MICROANALYSIS OF RAPIDLY-FROZEN TISSUE IN THE FIELD EMISSION STEM

直接冷冻小鼠肝脏的冷冻干燥、冷冻切片， 脑已经制备并通过低剂量暗场表征 扫描透射电子显微镜（STEM）。 这些改进的 冷冻切片的图像与传统塑料切片的图像相当。 路段 它们足够薄，可以使用现有的暗场技术 （针对厚度依赖非线性进行了修改），以便测量 干质量分数的个别细胞器，从而推断其 含水量 数字STEM成像，结合电子和 X射线光谱学具有重要的生物学应用，如图所示。 浦肯野神经元钙调节的研究。 钙 浓度每单位干重的树突状隔室是 通过能量色散X射线的峰值/连续方法测定 X射线能谱（EDXS），它必然夸大元素 由于光束引起的质量损失。 干重含量 低剂量下的细胞器和 高剂量分析用于校正元素浓度， 质量损失 结果表明，主要的钙储存细胞器 在浦肯野细胞树突是内质网，其中有 有两种类型，以钙的含量来区分。 这些 技术正在应用于表征元素和离子 分布在其他组织中，包括肝细胞和原生动物 寄生虫贾第虫

Freeze-dried, ultrathin cryosections of directly frozen mouse liver and brain have been prepared and characterized by low-dose dark-field scanning transmission electron microscopy (STEM). These improved cryosections gave images comparable to those from conventional plastic sections. They were thin enough to use established dark-field techniques (modified for thickness-dependent nonlinearities) in order to measure the dry mass fraction of individual organelles, and hence to deduce their water content. Digital STEM imaging, in combination with electron and x-ray spectroscopy, has important biological applications, as illustrated by studies on calcium regulation in Purkinje neurons. Calcium concentrations per unit dry weight of dendritic compartments were determined by the peak/continuum method of energy-dispersive x-ray spectroscopy (EDXS), which necessarily overstates elemental concentrations because of beam-induced mass loss. The dry-mass content of organelles at low dose and the percent of dry mass retained after analysis at high dose were used to correct elemental concentrations for mass loss. Results indicated that the major calcium storage organelle in Purkinje cell dendrites is the endoplasmic reticulum, of which there are two types, distinguished by their levels of calcium. These techniques are being applied to characterize elemental and ionic distributions in other tissues, including hepatocytes and the protozoan parasite Giardia.