We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.

• Keywords
• Backscatter electron imaging, High resolution scanning electron microscopy (HRSEM), Immunolabelling, Monte Carlo simulations, Scanning transmission electron microscopy, Simultaneous detection of multiple immunogold markers,
• Publication type
• Journal Article MeSH

The aim of the study is co-localization of N-glycans with fucose attached to N-acetylglucosamine in α1,3 linkage, that belong to immunogenic carbohydrate epitopes in humans, and N-glycans with α1,6-core fucose typical for mammalian type of N-linked glycosylation. Both glycan epitopes were labelled in cryosections of salivary glands isolated from the tick Ixodes ricinus. Salivary glands secrete during feeding many bioactive molecules and influence both successful feeding and transmission of tick-borne pathogens. For accurate and reliable localization of labelled glycans in both fluorescence and scanning electron microscopes, we used carbon imprints of finder or indexed EM grids on glass slides. We discuss if the topographical images can provide information about labelled structures, the working setting of the field-emission scanning electron microscope and the influence of the detector selection (a below-the-lens Autrata improved YAG detector of back-scattered electrons; in-lens and conventional Everhart-Thornley detectors of secondary electrons) on the imaging of gold nanoparticles, quantum dots and osmium-stained membranes.

• MeSH
• Staining and Labeling methods   MeSH
• Cryoelectron Microscopy instrumentation   methods   MeSH
• Microscopy, Fluorescence instrumentation   methods   MeSH
• Ixodes * metabolism   ultrastructure   MeSH
• Microscopy, Electron, Scanning instrumentation   methods   MeSH
• Arthropod Proteins metabolism   MeSH
• Proteoglycans metabolism   MeSH
• Glass MeSH
• Salivary Proteins and Peptides metabolism   MeSH
• Salivary Glands * metabolism   ultrastructure   MeSH
• Carbon MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arthropod Proteins MeSH
• Proteoglycans MeSH
• Salivary Proteins and Peptides MeSH
• Carbon MeSH

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

• MeSH
• Actins metabolism   MeSH
• Staining and Labeling methods   MeSH
• Cell Nucleus MeSH
• Microscopy, Electron MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• HeLa Cells MeSH
• Immunohistochemistry methods   MeSH
• Nuclear Proteins metabolism   MeSH
• Metal Nanoparticles chemistry   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Nucleophosmin MeSH
• Cell Cycle Proteins MeSH
• Antibodies immunology   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   MeSH
• Carrier Proteins metabolism   MeSH
• Gold chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Nuclear Proteins MeSH
• Nucleophosmin MeSH
• Cell Cycle Proteins MeSH
• Antibodies MeSH
• Ribonucleoproteins, Small Nuclear MeSH
• SMC2 protein, human MeSH Browser
• Carrier Proteins MeSH
• Gold MeSH