Multiphoton Confocal Laser Scanning-Microscope

所要求的显微镜系统能够实现多光子激发，用于活体组织深处的图像采集、实时成像和发射采集，无需滤光片，并结合标准的共焦实时成像产生直接的光谱信息。因此，该系统将有助于在活体、原位和单细胞中研究生理和临床相关的过程和机制。基于此，我们计划对人类诱导的多能来源的心肌细胞的兴奋-收缩系统进行详细的三维重建，并用无标记光学方法研究其收缩行为。我们还将研究在离体窦房结中心脏冲动产生和传播的机制。此外，我们将同时在三个不同的隔室(即胞浆、线粒体和内质网)研究“整装”垂体腺标本中促性腺激素细胞的亚细胞钙信号。这些研究的结果将深入了解腺体内激素分泌细胞群的时空组织，并阐明这些细胞同步释放适当激素脉冲的机制，这取决于动物的荷尔蒙状态。我们还计划研究感觉细胞在呼吸道上皮细胞中的作用。在其他项目中，我们计划研究感觉细胞在呼吸道上皮细胞及其瞬时受体电位通道中的作用。我们的目标是解决导致刺激这些感觉上皮细胞的详细机制。此外，我们将讨论它们在先天免疫和获得性免疫中的直接作用，以及在将感知传递到神经系统中的直接作用。为此，我们将使用各种表达基因编码的钙传感器的转基因小鼠模型，并在原位和体内进行钙成像。

The requested microscope system enables multi photon excitation for image acquisition deep within living tissue, realtime imaging and emission acquisition that is filter-free and yields direct spectral information in combination with standard confocal realtime imaging. This system will therefore facilitate the investigation of physiologically and clinically relevant processes and mechanisms in vivo, in situ and in single cells. Based on this, we plan to generate a detailed three dimensional reconstruction of the excitation-contraction system in human induced pluripotent-derived cardiac myocytes and study their contraction behavior with a label-free optical approach. We will also study the mechanisms underlying cardiac impulse generation and propagation in the isolated sinoatrial node in situ. Furthermore, we will investigate subcellular calcium signals in gonadotrope cells in “whole mount” pituitary gland preparations in three different compartments simultaneously (i.e. in the cytosol, the mitochondria and the endoplasmic reticulum). Results from these studies will provide insight into the spatiotemporal organization of hormone-secreting cell populations within the gland and shed light on the mechanisms underlying the synchronisation of these cells to release appropriate hormone pulses depending on the hormonal status of the animal. We also plan to investigate the role of sensory cells in the airway epithelium. In other projects we plan to investigate the role of sensory cells in the airway epithelium and their transient receptor potential channels. We aim at resolving the detailed mechanisms leading to stimulation of these sensory epithelial cells. Moreover we will address their direct role in innate and adaptive immunity and in transmission of perception to the nervous system. For this we will employ various transgenic mouse models expressing genetically encoded calcium sensors and calcium imaging in situ and in vivo.