ANALYSIS OF MICROCIRCULATORY HEMODYNAMICS IN MYOCARDIUM USING A NEW INTRAVITAL NEAR-INFRARED FLUORESCENCE MICROSCOPE SYSTEM

由于吲哚菁绿色（ICG）已被证明在近红外区域发射荧光，其荧光由于波长较长而被期望提供关于深部组织微循环的信息。我们尝试建立一种红外ICG活体电视显微镜系统，以观察和定量心肌和骨骼肌等较厚组织的微循环，并对ICG荧光进行了一系列体外荧光光谱分析，确定了在显微镜下观察ICG荧光的最佳条件。发射光谱在780~840 nm之间，发射峰波长随ICG浓度和pH值的变化而变化，荧光强度在ICG浓度为2.0~5.0 μ g/ml和pH值为8~9时达到最大值。为了在体内观察ICG荧光，对市售的红外显微镜系统进行了改进，以便配备足够长的显微镜支架和两个不同的照明光源，即，卤素灯或激光束。通过CCD电视摄像机对大鼠肠系膜、骨骼肌和心肌的微血管进行ICG荧光活体显微镜观察，发现直径在15 - 45 μ m范围内的微血管和肌肉表面下约200 - 500 μ m的位置被可视化，尽管与背景的对比度不令人满意。光学系统的修改，如引入具有更高能量的光源和最佳组合的过滤器正在进行中，以实现研究跳动的心肌的微血流动力学的最终目标。

As indocyanine green (ICG) has been shown to emit fluoresence in the near infrared region, its fluorescence is expected to provide information on the deep tissue microcirculation because of the longer wavelength. We attempted to develop an infrared ICG intravital television microscope system to visualize and quantify the microcirculation of relatively thick tissues such as myocardium and skeletal muscles.To determine optimal conditions for visualizing ICG fluorescence under a microscope, a series of in vitro spectrofluorometric analyses of dye solution were performed.ICG fluoresence had its peak spectral excitation at a wavelength of 765 nm, and emission spectrum with a broad band between 780 and 840 nm, within which a peak emission wavelength varied considerably depending on the ICG concentration and pH. The intensity of fluorescence showed a maximal value in the ICG concentration range at around 2.0 - 5.0 ug/ml and pH 8-9. HPLC analyses also revealed that ICG predominantly fluoresced when bound to albumin.To visualize ICG fluorescence in vivo, a commercially available infrared microscope system was modified so as to be equipped with a long enough microscope stand and two different light sources for illumination, i.e., a halogen lamp or a laser beam. Intravital microscopic observation of the ICG fluorescence through a CCD TV camera with microvasculatures of the mesentery, skeletal muscle and myocardium of the rats revealed that microvessels over a range of 15 to 45 um in diameter and location about 200 - 500 um beneath the muscle surface were made to be visualized, although the contrast against background is not satisfactory sharp. Modification of the optical system, such that introducing a light source with higher energy and the bests uited filters are under way to fulfill the ultimate goal for studying the microhemodynamics of the beating heart muscles.