Ausserer W A, Chandra S, Morrison G H
Department of Chemistry, Cornell University, Ithaca, NY 14853-1301.
J Microsc. 1989 Apr;154(Pt 1):39-57. doi: 10.1111/j.1365-2818.1989.tb00566.x.
The effects of progressive ion beam bombardment on freeze-fractured, freeze-dried cultured cells during ion microscopic (SIMS) analysis were studied with scanning electron microscopy (SEM) and ion microscopy. The freeze-fracture, freeze-dry sample preparation method was generally found to preserve cell morphology to a level far exceeding the spatial resolution of the ion microscope, with splitting at the nuclear envelope being the most commonly observed artefact. SEM monitoring of surface topography of an NRK-49F fibroblast after various ion bombardment doses showed relatively uniform erosion of cellular material, with some apparent selective retention of small cytoplasmic granules. Prolonged bombardment produced no detectable lateral elemental translocation. 41K+/24Mg+ signal ratios from Swiss 3T3 fibroblasts and RBL rat basophilic leukaemia cells were shown to vary generally by less than 10% during the course of extended ion bombardment. GM0415 human skin fibroblasts containing engorged lysosomes characteristic of Hurler's Syndrome were used to evaluate the effects of ion bombardment during a typical analysis session, where ion images of 39K+, 23Na+, 40Ca+ and 24Mg+ are sequentially recorded. This cell line was chosen as a worst-case system, because these cells are often thinly spread and possess extreme surface topography. Thin cell edges were shown sometimes to sputter away during analysis, giving misleadingly low ion signals from these regions in some 24Mg+ micrographs. Various non-uniform sputtering phenomena occurring in the submicrometre spatial domain had little or no measurable impact on local intensities in ion micrographs, indicating that freeze-dried, freeze-fractured cells are sampled in a sufficiently uniform fashion that quantitative ion microscopic evaluations of intracellular elemental levels in the general cytoplasmic or nuclear regions are feasible.
利用扫描电子显微镜（SEM）和离子显微镜研究了在离子显微镜（SIMS）分析过程中，渐进式离子束轰击对冷冻断裂、冷冻干燥的培养细胞的影响。通常发现，冷冻断裂、冷冻干燥的样品制备方法能够将细胞形态保存到远远超过离子显微镜空间分辨率的水平，其中核膜处的分裂是最常见的假象。对NRK - 49F成纤维细胞在不同离子轰击剂量后的表面形貌进行SEM监测，结果显示细胞物质受到相对均匀的侵蚀，一些小的细胞质颗粒有明显的选择性保留。长时间轰击未产生可检测到的横向元素迁移。结果表明，在长时间离子轰击过程中，瑞士3T3成纤维细胞和RBL大鼠嗜碱性白血病细胞的41K + / 24Mg +信号比总体变化小于10%。GM0415人皮肤成纤维细胞含有黏多糖贮积症Hurler综合征特征性的肿胀溶酶体，用于评估在典型分析过程中离子轰击的影响，在此过程中依次记录39K +、23Na +、40Ca +和24Mg +的离子图像。选择该细胞系作为最坏情况系统，因为这些细胞通常铺展得很薄且具有极端的表面形貌。分析过程中有时会发现薄的细胞边缘溅射掉，这在一些24Mg +显微照片中会给出这些区域误导性的低离子信号。在亚微米空间域中出现的各种不均匀溅射现象对离子显微照片中的局部强度几乎没有可测量的影响，这表明冷冻干燥、冷冻断裂的细胞是以足够均匀的方式进行采样的，因此对一般细胞质或核区域内细胞内元素水平进行定量离子显微镜评估是可行的。
