Confocal Laser Scanning Microscope (CLSM) with Fluorescence Lifetime Imaging Microscopy (FLIM)

蛋白质/蛋白质或蛋白质/核酸(DNA或RNA)相互作用在理解复杂的细胞和分子生物学网络中发挥着基础性作用。两个或多个荧光标记相互作用对之间的共定位研究可能表明潜在的分子相互作用，但受到光的分辨率(250 nm侧向，500 nm轴向或去卷积125 nm侧向，200 nm轴向)的限制。为了区分局部共定位和分子相互作用(距离小于10 nm)，我们希望在IMB的显微镜核心设施申请设备来建立薄膜共振技术(荧光寿命成像显微镜-Förster Resonanz Energie Transfer)。Flim已成为表征分子相互作用和感知当地环境条件的有力工具。FOLIM直接测量荧光团的激发态寿命(荧光寿命)。荧光寿命不仅对荧光团的局部微环境(pH、温度)非常敏感，而且对FRET等能量传递过程也非常敏感。结合Flim的最新技术进步，这允许更高的成像速度，使Flim成为最适合通过Flim-FRET在高空间和时间分辨率下定量探测分子相互作用的技术。分子生物学研究所(IMB)提供装备精良的核心设施，并为IMB的科学家和美因茨的科学界提供服务。显微镜和组织学核心设施(MHCF)提供对不同高科技显微镜的访问。我们希望为IMB的科学家以及大学医学中心和约翰尼斯·古滕贝格大学的研究人员提供共焦显微镜领域的最新技术，用于他们的研究和教学。该领域有许多令人兴奋的创新，例如图形卡(GPU)支持的反卷积算法，可提供即时反卷积或改进的激光技术和探测器灵敏度。Flim提供了额外的功能，如通过消除自发荧光来改善图像质量，利用重叠光谱对荧光团进行多路复用，测量分子相互作用或使用FRET传感器评估细胞新陈代谢。所要求的带有荧光寿命成像显微镜(FLIM)的共聚焦激光扫描显微镜应该取代已经使用了10年的两个共聚焦显微镜。目前的共焦系统每年使用时间超过2000小时，是我们的工作马匹。我们希望保留目前的共焦系统之一，以减少工作量。

Protein/protein or protein/nucleic acid (DNA or RNA) interaction play a fundamental role in the understanding of complex cell- and molecular biological networks. Colocalization studies between two or more fluorescently tagged interaction partners could indicate potential molecular interaction, but are limited by the resolution of light (250 nm lateral, 500 nm axial or deconvolved 125 nm lateral, 200 nm axial). In order to discriminate between local colocalization and molecular interaction (distances of less than 10 nm), we would like to apply for equipment to establish the FLIM-FRET technique (Fluorescence Lifetime Imaging Microscopy - Förster Resonanz Energie Transfer) at IMB’s microscopy core facilities. FLIM has emerged as a powerful tool for the characterization of molecular interaction and the sensing of local environmental conditions. FLIM directly measures the lifetime of the excited state of fluorophores (fluorescence lifetime). Fluorescence lifetime is very sensitive not only to the local microenvironment of the fluorophore (pH, temperature), but also to energy transfer processes such as FRET. Combined with the recent technological advances in FLIM, this allows for much higher imaging speeds, making FLIM the most suitable technique for probing molecular interaction quantitatively via FLIM-FRET in high-spatial and temporal resolution. The Institute of Molecular Biology (IMB) provides excellent equipped Core Facilities and offers service to IMB scientists as well as the scientific community in Mainz. The Microscopy and Histology Core Facility (MHCF) offers access to different high-tech microscopes. We would like to provide the scientists at IMB, as well as researchers from the university medical center and the Johannes Gutenberg University, access to the latest technique in the field of confocal microscopy for their research and teaching. There are quite a few exciting innovations in the field, e.g. graphic card (GPU) supported deconvolution algorithms that provide deconvolution on the fly or improved laser techniques and detector sensitivity. FLIM offers additional functions like improved image quality by removing autofluorescence, multiplexing of fluorophores with overlapping spectra, measuring molecular interaction or assessing cellular metabolism with FRET sensors. The requested confocal laser scanning microscope with fluorescence lifetime imaging microscopy (FLIM) should replace two confocal microscopes that are already 10 years old. With a usage time of more than 2000 annual hours, the current confocal systems are our working horses. We would like to keep one of the current confocal systems to reduce the workload.