High pressure freezer with a light and electrical stimulation

带光和电刺激的高压冷冻机

• 批准号: 436509545
• 金额: --
• 依托单位: Albert-Ludwigs-Universität Freiburg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/436509545?language=en
• 关键词: pressure; freezer; light; electrical; stimulation

We hereby apply for a high pressure freezer with a light and electrical stimulation unit that is integrated into the freezer chamber. The requested device is a crucial research equipment that we would like to combine with our 3D electron tomographic imaging efforts to tackle the ‘dynamic ultrastructure’ of cardiomyocytes - cells whose function is to contract and relax, once every second of the human life. Our proposal builds on a solid track record of developing and utilising advanced imaging tools for monitoring cardiac ultrastructure and function in general, and on an actual test of the suitability of the here requested device for our studies.Our previous work was limited to chemically fixed preparations, where the limited temporal resolution (in the range of minutes rather than milliseconds) limited our ability to make statements on the functional relevance of observed 3D nanoscopic changes in cardiomyocyte subcellular domains during mechanical deformation. Millisecond-resolved 3D reconstructions of cardiac ultrastructure during the physiologically initiated (i.e. action-potential-triggered) contractile cycle will allow us to address crucial questions of fundamental relevance (e.g. length-dependent membrane integration of caveolae, convective T-tubular fluid transport, spatially-determined gain of Ca2+-induced Ca2+-release in excitation-contraction coupling). The requested device will allow us to conduct principally new research (contraction-cognisant nano-micro-macro integration) and drive insight into clinically relevant cardiovascular physiology (as – thanks to the built-in electrical stimulator – the system can be used with genetically non-modified tissue, including human samples, to explore disease-related remodelling). At the same time, we will create a pipeline for studying the ultrastructure of contracting cells within a true-to-nature temporal frame, and provide others with a unique reference source of 3D nm-resolution information on cardiac cell ultrastructure in different species, cardiac regions, contraction states, and patho-physiological conditions. These insights will be suitable for applications, from computational modelling of excitation-contraction coupling to the functional interpretation of molecular signalling pathways in the context of cell compartmentalization.The new research device will be operated at the Institute for Experimental Cardiovascular Medicine in Freiburg, in close proximity to the in-house cardiovascular tissue biobank, cell isolation and culture facilities, freeze-substitution equipment, molecular biology laboratory, and advanced multiphoton / confocal microscopy platform.We believe to be in a unique position with regard to track record, technical expertise, scientific abilities, and pilot data, to develop the tools and techniques required for millisecond-accurate 3D characterisation of cardiomyocyte nanostructure dynamics during the cycle of action-potential-induced contraction and relaxation.

我们在此申请一种集成在冷冻室中的具有光和电刺激单元的高压冷冻箱。所要求的设备是一种关键的研究设备，我们希望将其与我们的3D电子断层成像努力相结合，以解决心肌细胞的“动态超微结构”--心肌细胞的功能是收缩和放松，人类生命中每一秒就有一次。我们的建议建立在开发和利用先进的成像工具来监测心脏超微结构和功能方面的坚实记录，以及对这里要求的设备是否适合我们的研究的实际测试。我们之前的工作仅限于化学固定的准备，其中有限的时间分辨率(在几分钟而不是几毫秒的范围内)限制了我们就在机械变形期间观察到的心肌亚细胞亚细胞域的3D纳米变化的功能相关性发表声明的能力。生理启动(即动作电位触发)收缩周期中心脏超微结构的毫秒分辨率三维重建将使我们能够解决基本相关的关键问题(例如，腔泡与膜的长度相关的整合，对流的T管流体运输，空间决定的钙离子诱导的钙释放的增益-兴奋-收缩耦合)。所要求的设备将允许我们主要进行新的研究(收缩-认知纳米-微观-宏观整合)，并推动对临床相关心血管生理学的洞察(由于内置的电刺激器，该系统可以用于包括人体样本在内的非转基因组织，以探索与疾病相关的重构)。同时，我们将建立一个在真实的时间框架内研究收缩细胞超微结构的管道，并为其他人提供关于不同物种、心脏区域、收缩状态和病理生理条件下心脏细胞超微结构的独特的3D nm分辨率信息的参考源。这些见解将适用于从兴奋-收缩耦合的计算模型到在细胞区划的背景下对分子信号通路的功能解释的应用。新的研究设备将在弗莱堡的实验心血管医学研究所操作，靠近内部的心血管组织生物库、细胞分离和培养设施、冷冻替代设备、分子生物学实验室和先进的多光子/共聚焦显微镜平台。我们相信，在跟踪记录、技术专长、科学能力和试点数据方面处于独特的地位，开发在动作电位诱导的收缩和松弛周期中毫秒级精确的心肌纳米结构动力学的3D表征所需的工具和技术。