An upright confocal microscope for multidisciplinary research

用于多学科研究的正置共焦显微镜

基本信息

批准号：
BB/R014361/1
负责人：
Christoph Ballestrem
金额：
$ 36.03万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR014361%2F1
关键词：
upright confocal microscope multidisciplinary research

项目摘要

The desire to visualise cellular structures and processes has been a central aim of biologists ever since the development of the light microscope and the advances in cell biology are intrinsically linked to the advances in microscope technology. The development of synthetic fluorescent probes made it possible to visualize the location of individual proteins and complexes within the cell and the development of different coloured probes allowed multiple proteins to be studied at the same time. This gives an insight not only into the localization of the proteins within the cell but also their interactions with other proteins. Along with this, the development of the laser scanning confocal microscope, using a detection pinhole to reject out of focus light, allowed researchers to see these fluorescent probes inside thick samples and obtain a "free from blur" optical section and three-dimensional model of the sample. The ability to visualize protein dynamics in live cells was made possible with the discovery and subsequent sequencing of the green fluorescent protein from jelly fish. Using genetic engineering it is possible to form a protein chimera in which a protein of interest is fused to this fluorescent protein. The now fluorescently tagged protein of interests can then be expressed and observed in live cells. With the development of coloured variants of the jelly fish protein (and coral proteins), it has become possible to follow multiple different proteins inside living cells, tissues and even whole organism and has allowed researchers to use optical approaches to gain an understanding of how proteins interact, how cells communicate and how cells and tissues react to the their external environment. Microscopes can come as essentially two models with respect to access to the sample to be imaged. An inverted model accesses the sample plated in dishes from the bottom and high resolution imaging needs imaging through thin transparent surfaces such as glass. An upright microscope accesses samples placed in dishes directly from the top with the use of lenses that can be "dipped" (hence dipping lenses) in the culture media. The use of this approach enables imaging of samples without additional interface which can be thick or opaque etc. Such approach allows imaging of samples that grow in a three-dimensional environment resembling their natural in vivo environment. Such environments can be original tissue or in-vivo like engineered biomaterials. These new developments provide a realistic insight into the role of how cells behave in their environment in health and disease, and an upright confocal microscope provides the ideal platform and critical for imaging cells under such modified environments.Whist we currently have a Leica SP5 upright confocal microscope, it is at the end of its useful life and lacks sensitivity which is critical for imaging combined with new other technologies such as CRISPR, where single copies of fluorescently labelled protein genes are targeted to specific locations within the genome of cells and organisms. Although this targeted approach offers enormous potential for understanding the role of individual proteins in the cell, their level of expression is often so low that the resulting fluorescent signal is very weak. The latest generation of upright confocal microscopes provide the ability to perform these sophisticated multi-colour microscope experiments even on thick samples due to their improved light efficiency and detector sensitivity. Here we propose to replace our old out-dated upright microscope with a new state-of-the-art Leica SP8 upright confocal microscope. This will allow improved delivery of a core service to a productive set of around 67 well-funded research groups who heavily use the current instrument and will provide them with access to a system with improved flexibility, improved sensitivity and improved resolution.

自从光学显微镜的发展和细胞生物学的进步以来，可视化细胞结构和过程的渴望一直是生物学家的核心目的，与显微镜技术的进步本质上联系在一起。合成荧光探针的发展使得可以看到单个蛋白质和复合物在细胞内的位置，并且不同彩色探针的发展使得可以同时研究多种蛋白质。这不仅可以深入了解细胞中蛋白质的定位，还可以洞悉它们与其他蛋白质的相互作用。随之而来的是，使用检测针孔从聚焦光中拒绝激光扫描共聚焦显微镜的发展，使研究人员可以在厚样品内看到这些荧光探针，并获得“无模糊”光学部分和三维模型样本。通过发现和随后对果冻鱼的绿色荧光蛋白的测序，使活细胞中蛋白质动力学的能力成为可能。使用基因工程，可以形成蛋白质嵌合体，其中感兴趣的蛋白质与该荧光蛋白融合在一起。然后可以在活细胞中表达并观察到现在的荧光标记的蛋白质。随着果冻鱼蛋白（和珊瑚蛋白）的彩色变体的发展，已经有可能跟随活细胞，组织甚至整个生物体中的多种不同的蛋白质，并允许研究人员使用光学方法来了解蛋白质相互作用，细胞如何通信以及细胞和组织如何对其外部环境做出反应。对于访问要成像的样品，显微镜基本上可以是两个模型。倒置的模型可通过透明表面（例如玻璃）访问底部和高分辨率成像成像成像成像中的样品。直立的显微镜通过使用可以在培养基中“浸入”（因此浸入镜片）的镜头直接从顶部放置在菜肴中的样品。这种方法的使用实现了没有其他接口的样品成像，这些界面可能是厚或不透明的等。这种方法可以成像在三维环境中生长的样品，类似于其自然体内环境。这样的环境可以是原始的组织或类似工程生物材料的体内。这些新的发展提供了对细胞在健康和疾病中的作用的作用的现实见解，而直立的共聚焦显微镜为这种修改的环境下的成像细胞提供了理想的平台和至关重要的平台。WhistWhist我们目前有Leica SP5直立的共焦显微镜，它正处于其用途寿命的尽头，缺乏灵敏度，这对于与新技术（例如CRISPR）结合成像至关重要，其中荧光标记的蛋白质基因的单个副本针对细胞和生物体基因组中的特定位置。尽管这种靶向方法具有理解单个蛋白在细胞中的作用的巨大潜力，但它们的表达水平通常如此之低，以至于所得的荧光信号非常弱。最新一代的直立共聚焦显微镜提供了能够，即使在厚样品上进行这些复杂的多色显微镜实验，由于其光效率提高了和检测器敏感性。在这里，我们建议用新的最先进的Leica SP8直立共聚焦显微镜代替旧的户外直立显微镜。这将允许改善大约67个富有资金的研究小组的富有生产力的核心服务，这些研究小组大量使用当前的仪器，并将为他们提供具有提高灵活性，提高灵敏度和提高分辨率的系统的访问。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Tensin3 interaction with talin drives the formation of fibronectin-associated fibrillar adhesions.

DOI：
10.1083/jcb.202107022
发表时间：
2022-10-03
期刊：
The Journal of cell biology
影响因子：
0
作者：
通讯作者：

Applying Tensile and Compressive Force to Xenopus Animal Cap Tissue.

对爪蟾动物帽组织施加拉力和压力。

DOI：
10.1101/pdb.prot105551
发表时间：
2020
期刊：
Cold Spring Harbor protocols
影响因子：
0
作者：
Goddard GK
通讯作者：
Goddard GK

Circadian control of the secretory pathway maintains collagen homeostasis.

DOI：
10.1038/s41556-019-0441-z
发表时间：
2020-01
期刊：
Nature cell biology
影响因子：
21.3
作者：
Chang J;Garva R;Pickard A;Yeung CC;Mallikarjun V;Swift J;Holmes DF;Calverley B;Lu Y;Adamson A;Raymond-Hayling H;Jensen O;Shearer T;Meng QJ;Kadler KE
通讯作者：
Kadler KE

Abrogation of TGF-beta signalling in TAGLN expressing cells recapitulates Pentalogy of Cantrell in the mouse.