Kitzberger František, Yang Shun-Min, Týč Jiří, Bílý Tomáš, Nebesářová Jana
Laboratory of Electron Microscopy, Institute of Parasitology, Biology Centre CAS, 370 05, České Budějovice, Czech Republic.
Faculty of Science, University of South Bohemia, 370 05, České Budějovice, Czech Republic.
Sci Rep. 2024 May 2;14(1):10150. doi: 10.1038/s41598-024-60314-0.
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.
我们提出了一种用于同时检测超薄切片两侧金纳米颗粒的强大方法。该方法采用在扫描透射电子显微镜（STEM）模式下运行的高分辨率扫描电子显微镜（HRSEM），并结合背散射电子（BSE）检测。在精确选择的加速电压下，在STEM和BSE成像过程中同时记录图像。在适当的成像条件下，可以清楚地区分金纳米颗粒在顶部或底部的位置，因此表明该方法适用于使用金纳米颗粒（NPs）作为标记的多重免疫标记。上下金纳米颗粒之间的差异非常大，以至于可以应用通用软件工具（如图像J）来实现它们的自动区分。展示了切片厚度、探测器设置和加速电压对所得图像的影响。我们的实验结果与通过蒙特卡罗（MC）模拟在计算机上建模的结果一致。
