Vos Yoram, Lane Ryan I, Peddie Chris J, Wolters Anouk H G, Hoogenboom Jacob P
Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, Delft2628CJ, The Netherlands.
Electron Microscopy STP, The Francis Crick Institute, 1 Midland Road, LondonNW1 1AT, UK.
Microsc Microanal. 2021 Feb;27(1):109-120. doi: 10.1017/S1431927620024745.
The authors present the application of a retarding field between the electron objective lens and sample in an integrated fluorescence and electron microscope. The retarding field enhances signal collection and signal strength in the electron microscope. This is beneficial for samples prepared for integrated fluorescence and electron microscopy as the amount of staining material added to enhance electron microscopy signal is typically lower compared to conventional samples in order to preserve fluorescence. We demonstrate signal enhancement through the applied retarding field for both 80-nm post-embedding immunolabeled sections and 100-nm in-resin preserved fluorescence sections. Moreover, we show that tuning the electron landing energy particularly improves imaging conditions for ultra-thin (50 nm) sections, where optimization of both retarding field and interaction volume contribute to the signal improvement. Finally, we show that our integrated retarding field setup allows landing energies down to a few electron volts with 0.3 eV dispersion, which opens new prospects for assessing electron beam induced damage by in situ quantification of the observed bleaching of the fluorescence following irradiation.
作者介绍了在集成荧光和电子显微镜中，在电子物镜和样品之间应用减速场的情况。减速场增强了电子显微镜中的信号收集和信号强度。这对于为集成荧光和电子显微镜制备的样品是有益的，因为为了保留荧光，与传统样品相比，添加以增强电子显微镜信号的染色材料的量通常较低。我们展示了通过应用减速场，80纳米包埋后免疫标记切片和100纳米树脂中保留的荧光切片的信号增强。此外，我们表明调整电子着陆能量特别改善了超薄（50纳米）切片的成像条件，其中减速场和相互作用体积的优化都有助于信号改善。最后，我们表明我们的集成减速场设置允许着陆能量低至几电子伏特，色散为0.3电子伏特，这为通过原位量化辐照后观察到的荧光漂白来评估电子束诱导损伤开辟了新前景。
