Fixation and Drying Protocols for the Preparation of Cell Samples for Time-of-Flight Secondary Ion Mass Spectrometry Analysis
Fixation and Drying Protocols for the Preparation of Cell Samples for Time-of-Flight Secondary Ion Mass Spectrometry Analysis
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DOI：
10.1021/ac900636v
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发表时间：
2009-09-01
影响因子：
7.4
通讯作者：
Sjovall, Peter
中科院分区：
文献类型：
--
作者：
Malm, Jakob;Giannaras, Dimitrios;Sjovall, Peter
飞行时间二次离子质谱（TOF - SIMS）是一种用于生物细胞亚细胞化学分析的有前景的工具。然而，为了获得相关信息，用于样品制备的方法至关重要。在这项工作中，我们使用了飞行时间二次离子质谱、扫描电子显微镜（SEM）和干涉反射显微镜（IRM）来研究不同的固定和干燥方法对附着在硅表面的人成纤维细胞（hTERT）的形态和化学结构的影响。具体而言，研究了两种固定技术（戊二醛化学固定和骤冷冷冻固定）以及两种干燥技术（冷冻干燥和醇替代干燥）。经确定，冷冻固定后进行冷冻干燥可产生具有保留的细胞形态、完整的细胞膜以及质膜上保留的钠/钾离子浓度梯度的干燥细胞。通过在冷冻固定前用甲酸铵（AF）水溶液洗涤样品，可以抑制干燥过程中样品表面盐分的积累。干涉反射显微镜测量表明，在用甲酸铵洗涤过程中细胞形态得以保留，尽管出现了一些肿胀。与冷冻固定相比，戊二醛固定的细胞在扫描电子显微镜下显示细胞表面结构更精细，在飞行时间二次离子质谱中脂质分布相似，但钠/钾离子梯度未得到保留。经确定，醇干燥会显著去除细胞膜磷脂，尽管使用四氧化锇作为后固定剂可降低这种影响。
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a promising tool for subcellular chemical analysis of biological cells. However, to obtain relevant information, the method used for sample preparation is critical. In this work, we have used TOF-SIMS, scanning electron microscopy (SEM), and interference reflection microscopy (IRM) to study the effects of different fixation and drying methods on the morphology and chemical structure of human fibroblast cells (hTERT) adhered to a silicon surface. Specifically, two fixation techniques (chemical fixation with glutaraldehyde and cryofixation by plunge freezing) and two drying techniques (freeze drying and alcohol substitution drying) were investigated. Cryofixation followed by freeze drying was determined to produce dried cells with preserved cell morphology, intact cell membranes, and retained sodium/potassium ion concentration gradients across the plasma membrane. By washing samples in ail aqueous solution of ammonium formate (AF) before cryofixation, the accumulation of salts on the sample surface during drying could be suppressed. IRM measurements showed that the cell morphology was preserved during washing with ammonium formate, although some swelling occurred. Compared with cryofixation, cells fixed with glutaraldehyde showed finer structures on the cell surface in SEM and similar lipid distributions in TOF-SIMS, but the sodium/potassium ion gradients were not retained. Alcohol drying was determined to remove cell membrane phospholipids significantly, although the use of osmium tetroxide as a post-fixative was shown to decrease this effect.