French S A, Territo P R, Balaban R S
Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892-1061, USA.
Am J Physiol. 1998 Sep;275(3):C900-9. doi: 10.1152/ajpcell.1998.275.3.C900.
Fluorescent determinations of NADH in porcine heart mitochondria were subject to significant errors caused by alterations in inner filter effects during numerous metabolic perturbations. These inner filter effects were primarily associated with changes in mitochondrial volume and accompanying light scattering. The observed effects were detected in a standard commercial fluorometer with emission orthogonal to the excitation light path and, to a lesser extent, in a light path geometry detecting only the surface fluorescence. A method was developed to detect and correct for inner filter effects on mitochondrial NADH fluorescence measurements that were independent of the optical path geometry using an internal fluorescent standard and linear least-squares spectral analysis. A simple linear correction with the inner fluorescence reference was found to adequately correct for inner filter effects. This approach may be useful for other fluorescence probes in isolated mitochondria or other light-scattering media.
在猪心脏线粒体中，通过荧光法测定烟酰胺腺嘌呤二核苷酸（NADH）时，由于在众多代谢扰动过程中内滤光效应发生改变，会产生显著误差。这些内滤光效应主要与线粒体体积的变化以及随之而来的光散射有关。在发射光与激发光路正交的标准商用荧光计中检测到了观察到的效应，并且在仅检测表面荧光的光路几何结构中也在较小程度上检测到了该效应。开发了一种方法，使用内部荧光标准和线性最小二乘光谱分析来检测并校正内滤光效应，该效应与线粒体NADH荧光测量有关，且与光路几何结构无关。发现使用内部荧光参考进行简单的线性校正就足以校正内滤光效应。这种方法可能对分离的线粒体或其他光散射介质中的其他荧光探针有用。
