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被解决到分子细节，但力量在细胞命运决定中的作用才刚刚开始被解决。已知底物的弹性对培养细胞的分化途径有重要影响。由于缺乏分子工具和具有必要渗透深度的显微镜，在有机环境中对决定性元素的直接测量受到限制。基于Crispr/Cas9的基因组编辑技术的进展，基因编码力传感器的建立，以及多光子共聚焦显微镜，特别是荧光寿命成像（FLIM）的进展，为在发育、再生和衰老的生物体中进行体内分析打开了大门。鱼类视网膜干细胞生态位（CMZ）的建立和维持是我们多年来研究的重点，似乎关键依赖于力。CMZ位于神经视网膜和视网膜色素上皮之间的界面，位于一个突出的“弯曲区”。随着仪器的应用，我们（除其他点外）旨在解决在生物体环境中作用于干细胞和祖细胞的物理力的作用。这将通过使用基因编码的力传感器（例如Talin-FRET传感器，vinculin-FRET传感器，Abl-kinase fret传感器）促进，通过同源定向修复将其集成到模型的基因组位点中。应用的仪器将允许通过FRET信号的力相关损失来测量作用力。克隆分析将促进力与干细胞和祖细胞发育命运的相关性。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。这将通过直接和定向的力应用于类器官来实现，以解决它们在脱离环境的情况下的行动。所申请的工具将是进行这些分析和结束争论循环的关键。该仪器将是COS的四个（初级）小组取得科学进展的强制性先决条件。莱姆克实验室专门从事几种不同昆虫物种的细胞骨架和运动蛋白的高分辨率成像，具有很强的自荧光。FLIM允许从实际信号中区分这种自身荧光，从而使跨物种比较成为可能。Centanin， Bageritz和Weinhardt实验室都依赖于快速多色采集具有重叠发射光谱的大量荧光团。FLIM显微镜有利于大量的共标记探针，使精确的假设检验以及减少所需的实验和动物总数。使用所应用的工具将确保所有用户群体都能在具有国际竞争力的水平上开展项目。

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.