Backscattered Electron Imaging of the Colloidal Gold Marker on Cell Surfaces

Harven, Étienne de

doi:10.1016/b978-0-12-333927-0.50013-1

Cited by 18 publications

(11 citation statements)

References 28 publications

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“…Confirming earlier observations by Walther & Muller (1985) we reported recently on the successful imaging of 5 nm gold markers with the field emission SEM or, after 8 rain silver enhancement with the LaB6 SEM (JEOL-JSM#840) (de Harven & Soligo, 1989). However, we could not convincingly repeat our double labelling observations with 5 and 15 nm gold markers since, in the conditions of our silver enhancement experiments, the sizes of the markers were close to overlap, preventing us from unambiguously analysing the data.…”

Section: Discussionsupporting

confidence: 87%

Double labelling of cell surface antigens with colloidal gold markers

1990

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Particles of colloidal gold of different diameters (15 nm and 40) have been used to distinctively label different surface antigens expressed on the surface of human peripheral blood B and T lymphocytes. Silver enhancement has been used to facilitate the observation of the gold particles. Observations were carried out in the backscattered electron imaging mode of the scanning electron microscope. Two different methods have been compared: in Method I the two antigens have been identified by monoclonal antibodies of different classes (IgG and IgM); in Method II monoclonal antibodies of the same subclass were used but the ligands were different (goat anti-murine Ig versus biotin/streptavidin). Some cross-reactivity was observed with Method I, but not with Method II.

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Section: Discussionsupporting

confidence: 87%

Double labelling of cell surface antigens with colloidal gold markers

1990

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“…Further advantages of a lower V, include smaller radiation damage volume, lower charging of the sample, less contamination on the specimen, and only minimum delocalization of the SE signal (Pawley and Erlandsen 1989), which was particularly true in the case of the noncoated specimens in our study. Harven and Soligo 1989, Hodges et al 1987, Walther and Muller 1988, when looking for gold labels, regard platinum coating of biological specimens of < 20 nm as unsuitable when using the BSE signal. This would indeed ap-pear to be an unfavorable method on account of the adjacent chemical groups.…”

Section: Discussionmentioning

confidence: 96%

“…Comparing the SE image of immunomarked surface areas with the BSE image (V, above 2 kV but below 15 kV), we are able to clearly identify markings in the BSE image because of the significant material contrast between biological matter and the gold label. Any attempt to quantify gold particles, therefore, depends on the BSE image (De Harven and Soligo 1989), an important point being whether one requires a surface distribution or an overall distribution of the markers involved. By varying the V, voltage one is able to detect gold particles on the surface and, in addition, at various layers within the cell (Albrecht er al.…”

Section: Discussionmentioning

confidence: 99%

Imaging of immunolabeled membrane receptors in uncoated sem specimens

Heinzmann¹,

Reininger

Autrata

et al. 1994

Scanning

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Summary: Epidermal growth factor receptors (EGFR) were labeled with I0 n m immunogold and examined on uncoated specimens of A43 I human epidermoid carcinomacells. A field emission gun and a high-sensitivity YAG ring detector were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes with a low accelerating voltage (V,). At V0=2 kV, the SE and BSE signals were too weak to identify all markers, while at Vc,=3-7 kV labeling was observed unambiguously in both the SE and BSE modes with smaller and higher working distances. Increaing the V, to above 7 kV sometimes provokes instability of the specimens. A V,, of> 10 kV produces charging artifacts in the SE image, but permits a BSE image of the gold markers providing additional topographic information. In conclusion, immunogold labeling can be used with good results for uncoated specimens.

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“…Coating of biological samples with platinum has also been previously thought to be incompatible with the detection of gold probes of less than 20 nm (3,7,12,13).…”

Section: Introductionmentioning

confidence: 99%

“…Attempts to develop quantitative methods for backscatter detection of colloidal gold markers have been reported (3,12), but until recently have been limited by the necessity to use large gold particles (>15 nm) and high accelerating voltages (>lo kV) for backscatter imaging of the gold probe. Coating of biological samples with platinum has also been previously thought to be incompatible with the detection of gold probes of less than 20 nm (3,7,12,13).…”

Section: Introductionmentioning

confidence: 99%