CHARACTERIZATION OF FROZEN-HYDRATED SPECIMENS BY EELS

我们开发的基于电子的水成图技术 扫描电子的能量损失谱(EELS) 显微镜(STEM)已进一步增强，以绘制亚细胞 冷冻水合生物冰冻切片中水的分布。这个 新的方法利用光谱成像，从而将EELS数据 在每个像素处并行采集。需要几个操作才能 处理光谱，包括：减去探测器的暗光 电流，由探测器的点扩展函数反卷积，去除 复数次非弹性散射，并对支撑膜进行修正。这个 得到的单次散射分布符合标准 每个像素的参考光谱，以及水分含量可以从 拟合系数。尽管暗视场或明视场图像 从水化的冰冻切片中显示出最小的结构，加工后的鳗鱼 图像显示了由于水分含量的变化而产生的强烈对比。 冷冻鳗鱼也被用来确定薄层的厚度， 水化组织切片，从而提供了关于 冷冻切割过程本身。

The water-mapping technique that we developed based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been further enhanced to map the subcellular distribution of water in frozen-hydrated biological cryosections. The new approach makes use of spectrum imaging, whereby EELS data are collected in parallel at each pixel. Several operations are required to process the spectra, including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering, and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the dark-field or bright-field image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Cryo-EELS has also been applied to determine the thickness of thin, hydrated tissue sections, thus providing information about the limits of the cryosectioning process itself.