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.

Faculty of Science Charles University 128 00 Prague 2 Czech Republic

Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Laboratory of Electron Microscopy Institute of Parasitology Biology Centre CAS 370 05 České Budějovice Czech Republic

Laboratory of Evolutionary Protistology Institute of Parasitology Biology Centre CAS 370 05 České Budějovice Czech Republic

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