated with carbon were used as specimen support. The grids were prepared as described by Fukami and Adachi [24]. The film-coated grids were made hydrophilic (negatively charged) by glow discharging for 30 s, prior to the specimen preparations. The specimen preparation was carried out in a flow of humid air. The grids (held in fine forceps) were mounted on a simple guillotinelike frame 13 cm above a 4 ml recipient of liquid ethane cooled by liquid nitrogen. 3 ml of parotid saliva was applied to the grid and excess fluid drained o immediately with a filter paper until a
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thin film remained. The grid was then plunged into the liquid ethane to instantaneously vitrify the sample, then transferred to liquid nitrogen and further to the microscope using a Gatan 626 cryotransfer system. The specimens were examined at −170°C using a Philips CM12 transmission electron microscope operating at 80 kV. Low magnification (×3.400) and binoculars, or a video screen, were used when examining the samples to minimise the exposure of the specimens to electron irradiation. Representative electron micrographs ( Kodak SO-163) were taken at magnifications of ×17.000 at 1 s exposure time and finally developed for 12 min in a Kodak Developer D-19 at full strength.
2.4. Transmission electron microscopy and negative staining Standard Formvar carbon-coated copper grids (200 mesh) were used as specimen support. The copper grids were prepared as described by Forte and Nordhausen [10]. The film-coated grids were positively ionised by glow discharging for 30 s immediately prior to specimen preparations. 5 ml of the prepared samples was immediately after sampling applied to the grids, and excess fluid drained o with a filter paper after 2 min. The specimens were then negatively stained with a 2% ammonium molybdate solution for 1 min (pH 7.8, 0.22 mm filtered). Excess stain was drained o until a thin film remained and the specimens were airdried. Specimens were prepared directly from freshly collected parotid saliva, from 0.45 mm filtered parotid saliva (MillexA-HV filters with DuraporeA membranes), from glutaraldehyde fixed parotid saliva, and from clarified whole saliva. A further three specimens were prepared from glutaraldehyde fixed human milk samples obtained by adding 100 ml of the fixed human milk to 3 ml samples of SMUF buer (pH 6.7), and three specimens were prepared from glutaraldehyde fixed bovine milk samples obtained by adding 50 ml of the fixed bovine milk to 3 ml samples of SMUF buer. Specimens were also prepared from human and bovine milk samples obtained by adding 100 ml of freshly collected human milk and
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50 ml of freshly collected bovine milk respectively to 3 ml samples of SMUF buer. The specimens were examined using a Philips CM12 transmission electron microscope operating at 80 kV. Representative electron micrographs were taken at magnifications of ×10.000 to ×28.000.
3. Results The results of the transmission electron microscopy are shown as representative micrographs presented in Figs. 1–7. The micrographs are judged to represent the main findings after a thorough scanning of all the prepared specimens. Transmission electron microscopy of negatively stained specimens revealed globular structures in human parotid saliva in the size range about 100–400 nm in diameter. The globular structures appeared predominantly as single units from 90–180 nm ( Fig. 1), or in clusters with a total size of about 500–900 nm, each cluster comprising up to about 10–15 units or more ( Fig. 2). These salivary globular structures seemed to consist of smaller sub-units in that the structures exhibited a
multi-globular structure or a ‘‘raspberry-like’’ appearance (Figs. 1 and 2). Such smaller sub-units with a size of about 40–50 nm were also observed as single units in the clusters and in the background of the micrographs (arrows in Figs. 1 and 2). Similar globular structures of about 100–350 nm in diameter appearing as single units, or in clusters, were also observed in the micrographs from the cryo-TEM (Fig. 3). Smaller units of about 50 nm are also observed in these clusters and, furthermore, small units of about 20 nm scattered all over the background. Some of the globular structures in the cryo-TEM showed signs of degradation in that a number of vacuoles were observed to be formed during the examination of the specimens. Electron micrographs of specimens prepared from glutaraldehyde fixed parotid saliva showed similar globular structures of about 190–350 nm ( Fig. 4). However, the background appeared to be more grainy, or variegated, mostly consisting of small units of 20–30 nm. Micrographs of whole saliva showed similar multi-globular structures of about 100–200 nm, appearing in clusters and surrounded by abundant small units of 30–40 nm ( Fig. 5).
Fig. 1. Transmission electron micrograph of freshly collected parotid saliva, negatively stained with 2% ammonium molybdate for 1 min. The globular structures of about 90–180 nm in diameter appear as single units. The structures have a multi-globular structure consisting of sub-units of 40–50 nm, also observed as single units in the micrograph (arrows). Bar represents 200 nm.
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Fig. 2. Transmission electron micrograph of freshly collected parotid saliva, negatively stained with 2% ammonium molybdate. The globular structures of about 100–400 nm in diameter appear in clusters of 10–15 units with a total size of 500–900 nm. The structures appear to be multi-globular, consisting of sub-units of 40–50 nm, also observed as single units in the clusters and in the background of the micrograph (arrows). Bar represents 200 nm.
Fig. 3. Cryo-transmission electron micrograph of freshly obtained human parotid saliva instantaneously vitrified in liquid nitrogen. The globular structures of 100–350 nm appear in a cluster of 10–15 units. Smaller units of about 50 nm are observed in the cluster as well as small globular structures of about 20 nm all over the background. Bar represents 200 nm.
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Fig. 4. Transmission electron micrograph of parotid saliva fixed with 2% glutaraldehyde prior to negative staining with 2% ammonium molybdate. Salivary multi-globular structures of 190–350 nm in diameter may be observed consisting of sub-units of 30–40 nm. The grainy background comprises smaller units of about 20–30 nm. Bar represents 200 nm.
Fig. 5. Transmission electron micrograph of freshly obtained whole saliva clarified at 2000×g for 10 min and negatively stained with 2% ammonium molybdate. Multi-globular salivary structures of about 100–200 nm may be seen surrounded by numerous small units of 30–40 nm. Bar represents 200 nm.
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Globular structures, i.e. the casein micelles, were observed in specimens prepared from both human (Fig. 6) and bovine milk (Fig. 7), and they appeared to be similar to the globular structures observed in the salivary samples. The casein micelles appeared partly in clusters and partly as single structures. These casein micelles also exhibited a multi-globular appearance consisting of subunits of about 20 nm, similar to the salivary globular structures. The casein micelles appeared to be smaller than the salivary structures, i.e. mostly in the size range about 80–120 nm in diameter, the largest observed to be about 140 nm. Furthermore, the casein micelles of human milk (Fig. 6) and fixed bovine milk samples were predominantly observed as single structures.
4. Discussion In this study, globular structures consistent with salivary micelle-like structures were demonstrated in human saliva by TEM. Cryo-TEM of vitrified saliva specimens and TEM of negatively stained samples showed similar globular structures in par-
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otid and whole saliva. Casein micelles were demonstrated by TEM in both human and bovine milk samples, and these structures appeared to be similar to the multi-globular structures observed in human saliva. Cryo-TEM allows parotid saliva to be examined by TEM without the addition of any chemicals or stains, and with only minimal sample handling of the freshly obtained samples, as described by Dubochet et al. [13]. The specimen preparations were performed in a flow of humid air to reduce evaporation and drying eects as described by Cyrkla et al. [12], and were then transferred to the microscope in liquid nitrogen without any devitrification. Therefore, artefact formations may be negligible when using cryo-TEM, and a method for specimen preparation providing instantaneous vitrification of the freshly collected samples. The globular structures observed by the cryoTEM appeared to be similar to the globular structures demonstrated in parotid saliva by TEM in a previous study [3], and similar to the globular structures demonstrated in negatively stained saliva specimens in the present study. The fact that fewer globular structures were observed by the
Fig. 6. Transmission electron micrograph of freshly obtained human milk negatively stained with 2% ammonium molybdate. Fat was removed by centrifugation prior to the specimen preparation. The casein micelles with a size of about 80–120 nm are similar to the salivary globular structures, i.e. multi-globular, consisting of sub-units of about 20 nm. Bar represents 200 nm.
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Fig. 7. Transmission electron micrograph of freshly obtained bovine milk fixed with 2% glutaraldehyde prior to negative staining with 2% ammonium molybdate. The multi-globular casein micelles appear predominantly as single units with a size of about 80–120 nm, and appear to be similar to the human casein micelles and the salivary micelle-like structures. Fat was removed by centrifugation prior to specimen preparation. Bar represents 200 nm.
cryo-TEM may be due to the specimen preparation. In specimens prepared with negative staining, the salivary proteins were allowed to adsorb to the grids for 2 min prior to removal of excess fluid and staining. In some cryo-electron micrographs, small vacuoles were observed inside the globular structures. These were observed to form during the examination of the specimens, indicating the onset of boiling of organic material due to excessive electron irradiation. TEM of negatively stained parotid saliva samples demonstrated globular structures of about 40–400 nm appearing in clusters, or as single units. This is consistent with the size measurements of these structures as estimated by photon correlation spectroscopy (PCS ), also indicating polydispersity of the structures [8]. This study suggests a time dependent increase of the globular structures as the mean hydrodynamic diameter increased after sampling. This observation may be caused by the fact that the structures aggregate into clusters as shown in the micrographs, also indicating a spherical morphology of the structures [10]. Similar globular structures were observed in
saliva specimens prepared from 0.45 mm filtered saliva samples and in glutaraldehyde fixed saliva as in specimens prepared from freshly obtained parotid saliva, indicating that the structures are resistant to dierent sample handling procedures. Furthermore, the globular structures did not seem to be dependent on dierent salivary flow rates as they appeared in all specimens from all the subjects. The micrographs from the glutaraldehyde fixed parotid saliva samples showed similar globular structures, however, the background appeared to be more grainy or variegated. The variegated background appeared in repeated experiments, and may represent proteinaceous material visualised in TEM due to the glutaraldehyde fixation, also shown in the cryo-TEM as the small units of about 20 nm in the background of these micrographs. Multi-globular structures surrounded by small units were also found in the micrographs of whole saliva, indicating that proteinaceous aggregates in the form of such globular, or micelle-like, structures may be an important fraction of human saliva. TEM of both human and bovine milk showed
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numerous multi-globular structures consistent with the casein micelles. The amphiphilic nature and phosphorylation of the casein molecules result in interactions involving calcium phosphates, forming spherical complexes of 100–600 nm consisting of sub-units of 20–30 nm [14–18,25–27]. These casein micelles have been extensively studied and demonstrated by TEM of milk specimens prepared with dierent techniques, all showing spherical structures with a raspberry-like appearance [14–18]. The casein micelles demonstrated in the present study appear to be similar to the casein micelles previously described. Based on the electron micrographs, size measurements of the casein micelles vary from 20 to about 140 nm. These measurements correspond to size calculations as obtained by PCS, also indicating polydispersity of the particles [25,26 ]. Smaller discrepancies in the size measurements are of little significance as the casein micelles may vary in size between individuals, and the fact that the PCS analysis may underestimate the fraction of smaller particles [25]. Also, the human casein micelles appeared to vary in size from about 30 to 200 nm in diameter and with a more irregular shape, as observed in the micrographs. The casein micelles appeared to be similar in specimens prepared from glutaraldehyde fixed samples and in specimens from freshly obtained samples. However, the casein micelles in glutaraldehyde fixed bovine milk samples appeared predominantly as single structures, and it therefore seems that the fixation of the casein micelles may prevent aggregation into clusters. It has been shown that approximately 70% of the parotid salivary proteins comprise amphiphilic proteins, of which the proline-rich proteins constitute a major component [28]. Several of these proteins have been characterised, and shown to exhibit structural similarities to the casein molecules of milk [19–22]. Due to their phosphorylation and amphiphilic nature, these salivary proteins may also interact and form similar aggregates, or globular structures. Therefore, the globular structures demonstrated in this study both by cryoTEM and TEM of negatively stained saliva samples most probably represent salivary micellelike structures. Calcium has also been shown to be
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of importance, as the addition of calcium complexing agents like EDTA and pyrophosphate causes disintegration of the structures [3]. Previously, human saliva has been examined by Glantz and coworkers [5–7]. TEM of specimens prepared from whole saliva have demonstrated dierent globular structures observed lying within a network structure. Some of these structures appear to be similar to the globular structures demonstrated in this study. The acquired enamel pellicle has also been examined by TEM, showing the presence of globular structures of 100–200 nm in diameter, consistent with the salivary structures demonstrated in the present study [2,29]. These structures may be adsorbed to the enamel surfaces as an important fraction of the acquired pellicle, thus accounting for the globular morphology, as previously reported [1,2]. The negative staining technique for TEM has been extensively described by Forte and Nordhausen [10] and Spiess et al. [9]. The biological material has to be free in suspension, available at a minimum concentration, and exhibit anity for the supporting material. Saliva is assumed to fulfil these requirements. The ammonium molybdate solution is isotonic and exhibits a pH of 7.0–7.4, which is convenient when studying biological fluids like saliva with a neutral pH. It is assumed that the stain solution is able to occupy hydrated regions in and around the structures to be studied, the degree of stain penetration into the structures being dependent on local charges and degree of hydration. The stain penetration into hydrated regions of the salivary structures was demonstrated as the structures appeared to be multi-globular. This sub-division of the structures was not observed in the cryo-TEM. The negative staining technique may, therefore, be very useful when studying salivary protein structures. Controls of the negative staining technique were performed deleting sample application to the grids. These results showed that the stain solution did not contain impurities, and that the grids did not contain artefacts in the supporting membrane, e.g. hydrophobic regions staining unevenly. All specimens were processed immediately after sample collection and examined promptly to minimise the possibilities of artefacts. Thus, pH rise due to air
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exposure is unlikely, and particles resembling calcium phosphate precipitates or distorted particles were not observed. Furthermore, haphazard clumping of the structures was not observed, rather an organised aggregation into clusters dependent on their spherical geometry [10]. Saliva was collected from four subjects, and a previous study indicates that the structures appear in parotid saliva samples collected from several individuals [8]. The milk samples were diluted in SMUF buers, as described, to obtain samples with a micellar concentration suitable for studying in the electron microscope. The casein micelles are known to be resistant to dierent sample handling techniques, including heat treatment, acidification, centrifugation and fixation with glutaraldehyde [17,18,30]. Filtration of the saliva samples was performed using 0.45 mm Millex-HVA filters which are known to adsorb only negligible amounts of proteins [31]. The glutaraldehyde solution was suspended to a 4% concentration in a SMUF buer [23] at pH 6.7 to avoid pH drops in the samples. It has been shown that glutaraldehyde fixation does not influence the pH of biological material, and that glutaraldehyde preserves the micellar structures better than formaldehyde and osmium tetroxide fixation [30]. Controls verified that the pH remained stable in the samples after glutaraldehyde addition, consistent with the known buering capacity of the saliva and the SMUF buer. Human saliva and the acquired enamel pellicle have been claimed to exhibit important functions in oral physiology, i.e. maintaining calcium supersaturation of saliva, bacterial agglutination, lubrication of oral tissues and remineralization of teeth [20,32–34]. The salivary globular structures demonstrated by TEM in this study may be of importance in these aspects.
Acknowledgment This work was supported by the Research Council of Norway, Grant No. 107573/320.
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