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

通过设计一种新的双狭缝激光束外延照明系统，我们研制了一种用于器官微循环三维观察的荧光显微镜。两束激光通过光学透镜转换成厚度为28 ~ 60微米的细缝光束，照射到组织上，在物镜焦平面处相交。这种交叉照明的示踪剂发出的荧光被显微镜通过滤光片拾取，在交叉区上下都没有示踪剂发出的“失焦”噪声。体外实验结果表明，利用填充荧光示踪剂和骨骼肌组织的玻璃微移液管（20-80 μ m ID），在距离表面100和200 μ m深度处，示踪剂在交叉区激发均匀，光在组织中的散射使光束厚度增加1.7倍和2.5倍。兔腱肌的体内实验表明，该系统可以实现微血管断层扫描，具有足够的垂直区选择性和空间分辨率来重建其三维映射，并长期监测示踪剂从单个毛细血管渗漏到周围组织，具有足够的定量可靠性来确定毛细血管渗透率及其沿通道的异质性。

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.