Confocal laser scanning microscope

为了研究吕贝克大学的神经科学问题，我们想申请一台共聚焦激光扫描显微镜。该设备将使成像实验，目前不能在校园内进行。我们是一组研究神经生物学回路和不同神经细胞类型在外周和CNS之间界面的相互作用的研究人员。由于所有申请人都在同一研究大楼工作，因此有最佳的无障碍条件和使用仪器。该显微镜将取代在同一地点使用了15年的共焦显微镜。拟议的显微镜将能够成像非常快的细胞过程，如细胞内信使Ca2+或cAMP的三维动态。此外，将在同一样品中检测不同的细胞标志物，并以高通量对特定脑结构（例如血管）进行定量分析。正在研究的界面包括血管系统的细胞，形成血脑屏障一部分的专门的神经胶质细胞，以及处理外周信号如伤害性刺激或激素的神经元。各种研究问题处理周边和中央信号的相互作用将与显微镜的帮助下回答。对于计划中的实验，显微镜必须具有优化的激发和发射技术，并可靠地区分具有相似荧光光谱的荧光团。与灵敏的检测系统一起，这是朝着高灵敏度的特定信号检测迈出的一大步。快速成像是重要的，首先是在时间上分辨秒范围内的三维细胞过程，其次是能够以非常好的空间分辨率实现高样品通量。许多制剂的计划定量分析只能通过高通量成像实现，而这又只能通过高速实现。除了传感器的灵敏度外，扫描速度对图像采集所需的时间也起着决定性的作用。我们申请的显微镜具有kHz范围内的扫描频率，因此非常适合三维样品的高通量成像和活细胞动态过程的快速成像。总的来说，我们将使用拟议的共聚焦显微镜来研究CNS细胞的亚细胞动力学，并进行高通量成像，最终找到神经生理学和神经疾病的新机制和相关性。

To investigate neuroscientific questions at the University of Lübeck, we want to apply for a confocal laser scanning microscope. This device will enable imaging experiments that currently cannot be performed on campus. We are a group of researchers working on neurobiological circuits and the interactions of different neural cell types at the interfaces between the periphery and the CNS. Since all applicants work in the same research building, there are optimal conditions for accessibility and use of the instrument. The microscope will replace a 15-year-old confocal microscope at the same location. The proposed microscope will be able to image very fast cellular processes such as the dynamics of the intracellular messengers Ca2+ or cAMP in three dimensions. In addition, different cell markers will be detected in the same sample and quantitative analyses of specific brain structures, e.g. blood vessels, will be carried out with high throughput. The interfaces being studied include cells of the vascular system, specialised glial cells that form part of the blood-brain barrier, and neurons that process peripheral signals such as nociceptive stimuli or hormones. Various research questions dealing with the interaction of peripheral and central signals will be answered with the help of the microscope. For the planned experiments, the microscope must have optimised excitation and emission techniques and reliably distinguish fluorophores with similar fluorescence spectra. Together with a sensitive detection system, this is a big step towards specific signal detection with high sensitivity. Fast imaging is important, firstly to temporally resolve three-dimensional cellular processes in the seconds range, and secondly to enable a high sample throughput with very good spatial resolution. The planned quantitative analyses of many preparations are only possible with high-throughput imaging, and this in turn can only be achieved with high speed. In addition to the sensitivity of the sensors, the scanning speed plays a decisive role in the time required for image acquisition. The microscope we are applying for has a scanning frequency in the kHz range and is thus ideally suited for high-throughput imaging of three-dimensional samples and rapid imaging of dynamic processes in living cells. Overall, we will use the proposed confocal microscope to investigate the subcellular dynamics of CNS cells and to perform high-throughput imaging, ultimately to find new mechanisms and correlations in neurophysiology and neurological diseases.