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.

• Klíčová slova
• Backscatter electron imaging, High resolution scanning electron microscopy (HRSEM), Immunolabelling, Monte Carlo simulations, Scanning transmission electron microscopy, Simultaneous detection of multiple immunogold markers,
• Publikační typ
• časopisecké články MeSH

To understand the response of the Lyme disease spirochete Borrelia burgdorferi exposed to stress conditions and assess the viability of this spirochete, we used a correlative cryo-fluorescence and cryo-scanning microscopy approach. This approach enables simple exposition of bacteria to various experimental conditions that can be stopped at certain time intervals by cryo-immobilization, examination of cell viability without necessity to maintain suitable culture conditions during viability assays, and visualization of structures in their native state at high magnification. We focused on rare and transient events e.g., the formation of round bodies and the presence of membranous blebs in spirochetes exposed to culture medium, host sera either without or with the bacteriolytic effect and water. We described all crucial steps of the workflow, particularly the influence of freeze-etching and accelerating voltage on the visualization of topography. With the help of newly designed cryo-transport device, we achieved greater reproducibility.

• Klíčová slova
• Borrelia burgdorferi, Lyme disease, cryo-fluorescence, cryo-scanning electron microscopy, pleomorphism, round body, viability staining,
• Publikační typ
• časopisecké články 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
• barvení a značení metody   MeSH
• elektronová kryomikroskopie přístrojové vybavení   metody   MeSH
• fluorescenční mikroskopie přístrojové vybavení   metody   MeSH
• klíště * metabolismus   ultrastruktura   MeSH
• mikroskopie elektronová rastrovací přístrojové vybavení   metody   MeSH
• proteiny členovců metabolismus   MeSH
• proteoglykany metabolismus   MeSH
• sklo MeSH
• slinné proteiny a peptidy metabolismus   MeSH
• slinné žlázy * metabolismus   ultrastruktura   MeSH
• uhlík MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny členovců MeSH
• proteoglykany MeSH
• slinné proteiny a peptidy MeSH
• uhlík MeSH