Shibata M, Kawamura T, Sohirad M, Kamiya A
Institute of Medical Electronics, Faculty of Medicine, University of Tokyo, Japan.
Microvasc Res. 1995 May;49(3):300-14. doi: 10.1006/mvre.1995.1026.
By designing a new epi-illumination system with dual slit laser beams, we have developed a fluorescence microscope for tomographic observations of in vivo microcirculation. Two beams of an Argon ion laser were converted into thin slit beams of thickness 28 to 60 microns by optical lenses and were illuminated onto tissue to intersect at the focal plane of the objective. The fluorescent light emitted from a FITC 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 microns i.d.) filled with FITC-dextran 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 microns 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 three-dimensional 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.
通过设计一种具有双缝激光束的新型落射照明系统，我们开发了一种用于体内微循环断层观察的荧光显微镜。氩离子激光的两束光通过光学透镜被转换为厚度为28至60微米的细缝光束，并照射到组织上，在物镜的焦平面处相交。通过这种交叉照明从FITC示踪剂发出的荧光由显微镜通过滤光片收集，在交叉区域上方和下方的示踪剂没有“离焦”噪声。使用填充有FITC-葡聚糖的玻璃微量移液器（内径20 - 80微米）和骨骼肌组织进行的体外实验结果表明，在交叉区域示踪剂激发均匀，并且由于组织中的光散射，在距表面100和200微米深处光束厚度分别增加到1.7倍和2.5倍。在兔薄肌中的体内测试表明，该系统可以实现微血管断层成像，具有足够的垂直区域选择性和空间分辨率以重建其三维图谱，并且可以长期监测示踪剂从单个毛细血管泄漏到周围组织的情况，具有足够的定量可靠性来确定毛细血管通透性及其沿通道的异质性。
