Intra-vital microscopy using non-linear optical techniques

这些研究的目的是开发成像技术来监测体内的亚细胞结构和过程。使用的主要方法是非线性光学显微镜技术。我们一直在系统地开发一种活体光学显微镜系统，该系统适用于生物组织和结构，而不是迫使动物登上传统的显微镜舞台。在过去的一年中取得了以下主要发现：1)微创双光子激发荧光显微镜(TPEFM)正在使用各种外源性和内源性荧光探针，在正常活体条件下，研究细胞内、完整动物体内的亚细胞代谢过程。我们继续改进这种方法的技术，扩展了我们的快速z聚焦系统，采用了使用六脚压电定位工作台的完整X-Y-Z运动校正方案。这一阶段已经完全融入了显微镜的操作中。还开发了运动补偿算法来补偿面内位移，从而获得更高的信噪比性能。利用这些方法，人们开始研究运动、低氧和各种基因操作的生理效应。2)用TPEFM确定了猪冠状动脉和小鼠主动脉壁的大分子结构。这些数据首次揭示了这些结构中胶原和弹性蛋白的完整三维微结构。我们发现，位于血管分支点的独特暴露的多聚糖与低密度脂蛋白特异性结合。这种结合是高度合作的，最初反映了与大分子的静电相互作用，随后是疏水自结合，导致了合作行为。低密度脂蛋白与血管壁结合的稳态动力学显示，拐点约为150 mg/dl，接近血浆值，此时胆固醇变化的非线性影响反映在非线性临床结果中。低密度脂蛋白与大分子结合的高度协作性可能是这种现象的部分原因。我们目前正在使用这种方法来绘制肾动脉的整个大分子结构图，肾动脉是一种对动脉粥样硬化高度敏感的血管。3)利用TPEFM的固有性质，我们开发了一种成像方案，该方案在成像实验期间收集几乎所有来自探测器的发射光。这种称为总发射检测(TED)的方法使用了一个镜面系统，在TPEFM过程中将所有发射的光重新定向到分离的光电倍增管，使实验的信噪比提高了5倍以上，时间效率提高了25倍。显然，这种方法是目前对任何荧光探针成像最有效的方法。

The purpose of these studies was to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. The major approach used was non-linear optical microscopy techniques. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. The following major findings were made over the last year: 1) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) is being used to study sub-cellular metabolic processes within cells, in intact animals, under normal in vivo conditions using various exogenous and intrinsic fluorescent probes. We have continued to make improvements in the technology of this approach by expanding our rapid z-focusing system with a full X-Y-Z motion correction scheme using a hexa-pod piezo-electric positioning stage. This stage has been completely integrated into the operation of the microscope. Motion compensation algorithms have been also developed to compensate for in-plane displacements resulting in higher signal to noise performance. Using these approaches studies on the physiological effects of exercise, hypoxia and various genetic manipulations are being initiated. 2) Using TPEFM we have defined the macromolecular structure of the arterial wall of the porcine coronary and murine aorta. These data reveal, for the first time, the full 3 dimensional microstructure of collagen and elastin in these structures. We discovered that the uniquely exposed polyglycans located at the vessel branch points specifically bind LDL. This binding is highly cooperative, initially reflecting an electrostatic interaction with the macromolecules followed by a hydrophobic self association, resulting in the cooperative behavior. The steady state kinetics of LDL binding to vessel walls reveal an inflection point at approximately 150 mg/DL, close to the plasma value where non-linear impact of changes in cholesterol are reflected in non-linear clinical outcomes. The highly cooperative nature of LDL binding to macromolecules could be partially responsive for this phenomenon. We are currently using this approach to map the entire macromolecular structure of the renal artery, a highly susceptible vessel to atherosclerosis. 3) Using the inherent nature of TPEFM we have developed an imaging scheme that collects nearly all of the emitted light from a probe during the imaging experiment. This approach termed Total Emission Detection (TED) uses a mirror system that redirect all of the emitted light to separate photomultiplier tube during the TPEFM process increasing the signal to noise of the experiment by over a factor 5 and the time efficiency by a factor of 25. Clearly, this approach is currently the most efficient method of imaging any fluorescent probe.