An upright confocal microscope for multidisciplinary research

用于多学科研究的正置共焦显微镜

• 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

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与显微镜技术的进步内在地联系在一起以来，可视化细胞结构和过程的愿望一直是生物学家的中心目标。合成荧光探针的发展使人们有可能可视化单个蛋白质和复合体在细胞内的位置，而不同颜色的探针的发展使同时研究多种蛋白质成为可能。这不仅提供了对蛋白质在细胞内的定位的洞察，也提供了它们与其他蛋白质的相互作用的洞察。随之而来的是激光扫描共聚焦显微镜的发展，利用探测针孔来阻挡离焦光，使研究人员能够看到厚样品中的这些荧光探针，并获得样品的“无模糊”光学切片和三维模型。随着水母绿色荧光蛋白的发现和随后的测序，显示活细胞中蛋白质动态的能力成为可能。利用基因工程，有可能形成一种蛋白质嵌合体，在这种嵌合体中，感兴趣的蛋白质与这种荧光蛋白质融合。现在荧光标记的目标蛋白可以在活细胞中表达和观察。随着水母蛋白(和珊瑚蛋白)的彩色变体的发展，在活细胞、组织甚至整个生物体内跟踪多种不同的蛋白质成为可能，并使研究人员能够使用光学方法来了解蛋白质如何相互作用、细胞如何交流以及细胞和组织如何对外部环境做出反应。显微镜基本上可以作为两种模型来获取要成像的样本。倒置模型从底部进入镀盘中的样品，高分辨率成像需要通过薄的透明表面(如玻璃)成像。直立显微镜可以直接从顶部取放在培养皿中的样品，使用的透镜可以在培养基中“浸泡”(因此浸泡透镜)。使用这种方法可以对样品成像，而不需要额外的界面，这些界面可能是厚的或不透明的等。这种方法允许对生长在类似于其体内自然环境的三维环境中的样品进行成像。这样的环境可以是原始组织或体内类似的工程生物材料。这些新的发展为细胞在其环境中的行为在健康和疾病中的作用提供了现实的洞察，而立式共焦显微镜为在这种改进的环境下对细胞进行成像提供了理想的平台和关键。虽然我们目前拥有徕卡SP5立式共焦显微镜，但它的使用寿命即将结束，并且缺乏灵敏度，这对于与CRISPR等新的其他技术相结合是至关重要的，在CRISPR中，荧光标记的蛋白质基因的单副本针对细胞和生物体基因组中的特定位置。尽管这种有针对性的方法为了解单个蛋白质在细胞中的作用提供了巨大的潜力，但它们的表达水平往往很低，以至于产生的荧光信号非常弱。最新一代的直立式共聚焦显微镜由于其更高的光效和探测器灵敏度，即使在厚样品上也能够进行这些复杂的多色显微镜实验。在这里，我们建议用最先进的徕卡SP8立式共聚焦显微镜取代我们过时的直立式显微镜。这将改善向资金充足的大约67个研究小组提供核心服务的工作，这些小组大量使用目前的仪器，并将使他们能够使用一个灵活性更高、灵敏度更高和分辨率更高的系统。

项目成果

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

Tensin3与塔林的相互作用驱动了与纤连蛋白相关的原纤维粘附的形成。

• DOI: 10.1083/jcb.202107022
• 发表时间: 2022-10-03
• 期刊: The Journal of cell biology

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
• 作者: Goddard GK

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

• DOI: 10.1038/s41598-018-21948-z
• 发表时间: 2018-02-26
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Aldeiri B;Roostalu U;Albertini A;Behnsen J;Wong J;Morabito A;Cossu G
• 通讯作者: Cossu G

LRRC8A is dispensable for a variety of microglial functions and response to acute stroke.

• DOI: 10.1002/glia.24156
• 发表时间: 2022-06
• 期刊: Glia
• 影响因子: 6.2

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