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Development of a high-sensitive three-dimensional fluorescence stopped-flow

高灵敏三维荧光停流装置的研制

基本信息

批准号：
04557114
负责人：
ORII Yutaka
金额：
$ 10.94万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

Fortified with an image-intensifier in front of the detector and using a 150 W Xenon lamp for excitation, an instrument that can record 512 fluorescence spectra with a time resolution of 10 ms was constructed. By introduction of a Xenon flash lamp (Hamamatsu Photonics, L-4633,1 microsecond pulse width), the time resolution was improved to 1 msec. This allows the excitation of a sample solution at pre-programd times with a 1-ms time interval at least after initiation of the reaction, and consecutive 173 fluorescence spectra are recorded. This instrument can be set up to follow the fluorescence change with time at a fixed wavelength, and the software developed determines the kinetic parameter easily.Recorded fluorescence spectra can be displayd three-dimensionally with wavelength, reaction time, and fluorescence intensity as orthogonal axs, respectively. The fluorescence intensity can be converted into pseudo-colors and expressed by contours, too. The softwares for data analysis and disp … More lay were developed by using various software tools for numerical analysis, especially MATLAB.Usually 512*512 data points collected as 512 consecutive spectra are processed by the SVD analysis to obtain the minimal number of the component spectra. With an assumed reaction model that descrives all of the participating reaction species and the initial values for microscopic rate constants for the model, the formation and decay of the reaction intermediates are derived by solving differential equations, and stored in a matrix. The spectra of the reaction intermediates are derived by using pseudoinverse, and the predicted spectral changes are constructed using the time matrix as well as the derived spectra. Optimization is then carried out to minimize the residuals between the experimental and calculated spectra to derive the kinetic constants and the spectra of the intermediates.This technnique can be applied universally to time-resolved spectral change as proved in this study. An application of this technique to the antigen-antibody reaction will be promoted by selecting appropriate reactions. Less

在探测器前加装像增强器，用150W氙灯激发，建立了一台可记录512个荧光光谱、时间分辨率为10ms的仪器。通过引入氙灯(滨松光电，L-4633，1微秒脉冲宽度)，时间分辨率提高到1毫秒。这允许至少在反应开始之后以1ms的时间间隔以预先编程的时间激发样品溶液，并记录连续的173个荧光光谱。该仪器可以在固定的波长下跟踪荧光随时间的变化，开发的软件可以方便地确定动力学参数，记录的荧光光谱可以分别以波长、反应时间和荧光强度为正交轴进行三维显示。荧光强度可以转换为伪彩色，也可以用等高线表示。数据分析与显示软件…利用各种软件工具，特别是matlab，开发了更多的Layer。通常，对512个连续光谱采集的512×512个数据点进行奇异值分解(SVD)分析，以获得最小数目的成分光谱。通过一个描述所有参与反应物种的假设反应模型和模型的微观速率常数的初始值，通过求解微分方程组得到反应中间体的形成和衰变，并将其存储在矩阵中。利用伪逆方法得到反应中间体的光谱，并利用时间矩阵和导出的光谱构造预测的光谱变化。然后进行优化，使实验光谱和计算光谱之间的残差最小，从而得到中间体的动力学常数和光谱。该技术可普遍适用于本研究证明的时间分辨光谱变化。通过选择合适的反应，将促进该技术在抗原-抗体反应中的应用。较少