Lustyik G, Nagy I
Fritz Verzár International Laboratory for Experimental Gerontology, University Medical School, Debrecen, Hungary.
Scanning Microsc. 1988 Mar;2(1):289-99.
In this paper we give a brief outline of our bulk specimen technique developed to measure intracellular water concentration in frozen-hydrated biological specimens by means of energy dispersive X-ray microanalysis. Fractured surface of the deep-frozen tissue samples is analyzed in an electron microscope (a specimen area of 15 x 11.5 micron is scanned) using 20 kV accelerating voltage and 1-5 pA effective beam current (measured in the specimen). Strong electric charging, which is the main problem associated with the low temperature X-ray microanalysis of frozen-hydrated specimens, is reduced by choosing optimum temperature range for the measurements (170-185 K) and by etching a thin surface layer on specimen surface. The main advantage of the method over other X-ray microanalytical techniques using sections and bulk specimens for water and dry-mass content determinations in cells (which are shortly reviewed) is the simple specimen preparation, the easy sample handling and the good stability of specimen during measurements. The main disadvantage is the poor spatial resolution as compared to the analysis of sections. Measurements with our method provided meaningful results of the change in intracellular water contents in various postmitotic cells of rats dependent on age. The observed decline of the intracellular water contents results in increased ionic strength and slower diffusion in old cells than in young ones. These effects may be implicated in senescent deterioration of cell metabolism.
在本文中，我们简要概述了我们开发的大块标本技术，该技术通过能量色散X射线微分析来测量冷冻水合生物标本中的细胞内水浓度。在电子显微镜下分析深度冷冻组织样本的断裂表面（扫描15×11.5微米的标本区域），使用20 kV加速电压和1 - 5 pA有效束流（在标本中测量）。通过选择测量的最佳温度范围（170 - 185 K）并在标本表面蚀刻一层薄表面层，减少了强电荷积累，这是与冷冻水合标本的低温X射线微分析相关的主要问题。与其他使用切片和大块标本进行细胞内水和干物质含量测定的X射线微分析技术（简要回顾）相比，该方法的主要优点是标本制备简单、样品处理容易以及测量期间标本稳定性良好。主要缺点是与切片分析相比空间分辨率较差。用我们的方法进行的测量提供了大鼠各种有丝分裂后细胞中细胞内水含量随年龄变化的有意义结果。观察到的细胞内水含量下降导致老年细胞中的离子强度增加且扩散比年轻细胞慢。这些效应可能与细胞代谢的衰老退化有关。
