Kucera P, de Ribaupierre Y, de Ribaupierre F
J Microsc. 1979 Jul;116(2):173-84. doi: 10.1111/j.1365-2818.1979.tb00200.x.
A method for automated collection of various specific data from an entire microscopical preparation and their quantitative evaluation is described. Its application to the study of neuronal connections is discussed in some detail. Brain sections are scanned using a computer-controlled microscope for reflectance, fluorescences or absorbance signals. Two illuminating beams are used, one of them being amplitude modulated. By means of a synchronous detection the two signals are recorded simultaneously: for example, in an autoradiograph, the reflectance (measuring the density of the silver grains in emulsion) and the absorbance (allowing to localize the underlying counterstained cells). The data are stored in a computer. Various off-line processing schemes allow the reconstruction of the data with respect to the corresponding spatial coordinates. Thus pseudo-three-dimensional, analogue or digital, graphic displays may be obtained in which the patterns of neuronal connections can be recognized and interpreted. A method for the detection of weakly labelled nerve fibres based on digital filtering is presented. The whole processing for a frontal section of the mouse brain (7 X 10 mm area) takes less than 1 h. In addition to the evaluation of microscopically labelled material (grains of autoradiographs, horseradish peroxidase, nucleic acids) the technique described has been successfully used for the study of naturally fluorescent intracellular components in living tissue cultures.
本文描述了一种从整个显微镜标本中自动收集各种特定数据并进行定量评估的方法。详细讨论了该方法在神经元连接研究中的应用。使用计算机控制的显微镜对脑切片进行反射率、荧光或吸光度信号扫描。使用两束照明光，其中一束进行幅度调制。通过同步检测同时记录两个信号：例如，在放射自显影片中，反射率（测量乳剂中银颗粒的密度）和吸光度（用于定位下层的对比染色细胞）。数据存储在计算机中。各种离线处理方案允许根据相应的空间坐标重建数据。因此，可以获得伪三维、模拟或数字图形显示，其中可以识别和解释神经元连接模式。提出了一种基于数字滤波检测弱标记神经纤维的方法。对小鼠脑额叶切片（面积为7×10毫米）的整个处理过程耗时不到1小时。除了评估显微镜下标记的物质（放射自显影片的颗粒、辣根过氧化物酶、核酸）外，所描述的技术已成功用于研究活组织培养中天然荧光细胞内成分。
