MICROANALYSIS OF RAPIDLY-FROZEN TISSUE IN THE FIELD EMISSION STEM

直接冷冻的小鼠肝脏和肝脏的冷冻干燥超薄切片 已经制备了大脑，并以低剂量暗视野为特征 扫描电子显微镜(STEM)。这些改进 冷冻切片得到的图像与传统塑料的图像相当 横断面。它们足够薄，可以使用现有的暗场技术 (针对厚度相关的非线性进行了修改)，以便测量 单个细胞器的干质量分数，从而推导出它们的 水分含量。数字STEM成像，结合电子和 如图所示，X射线光谱学具有重要的生物学应用。 通过对浦肯野神经元钙调节的研究。钙 单位干重树枝状隔室的浓度为 用能量色散x射线的峰/连续谱方法测定 光谱学(EDXS)，这必然夸大了元素 由于束流引起的质量损失而产生的浓缩性。干重含量 在低剂量下细胞器的数量，以及在 使用高剂量分析来校正元素浓度 质量损失。结果表明，主要的钙储存细胞器 浦肯野细胞中的树突是内质网，其中 是两种类型，根据它们的钙水平来区分。这些 正在应用技术来表征元素和离子 在其他组织中的分布，包括肝细胞和原虫 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.