Two-photon-confocal microscope incl. FLIM detectors

包括 FLIM 探测器的双光子共焦显微镜

• 批准号: 518559509
• 金额: --
• 依托单位: Ruprecht-Karls-Universität Heidelberg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2023
• 资助国家: 德国
• 起止时间: 2022-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/518559509?language=en
• 关键词: Two; photon; confocal; microscope; incl.

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.

虽然生化和遗传对增殖和分化的影响已经深入到分子细节，但力在细胞命运决定中的作用才刚刚开始得到解决，众所周知，基质的弹性对细胞的分化途径有至关重要的影响。由于缺乏具有必要穿透深度的分子工具和显微镜，基于 Crispr/Cas9 的基因组编辑的进展、基因编码力传感器的建立以及结合，对培养细胞中决定性元素的直接测量受到限制。多光子研究进展共聚焦显微镜，特别是荧光寿命成像（FLIM）为发育中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（CMZ）的建立和维持是我们的重点。多年来的研究表明，CMZ 位于神经视网膜和视网膜色素上皮之间的界面，并且位于一个显着的“弯曲区”。 （除其他外）旨在解决体内生物体中物理力作用于干细胞和祖细胞的作用，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）来促进。传感器（Abl 激酶 FRET 传感器），通过同源定向修复整合到模型的基因组位点中。所应用的仪器将允许通过 FRET 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发育命运的相关性，最终将允许直接测试体内分析所证实的假设，这将通过直接和定向的力应用来实现。所使用的工具对于进行这些分析和结束争论循环至关重要，该工具将是四个（初级）小组取得科学进步的强制性先决条件。 COS. Lemke 实验室专门研究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，具有强的自发荧光，可以将这种自发荧光与实际信号区分开来，从而实现否则不可能进行的跨物种比较。 Centanin、Bageritz 和 Weinhardt 实验室都依赖于通过重叠的 FLIM 显微镜快速获取大量荧光团，从而促进大量荧光团的采集。共同标记的探针，能够进行精确的假设检验，并减少所需的实验和动物总数，这将确保所有用户群体都可以从事具有国际竞争力的项目。