Andrews S B, Buchanan R A, Leapman R D
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-4062, USA.
Scanning Microsc Suppl. 1994;8:13-23; discussion 23-4.
The availability of a cryotransfer stage, highly efficient electron energy loss spectrometers, and ultra-thin-window energy-dispersive x-ray spectrometers for the VG Microscopes HB501 field-emission scanning transmission electron microscope (STEM) provides this instrument with the potential for high resolution biological microanalysis. Recent technical advances offer cryosections that are thin enough to take advantage of the analytical capabilities of this microscope. This paper first discusses the quantitative characterization of freeze-dried, ultrathin cryosections of directly frozen liver and brain by low-dose dark-field STEM imaging. Such images reveal high-quality sections with good structural detail, mainly due to reduced preparation artifacts and electron beam damage. These sections are thin enough for dark-field mass analysis, so that the mass of individual organelles can be measured in situ, and their water content deduced. This permits the measurement of mass loss-corrected subcellular elemental concentrations. The results suggest several new applications for cryosections as illustrated by data on synaptic activity-dependent calcium regulation in Purkinje cells of mouse cerebellum. Low-dose mass analysis of cryosections in combination with x-ray and electron spectroscopy is a promising approach to quantitating physiological changes in mass distribution and elemental composition.
VG 显微镜 HB501 场发射扫描透射电子显微镜（STEM）配备了低温传输台、高效电子能量损失谱仪和超薄窗口能量色散 x 射线谱仪，这使得该仪器具备了进行高分辨率生物微分析的潜力。最近的技术进步提供了足够薄的冷冻切片，能够利用这台显微镜的分析能力。本文首先讨论了通过低剂量暗场 STEM 成像对直接冷冻的肝脏和大脑的冻干超薄冷冻切片进行定量表征。此类图像显示出具有良好结构细节的高质量切片，这主要归因于制备伪像和电子束损伤的减少。这些切片足够薄，可用于暗场质量分析，从而能够原位测量单个细胞器的质量，并推断其含水量。这使得能够测量经过质量损失校正的亚细胞元素浓度。结果表明冷冻切片有几种新的应用，如小鼠小脑浦肯野细胞中突触活动依赖性钙调节的数据所示。冷冻切片的低剂量质量分析与 x 射线和电子光谱相结合，是定量分析质量分布和元素组成生理变化的一种有前景的方法。
