Brandes R, Figueredo V M, Camacho S A, Weiner M W
Department of Medicine (Cardiology), University of California, San Francisco.
Am J Physiol. 1994 Jun;266(6 Pt 2):H2554-67. doi: 10.1152/ajpheart.1994.266.6.H2554.
Fluorescence studies of NADH and various dyes in tissue are complicated by changes in light absorption, causing spurious results. Methods were developed to reduce and compensate for changes in light absorption in perfused rat hearts, subjected to normoxia and hypoxia. Isosbestic wavelengths were determined (i.e., absorption independent of oxygenation) in the whole ventricular wall and in the epicardium using transmitted and reflected light, respectively. Isosbestic wavelengths were found at approximately 385, 427, 455, 510, and 525 nm, similar in the epicardium, throughout the ventricle, in beating and arrested hearts, although the exact wavelengths varied among experiments. Furthermore, absolute light absorption was identical in the epicardium and endocardium. At nonisosbestic wavelengths, the effect of changing light absorption on fluorescence was quantified at various detection wavelengths using a reference dye. New correction methods were also developed and used to correct indo 1 fluorescence ratios for changing absorption so that results were independent of detection wavelengths. These methods can be used to greatly reduce artifacts due to changing tissue light absorption in a variety of fluorescence experiments.
组织中烟酰胺腺嘌呤二核苷酸（NADH）和各种染料的荧光研究因光吸收变化而变得复杂，从而导致虚假结果。已开发出方法来减少和补偿灌注大鼠心脏在常氧和缺氧条件下光吸收的变化。分别使用透射光和反射光在整个心室壁和心外膜中确定等吸收波长（即与氧合无关的吸收）。在大约385、427、455、510和525纳米处发现了等吸收波长，在心外膜、整个心室、跳动和停搏的心脏中相似，尽管确切波长在不同实验中有所变化。此外，心外膜和心内膜的绝对光吸收是相同的。在非等吸收波长处，使用参考染料在各种检测波长下对光吸收变化对荧光的影响进行了量化。还开发了新的校正方法并用于校正因吸收变化而产生的indo 1荧光比率，以使结果与检测波长无关。这些方法可用于在各种荧光实验中大大减少因组织光吸收变化而产生的伪像。
