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)已得到进一步发展，以绘制亚细胞 冷冻水合生物冰冻切片中水的分布。 以前，水的测定方法是间接的，因为它们 要求冰冻切片首先脱水。新方法 利用光谱成像技术，收集鳗鱼数据 在每个像素处平行。需要几个操作才能处理 光谱，包括探测器暗电流的减法； 通过检测器的点扩展函数进行反卷积；去除复数 非弹性散射；支撑膜的修正。这个 得到的单次散射分布符合标准 每个像素处的参考光谱，可以确定水分含量 从拟合系数中得出。尽管暗场或 来自水合冷冻切片的亮场图像显示最小 结构，处理后的鳗鱼图像由于以下原因显示出强烈的对比度 水分含量的变化。

The water-mapping technique that we developed, based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM), has been developed further to map subcellular distribution of water in frozen-hydrated biological cryosections. Previously, methods for water determination were indirect in that they required the cryosections to be dehydrated first. 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.