DEVELOPMENT OF INTRAVITAL LASER MICROSCOPE FOR VISUALIZATION OF MICROVASCULAR PO_2 DISTRIBUTION

为了阐明氧在微血管中的传输和氧在骨骼肌中的分布，我们设计了一台具有氧依赖磷光猝灭技术的活体激光显微镜。将Pd-卟啉溶液注入全身血液，用光电倍增管捕捉由N_2/染料脉冲激光激发的组织面积为15μm的磷光。体外和体内氧分压(PO2)测量表明，该方法具有无创、准确、反应时间快等优点。将该技术应用于大鼠提睾肌微循环中的PO2测量。测量动脉血氧饱和度和静脉血氧分压，并按分支顺序进行分类。不同级别的平均动脉血氧分压随血管分支的增加而降低(A1：72.4、A2：51.8和A3：44.1 mm Hg)，而静脉血氧分压与血管级别无关(V1：27.5、V2：28.6和V3：32.0 mm Hg)。与不同级别小动脉相邻的间质PO2显著低于血管内PO2(I1：47.8、I2：35.8和I3：24.4)。综上所述，本研究表明，磷光猝灭激光显微镜可以用来阐明O_2在微血管中的传输和O_2在组织中的分布，尽管目前还需要更多关于组织中P_2值的区域性数据。使用激光激发磷光探头可以使较小面积的组织被照亮(&lt；10μm)，如果组织损伤的可能性被详细阐明的话。此外，我们还发现不同级别小动脉的PO_2水平有较大的降低，在血与组织的交界处也有较大的PO_2梯度。这些发现表明，毛细血管并不是向周围组织供应氧气的唯一来源。

To clarify the oxygen transport across the microvessed and oxygen distribution in the skeletal muscle, we have newly designed an intravital laser microscope with oxygen-dependent quenching of phosphorescence technique. Pd-porphyrin solution was injected into the systemic blood, and its phosphorescence excited by N_2/dye pulse laser in the tissue area of 15 μm in diameter was captured by a photomultiplier. In vitro and in vivo oxygen tension (pO_2) measurements revealed the efficiency of this method because of its non-invasiveness, accuracy, and rapid response time. This technique was applied to pO_2 measurements in the rat cremaster muscle microcirculation. The arteriolar and venular pO_2 measurements were performed at the several regions classified by branching order. The mean arteriolar pO_2 of different orders decreased according to increase the branching (A1 : 72.4, A2 : 51.8 and A3 : 44.1 mmHg), however venular pO_2 were independent on vessel orders (V1 : 27.5, V2 : 28.6 and V3 : 32.0 mmHg). Interstitial pO_2 adjacent to arterioles of different orders were significantly lower than those of intravascular pO2 (I1 : 47.8, I2 : 35.8 and I3 : 24.4 mmHg). In conclusion, the present study has shown that the phosphorescence quenching laser microscope can be used to clarify the transport of O_2 across microvessels and the distribution of O_2 in tissues, although more regional data on pO_2 values in tissues are required in the current. The use of lasers to excite phosphorescent probes enables a smaller area of tissue to be illuminated (<10μm), if the potential for tissue damage is clarified in detail. In addition, we found a large reduction in pO_2 levels of different order arterioles as well as a large pO_2 gradient at the interface between blood and tissue. These findings suggest that capillaries are not the sole source of oxygen supply to surrounding tissue.