Intra-vital microscopy using non-linear optical techniques

这些研究的目的是发展成像技术来监测亚细胞结构和过程，在体内。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适应生物组织和结构的体内光学显微镜系统，而不是强迫动物在传统的显微镜台上。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）被用于研究细胞内的亚细胞代谢过程，在完整的动物，在正常的体内条件下，使用各种外源性和内在荧光探针。我们继续改进这种方法的技术，通过使用六pod压电定位平台扩展我们的快速z聚焦系统，采用完整的X-Y-Z运动校正方案。这一阶段已完全融入显微镜的操作中。运动补偿算法也被开发来补偿平面内的位移，从而提高信噪比性能。利用这些方法，人们开始研究运动、缺氧和各种基因操作对生理的影响。2)利用TPEFM确定了猪冠状动脉和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蛋白和弹性蛋白的完整三维微观结构。我们发现，位于血管分支点的独特暴露的多聚糖特异性地结合LDL。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，然后是疏水的自结合，导致合作行为。LDL与血管壁结合的稳态动力学揭示了一个拐点，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同特性可能部分地响应了这一现象。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，肾动脉是一种极易发生动脉粥样硬化的血管。3)利用TPEFM的固有特性，我们开发了一种成像方案，该方案在成像实验期间几乎收集了探测器发射的所有光。这种方法被称为总发射检测（TED）使用一个反射系统，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，将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.