Application of Colloidal Gold in Electron Microscope
Electron microscopy is essential when studying the complex structure of cells and organelles and studying cellular biological processes. Colloidal gold and its various derivatives have been the most widely used antigen markers in biological electron microscopy. These particles can be attached to many traditional biological probes including antibodies, lectins, superantigens, glycans, nucleic acids, and receptors. Due to their different sizes, these particles are easily distinguished under an electron microscope, allowing multiple simultaneous labeling experiments. In 1971, colloidal gold was first introduced into transmission electron microscopy (TEM) as a special marker. Then in 1975, Horisberger and his colleagues developed the colloidal gold method for scanning electron microscopy (SEM), and in 1979 for fluorescence microscopy. In 1979, the use of X -rays to map gold particles was reported.
In SEM, the colloidal gold method has been used to label many cells, including yeast, erythrocytes, platelets, hepatocytes, and milk fat globules. One advantage of SEM is that one can observe the details of a relatively large area (Figure 1). Unlike other traditional SEM markers, gold markers are good emitters of secondary electrons, which enables the observer to locate gold particles on the surface of cells without metal coatings. Currently, the most convenient gold markers are around 50 nm in size, although particles as small as Au22 have also been found.
Figure 1: Pre-embedded SEM of HER2 nano immunogold labels in SKBR3 cells. SKBR3 cells were fixed regardless of (A) incubation with anti-HER2 nanobodies (B, D) or anti-HER2 antibody trastuzumab (C). Immunolabeling was performed in a mixture of cold fish gelatin and acetylated bovine serum albumin. Subsequently, cells were incubated with secondary antibodies and 15 nm of protein A-conjugated gold particles. Samples were subjected to critical point drying and electron microscopy as described in Materials and Methods. Scale: Left panel: A: 1 µm, B, C: 0.5 µm, D: 0.2 µm; Right panel: A: 250 nm, BD: 100 nm.
In TEM, gold markers can be easily identified due to their opacity to electrons and their characteristic shape. Furthermore, the choice of particle size depends on the magnification used and consideration of the steric hindrance of the binding site. accessibility. Au5 and Au20 particles are the particles most commonly used to label viruses, bacteria, yeast, plant cells, and various animal cells (Figure 2).
Figure 2: TEM of pre-embedded nanobody for HER2 immunogold labeling in SKBR3 cells. SKBR3 or MDA-MB-231 cells were fixed with 4% (w/v) formaldehyde and processed for immunolabelling. As a negative control, the primary antibody was omitted (A) or MDA-MB-231 cells lacking HER2 expression were used (B). SKBR3 cells were fixed with (C, E) or (D, F) formaldehyde and treated with anti-HER2 nanobody 11A4 (VHH) (C, D) or trastuzumab (mAb) (E, F) Incubation. Scale = 500 nm.
Immuno-electron microscopy is one of the best methods for detecting and localizing proteins in cells and tissues. This approach can be applied to virtually every species of unicellular and multicellular organisms, often providing unexpected insights into the linkages between structure and function. The application of primary antibodies bound to gold particles allows high-resolution detection and localization of multiple antigens on and within cells. However, the successful application of immunoelectron microscopy is determined by the following factors: 1) the preservation of protein antigenicity; 2) the ability of the antibody to infiltrate the whole cell; 3) the specificity of recognition between the antigen and the primary antibody. In addition, appropriate handling of biological samples, such as fixation, is required, which involves the proper selection of embedding resins and specific antibodies against the target molecule.
While conventional electron microscopy cannot provide information about specific molecules, immunogold labeling can establish a high-resolution link between visible structures, specific in situ localization, and molecular distribution. In this light, there is no doubt that the application of colloidal gold particles represented a major event in the improvement of immunochemical methods. During the process of introducing colloidal gold into immunocytochemistry, many protocols were developed. Generally, they are TEM-based and include immunolabeling after resin embedding or immunostaining prior to the process.
Although colloidal gold has several uses in histochemistry, its primary application is in cytochemistry using SEM and TEM. This approach is common and relies on a wide variety of macromolecules that can attach to gold particles. Furthermore, gold labels can be prepared quickly and cheaply with little non-specific adsorption and can be quantified by various methods. They are also ideal for multiple labeling experiments select. Finally, by using colloidal gold under an electron microscope, intracellular antigens can be clearly identified.
References
1. Horisberger, M. (1981). Colloidal gold as a cytochemical marker in electron microscopy. Gold Bulletin, 14(3), 90-94.https://link.springer.com/article/10.1007/BF03216735
2. De Paul, A. L., Mukdsi, J. H., Petiti, J. P., Gutiérrez, S., Quintar, A. A., Maldonado, C. A., & Torres, A. I. (2012). Immunoelectron microscopy: a reliable tool for the analysis of cellular processes. In Applications of Immunocytochemistry. IntechOpen. https://sc.panda321.com/#google_vignette
3. Horisberger, M., Rosset, J., & Bauer, H. (1975). Colloidal gold granules as markers for cell surface receptors in the scanning electron microscope. Experientia, 31(10), 1147-1149. https://link.springer.com/article/10.1007/BF02326761
4. Kijanka, M., van Donselaar, E. G., Müller, W. H., Dorresteijn, B., Popov-Čeleketić, D., El Khattabi, M., & Post, J. A. (2017). A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy. Journal of structural biology, 199(1), 1-11.https://doi.org/10.1016/j.jsb.2017.05.008