A QUANTIFICATION OF MACROMOLECULAR TRANSPORT ACROSS THE CAPILLARY WALL USINGLASER FLUORESCENCE MICROSCOPE

我们通过设计一种简单的双激光束外延照明技术，研制了一种新型的长工作距离（39 mm）荧光活体显微镜，用于在大视野范围内观察微循环。交叉照明，其中一对激光束对称地放置在物镜的两侧，使它们在物镜的焦平面相交，被用来产生入射光在物镜平面上的均匀分布。体外实验使用已知浓度的异硫氰酸荧光素右旋糖酐（FITC-dextran；分子量= 70000）溶液，证实示踪剂在宽视野（约30 mm^2）内均匀激发，荧光强度与示踪剂浓度呈线性相关（r = 0.999），范围在5 μ mol/L至25 μ mol/L之间。对仓鼠颊袋微循环的体内观察表明，该技术与明场交叉照明相比具有高对比度的优点。该显微镜照明器可用于评估生理和炎症条件下的血管通透性，具有足够的定量可靠性，可确定微血管网络所有部分的示踪剂浓度。此外，该技术的工作距离较远，对于应用于人体甲襞毛细血管镜检查具有相当大的优势。

We have developed a new fluorescence intravital microscope of long working distance (39 mm) for the observation of microcirculation in a wide visual field by designing a simple epi-illumination technique with dual laser beams. Cross-illumination, in which a pair of laser beams is symmetrically placed on either side of the objective such that they intersect at the focal plane of the objective, was employed to produce uniform distribution of the incident light in the object plane. In vitro experiments using a fluorescein isothiocyanate dextran (FITC-dextran ; molecular weight = 70,000) solution of known concentration confirmed uniform tracer excitation in a wide visual field (approximately 30 mm^2), and a linear correlation between fluorescence intensity and tracer concentration (r = 0.999), ranging between 5 mumol/L and 25 mumol/L.In vivo observations in the microcirculation of a hamster cheek pouch indicated that the present technique had the advantage of high contrast compared with the image obtained by bright-field trans-illumination. This microscope illuminator may prove useful for the evaluation of vascular permeability under physiological and inflammatory conditions, with sufficient quantitative reliability to determine tracer concentrations in all parts of the micro vascular network. Furthermore, a long working distance in this technique could have considerable advantages for the application to nail-fold capillaroscopy in humans.