Development of a system for the continuous and simultaneous measurement of vascular intracellular signalings and metabolism

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The only successful technique for "the continuous and simultaneous monitoring of the intracellular signalings, function and metabolism of blood vessels, without destructing the tissue structure" must be the front-surface fluorimetry. In the first year of this study, we have newly developed a system to carry out 3 sets of dual-wavelength front-surface fluorimetries simultaneously. Followings are characteristic features of the system: (1) In collaboration with the Japan Spectroscopic Co. (JASCO, Tokyo, Japan), we have developed a "shuttering system" which controls the on/off of the excitation lights from 3 sets of fluorimeters( CAM-OF), When 1 fluorimeter is on, the other 2 are off. Thus, it is possible to obtain the information of emission of every fluorimeter for 1s every 3s, for 2s every 6s, for 4s every 12s, for 8s every 24s, or for 16s every 48s. (2) In collaboration with FUJITOK Co. (Tokyo, Japan), we have specifically designed and made the fiber-optic light guide for front-surface fluorimetry. The light guide utilizes 3 sets of quartz fibers and 3 sets of glass fibers, which are connected to 3 light sources and 3 photomultipliers at one end," respectively. Fibers are concentrically arranged at the common end, which faces to the samples. The quartz fibers are arranged in an inner circle, whereas the glass fibers are arranged in an outer circle. We also made a light guide with fibers arranged at random at the common end. Using this system, we tried to measure [Ca^<2+>]I (fura-2; Em: 500nm, Ex: 340/380nm) and (pH)I (BCECF; Em; 540nm, Ex: 500/450nm), simultaneously, and found out that 500nm Em-light for BCECF interfered the 500nm Ex-light for fura-2. In the second year, we have modified the "shuttering system", which will solve this problem. Using this system, we have successfully carried several studies about the Ca^<2+> signalings in the vascular smooth muslcs.