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）几乎影响所有生物过程。长时间的高静水压力暴露是我们地球生物圈的规则而不是例外。了解HP对活细胞或整个生物体的影响需要受控的压力条件和观察诱导变化的能力，以了解生物体如何对抗或适应压力增加的环境。第一项任务在技术上具有挑战性，例如，密封实验以抵抗高达几百MPa的压力梯度。第二项任务要求将联合收割机HP条件与光学技术相结合，在细胞和组织的生物物理研究的情况下，作为显微镜技术。活细胞显微镜已经发展到非常先进的水平，然而，HP很少得到解决。从这样的HP实验中，可以获得活细胞对高压应激的特异性分子反应，这是其他生物物理技术所不能获得的。我们的目标是联合收割机显微镜和工程专业知识，开发一种新的HP容器，适用于多光子成像下的活细胞HP。新的光学室将被设计为承受高达400 MPa的压力，这代表了真核和原核细胞感兴趣的广泛压力范围。我们将采用新的光学梯度折射率透镜技术直接进入压力容器，而不是使用玻璃窗和外部透镜。然后，该系统将被应用于研究（i）场刺激的哺乳动物骨骼肌细胞中的Ca2+瞬变和（ii）在从大气压到深海环境的各种环境压力分布和“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的压力引起的骨骼肌兴奋耦合的Ca2+稳态的改变。利用二次谐波显微镜的肌球蛋白，我们将确定压力阈值和肌细胞肌节解体的动力学。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，可以解释哺乳动物的主要接触限值。建立新的显微镜技术将提供一种新的工具，以扩大当代显微镜新的压力视野和光学研究生物物理细胞过程中的不同类型的细胞，这将提供新的场所，高压生物物理在许多生物体的不同复杂性。

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.