Jonker A, Geerts W J, Chieco P, Moorman A F, Lamers W H, Van Noorden C J
Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.
Histochem J. 1997 May;29(5):347-64. doi: 10.1023/a:1026434816947.
The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetic in difficult imaging situations, such as low fluorescence signals and high absorbance signals.
本综述为建立一个图像分析系统提供了一个起点，该系统用于细胞制剂、组织切片或凝胶中的定量密度测定以及吸光度或荧光测量。描述了仪器设置指南，这些指南对于图像分析在细胞光度法和细胞荧光测定法中的有效应用至关重要。介绍了电荷耦合器件（CCD）相机工作机制的一般原理以及改善相机性能的一般方法。由于其对有效细胞计数的重要性，显微镜和宏观物体照明的优化受到特别关注。列出了定量测量中的误差来源，并描述了为优化系统使用而调整CCD相机、图像采集卡和照明的分步图表。针对诸如低荧光信号和高吸光度信号等困难成像情况，给出了改进图像算法的建议。
