Confocal laser scanning microscope

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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.