Direct visualization of colloidal gold‐bound molecules and a cell‐surface receptor by ultrahigh‐resolution scanning electron microscopy

Tánaka, Keiichi; Mitsushima, Akira; Yamagata, Noboru; Kashima, Yuzuru; Takayama, Hisao

doi:10.1111/j.1365-2818.1991.tb03103.x

Cited by 11 publications

(5 citation statements)

References 11 publications

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“…A recent study demonstrated the SEM examination of uncoated biological specimens, using the plasma-ion shower method for which resolution in the nanometer range was obtained (10). Single colloidal gold-bound molecules have been directly visualised by ultra-high-resolution SEM using staining with Osmium by the OTO method (11,12). Compared with these studies, our approach employs commonly used instrumentation and not staining procedures that affect the shape of the sample.…”

Section: Discussionmentioning

confidence: 99%

Electron density imaging of protein films on gold-particle surfaces with transmission electron microscopy

1999

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Section: Discussionmentioning

confidence: 99%

Electron density imaging of protein films on gold-particle surfaces with transmission electron microscopy

1999

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“…Therefore, we prefer the CC plates to another substrate for our observations. Spreading molecules on CC plates (Tanaka et al, 1989(Tanaka et al, , 1991 allows some molecules to be observed without charging effects, but is not generally applicable. We have therefore taken the approach of using a solution as a source of impregnating or staining metal, to decrease charging and improve the electron yield.…”

Section: Discussionmentioning

confidence: 99%

Application of high‐resolution scanning electron microscopy to biological macromolecules

Nakadera

Mitsushima

Tánaka

1991

Journal of Microscopy

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SUMMARY The development of ultrahigh‐resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequate preparation methods have not yet been established. Although it has been possible to observe some molecules after they have been spread on a carbon substrate, this method has not proved suitable for other molecules which exhibit lower contrast, or are more susceptible to damage by the electron beam. In this study we have applied heavy‐metal impregnation methods using phosphotungstic acid, uranyl acetate, or osmium tetroxide mordanted by tannic acid. In addition, contamination due to the electron beam was reduced by improving the vacuum in the specimen chamber, and by the use of a heated specimen stage. Using these measures, haemocyanin, ferritin, apoferritin, thyro‐globulin and immunoglobulin M were successfully imaged. Ultrahigh‐resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules.

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“…In this way, information about the receptor's distribution can be obtained indirectly by imaging the receptor-antibody immune complex, and the characterization and orientation of the receptor can be seen indirectly. The receptor distribution on the cell membrane surface can then be depicted visually, and quantitative analysis of the number of receptors can be carried out (Tanaka et al 1991). In this study we used AFM at a nanometer scale to investigate the distribution of VEGFR-2 with immune complex labeling and to determine its receptor density on the glioma microvascular endothelial cell surface.…”

Section: Introductionmentioning

confidence: 99%

A study of the Distribution and Density of the VEGFR-2 Receptor on Glioma Microvascular Endothelial Cell Membranes

et al. 2011

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The purpose of the study was to examine the nanoscale distribution and density of the VEGFR-2 membrane receptor on the endothelial cell surface of glioma microvasculature. Immunofluorescence and atomic force microscopy combined with immunogold labeling techniques were used to characterize and determine the position of the glioma microvasculature endothelial cell surface receptor VEGFR-2. We aimed to indirectly detect the distribution of VEGFR-2 on the cell membrane at the nanoscale level and to analyze VEGFR-2 quantitatively. Immunofluorescence imaging showed a large amount of VEGFR-2 scattered across the endothelial cell surface; atomic force microscopy imaging also showed two globular structures of different sizes scattered across the endothelial cell surface. The difference between the average diameter of the small globular structure outside the cell surface (43.67 ± 5.02 nm) and that of IgG (44.61 ± 3.19 nm) was not statistically significant (P > 0.05). The three-dimensional morphologies of the small globular structure outside the cell surface and IgG were similar. The difference between the average diameter of the large globular structure outside the cell surface (74.19 ± 9.10 nm) and that of IgG-SpA-CG (74.54 ± 15.93 nm) was also not statistically significant (P > 0.05). The three-dimensional morphologies of this large globular structure outside the cell surface and IgG-SpA-CG were similar. The total density of these two globular structures within the unit area was 92 ± 19 particles μm(2). No globular structures were seen on the cell surface in the control group. The large globular structure on the surface of glioma microvascular endothelial cells was categorized as a VEGFR-2-IgG-SpA-CG immune complex, whereas the small globular structure was categorized as a VEGFR-2-IgG immune complex. The positions of the globular structures were the same as the positions of the VEGFR-2 molecules. A large amount of VEGFR-2 was scattered across glioma microvascular endothelial cell surfaces; the receptor density was about 92 per square micron.

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Direct visualization of colloidal gold‐bound molecules and a cell‐surface receptor by ultrahigh‐resolution scanning electron microscopy

Cited by 11 publications

References 11 publications

Electron density imaging of protein films on gold-particle surfaces with transmission electron microscopy

Electron density imaging of protein films on gold-particle surfaces with transmission electron microscopy

Application of high‐resolution scanning electron microscopy to biological macromolecules

A study of the Distribution and Density of the VEGFR-2 Receptor on Glioma Microvascular Endothelial Cell Membranes

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