Confocal laser scanning microscope

申请人研究心力衰竭和其他获得性和遗传性心脏病中收缩、电生理和代谢失调的细胞和遗传机制。最重要的是细胞钙（Ca）处理的研究，其精确调节对于适当的兴奋-收缩耦合和线粒体代谢的相关适应至关重要。在这方面，申请人已经在分离重要心肌细胞方面拥有丰富的经验，这些细胞可以接受广泛的显微测量方法。到目前为止，这些技术包括聚光荧光显微镜，它可以利用光电倍增管、外加电场刺激、膜片钳技术和/或单细胞力测量来分析各种染料的整体细胞荧光。心力衰竭的一个中心缺陷是心肌细胞肌浆网（SR）的钙储存减少了钙负荷，这可以解释为（除其他因素外）钙从SR钙释放通道（Ryanodine Receptors 2 (RyR2)）“泄漏”。这种来自RyR2的Ca泄漏使SR自发释放Ca（所谓的Ca火花），这被认为是心律失常的重要触发因素。因此，使用钙敏感荧光染料在细胞的“静息期”（即舒张期）对钙火花进行荧光显微镜检测是确定心肌细胞中RyR2开放概率和相关的心律失常电位的关键方法。这需要高的时间和空间分辨率，并且由于细胞质内Ca火花的空间范围小，因此需要对狭窄定义的焦平面进行孤立观察。这些要求不能满足传统的荧光显微镜，但需要使用共聚焦激光扫描显微镜（CLSM）。CLSM的另一个重要应用是使用荧光标记抗体对心肌样本、分离的成人心肌细胞和从诱导多能干细胞（iPSC-CM）中获得的心肌细胞进行（免疫）组织学检查。这允许精确测定表达，亚细胞定位，以及参与钙处理和心肌细胞代谢的关键蛋白的相互作用。利用CLSM计划开展的基础科学项目旨在揭示改善心肌收缩力和治疗心律失常的新的潜在治疗机制。

The applicants investigate the cellular and genetic mechanisms of contractile, electrophysiological, and metabolic dysregulation in heart failure and other acquired and inherited heart diseases. Of central importance is the investigation of cellular calcium (Ca) handling, the precise regulation of which is crucial for proper excitation-contraction coupling and the associated adaptation of mitochondrial metabolism. In this regard, the applicants already have extensive experience in isolating vital cardiomyocytes that can be subjected to a broad array of microscopic measurement methods. These include, thus far, epifluorescence microscopes, which can analyze the overall cell fluorescence of various dyes using photomultipliers, coupled with electrical field stimulation, patch-clamp technology, and/or single-cell force measurement. A central defect in heart failure is that the Ca stores of cardiac muscle cells, the sarcoplasmic reticulum (SR), have reduced Ca loading, which is explained (among other factors) by a "leak" of Ca from the SR Ca release channels, the Ryanodine Receptors 2 (RyR2). Such a Ca leak from RyR2 enables spontaneous Ca release from the SR (so-called Ca sparks), which are considered important triggers for arrhythmias. The fluorescence microscopic detection of Ca sparks during the "resting phase" of the cell (i.e., diastole) using Ca-sensitive fluorescent dyes is therefore a key method for determining the open probability of RyR2 and the associated proarrhythmic potential in cardiac muscle cells. This requires high temporal and spatial resolution and, due to the small spatial extent of Ca sparks within the cytosol, the isolated observation of a narrowly defined focal plane. These requirements cannot be met with conventional epifluorescence microscopy, but require the use of a confocal laser scanning microscope (CLSM). Another important application of CLSM lies in the (immuno)histological examination of myocardial samples, isolated adult cardiomyocytes, and cardiomyocytes obtained from induced pluripotent stem cells (iPSC-CM) using fluorescence-labeled antibodies. This allows for the precise determination of the expression, subcellular localization, and interaction of key proteins involved in Ca handling and cardiomyocyte metabolism. The fundamental scientific projects planned using CLSM aim to reveal new potential therapeutic mechanisms for improving myocardial contractility and treating cardiac arrhythmias.