DEVELOPMENT OF SLIT LASER MICROSCOPE FOR THREE DIMENSIONAL OBSERVATION OF ORGAN MICROCIRCULATION

通过设计一种新的双缝激光落射照明系统，我们研制了一种用于器官微循环三维观察的荧光显微镜。通过光学透镜将两个激光束转换成厚度为28至60 μ m的薄狭缝光束，并照射到组织上以在物镜的焦平面处相交。通过该交叉照明从示踪剂发射的荧光由显微镜通过滤光器拾取，没有来自交叉区上方和下方的示踪剂的“焦点外”噪声。通过使用填充有荧光示踪剂和骨骼肌组织的玻璃微量移液器（20-80 μ m ID）的体外实验的结果证明了在交叉区中的均匀示踪剂激发和由于在组织中的光散射而导致的光束厚度的增大在距离表面100和200 μ m的深度处分别为1.7和2.5倍。在体实验表明，该系统可实现微血管断层扫描，具有足够的垂直区域选择性和空间分辨率，可重建微血管的三维图像，并可长期监测示踪剂从单个毛细血管向周围组织的渗漏，具有足够的定量可靠性，可确定毛细血管的通透性及其沿通道的沿着异质性。

By designing a new epi-illumination system with dual slit laser beams, we have developed a fluorescence microscope for three dimensional (3D) observations of organ microcirculation. Two laser beams were converted into thin slit beams of thickness 28 to 60 mum by optical lenses and were illuminated onto tissue to intersect at the focal plane of the objective. The fluorescent light emitted from tracer by this cross-illumination was picked up by the microscope through a filter, with no "out of focus" noise from the tracer above and below the crossing zone. The results of in vitro experiments by using glass micropipettes (20-80 mum ID) filled with fluorescent tracer and skeletal muscle tissue certified uniform tracer excitation in the crossing zone and the augmentation of the beam thickness due to light scattering in the tissue to be 1.7 and 2.5 times at depths of 100 and 200 mum from the surface, respectively. In vivo tests in the rabbit tenuissimus muscle revealed that this system can realize microvasculature tomography, with adequate vertical zone selectivity and spatial resolution to reconstruct its 3D mapping, and long-term monitoring of tracer leakage from a single capillary to the surrounding tissue, with sufficient quantitative reliability to determine the capillary permeability and its heterogeneity along the channel.