Skeletal Muscle Function under Pressure Elucidating Mechanisms of Muscle Ca2+ Signaling Failure and Ultrastructure Disintegration using a novel High-Pressure Multiphoton Microscopy Technology

高压（HP）几乎影响所有的生物过程。对于我们地球上的大多数生物圈来说，长时间的高静水压力暴露是规律而不是例外。了解hp对活细胞或整个生物体的影响需要控制压力条件和观察诱导变化的能力，以便了解生物体如何抵消或适应增加的压力环境。第一项任务在技术上具有挑战性，例如，在环境压力下密封实验，承受高达数百兆帕的压力梯度。第二项任务需要将HP条件与光学技术结合起来，在细胞和组织的生物物理研究中使用显微镜技术。活细胞显微镜已经发展到非常先进的水平，然而，HP很少被解决。从这样的高压实验中，可以获得其他生物物理技术无法获得的高压胁迫特异性活细胞的分子反应。我们的目标是结合显微镜和工程专业知识，开发一种适用于HP下活细胞多光子成像的新型HP容器。这种新型光学室将被设计成能够承受高达400mpa的压力，这代表了真核和原核细胞感兴趣的广泛压力范围。我们将把新的光学梯度折射率透镜技术直接应用到压力容器中，而不是使用玻璃窗和外部透镜。然后，该系统将应用于研究(i)场刺激哺乳动物骨骼肌细胞中的Ca2+瞬态和（ii）在从大气到深海环境的各种环境压力剖面和“压力X暴露时间”产品下静止细胞中的静息Ca2+水平。这将揭示新的生物物理见解到压力诱导的骨骼肌兴奋耦合Ca2+稳态的改变。使用二次谐波代显微镜肌球蛋白，我们将确定压力阈值和肌肉细胞的肌节解体动力学。这种方法可以研究压力引起的骨骼肌器官损伤的具体机制，从而解释哺乳动物的主要暴露极限。建立的新显微镜技术将提供一种新的工具，将当代显微镜扩展到新的压力范围，并对不同细胞类型的生物物理细胞过程进行光学研究，这将为许多不同复杂生物体的高压生物物理学提供新的途径。

High Pressure (HP) affects virtually all biological processes. Prolonged high hydrostatic pressure exposures are the rule rather than the exception for most of our earths biosphere. Understanding HPs impact on living cells or whole organisms requires controlled pressure conditions and the ability to observe the induced changes in order to understand how organisms counteract or adapt to increased pressure environments. The first task is technically challenging, e.g. sealing the experiment against ambient pressure withstanding pressure gradients of up to several hundred MPa. The second task requires to combine HP conditions with optical technologies, in the case of biophysical studies of cells and tissues as microscopy techniques. Live cellmicroscopy has been developed to very advanced levels however, HP has rarely been addressed. From such HP experiments, molecular reactions of viable cells that are specific to high pressure stress can be obtainedthat are not available by other biophysical techniques. Our goal is to combine microscopy and engineering expertise to develop a novel HP vessel suitable for multiphoton imaging of living cells under HP. The novel optical chamber will be designed to withstand pressures up to 400 MPa that represent a wide pressure range interesting for eukaryotic and prokaryotic cells. We will implement new optical gradient-index lens technology directly into the pressure vessel rather than using glass windows and external lenses. Then, the system will be applied to investigate (i) Ca2+ transients in field-stimulated mammalian skeletal muscle cells and (ii) resting Ca2+ levels in quiescent cells under various ambient pressure profiles from atmospheric to deep seaenvironments and "pressure X exposure time" products. This will reveal novel biophysical insights into the pressure-induced alterations of excitation-coupled Ca2+ homeostasis in skeletal muscle. Using second-harmonic generation microscopy of myosin, we will determine pressure thresholds and dynamics of sarcomere disintegration for muscle cells. This approach allows to study specific mechanisms for pressure-induced organ damage to skeletal muscle that can explain prime exposure limits for mammals. Theestablished new microscopy technique will provide a novel tool to extend contemporary microscopy to new pressure horizons and to optically study biophysical cell processes for different cell types which will offer newvenues for high pressure biophysics in many organisms of varying complexity.