Confocal laser scanning microscope with fast fluorescence lifetime imaging module

使用共聚焦激光扫描显微镜（CLSM）对荧光标记的细胞结构进行成像是在各种生物背景下以高空间和时间分辨率研究细胞过程的关键方法之一。在我们自己的研究中，我们采用了广泛的方法组合，这些方法严重依赖于共聚焦显微镜，例如荧光标记蛋白质的定位和表达研究，Ca 2+，pH或膜微粘度的荧光指示剂的实时成像，荧光标记内膜和生物冷凝物隔室的形成和动力学，和Förster共振能量转移（FRET）为基础的技术来研究复杂的解离和蛋白质-蛋白质相互作用的动力学。总的来说，如果不使用共聚焦激光扫描显微镜，申请人实验室的研究是不可能的。通过这项拨款申请，我们的目标是取代2010年购买的现有CLSM，该CLSM现已过时，易于维修且维护不经济，这是我们正在进行的基于成像的研究的限制因素。未来的研究目标将需要同时使用多种荧光探针。因此，未来的系统必须能够并行操作多达四个不同的荧光通道，荧光激发和检测的规格范围从紫外到近红外光谱区域。此外，所需的仪器应该能够同时获取强度和寿命信息。荧光寿命信息的采集是必需的应用程序，如扣除自发荧光光谱重叠的荧光报告，细胞pH值和膜微粘度的成像，酶复合物的形成和蛋白质-蛋白质相互作用的动态测量。因此，该系统需要脉冲激光源和能够在光子计数模式下操作的高灵敏度检测器。对于小于500 µm的细胞结构（内体、膜结构域和生物凝聚物的子域）的动态成像，有必要以高空间和时间分辨率对3D体积进行成像。因此，仪器必须配备超快扫描选项，并结合自适应图像处理软件，可以实时计算图像。我们对配备快速荧光寿命成像模块和超快扫描光学器件的CLSM的需求无法由内部或校园中央核心成像设施（NIC）中的其他显微镜系统提供，这是由于申请人团体所需的用户时间量以及技术限制，例如脉冲激光源的规格和对超快荧光寿命采集的需求。总的来说，申请人群体当前和未来研究目标的实现将严重依赖于所要求的共聚焦激光扫描显微镜的获得

Imaging of fluorescently labelled cellular structures using confocal laser scanning microscopy (CLSM) is one of the key methodologies to study cellular processes with high spatial and temporal resolution in various biological contexts. In our own research, we employ a broad portfolio of methods that rely heavily on confocal microscopy, such as localisation and expression studies of fluorescently labelled proteins, live imaging of fluorescent indicators of Ca2+, pH or membrane microviscosity, formation and dynamics of fluorescently labelled endomembrane and biocondensate compartments, and Förster resonance energy transfer (FRET)-based techniques to study the kinetics of complex dissociation and protein-protein interactions. Overall, research in the applicant labs would not be possible without the use of confocal laser scanning microscopy. With this grant application we aim to replace an existing CLSM purchased in 2010 which is now outdated, prone to repair and uneconomical to maintain, which is a limiting factor for our ongoing imaging-based research. Future research objectives will require the simultaneous use of multiple fluorescent probes. Therefore, the future system must be able to operate up to four different fluorescence channels in parallel, with specifications for fluorescence excitation and detection ranging from the ultraviolet to the near-infrared region of the spectrum. In addition, the required instrument should be able to simultaneously acquire intensity and lifetime information. The acquisition of fluorescence lifetime information is required for applications such as subtraction of autofluorescence that spectrally overlaps with fluorescent reporters, imaging of cellular pH and membrane microviscosity, and dynamic measurements of enzyme complex formation and protein-protein interactions. The system therefore requires a pulsed laser source and highly sensitive detectors capable of operating in photon-counting mode. For dynamic imaging of cellular structures smaller than 500 µm (subdomains of endosomes, membrane domains and bio-condensates), it will be necessary to image 3D volumes with high spatial and temporal resolution. Therefore, the instrument must be equipped with an ultra-fast scanning option combined with adaptive image processing software that can compute images on the fly. Our demand for a CLSM equipped with a fast fluorescence lifetime imaging module and ultra-fast scan optics cannot be served by other microscope systems in house or in the central core imaging facility on campus (NIC) due the amount of user time that will be required by the applicant groups and due to technical limitations, such as the specification of the pulsed laser sources and the demand for ultra-fast fluorescence lifetime acquisition. Overall, the realisation of the current and future research goals of the applicant groups will depend critically on the acquisition of the requested confocal laser scanning microscope