Akiba Keiichirou, Tamehiro Katsuyuki, Matsui Koki, Ikegami Hayata, Minoda Hiroki
Department of Applied Physics, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo, 184-8588, Japan.
Sci Rep. 2020 Oct 15;10(1):17342. doi: 10.1038/s41598-020-74367-4.
Green fluorescent protein (GFP) and its variants are an essential tool for visualizing functional units in biomaterials. This is achieved by the fascinating optical properties of them. Here, we report novel optical properties of enhanced GFP (EGFP), which is one of widely used engineered variants of the wild-type GFP. We study the electron-beam-induced luminescence, which is known as cathodoluminescence (CL), using the hybrid light and transmission electron microscope. Surprisingly, even from the same specimen, we observe a completely different dependences of the fluorescence and CL on the electron beam irradiation. Since light emission is normally independent of whether an electron is excited to the upper level by light or by electron beam, this difference is quite peculiar. We conclude that the electron beam irradiation causes the local generation of a new redshifted form of EGFP and CL is preferentially emitted from it. In addition, we also find that the redshifted form is rather robust to electron bombardment. These remarkable properties can be utilized for three-dimensional reconstruction without electron staining in focused ion beam/scanning electron microscopy technology and provide significant potential for simultaneously observing the functional information specified by super-resolution CL imaging and the structural information at the molecular level obtained by electron microscope.
绿色荧光蛋白（GFP）及其变体是可视化生物材料中功能单元的重要工具。这是通过它们迷人的光学特性实现的。在此，我们报告了增强型绿色荧光蛋白（EGFP）的新型光学特性，EGFP是野生型GFP广泛使用的工程变体之一。我们使用光与透射电子显微镜联用技术研究了电子束诱导发光，即阴极发光（CL）。令人惊讶的是，即使是来自同一标本，我们也观察到荧光和CL对电子束辐照的依赖性完全不同。由于发光通常与电子是被光激发到高能级还是被电子束激发无关，这种差异非常奇特。我们得出结论，电子束辐照会导致局部产生一种新的红移形式的EGFP，并优先从其发射CL。此外，我们还发现红移形式对电子轰击相当稳健。这些显著特性可用于聚焦离子束/扫描电子显微镜技术中无需电子染色的三维重建，并为同时观察超分辨率CL成像指定的功能信息和电子显微镜获得的分子水平结构信息提供了巨大潜力。
