Journal of Histochemistry and Cytochemistry, Vol. 47, 1487-1494, November 1999, Copyright © 1999, The Histochemical Society, Inc.


TECHNICAL NOTE

Observation of Fibronectin Distribution on the Cell Undersurface Using Immunogold Scanning Electron Microscopy

Tetsuya Gotoa,b, Kung S. Wonga, and Donald M. Brunettea
a Department of Oral Biological and Medical Sciences, Faculty of Dentistry, The University of British Columbia, Vancouver, BC, Canada
b Department of Oral Anatomy, Kyushu University, Fukuoka, Japan

Correspondence to: Tetsuya Goto, Dept. of Oral Anatomy, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or gold–palladium and observed by SEM. Using backscattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface. (J Histochem Cytochem 47:1487–1493, 1999)

Key Words: cell undersurface, scanning electron microscopy, glow discharge etching, immunogold, fibronectin


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Several methods using colloidal gold have been developed to visualize surface markers by scanning electron microscopy (SEM) (Horisberger et al. 1975 ; De Harven et al. 1984 ). These methods have been further improved using back scattered electron imaging (Tanaka et al. 1988 ; Walther and Muller 1988 ; Goode and Maugel 1997) or ultra-high-resolution SEM (Tanaka et al. 1991 ). Colloidal gold has been used as a surface marker in immuno-SEM and has obvious advantages for study of the three-dimensional distribution of cell surface antigens that cannot be readily demonstrated by light or transmission electron microscopy.

In investigating cell adhesion molecules, SEM and immunogold have been used very effectively to show the distribution of cell adhesion molecules on the dorsal surface (Trejdosiewicz et al. 1981 ; Abiko and Brunette 1993 ). However, the distribution of the cell adhesion molecules on the cell undersurface is more relevant to their function.

The use of immuno-SEM on the cell's ventral surface requires methods to separate the cells from the substrate so that the undersurface is exposed. Initially Nermut developed a method for observation of cell undersurface and succeeded in immunogold staining by ethylene diamino tetraacetic acid (EDTA)–gelatin (Nermut and Burt 1991 ; Nermut et al. 1993 ; Nermut 1995 ). Recently Richards et al. 1993 developed a method for investigation by SEM of the ventral cell surface embedded in resin. In brief, they used LR White Resin for embedding cells that had been cultured on plastic coverslips (Thermanox). The use of plastic coverslips enabled them to be separated from the embedding resin. A second aspect of their method was the use of glow discharge for removing the resin that covered the ventral cell surface.

In this study, the SEM/immunogold method and the glow discharge surface etching method were combined to observe the distribution of fibronectin (FN) on the ventral cell surface by SEM for cells cultured on titanium. Only a few studies have examined the cell undersurface facing biomaterials (Richards et al. 1995 ). The extension of the method to cells cultured on biomaterials, such as titanium, is important because it allows direct examination of the distribution of proteins that the cells use in attaching to these surfaces.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Cell Culture
Fibroblasts were isolated from outgrowths of human gingiva as described by Brunette et al. 1976 . The cells were cultured on tissue culture plastic (Falcon; Cockeysville, MD) in alpha minimal essential medium (MEM) (Terry Fox Media Lab; Vancouver, BC, Canada) supplemented with antibiotics [penicillin G (Sigma; St Louis, MO) 100 µg/ml, gentamicin (Sigma) 50 µg/ml, amphotericin B (Fungizone; Gibco, Grand Island, NY) 3 µg/ml] and 15% serum (calf supreme serum; Gibco) at 37C in a humidified atmosphere of 95% air, 5% CO2. Fibroblasts were removed from the growth surface by a trypsin solution [0.25% trypsin (Gibco) and 0.1% glucose dissolved in citrate–saline (pH 7.8)]. The cells were resuspended in a medium containing 5% serum at a cell population density of 1 x 104/cm2. The cells were cultured for 5 or 24 hr on silicon wafers that were coated with 50 nm of titanium to facilitate cell attachment. The titanium-coated substrates were cleaned by ultrasonication for 20 min in a detergent (7X; Flow Labs, McLean, VA) specifically formulated for tissue culture. Titanium surfaces were used because this is a biocompatible material widely utilized. The silicon wafer resisted bending to a greater extent than glass coverslips, which made removal of the substrate easier; however, any rigid material could be used.

Fixation and Embedding
The samples were first rinsed in 0.1 M PBS and fixed in 0.05% glutaraldehyde and 3.2% paraformaldehyde in PBS for 10 min at room temperature. After rinsing in PBS, the fixed cell cultures were dehydrated through a series of 50, 70, and 95% ethanol for 5 min, followed by LR Gold (Electron Microscopy Sciences; Fort Washington, PA) for 30 min to allow complete infiltration of the resin into the cells (Figure 1). The samples were taken out of LR Gold, and the gelatin capsules were put on it and sealed using rapid bonding adhesive (Aron Alpha; Kohishi Inc., Osaka, Japan) between the capsule and the substrate. Fresh LR Gold was poured into them and polymerized in a UV C1 Cryo Chamber (Ted Pella; Redding, CA) at -20C for 6 hr (Figure 1 Figure 2).



View larger version (38K):
[in this window]
[in a new window]
 
Figure 1. Schematic of cell treatment. Details are described in Materials and Methods.



View larger version (119K):
[in this window]
[in a new window]
 
Figure 2. SEM images of the undersurface of 24-hr cultured human gingival fibroblasts embedded in LR Gold resin. (a) Fourteen-kV accelerating voltage image displaying fibroblasts embedded in resin without glow discharge etching. Arrows show region where the resin was left on the cell surface. (b) Twenty-six-kV accelerating voltage image. After 15 min of glow discharge etching, the outline of the cells is clear. Arrow shows the impression of the nucleus. Bars = 25 µm. (c) Higher-power magnification image at 14-kV accelerating voltage at the regions close to the cell edge that were etched for 20 min. Longitudinal fibrillar lines (arrows) and lateral fibrillar lines (arrowhead) were observed. Bar = 5 µm.

Glow Discharge Etching and SEM Immunostaining
Titanium substrates were removed by inserting a sharp knife between the resin and the substrate to separate the substrate (Figure 1 Figure 2 Figure 3). The resin blocks were washed and placed in an argon gas glow discharge chamber fabricated according to the design of Aebi and Pollard 1987 . Glow discharge was carried out at about 3.0 kV at a pressure of 1.0 x 10-7 Pa. Resin blocks were etched for 0, 5, 10, 15, or 20 min (Figure 1 Figure 2 Figure 3 Figure 4). The specimens were washed with PBS and immersed for 30 min at 37C in PBS containing 1.0% bovine serum albumin to block nonspecific staining. The specimens were then incubated with a mouse monoclonal antibody to human fibronectin (clone no. FN-3E2, Sigma; 1:40 dilution) for 1.5 hr at 37C. After brief washing in PBS, the specimens were incubated with colloidal gold-labeled goat anti-mouse IgG (30-nm gold particles; 1:20 dilution) (Ted Pella) for 1 hr at 37C (Figure 1 Figure 2 Figure 3 Figure 4 Figure 5). The samples were washed with deionized water, postfixed with 0.5% osmium tetroxide for 5 min, dehydrated in graded alcohols, and critical point-dried with CO2. Then they were lightly sputter-coated with carbon or coated for 1.5–2 min with gold–palladium to observe the ultrastructure of the cell surface (Figure 1 Figure 2 Figure 3 Figure 4 Figure 5). The specimens were examined in a Leica 260 stereoscan scanning electron microscope (Cambridge, MA) equipped with both Lab-6 and tungsten filaments. Images were obtained via secondary electron or backscattered electron (SE or BSE) detection at a working distance of 15–25 mm and at an accelerating voltage of 14–26 kV. BSE images were detected by a four-quadrant solid-state detector (KE Development; Cambridge, UK).



View larger version (84K):
[in this window]
[in a new window]
 
Figure 3. Fluorescence images of a rounded fibroblast at 5 hr after seeding (a) and a 24-hr cultured well-spread fibroblast (b). Cells embedded in LR Gold resin were glow discharge etched for 15 min and stained by fluorescein-5-thiosemicarbazide. No resin was left on the cell undersurface. (If any resin had remained, some dark spots would be observed.) Small processes were well preserved (arrows). Bars = 25 µm.



View larger version (108K):
[in this window]
[in a new window]
 
Figure 4. TEM images of a sample that was glow discharge etched, coated with gold–palladium, re-embedded in Epon and ultrathin-sectioned. LR, LR Gold resin; E, Epon. The black band between arrows in a is a gold–palladium-coated layer. Bar = 0.5 µm. (b) Higher-magnification image of the portion indicated by asterisk in a. With 15 min of etching, no resin is observed between the cell undersurface and a gold–palladium coated layer. Bar = 25 nm.



View larger version (131K):
[in this window]
[in a new window]
 
Figure 5. Cell undersurface immunogold SEM micrographs using the SE mode. The cells were cultured for 5 hr and the embedded cell surface was glow discharge etched for 15 min. The cells were immunogold-labeled using anti-fibronectin (FN) antibody and coated with gold–palladium for 1.5 min. Gold beads indicate the localization of FN. (a) Twenty-one-kV accelerating voltage image displaying localization of FN. FN distributes randomly on the entire cell undersurface, but a couple of aligned fibrils (arrows) are observed close to the edge of the cell. Some dark spots where few gold beads were localized are found close to the center of the cell (arrowheads). Very few gold beads are located on the resin outside the cell surface. Bar = 10 µm. (b) Higher-magnification image of the region framed in (a) at 17-kV accelerating voltage. Bright lines close to the cell edge consist of aligned gold beads (arrowheads). Bar = 2 µm. (c) Higher-magnification image of the region framed in b at 17-kV accelerating voltage. The distance between the two parallel bars is 30 nm. Bar = 300 nm. (d) Fourteen-kV accelerating voltage image. No FN-positive immunogold staining was observed on the undersurface of cells incubated with preabsorbed primary antibody. Bar = 10 µm.

Cell Fluorescent Staining
To find the optimal glow discharge etching time, cell surface and cytosolic glycoconjugates had been reacted with fluorescent hydrazides. Finally the cell surfaces and cytosol were visualized using epifluorescence (Wilchek et al. 1980). In this method, fixed specimens were oxidized in 4.2 mM periodate (BDH; Poole, UK) in PBS for 30 min on ice at 4C, followed by three PBS rinses and an hour of incubation at 37C with 10 mM fluorescein-5-thiosemicarbazide (Molecular Probes; Eugene, OR). After washing in PBS, the specimens were mounted on the slide glass and observed in a microscope equipped with epifluorescence [confocal laser scanning microscope (CLSM); Zeiss, Oberkochen, Germany]. The surface of the titanium substrate was also stained to determine whether or not the cells had remained on it.

Transmission Electron Microscopy
After analysis in the SEM, parts of the top layer of the resin block, containing the cells and the coated material, were removed and re-embedded in Epon. Ultrathin sections were cut transversely and mounted on nickel grids coated with collodion. The grids were counterstained with 2% uranyl acetate and examined in a transmission electron microscope (Phillips 300 TEM).

Immunostaining Controls
Controls were performed by omission of the primary antibody or by incubation with primary antiserum preabsorbed with a 1 mg/ml concentration of human fibronectin (Gelco Diagnostics; Shreveport, LA).

We used at least five samples for each experiment and the same controls were run each time.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Removing resin from the cell undersurface using glow discharge etching is directly dependent on etching duration. Figure 2a shows the cell undersurface of fibroblasts without etching. Although parts of the cell's undersurfaces were exposed, the outlines of the cells lacked clarity and some parts of the cell surface were still overlaid with resin. Cell undersurfaces after 15 or 20 min of flow discharge etching were well exposed (Figure 2b and Figure 2c). Periods of 5 or 10 min of glow discharge etching were not sufficient to clearly expose the undersurface of the cells. Figure 2b is an SEM image of fibroblasts undersurfaces. The culture was on a titanium-coated silicon wafer for 24 hr. The outline of the etched cells was clearer than that of non-etched cells. Figure 2c is a high-magnification image at the region close to the cell edge. The edge of the cell was well exposed, and longitudinal fibrils were observed. Close to the edge of the cell there were tiny lateral fibrillar lines. To confirm whether or not the cell undersurfaces were really exposed, the cells were stained using fluorescein-5-thiosemicarbazide and cross-sectioned for TEM after SEM observation. After 15 min of glow discharge etching, the entire cell undersurface was stained by membrane fluorescence. Both 5-hr cultured cells and 24-hr cultured cells were well stained throughout the entire cell surfaces, and no resin could be observed on the cell undersurfaces (Figure 3a and Figure 3b). Fluorescence staining also indicated that some small cell processes were well preserved. Furthermore, the surface of titanium substrates that flaked off from the resin was stained to determine whether or not the cells were remained on it. Although the cells on the substrate were embedded in resin, they sometimes maintained their attachment to the substrate (in this case embedded materials were not separated between the cell undersurface and the substrate, but between the cell upper surface and resin), particularly when the cells were cultured for 24 hr after seeding. After staining of the substrates, only the resin blocks for which few cells remained on the replicated substrates were used for immunostaining. Figure 4a and Figure 4b are TEM cross-sections of a cell after 15 min of glow discharge etching, SEM observation, re-embedding in Epon, and ultrathin sectioning (without immunogold staining). The fibroblast was embedded in LR Gold resin and its undersurface was coated with gold–palladium. High-power examination showed that no resin was left between the cell surface and the gold sputter-coating (Figure 4b). It therefore appears that 15 min of glow discharge etching was enough to expose the cell undersurface.

On the undersurface of the 5-hr cultured cells, some dark spots with fewer gold beads were observed near the center of the cell (Figure 5a) and the gold beads appeared to be randomly distributed. At the edge of the cell, however, gold beads were concentrated and some appeared to be in linear arrays (Figure 5a and Figure 5b). The size of the gold beads measured on high-power images was in the range of 40–50 nm, which was conceivably the size of gold beads used for immunostaining after slight gold–palladium coating (Figure 5c). In controls, no FN-positive immunogold staining was observed on undersurfaces of cells incubated with preabsorbed primary antibody (Figure 5d).

Because the 24-hr cultured fibroblasts had a lot of fibrils on their undersurfaces (see Figure 2b and Figure 2c), it was difficult to identify the gold beads. Therefore, BSE detection, in which contrast depends on the atomic number, was used to recognize the gold marker in high contrast. Thus, the combination of SE and BSE images would provide a comprehensive understanding of FN distribution.

In this study, we mainly used SE imaging for the observation of immunolabeling on 5-hr cultured cells because the high energy of BSE often melted the surface of specimens embedded in resin. When we obtained BSE images, we had to use the lowest possible accelerating voltage beam. SE imaging gave us satisfactory results with this large gold size.

By this cell undersurface immunogold staining technique, it is probable that 24-hr cultured fibroblasts had less FN on the cell undersurface than that on 5-hr cultured cells, and that no FN alignment was localized on the undersurface of 24-hr cultured cells (not shown).


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

A crucial process in immunostaining ventral cell surfaces is removal of the resin left on the cell surfaces. There are several dry etching techniques, including Ar+ ion beam, O2+ ion beam, and O2 radiofrequency electrodeless discharging (Linton et al. 1984 ). An Ar+ ion beam was used in this study because it does not etch the surface by chemical effects, as observed in relative O2+ ion bombardment, but instead relies on physical sputtering. Furthermore, lipid-containing structures, such as the plasma membrane, are commonly etch-resistant relative to the cytoplasm (Humphreys and Henk 1979 ). We found that the resin on the cell surface was completely removed by 15-min or longer Ar+ etching without any apparent damage to the cell surface. Richards et al. 1993 showed that prolonged glow discharge can cause damage to the cell, but in our results there was no conspicuous damage up to 20-min Ar+ glow discharge. Another advantage of using glow discharge is the reduction of background labeling by colloidal gold. Squarzoni et al. 1990 reported that the glow discharge method reduced ion-specific absorption by inhibiting binding between the colloidal gold complex and the resin, which resulted in less nonspecific staining. As shown in Figure 5, very little nonspecific immunogold staining was observed outside the cell surface, on the resin surface. However, the immunolabeling became poor when the cell was etched for a longer period (60 min). In this case, the antigenicity would be decreased by damage to the cell surface, as reported by Richards et al. 1993 .

Several patterns of separation are conceivable when the embedded cells are separated from the substrate: (a) The cells are completely embedded in the resin and their undersurfaces are exposed. (b) The cells are split between the plasma membrane and the cytoplasm, and the cell membrane is left on the substrate. (c) The cells are completely left on the substrate. To determine which part of the cell was exposed, cell membrane and cytosolic staining and TEM cross sectional observation were used. Cell membrane and cytosolic staining clearly showed the cells morphology (Figure 3), and the continuity of the cell membrane was visible in the TEM image (Figure 4). These observations demonstrated that the surfaces observed by SEM were undoubtedly cell undersurfaces.

The undersurface of fibroblastic cells observed by SEM was reported by Richard et al. (1993). Some characteristic structures were reported, i.e., the nucleus, small processes on the cell surface, and stress fibers. In addition, the nuclear impressions and stress lines were observed, but no small processes on the surface were found. As noted by Richards et al. 1993 , some 24-hr cultured cells showed clear nuclear impressions but others did not, nor did, as a rule, cells cultured for 5 hr or less. These authors suggested that the appearance of the nuclear impression is probably due to morphological differences related to the phase of the cell cycle. Fine fibrillar structures were especially common in cells cultured for 76 hr. In this study two kinds of fibrillar structures were observed: thick longitudinal fibrils and tiny lateral fibrils close to the edge of the cells. Whether these fibrillar structures are cell processes or extracellular fibrils is not known. The TEM studies of Chen and Singer 1982 and Hedman et al. 1978 indicated that the small fibrils or processes were not seen on the cell undersurface but were found on the dorsal cell surface. In our study, small fibrils or processes were observed on the dorsal cell surface in the cross-sectional TEM image (Figure 4) but they were never found on the cell undersurface by SEM.

Immuno-SEM using colloidal gold has been widely used to show the distribution of membrane-bound molecules, such as cell adhesion molecules or receptors on the dorsal cell surface. However, the distribution of adhesion molecules on the ventral cell surface has not been shown by SEM because of the difficulty of exposing the surface. In this study, glow discharge etching and immunogold staining were combined to observe the topographic distribution of FN on the cell undersurface without losing the antigenicity or surface structure. A pattern of gold beads on the 5-hr cultured cells was observed. This was similar to the "string of beads" that was found on the dorsal cell surface of He117 fibroblasts by immuno-SEM using 45-nm colloidal-gold (Trejosiewicz et al. 1981). An abundant amount of FN on the 5-hr cultured cell undersurfaces was found, but relatively less on the undersurface of cells cultured for 24 hr. The change in the amount of FN bound on the cell undersurface at these times might result from the differences between cells that were still spreading and those that were well spread.


  Acknowledgments

Supported by Medical Research Council of Canada (grant no. 5-97617 to DMB).

We thank Lesley Weston for technical assistance.

Received for publication April 6, 1999; accepted July 13, 1999.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Abiko Y, Brunette DM (1993) Immunohistochemical investigation of tracks left by the migration of fibroblasts on titanium surfaces. Cell Mater 3:161-170

Aebi U, Pollard TP (1987) A glow-discharge unit to render electron microscope grids and other surfaces hydrophillic. J Electron Microsc 7:29-33

Brunette DM, Melcher AH, Moe HK (1976) Culture and origin of epithelium-like and fibroblast-like cells from porcine periodontal ligament explants and cell suspensions. Arch Oral Biol 21:393-400[Medline]

Chen W-T, Singer SJ (1982) Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol 95:205-222[Abstract]

De Harven E, Leung R, Christensen H (1984) A novel approach for scanning microscopy of colloidal gold-labelled cell surface. J Cell Biol 99:53-57[Abstract/Free Full Text]

Goode D, Maugel TK (1987) Backscattered electron imaging of immunogold-labelled and silver-enhanced microtubules in cultured mammalian cells. J Electron Microsc Tech 5:263-273

Hedman K, Vaheri A, Wartiovaara J (1978) External fibronectin of cultured human fibroblasts is predominantly a matrix protein. J Cell Biol 76:748-760[Abstract]

Horisberger M, Rosset J, Bauer H (1975) Colloidal gold granules as marker for cell surface receptors in the scanning electron microscope. Experientia 31:1147-1149[Medline]

Humphreys WJ, Henk WG (1979) Ultrastructure of cell organelles by scanning electron microscopy of thick sections surface-etched by an oxygen plasma. J Microsc 116:255-264[Medline]

Linton RW, Farmer ME, Ingram P, Sommer JR, Shelburne JD (1984) Ultrastructural comparison of ion beam and radiofrequency plasma etching effects on biological tissue sections. J Microsc 134:101-112[Medline]

Nermut MV (1995) Manipulation of cell monolayers to reveal plasma membrane surface for freeze-drying and surface replication. In Severs J, Sholton DM, eds. Rapid Freezing, Freeze Fracture, and Deep Etching. New York, Willey–Liss, 151-172

Nermut MV, Burt JS (1991) Method for the study of the cell underside in cultured cell monolayers. Exp Cell Res 192:311-314[Medline]

Nermut MV, Burt JS, Hirst EMA, Larjava H (1993) Distribution of avian integrin during the lifetime of chicken embryo fibroblasts in vitro: study by immunofluorescence microscopy and immuno electron microscopy. Micron 24:363-375

Richards RG, Lloyd PC, Rahn BA, Gwynn IAP (1993) A new method for investigating the undersurface of cell monolayers by scanning electron microscopy. J Microsc 171:205-213[Medline]

Richards RG, Rahn BA, ap Gwynn I (1995) Scanning electron microscopy of the undersurface of cell monolayers grown on metallic implants. J Mater Sci Mater Med 6:120-124

Squarzoni S, Zini N, Marinelli F, Maradli NM (1990) Reduction of background labelling in colloidal gold-enzyme reaction. Histochemistry 94:297-301[Medline]

Tanaka K, Akimoto Y, Ogura K, Yamagishi S, Hirano H (1988) Colloidal gold label observed with a high resolution backscattered electron imaging in mouse lymphocytes. J Electron Microsc 37:346-350[Medline]

Tanaka K, Mitshushima A, Yamagata N, Kashima Y, Takayama H (1991) Direct visualization of colloidal gold-bound molecules and a cell-surface receptor by ultrahigh-resolution scanning microscopy. J Microsc 161:455-461[Medline]

Trejdosiewicz LK, Smolira MA, Hodges GM, Goodman SL, Livingston DC (1981) Cell surface distribution of fibronectin in cultures of fibroblasts and bladder derived epithelium: SEM-immunogold localization compared immuno-peroxidase and immuno-fluorescence. J Microsc 123:227-236[Medline]

Walther P, Muller M (1988) Science of Biological specimen. Scan Microsc Int 195–201.

Wilichek M, Spiegel S, Spiegel Y (1980) Fluorescent regents for the labelling of glycoconjugates in solution and on cell surfaces. Biochem Biophys Res Commun 92:1215-1222[Medline]





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Goto, T.
Articles by Brunette, D. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Goto, T.
Articles by Brunette, D. M.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]