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

通过设计一种新的双缝激光外照系统，我们研制了一种用于器官微循环三维观察的荧光显微镜。通过光学透镜将两束激光转换成厚度为28~60微米的狭缝光束，并照射到组织上，使其在物镜的焦平面相交。这种交叉照明从示踪剂发出的荧光通过滤光片被显微镜拾取，示踪剂在交叉区上方和下方都没有“离焦”噪音。用填充荧光示踪剂的玻璃微管(20-80微米直径)和骨骼肌组织进行的体外实验结果证明，在距离表面100微米和200微米的深度，组织中的光散射引起的束流厚度增加了1.7倍和2.5倍，在交叉区有均匀的示踪剂激发。活体实验表明，该系统可以实现微血管断层成像，具有足够的垂直区选择性和空间分辨率来重建其三维标测，并可以长期监测单个毛细血管向周围组织的示踪剂泄漏，具有足够的定量可靠性来确定毛细血管的通透性及其沿通道的非均质性。

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.