Confocal laser scanning microscope with fast fluorescence lifetime imaging module

利用共聚焦激光扫描显微镜（CLSM）对荧光标记细胞结构进行成像是研究各种生物环境下高时空分辨率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，严重依赖于共聚焦显微镜，如荧光标记蛋白质的定位和表达研究，Ca2+， pH或膜微粘度的荧光指标的实时成像，荧光标记的内膜和生物凝聚室的形成和动力学，以及Förster共振能量转移（FRET）为基础的技术来研究复杂解离和蛋白质-蛋白质相互作用的动力学。总的来说，如果没有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。通过这笔拨款申请，我们的目标是更换2010年购买的现有CLSM，该CLSM现在已经过时，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个荧光探针。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，具有荧光激发和检测的规格，范围从光谱的紫外到近红外区域。此外，所需仪器应能同时获取强度和寿命信息。荧光寿命信息的获取是应用程序所必需的，如在光谱上与荧光报告重叠的自身荧光的减法，细胞pH和膜微粘度的成像，以及酶复合物形成和蛋白质-蛋白质相互作用的动态测量。因此，该系统需要一个脉冲激光源和能够在光子计数模式下工作的高灵敏度探测器。对于小于500µm的细胞结构（核内体亚域、膜域和生物凝聚体）的动态成像，将需要具有高空间和时间分辨率的三维体积成像。因此，仪器必须配备超快速扫描选项，并结合可动态计算图像的自适应图像处理软件。我们对CLSM的需求配备了快速荧光寿命成像模块和超快速扫描光学器件，由于申请人群体需要大量的用户时间，并且由于技术限制，例如脉冲激光源的规格和对超快速荧光寿命采集的需求，其他内部显微镜系统或校园中央核心成像设施（NIC）无法满足我们的需求。总的来说，实现当前和未来的研究目标的申请人群体将主要取决于所要求的共聚焦激光扫描显微镜的收购

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 With this grant application we aim to replace an existing CLSM purchased in 2010 which is now outdated, prone to repair and uneconomical to maintain, which is a limiting factor for our ongoing imaging-based research.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