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: 9560568
• 负责人: Robert Balaban
• 金额: $ 174.4万
• 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/9560568
• 关键词: Animals; Bibliography; Biological; Blood Vessels; Caliber; Cell physiology; Cells; Cellular Structures; Cellular biology; Clinical; Collaborations; Coupled; Coupling; Culicidae; Diffusion; Evaluation; Event; Excision; Glass; Goals; Green Fluorescent Proteins; Heart; Imaging Techniques; Injectable; Ion Exchange; Ions; Label; Laboratories; Malaria; Medicine; Microscope; Microscopy; Mitochondria; Modeling; Monitor; Mus; Muscle; Muscle Cells; Muscle Contraction; Muscle Fibers; Myocardium; Nature; Optics; Parasites; Physiology; Potassium; Potential Energy; Process; Proteins; Protons; Publishing; Reticulum; Risk; Signal Transduction; Skeletal Muscle; Skin; Sodium Chloride; Structure; System; Techniques; Technology; Tissues; design; heart cell; in vivo; malaria infection; new technology; prevent; trafficking; transmission process; two-photon

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) We have recently established that the mitochondria forms a reticulum across the skeletal muscle cell that permits the rapid transmission of potential energy for muscle contraction across the cell. This is critical for the fundamental energy support of muscle contraction. We published this year the mitochondrial reticulum structure of the heart cell which was distinctly different from the skeletal muscle cell. In the heart this network coupled more tightly across the long axis of the muscle rather than the short axis as in the skeletal muscle. We reasoned this to be due to the reduced diameter of the heart cell when compared to the heart muscle. We are currently attempting to look for the proteins associated with the formation of the mitochondria reticulum in heart cells. 2) This tight coupling of mitochondria across all of the muscle cells is also a risk. If one mitochondria fails, it could pull down the entire mitochondrial network just like a short circuit in a house. We have recently published (Power Grid Protection of the Muscle Mitochondrial Reticulum see bibliography) that a rapid fail safe system is in place that rapidly 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. This rapid distribution of energy within the muscle cell contrasts with earlier models relying on slow high energy metabolite diffusion and provides another parameter to evaluate in different clinical conditions. The mechanisms associated with this rapid removal of mitochondria from the network is an ongoing effort in the laboratory. 3) We have also published a modeled (The electrochemical transmission in I-Band segments of the mitochondrial reticulum: see bibliography) on how the electrical conduction occurs across the mitochondrial reticulum and reached the conclusion that the dominate ions, such as potassium, sodium and chloride must carry this current with appropriate proton-ion exchange systems. 4) Using our ability to monitor subcellular events rapidly in the living animal, we have initiated 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. Using genetically labeled parasites with green fluorescent protein and mice ( with a vessel wall fluorescent protein) we have been able to observe the first few seconds of the inoculation process and observe how the parasites behave under the skin. We hope these studies will reveal how the parasites find and penetrate blood vessels to initiate the malaria infection. This understanding may provide a new strategy in preventing the malaria infection immediately after the inoculation.

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。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。在过去的一年里，我们取得了以下主要发现：1)我们最近证实，线粒体在骨骼肌细胞之间形成一个网状结构，允许肌肉收缩的势能在细胞内快速传递。这对于肌肉收缩的基本能量支持至关重要。我们今年发表了心脏细胞的线粒体网状结构，这与骨骼肌细胞有明显的不同。在心脏，这个网络在肌肉的长轴上连接得更紧密，而不是像骨骼肌那样在短轴上连接得更紧密。我们推断这是由于与心肌相比，心脏细胞的直径变小了。我们目前正在尝试寻找与心肌细胞中线粒体网状结构形成相关的蛋白质。2)线粒体在所有肌肉细胞间的紧密耦合也是一种风险。如果一个线粒体出现故障，它可能会拖垮整个线粒体网络，就像房子里的短路一样。我们最近发表了(电网保护肌肉线粒体网状结构见参考文献)，一个快速故障安全系统已经到位，可以快速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的物理分离。这种肌肉细胞内能量的快速分布与早期依赖于缓慢的高能代谢物扩散的模型形成了对比，并为在不同的临床条件下评估提供了另一个参数。与这种从网络中快速移除线粒体相关的机制是实验室正在进行的一项努力。3)我们还发表了一个模型(线粒体I带节段中的电化学传递：参见参考文献)，说明了线粒体网状结构中的电传导是如何发生的，并得出结论，主要离子，如钾、钠和氯，必须通过适当的质子-离子交换系统来携带这种电流。4)利用我们在活动物体内快速监测亚细胞事件的能力，我们与约翰霍普金斯大学的辛尼斯博士开展了一项合作，通过模拟蚊子鼻子注射疟疾寄生虫，以监测疟疾寄生虫的传播。模拟的鼻子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在动物身上进行监测。使用带有绿色荧光蛋白的基因标记寄生虫和小鼠(带有管壁荧光蛋白)，我们已经能够观察到接种过程的前几秒钟，并观察寄生虫在皮肤下的行为。我们希望这些研究将揭示寄生虫是如何找到并穿透血管来启动疟疾感染的。这一认识可能为预防接种后立即感染疟疾提供一种新的策略。