Cell-dynamics Analyzing Apparatus with Multispectral Fluorescence Images Obtained by a Cooled-CCD Camera.

本课题旨在设计和制作一套由落射荧光显微镜、脉冲电机驱动的干涉滤光片交换器、冷CCD摄像机和32位微机组成的多光谱图像采集系统。滤光片交换器装置被设计成将四个干涉滤光片中的一个交替地插入到被设计成放置在物体和目镜之间的相对长的平行光路中。这四个滤光器是从一组11个带通滤光器中选择的，其半透射宽度为20纳米，覆盖400至600纳米的波长范围。交换由计算机控制的脉冲马达驱动，命令通过个人计算机的接口传递。荧光图像通过无噪声、超宽动态范围和超高灵敏度的冷却CCD相机获得。通过GPIB接口由个人计算机发出命令进行图像采集。用该系统对注入多种荧光染料的胚胎标本或经荧光探针固定染色的胚胎标本进行多光谱成像。利用Kawata等人提出的从多光谱图像中提取材料图像的程序对获得的图像进行分析，该程序执行荧光强度矩阵的计算。矩阵的特征值表示主成分的比率，并且该值有助于确定负责多光谱图像的组分化学品的数量。然后，知道了化学物质的光谱，就有可能从多光谱图像中重建材料图像。

This project aimed to design and make the multispectral image acquiring system consisting of an epifluorescence microscope, a pulse-motor driven interference-filter exchanger, a cooled-CCD camera, and a 32 bit personal computer. The filter exchanger apparatus was designed to insert one out of four interference filters alternatively into the relatively long parallel optical path designed to be placed between the object and eyepiece. The four filters were selected from a set of 11 bandpass filters with half transmittance width 20 nanometer covering the wave length range from 400 to 600 nanometer. The exchange was driven by a computer-controlled pulse motor for which commands were delivered through the interface of the personal computer. The fluorescence images were acquired through the noise-less, ultra-widely dynamically ranged and ultra-highly sensitive cooled-CCD camera. The image acquisition was performed by the commands from the personal computer through the GPIB interface. The embryonic preparation injected with multiple fluorescent dyes or fixed and stained with fluorescent probes were used for obtaining multiple spectral images with this system. The obtained images were analyzed by the programs to extract the material images from the multispectral images, such as proposed by Kawata et al. The programs performed the calculation of fluorescence intensity matrix. The eigen values of the matrix represented the ratios of principal components and the values helped to determine the number of the component chemicals which were responsible for the multispectral images. Then, knowing the spectra of the chemicals, it was possible to reconstruct the material images from multispectral images.