Advanced Confocal Laser-Scanning-Microscope

在本提案中，我们申请资助一个最先进的，快速和高灵敏度的共聚焦激光扫描显微镜（CLSM），具有超过衍射极限的空间（超）分辨率和光谱检测。生物学、化学和药剂学学院的研究单位以及Freie Universität Berlin的集成核心单位biosuperamol光学显微镜都将从该仪器的安装中受益匪浅，因为它将允许快速，但高度敏感的图像采集，依赖于高像素停留时间进行3D定量密度测量和生物样品和组织的高通量筛选，而不会影响信噪比。新的检测技术将提高横向分辨率，甚至更多的轴向分辨率，允许检测超出经典共聚焦显微镜衍射极限的物体。该仪器的多功能性体现在高扫描速度与改进的信噪比和增强的空间分辨率相结合，因此即使在大体积中也可以对亚细胞纳米结构（低于200纳米）进行快速原位筛选。高灵敏度的检测器元件允许在可见光谱中同时进行十多个荧光团的光谱检测。该系统依赖于不同荧光团的原位直接分离，因此不仅提供了单次扫描的高效多通道成像，而且还消除了（叶绿素-）自身荧光。此外，该新系统将提供通过Förster共振能量转移（FRET）检测分子相互作用的可能性，无论是通过光谱FRET还是荧光寿命成像（FLIM）。重要的是，该系统将弥补Freie Universität Berlin光学显微镜装置的巨大差距，因为目前没有其他系统将高速和超分辨率与光谱分解相结合。新的显微镜将被纳入DFG资助的核心设施biosuperamol，并位于SupraFAB研究大楼的实验室，该实验室针对主要仪器进行了优化。

In this proposal we apply for funding of a state-of-the-art, fast and highly sensitive confocal laser scanning microscope (CLSM), with spatial (super)resolution beyond the diffraction limit and spectral detection. Both research units at the faculty Biology, Chemistry and Pharmacy and the integrated core unit BioSupraMol Optical Microscopy of Freie Universität Berlin will highly benefit from installation of this instrument as it will permit fast, but highly sensitive image acquisition relying on a high pixel dwell time for quantitative densitometric measurements in 3D and for high-throughput screening of biological samples and tissues, without affecting the signal-to-noise ratio. The novel detection technology will improve the lateral, and even more the axial resolution, permitting the detection of objects beyond the diffraction limit of classical confocal microscopes. The versatility of the instrument is reflected by a high scanning speed in combination with an improved signal-to-noise ratio and enhanced spatial resolution, so that fast in situ screenings of subcellular nano-structures (below 200 nm) even in large volumes are possible. The highly sensitive detector elements permit simultaneous spectral detection of more than ten fluorophores in the visible spectrum. The system relies on direct de-mixing of the different fluorophores in situ and therefore offers not only efficient multichannel imaging with a single scan, but also elimination of (chlorophyll-) autofluorescences. In addition, the novel system will provide the possibility to detect molecule interactions via Förster Resonance Energy Transfer (FRET), either by spectral FRET or by Flurorescence Lifetime Imaging (FLIM). Importantly, the system will bridge a substantial gap at the optical microscopy units at Freie Universität Berlin, as no other system currently combines high speed and superresolution with spectral demixing. The new microscope will be incorporated in the DFG funded Core Facility BioSupraMol and be located in the laboratories of the research building SupraFAB which is optimized for majors instrumentations.