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

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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.