Confocal laser scanning microscope

申请人研究了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。最重要的是研究细胞钙（Ca）的处理，其精确调节是至关重要的适当的兴奋-收缩偶联和线粒体代谢的相关适应。在这方面，申请人已经在分离可以进行广泛的显微镜测量方法的重要心肌细胞方面具有丰富的经验。到目前为止，这些包括落射荧光显微镜，它可以使用光电倍增管分析各种染料的整体细胞荧光，再加上电场刺激，膜片钳技术和/或单细胞力测量。心力衰竭的中心缺陷是心肌细胞的Ca储存，即肌浆网（SR），具有减少的Ca负载，这（在其他因素中）通过Ca从SR Ca释放通道，即Ryanodine受体2（RyR 2）的“泄漏”来解释。RyR 2的这种Ca泄漏能够使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。在细胞的“静止期”（即，因此，使用Ca-敏感的荧光染料测定RyR 2的开放概率和心肌细胞中相关的促红细胞生成电位是一种关键方法。这需要高的时间和空间分辨率，并且由于胞质溶胶内Ca火花的空间范围小，需要对狭窄定义的焦平面进行隔离观察。这些要求不能满足与传统的落射荧光显微镜，但需要使用共聚焦激光扫描显微镜（CLSM）。CLSM的另一个重要应用在于使用荧光标记的抗体对心肌样品、分离的成体心肌细胞和从诱导多能干细胞（iPSC-CM）获得的心肌细胞进行（免疫）组织学检查。这允许精确测定参与Ca处理和心肌细胞代谢的关键蛋白的表达、亚细胞定位和相互作用。计划使用CLSM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.