Intra-vital microscopy using non-linear optical techniques

使用非线性光学技术的活体显微镜检查

• 批准号: 7969077
• 负责人: Robert Balaban
• 金额: $ 55.29万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969077
• 关键词: Animals; Biological; Blood Vessels; Blood capillaries; Cells; Cellular Metabolic Process; Cellular Structures; Cellular biology; Collection; Coupled; Data; Detection; Dimensions; Elements; Evaluation; Financial compensation; Fluorescence Microscopy; Fluorescent Probes; Generations; Goals; Hypoxia; Image; Imaging Techniques; In Vitro; Kidney; Light; Liver; Maintenance; Measurement; Medicine; Metabolic; Microcirculation; Microscope; Microscopy; Mitochondria; Monitor; Motion; Muscle; Muscle Mitochondria; Nature; Noise; Optics; Organ; Oxidation-Reduction; Oxygen; Oxygen measurement, partial pressure, arterial; Physiological; Physiology; Process; Proteins; Reactive Oxygen Species; Resolution; Rest; Scheme; Signal Transduction; Skeletal Muscle; Staging; Structure; Surface; System; Techniques; Technology; Three-Dimensional Imaging; Time; Tissues; capillary; computerized data processing; fluorescence imaging; imaging modality; improved; in vivo; insight; meter; minimally invasive; new technology; novel; oxidation; prevent; research study; response; three dimensional structure; two-photon

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.

这些研究的目的是开发成像技术来监测体内亚细胞结构和过程。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适用于生物组织和结构的体内光学显微镜系统，而不是将动物置于传统的显微镜平台上。在过去的一年里，取得了以下主要发现：1）微创双光子激发荧光显微镜（TPEFM）正在用于研究细胞内的亚细胞代谢过程，在完整的动物中，在正常的体内条件下，使用各种外源性和内源性荧光探针。我们通过使用来自Leica的新的共振TPEFM系统的全X-Y-Z运动校正方案来扩展我们的快速z聚焦系统，从而继续改进这种方法的技术，该系统使用图形处理单元（GPU）提供近真实的时间三维数据，以执行体内组织的近真实的时间三维运动校正。这种新颖的接口的一个真正的真实的时间3D成像技术与GPU提供了第一个真实的时间运动校正方案的活体显微镜。 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体内几个器官（包括肾脏、骨骼肌和肝脏）中细胞和血管结构的3D结构。这些结构的研究提供了无与伦比的意见，这种组织的微观结构，提供洞察微循环是如何耦合到功能的细胞元素。3)我们还应用该技术来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。发现位于毛细血管附近的线粒体比细胞内所谓的原纤维内区域的线粒体氧化程度更高。此外，我们证明了线粒体集中在血管旁区域相比，intrafrillar区域。这些观察结果导致了一种新的假设，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，而在其余的大多数细胞中非常低，导致静息时细胞PO 2总体较低。这种低细胞PO 2的维持可能有利于防止肌肉休息时产生活性氧或蛋白质氧化。目前正在尝试直接测量电池中的氧张力。 4)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从探针发出的光。这种方法被称为总排放检测（TED）。目前，我们已经修改了这个最初的概念，包括在体内测量兼容的表面收集计划。该系统已经显示出将来自体内荧光成像实验的信号收集提高2-4倍。显然，这种方法是目前在体外或体内成像任何荧光探针的最有效的方法。