Confocal laser scanning microscope

共焦激光扫描显微镜

• 批准号: 540575830
• 金额: --
• 依托单位: Julius-Maximilians-Universität Würzburg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2024
• 资助国家: 德国
• 起止时间: 2023-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/540575830?language=en
• 关键词: Confocal; laser; scanning; microscope

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.

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，申请人已经在分离活的心肌细胞方面拥有丰富的经验，可以对其进行广泛的显微测量方法。迄今为止，这些技术包括落射荧光显微镜，它可以使用光电倍增管结合电场刺激、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。心力衰竭的一个核心缺陷是心肌细胞肌浆网 (SR) 的 Ca 储存减少了 Ca 负荷，这可以通过 SR Ca 释放通道中的 Ca“泄漏”来解释（除其他因素外）。 Ryanodine 受体 2 (RyR2)。 RyR2 的这种 Ca 泄漏使得 SR 能够自发释放 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 Ca 敏感荧光染料在细胞“静息期”（即舒张期）期间对 Ca 火花进行荧光显微镜检测是确定心肌细胞中 RyR2 开放概率和相关致心律失常电位的关键方法。这需要高时间和空间分辨率，并且由于细胞质内 Ca 火花的空间范围较小，因此需要对狭窄定义的焦平面进行孤立观察。传统的落射荧光显微镜无法满足这些要求，但需要使用共焦激光扫描显微镜（CLSM）。 CLSM 的另一个重要应用在于使用荧光标记抗体对心肌样本、分离的成体心肌细胞以及从诱导多能干细胞 (iPSC-CM) 获得的心肌细胞进行（免疫）组织学检查。这使得能够精确测定参与 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潜在治疗机制。