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

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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.