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，使用徕卡的新型谐振TPEFM系统，提供接近实时的三维数据和图形处理单元（GPU），对体内组织进行接近实时的3D运动校正。这种具有GPU的真正实时3D成像技术的新颖接口为活体显微镜提供了第一个实时运动校正方案。2)使用这种运动补偿系统的早期版本，我们已经成功地确定了包括肾脏、骨骼肌和肝脏在内的几个器官的细胞和血管结构的3D结构。这些结构研究提供了这种组织微观结构的无与伦比的观点，为微循环如何与功能性细胞元件耦合提供了见解。3)我们还应用该技术在体内监测骨骼肌线粒体对整体缺氧的细胞内代谢反应。我们确定细胞内不同的线粒体池在静息肌肉中处于不同的氧化还原状态。位于毛细血管附近的线粒体被发现比所谓的纤维内区域的细胞深处的线粒体明显更氧化。此外，我们证明线粒体集中在血管旁区域，而不是在纤维内区域。这些观察结果导致了一个新的假设，即线粒体的分布有助于整个细胞的氧梯度，在血管结构附近高，在其他地方很低，大多数细胞导致静止的细胞PO2总体较低。当肌肉处于静止状态时，这种低细胞PO2的维持可能有利于防止活性氧或蛋白质氧化的产生。目前正在尝试直接测量细胞内的氧张力。4)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）。我们目前已经修改了这个最初的概念，包括一个与体内测量兼容的表面收集方案。该系统已被证明可以提高体内荧光成像实验的信号采集，提高了2-4倍。显然，这种方法是目前在体外或体内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 new resonant TPEFM system from Leica providing near real time 3 dimension data with a graphical processing unit (GPU) to perform near real time 3D motion correction of tissues in vivo. This novel interface of a true real time 3D imaging technique with a GPU provides the first real time motion correction scheme for intra-vital microscopy. 2) Using an earlier version of this motion compensation system we have been successful in determining the 3D structure of the cells and vascular structures in several organs including the kidney, skeletal muscle and liver, in vivo. These structural studies have provided unparalleled views of this tissue microstructure providing insight into how the microcirculation is coupled to the functional cellular elements. 3) We also applied this technology to monitor the intracellular metabolic responses of skeletal muscle mitochondria to global hypoxia, in vivo. We determined that different pools of mitochondria within the cell are poised at different redox states in the resting muscle. The mitochondria located adjacent to capillaries was found to the significantly more oxidized than mitochondria deep inside the cell in the so called intrafibrillar regions. In addition, we demonstrated that the mitochondria are concentrated in the paravascular regions compared to the intrafibrillar regions. These observations led to the novel hypothesis that the distribution of mitochondria is contributing to an oxygen gradient across the cell, high near the vascular structures and very low in the remaining, majority of the cell resulting in an overall low cellular PO2 at rest. This maintenance of a low cellular PO2 may be advantageous to prevent the generation of reactive oxygen species or protein oxidation when the muscle is at rest. Direct measurements of the oxygen tension in the cell are currently being attempted. 4) 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). We have currently modified this initial concept to include a surface collecting scheme compatible with in vivo measurements. This system has shown to improve the signal collection from fluorescence imaging experiments in vivo by a factor of 2-4 fold. Clearly, this approach is currently the most efficient method of imaging any fluorescent probe in vitro or in vivo.