The development of multi-color FISH for mRNA

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In order to construct multi-colored images in microscopy, labeling with fluorescence dyes is most practical methods. The purpose of this project is to establish a method for constructing multi-color (5 to 7) images of fluorescence in situ hybridization toward mRNA.The object was accomplished by combining filter method and hyperspectral imaging method. The methodological bases of this project are as follows ; (1) synthesis or selection of appropriate dyes or combination of dyes, (2) to establish labeling methods of nucleic acid probe, (3) design and manufacturing filter-sets for those dyes, (4) obtaining hyperspectral images, (5) model experiment of fluorescence in situ hybridization, (6) deconvolution of obtained spectra into component spectra. Alexa series dyes (488, 532, 546, 568, 594) are the most appropriate in the viewpoint of quenching, bleaching, signal-strength and the distribution of the spectra. PCR and elongation at 3' end using TDT showed efficient incorporation of dUTP-Alexa dyes. Two filter sets were designed for obtaining hyperspectral images of those dyes. One set was for Alexa488 and Alexa532. The other was for Alexa546, Alexa568 and Alexa594. Those filter sets enabled to get proper spectra of dyes from hyperspectral images. Fluorescence in situ hybridization toward mRNA of IFN-_<gamma> and IL-4 in mouse spleen showed very clear (far better than Antibody-stained) images. Model experiment of deconvolution of hyperspectral image showed complete isolation of signals from Alexa488, Alexa532, Alexa546 and AlexaS94. These resu1ts indicate multi-color FISH for mRNA is principally possible.