High pressure freezer with a light and electrical stimulation

我们在此申请一种高压冷冻器，其具有集成到冷冻室中的光和电刺激单元。所要求的设备是一个关键的研究设备，我们希望联合收割机与我们的三维电子断层成像工作相结合，以解决心肌细胞的“动态超微结构”-细胞的功能是收缩和放松，每一秒的人的生活。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的良好记录之上，并建立在对本文所要求的设备适用于我们研究的实际测试之上。我们以前的工作仅限于化学固定制剂，其中有限的时间分辨率（在几分钟而不是几毫秒的范围内）限制了我们对机械变形期间心肌细胞亚细胞结构域中观察到的3D纳米级变化的功能相关性进行陈述的能力。生理启动（即动作电位触发）收缩周期期间心脏超微结构的毫秒分辨3D重建将使我们能够解决基本相关的关键问题（例如，小窝的长度依赖性膜整合，对流T管流体运输，兴奋-收缩耦合中Ca 2+诱导的Ca 2+释放的空间确定增益）。所要求的设备将使我们能够进行主要的新研究（收缩认知纳米-微观-宏观集成），并深入了解临床相关的心血管生理学（由于内置的电刺激器，该系统可以与包括人体样本在内的非基因修饰组织一起使用，以探索疾病相关的重塑）。与此同时，我们将创建一个管道，用于在真实自然的时间框架内研究收缩细胞的超微结构，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3D nm分辨率信息的独特参考来源。这些见解将适用于从兴奋-收缩偶联的计算建模到细胞区室化背景下分子信号通路的功能解释等应用。新的研究设备将在位于弗赖堡的实验心血管医学研究所运行，靠近内部心血管组织生物库、细胞分离和培养设施、冷冻替代设备、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，可以开发出在动作电位诱导的收缩和舒张周期中对心肌细胞纳米结构动力学进行毫秒级精确3D表征所需的工具和技术。

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.