Study of the primary reaction process of firefly bioluminescence by measurements of its time-resolved absorption and emission spectra

本计画旨在利用光谱学的方法，以毫秒级的时间解析度来阐明萤火虫生物发光的主要反应过程。项目期限正式分为三年（2004年至2006年）。在第一个学期，有一个突出的技术问题，有关使用笼状ATP（CATP），ATP生产试剂的紫外光。实验结果表明，激光脉冲可以诱导CATP光化学分解生成ATP，但由于底物Ln的荧光强度太高，无法检测到生物发光的本征强度，且由于光谱峰的相似性，两者不能分离。第二阶段推迟了对装置的改进，寻找试剂制备的实验条件，使生物发光衰减曲线的带形更窄。然而，它的信号很难被制备的装置检测到。在上学期，田中博士被邀请作为一个专家的光谱测量的快速现象的时间毫秒秩序。采用机械快门防止Ln荧光对生物发光的微弱光的阻挡，采用光计数技术提高检测器的灵敏度，构建了适合于CATP检测的装置。因此，首次在毫秒时间尺度上观察到了激光脉冲触发生物发光的衰减曲线。作为该项目的成果，有两篇论文在某些国际期刊上发表，六篇论文出现在三本书中，其中一本是由Wada编辑的，并在国内年会和国际生物发光和化学发光研讨会上发表了17个主题。

This project has been planed to clarify the primary reaction process of firefly bioluminescence in msec time resolution by spectroscopic methods. Term of project was formally divided into three years (2004〜2006). In the first term, there was a conspicuous technical problem concerning the use of caged-ATP (CATP), an ATP producing reagent by UV light. Against expectation that a laser pulse can induce the bioluminescence by the photochemical decomposition of CATP to ATP, it was found that the fluorescent intensity of substrate luciferin (Ln) is too high to detect the intrinsic intensity of bioluminescence and the latter could not be separated from the former due to the similar spectral peaks each other. In the second term, the improvement of the device was postponed to search for experimental conditions of reagents' preparation so that band shape of the decay curve of bio-luminescence was much more narrow. However, its signal was hard to be detected by the prepared device. In the last term, Dr. Tanaka was invited as an expert on the spectroscopic measurement of fast phenomena in time of msec order. The equipment adequate to resolve CATP was constructed using the mechanical shutter to prevent Ln fluorescence from blocking the weak light of bioluminescence and photocounting technique to improve the sensitivity of the detector. Thus the decay curve of bioluminescence triggered by laser pulses was observed for the first time in the msec time scale. As the results of the project, there are two papers published in certain international journals, six papers appeared in three books one of which was edited by Wada and the publication of 17 subjects at the domestic annual meetings and the international symposiums on bioluminescence and chemiluminescence.