Advanced Confocal Laser-Scanning-Microscope

在这项提案中，我们申请资助一个最先进的，快速和高灵敏度的共焦激光扫描显微镜（CLSM），空间（超）分辨率超过衍射极限和光谱检测。这两个研究单位在学院生物学，化学和药学和集成核心单元BioSupraMol光学显微镜的自由大学柏林将高度受益于安装该仪器，因为它将允许快速，但高度灵敏的图像采集依赖于高像素停留时间的定量密度测量在3D和高通量筛选生物样品和组织，而不影响信噪比。新的检测技术将提高横向，甚至更多的轴向分辨率，允许超过经典共焦显微镜的衍射极限的对象的检测。该仪器的多功能性反映在高扫描速度与改善的信噪比和增强的空间分辨率相结合，使得即使在大体积中也可以快速原位筛选亚细胞纳米结构（低于200 nm）。高灵敏度的检测器元件允许同时光谱检测可见光谱中的十个以上的荧光团。该系统依赖于不同荧光团的原位直接去混合，因此不仅提供了有效的多通道成像与单次扫描，而且消除（叶绿素）自发荧光。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，该系统将弥合柏林自由大学光学显微镜单位的巨大差距，因为目前没有其他系统将高速和超分辨率与光谱分离相结合。新的显微镜将被纳入DFG资助的核心设施BioSupraMol，并位于研究大楼SupraFAB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.