Freire M, Boyde A
Department of Anatomy and Developmental Biology, University College London.
J Microsc. 1990 May;158(Pt 2):285-90. doi: 10.1111/j.1365-2818.1990.tb03002.x.
The tandem scanning reflected-light microscope (TSM) is a real-time, direct-view confocal microscope. Only those points in the specimen situated in the focal plane contribute information to the image. A Tracor Northern TMS with piezo-electric control of the objective lens was used to generate 3-D images from Golgi-impregnated hamster cerebral cortex. Stereoscopic pairs of images were recorded as 35-mm colour film transparencies by photographing while automatically through-focusing along inclined axes. Transferring the image via a TV camera to the computer, stereo-pairs were obtained by oblique through-focusing and summing, displaying maximum intensity data in each line of sight. Pseudocolour topographic displays were generated by assigning the pixel value in a zeta map image as the focal depth at which the back-scattered light signal was maximal. The TSM was also modified so that a conventional transmitted-light image with a large depth of field could be obtained simultaneously as the very shallow depth of field confocal back-scattered-light image seen at any focus level. The conventional image is a silhouette of the impregnated neurons: the top surface of the cell is not visible and the relationships of processes that cross over cell bodies cannot be discerned. TSM gives a high-contrast image. The Golgi precipitate over the neuronal surface is resolved as globular or ovoid, coloured particles. The smaller particles also cover the dendritic spines. All the confocal range (extended focus) image display methods satisfactorily demonstrated the 3-D arrangement of cell bodies and processes in the chosen volume.
串联扫描反射光显微镜（TSM）是一种实时、直视共聚焦显微镜。只有位于焦平面上的标本点才会为图像提供信息。使用一台带有物镜压电控制装置的Tracor Northern TMS，从经高尔基氏染色的仓鼠大脑皮层生成三维图像。通过沿倾斜轴自动聚焦拍摄，将立体图像对记录为35毫米彩色胶片透明片。通过电视摄像机将图像传输到计算机，通过倾斜聚焦和求和获得立体图像对，在每条视线中显示最大强度数据。通过将ζ图图像中的像素值指定为背散射光信号最大时的焦深，生成伪彩色地形图显示。TSM还经过了改进，以便在任何聚焦水平下，既能获得具有很浅焦深的共聚焦背散射光图像，同时又能获得具有大焦深的传统透射光图像。传统图像是经染色神经元的轮廓：细胞的顶面不可见，且无法分辨穿过细胞体的突起之间的关系。TSM能给出高对比度图像。神经元表面的高尔基氏沉淀物呈现为球形或卵形的彩色颗粒。较小的颗粒也覆盖在树突棘上。所有共聚焦范围（扩展聚焦）图像显示方法都令人满意地展示了所选体积内细胞体和突起的三维排列。
