Compensation for changes in tissue light absorption in fluorometry of hypoxic perfused rat hearts.
Compensation for changes in tissue light absorption in fluorometry of hypoxic perfused rat hearts.
复制标题
补偿缺氧灌注大鼠心脏荧光测定中组织光吸收的变化。
DOI：
10.1152/ajpheart.1994.266.6.h2554
复制
发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Weiner,MW
中科院分区：
文献类型：
--
作者：
Brandes,R;Figueredo,VM;Camacho,SA;Weiner,MW
组织中NADH和各种染料的荧光研究由于光吸收的变化而变得复杂，导致结果不真实。研究了减少和补偿灌注大鼠心脏在常氧和缺氧条件下光吸收变化的方法。利用透射光和反射光分别测定整个心室壁和心外膜的等吸波长（即不依赖氧合的吸收）。等吸波波长约为385、427、4555、510和525 nm，在心外膜、整个心室、跳动的心脏和停搏的心脏中都是相似的，尽管在不同的实验中确切的波长不同。此外，心外膜和心内膜的绝对光吸收完全相同。在非等吸波长，改变光吸收对荧光的影响是量化在不同的检测波长使用参考染料。还开发了新的校正方法，用于校正indo - 1荧光比，以改变吸收，使结果与检测波长无关。这些方法可用于大大减少由于改变组织光吸收在各种荧光实验的伪影。
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.