Confocal laser scanning microscope

为了研究<s:1>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。该设备将使目前无法在校园进行的成像实验成为可能。我们是一组研究神经生物学回路和不同类型的神经细胞在外周神经系统和中枢神经系统之间的界面的相互作用的研究人员。由于所有申请人都在同一栋研究大楼工作，因此有最佳的可达性和使用仪器的条件。该显微镜将在同一位置取代已有15年历史的共聚焦显微镜。提出的显微镜将能够成像非常快速的细胞过程，如细胞内信使Ca2+或cAMP的三维动力学。此外，在相同的样品中可以检测到不同的细胞标记物，并且可以对特定的大脑结构（例如血管）进行高通量的定量分析。正在研究的界面包括血管系统的细胞、构成血脑屏障一部分的特殊神经胶质细胞，以及处理诸如伤害性刺激或激素等外围信号的神经元。在显微镜的帮助下，将回答与外围和中心信号相互作用有关的各种研究问题。对于计划的实验，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。加上灵敏的检测系统，这是向高灵敏度特定信号检测迈出的一大步。快速成像非常重要，首先是在秒范围内暂时解决三维细胞过程，其次是在非常好的空间分辨率下实现高样品吞吐量。许多制剂的计划定量分析只能通过高通量成像来实现，而这反过来又只能通过高速来实现。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。我们申请的显微镜扫描频率在kHz范围内，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。总之，我们将使用所提出的共聚焦显微镜来研究中枢神经系统细胞的亚细胞动力学，并进行高通量成像，最终发现神经生理学和神经系统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.