Osakabe Haruyoshi, Suzuki Mihiro, Shimizu Toshiki, Minoda Hiroki
Department of Applied Physics and Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan.
Department of Applied Physics and Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan; Department of Applied Physics, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan.
Ultramicroscopy. 2025 Jan;268:114082. doi: 10.1016/j.ultramic.2024.114082. Epub 2024 Nov 25.
Fluorescent proteins exhibit fluorescence and photoconversion, which are used to study biological phenomena. Among these, enhanced green fluorescent protein (EGFP) emits cathodoluminescence when irradiated with electron beams; this phenomenon has numerous applications in new research tools for biological phenomena. However, bleaching during electron irradiation is a major problem. Generally, the presence of water is important for biological samples, and structural observations are often performed under cryogenic conditions. One of the advantages of cryogenic conditions is the stabilization of the sample due to cooling. However, it is unclear which factor is more effective: the presence of water molecules or cryogenic preservation. To explore the stabilizing factors of the sample structure, we prepared four environments around the sample-dry at room temperature, wet at room temperature, dry at low temperature, and under cryogenic conditions-and investigated the electron beam irradiation damage by measuring the fluorescence emission spectra. Emission intensity from EGFP was attenuated, and the peak was red-shifted by electron beam irradiation; however, the intensity attenuation was fast under dry conditions at low temperature and slow under wet conditions at room temperature. These results imply that sample cooling has no significant effect on the stability of the EGFP chromophore and that the presence of water molecules is extremely important.
荧光蛋白具有荧光和光转换特性，可用于研究生物现象。其中，增强型绿色荧光蛋白（EGFP）在电子束照射下会发出阴极荧光；这种现象在生物现象的新型研究工具中有着众多应用。然而，电子照射过程中的漂白是一个主要问题。一般来说，水的存在对生物样本很重要，结构观察通常在低温条件下进行。低温条件的优点之一是通过冷却使样本稳定。然而，尚不清楚哪个因素更有效：水分子的存在还是低温保存。为了探究样本结构的稳定因素，我们在样本周围制备了四种环境——室温干燥、室温湿润、低温干燥和低温条件下——并通过测量荧光发射光谱来研究电子束照射损伤。EGFP的发射强度会因电子束照射而减弱，且峰值会红移；然而，在低温干燥条件下强度衰减很快，在室温湿润条件下则很慢。这些结果表明，样本冷却对EGFP发色团的稳定性没有显著影响，而水分子的存在极其重要。
