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

在探测器前加像增强器，用150 W氙灯激发，制成了一台可记录512个荧光光谱的仪器，时间分辨率为10 ms。通过引入氙闪光灯（Hamamatsu Photonics，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后以1 ms的时间间隔在预编程时间激发样品溶液，并记录连续的173个荧光光谱。该仪器可在固定波长下跟踪荧光随时间的变化，所开发的软件可方便地确定动力学参数，记录的荧光光谱可分别以波长、反应时间和荧光强度为正交轴进行三维显示。荧光强度也可以转换为假彩色，并用等高线表示。数据分析与显示软件 ...更多信息 通常采用512*512个连续谱点作为512个连续谱点进行奇异值分解（SVD）分析，以获得最少数目的组分谱。与一个假设的反应模型，描述了所有的参与反应物种和初始值的微观速率常数的模型，反应中间体的形成和衰变的微分方程求解，并存储在一个矩阵中。反应中间体的光谱是通过使用伪逆推导出来的，预测的光谱变化是使用时间矩阵以及推导的光谱构建的。然后进行优化，以最小化实验和计算光谱之间的残差，以获得动力学常数和中间体的光谱.这种技术可以普遍适用于时间分辨光谱变化的研究证明。通过选择适当的反应，将促进该技术在抗原-抗体反应中的应用。少

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