Zupancic Gregor
Biotechnical Faculty, Department of Biology, University of Ljubljana, Vecna pot 111, P.O. Box 2995, 1001 Ljubljana, Slovenia.
Pflugers Arch. 2003 Oct;447(1):109-19. doi: 10.1007/s00424-003-1147-3. Epub 2003 Aug 12.
A method was developed for dynamic spectrophotometric measurements in vivo in the presence of non-specific spectral changes due to external disturbances. This method was used to measure changes in mitochondrial respiratory pigment redox states in photoreceptor cells of live, white-eyed mutants of the blowfly Calliphora vicina. The changes were brought about by exchanging the atmosphere around an immobilised animal from air to N2 and back again by a rapid gas exchange system. During an experiment reflectance spectra were measured by a linear CCD array spectrophotometer. This method involves the pre-processing steps of difference spectra calculation and digital filtering in one and two dimensions. These were followed by time-domain principal component analysis (PCA). PCA yielded seven significant time domain principal component vectors and seven corresponding spectral score vectors. In addition, through PCA we also obtained a time course of changes common to all wavelengths-the residual vector, corresponding to non-specific spectral changes due to preparation movement or mitochondrial swelling. In the final step the redox state time courses were obtained by fitting linear combinations of respiratory pigment difference spectra to each of the seven score vectors. The resulting matrix of factors was then multiplied by the matrix of seven principal component vectors to yield the time courses of respiratory pigment redox states. The method can be used, with minor modifications, in many cases of time-resolved optical measurements of multiple overlapping spectral components, especially in situations where non-specific external influences cannot be disregarded.
开发了一种方法，用于在存在因外部干扰导致的非特异性光谱变化的情况下进行体内动态分光光度测量。该方法用于测量活体、白眼突变家蝇（Calliphora vicina）光感受器细胞中线粒体呼吸色素氧化还原状态的变化。这些变化是通过快速气体交换系统将固定动物周围的空气换成氮气，然后再换回来实现的。在实验过程中，用线性电荷耦合器件（CCD）阵列分光光度计测量反射光谱。该方法涉及差光谱计算以及一维和二维数字滤波的预处理步骤。随后进行时域主成分分析（PCA）。PCA产生了七个显著的时域主成分向量和七个相应的光谱得分向量。此外，通过PCA我们还获得了所有波长共有的变化时间进程——残余向量，它对应于由于标本移动或线粒体肿胀导致的非特异性光谱变化。在最后一步，通过将呼吸色素差光谱的线性组合拟合到七个得分向量中的每一个来获得氧化还原状态时间进程。然后将所得的因子矩阵与七个主成分向量的矩阵相乘，以产生呼吸色素氧化还原状态的时间进程。该方法稍作修改后可用于许多时间分辨光学测量多个重叠光谱成分的情况，特别是在不能忽略非特异性外部影响的情况下。
