Two-photon-confocal microscope incl. FLIM detectors

虽然生物化学和遗传对增殖和分化的影响已经解决到分子细节，但细胞命运决定中的力的作用才刚刚开始解决。已知基质的弹性对培养细胞的分化途径有关键影响。由于缺乏具有必要穿透深度的分子工具和显微镜，对生物体中决定性元素的直接测量受到限制。基于Crispr/Cas9的基因组编辑的进展，遗传编码力传感器的建立，结合多光子共聚焦显微镜的进展，特别是荧光寿命成像（FLIM），为正在发育的再生和衰老生物体的体内分析打开了大门。鱼类视网膜干细胞生态位的建立和维持是我们多年来的研究重点，它的建立和维持似乎对力有着至关重要的影响。CMZ位于神经视网膜和视网膜色素上皮之间的界面处，并且位于突出的“弯曲区”中。随着仪器的应用，我们（除其他外）的目标是解决作用于干细胞和祖细胞在生物体背景下，在体内的物理力的作用。这将通过使用遗传编码的力传感器（例如Talin-FRET传感器、纽蛋白-FRET传感器、β-激酶FRET传感器）来促进，所述力传感器通过同源定向修复整合到模型的基因组基因座中。所申请的仪器将允许通过FRET信号的力依赖性损失来测量作用力。克隆分析将促进力与干细胞和祖细胞的发育命运的相关性。在实验室中建立的视网膜类器官最终将允许直接测试由体内分析证实的假设。这将通过对类器官直接和定向施力来实现，以解决它们在背景环境中的作用。所申请的工具对于进行这些分析和结束辩论至关重要。 该仪器将是COS四个（初级）小组取得科学进展的必要先决条件。Lemke实验室专门研究几种不同昆虫物种的细胞骨架和马达蛋白的高分辨率成像，具有强烈的自发荧光。FLIM允许从实际信号中区分这种自发荧光，从而实现否则不可能的跨物种比较。Centanin，Bageritz和Weinhardt实验室都依赖于快速多色采集大量具有重叠发射光谱的荧光团。FLIM显微镜有助于大量的共标记探针，从而实现精确的假设检验，并减少所需的实验和动物总数。获得所应用的工具将确保所有用户群体都能在国际竞争力的水平上实施项目。

While the biochemical and genetic impact on proliferation and differentiation has been addressed down to the molecular detail, the role of forces in cell fate decisions is just started to be addressed. It is known that the elasticity of the substrate crucially impacts on the differentiation route of cultured cells. The direct measurement of the decisive elements in the organismal context was limited by the absence of molecular tools and microscopes with the necessary penetration depth. The progress in Crispr/Cas9 based genome editing, the establishment of genetically encoded force sensors, combined with the progress in multi-photon confocal microscopy and here in particular the fluorescence life time imaging (FLIM) has opened the door for in vivo analyses in the developing regenerating and aging organism. The establishment and maintenance of the retinal stem cell niche (CMZ) in fish, a focus of our research for years, appears to be crucially dependent on forces. The CMZ is located at the interface between the neural retina and the retinal pigmented epithelium and resides in a prominent “bending zone”. With the instrument applied for we are (among other points) aiming to address the role of physical forces acting on the stem and progenitor cells in the organismal context, in vivo. This will be facilitated by the use of genetically encoded force sensors (e.g. Talin-FRET sensors, vinculin-FRET sensors, Abl-kinase FRET-sensors), integrated into the genomic loci of the model by homology directed repair. The instrument applied for will allow to measure acting forces by the force dependent loss of the FRET signal. Clonal analysis will facilitate the correlation of forces with the developmental fate of stem and progenitor cells. Retinal organoids established in the lab will eventually allow directly testing the hypotheses substantiated by the in vivo analyses. This will be achieved by direct and directed force application to the organoids to address their action in an out of context setting. The instrument applied for will be crucial to perform those analyses and close the loop of arguments. The instrument will be a mandatory prerequisite for the scientific progress of four (junior) groups at COS. The Lemke lab specializes in high-resolution imaging of cytoskeletal and motor proteins in several different insect species, with strong auto-fluorescence. FLIM allows the discrimination of such auto-fluorescence from the actual signal, enabling cross-species comparisons otherwise not possible. The Centanin, Bageritz, and Weinhardt labs all depend on fast multicolour acquisition of larger numbers of fluorophores with overlapping emission spectra. FLIM microscopy facilitates larger numbers of co-labelled probes, enabling precise hypothesis testing as well as reducing the total number of experiments and animals required. The access to the applied instrument will ensure that all user groups can pursue projects at a level that is internationally competitive.