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。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，主要发现如下：1)线粒体网中线粒体在肌肉细胞之间的紧密偶联对细胞有危险。如果一个线粒体出现故障，就会使整个线粒体网络瘫痪，就像房子里的短路一样。我们最近完成了一项研究，证明了一种快速故障安全系统可以从网络中移除受损的线粒体。我们目前的假设是，这种故障安全或断路器机制本质上是结构性的，代表了线粒体与网络和细胞骨架的物理解耦。这是基于观察到线粒体网处于紧张状态，它被细胞骨架拉伸以保持这些复杂的位置，直到检测到损伤，线粒体被释放并弹回其原始的球形结构。这一过程在线粒体功能局部破坏的肌肉细胞中被直接观察到。这增加了一个全新的调控方面，线粒体处理它的分布和结构，以满足细胞的需要。我们目前正在改进STED显微镜，以便在超分辨率光学显微镜（25纳米）下进行这些研究，以进一步表征这种细胞过程。2)利用我们快速监测活体动物亚细胞事件的能力，我们已经完成了与约翰霍普金斯大学Sinnis博士的合作，通过模拟蚊子的喙来监测注射后疟疾寄生虫的贩运。模拟喙是一种特殊设计的荧光玻璃吸管，我们可以用双光子激发显微镜对其进行监测。3)我们正在改进最初用于水car的多光子系统，以在完整的细胞系统上执行受激荧光和拉曼光谱。这将允许足够的信号噪声来测量在吸光度或拉曼光谱中检测到的发色团的亚细胞分布，大大提高我们对细胞代谢过程的区隔化的理解。

The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. 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) The tight coupling of mitochondria across muscle cells in the mitochondria reticulum is a risk to the cell. If one mitochondria fails, it could pull down the entire mitochondrial network just like a short circuit in a house. We have recently completed a study that demonstrates a rapid fail safe system is in place that removed damaged mitochondria from the network. Our current hypothesis is that this fail safe, or circuit breaker, mechanism is structural in nature representing the physical uncoupling of the mitochondria from the network and cytoskeleton. This is based on the observation that the mitochondrial reticulum is under tension, that is stretched by the cytoskeleton to hold these complex positions, until damage is detected and the mitochondria is released and springs back to its native spherical structure. This process has been directly observed in muscle cells during local disruption of mitochondrial function. This adds an entirely new regulatory aspect to the mitochondria dealing with it distribution and structure to meet cellular needs. We are currently modifying a STED microscope to conduct these studies at super resolution optical microscopy (25 nm) to further characterize this cellular process. 2) Using our ability to monitor subcellular events rapidly in the living animal, we have completed a collaboration with Dr. Sinnis at Johns Hopkins to monitor the trafficking of malaria parasites upon inject via a simulated mosquito proboscis. The simulated proboscis is a specially designed fluorescent glass pipet that we can monitor in the animal with a 2-Photon excitation microscope.3) We are modifying our multiphoton system originally used for water CARS to perform stimulated fluorescence and Raman spectroscopy on intact cellular systems. This will permit adequate signal to noise to hopefully measure the subcellular distribution of chromophores detected in absorbance or Raman spectroscopy greatly improving our understanding of the compartmentalization of cellular metabolic process.