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A multi-user light-sheet microscope for Bateson Centre researchers, University of Sheffield scientists, partners and collaborators

为贝特森中心研究人员、谢菲尔德大学科学家、合作伙伴和合作者提供的多用户光片显微镜

基本信息

批准号：
BB/M012522/1
负责人：
Tanya Whitfield
金额：
$ 46.73万
依托单位：
University of Sheffield
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM012522%2F1
关键词：
multi user light sheet microscope

项目摘要

Research in the Bateson Centre is concerned with how the body develops across the life course, from the embryo to the adult. We wish to understand the genetic and cellular mechanisms that direct the development of a single cell - the fertilised egg - into a fully functioning mature body. We use a number of different animal model systems in our research. These include the zebrafish, which has a transparent embryo and is ideal for imaging; the fruit fly Drosophila, with its superb genetics; the chick, which has a large embryo that is easy to manipulate; and the mouse, which, as a mammal, has the closest parallels to our own development.To follow dynamic processes, both during embryonic development and in the adult organism, we need to image samples in real time, using fluorescent proteins to light up different cells or tissues. Conventional microscopes, such as the laser scanning confocal microscope, are very good at detecting this fluorescence, but use a high intensity laser beam to illuminate the sample. This creates a number of problems, limiting the type of experiments that we can do. Firstly, the laser is focused onto a tiny area and scanned over the sample to build up a bigger image. This is time-consuming, and makes it difficult to image larger three-dimensional structures. The high intensity laser can also weaken or bleach the fluorescent signal, and can heat and damage cells. These factors limit the time for an experiment, and are likely to disrupt the very processes that we want to understand. Finally, because a conventional confocal microscope illuminates the sample from above, the image can be distorted by out-of-focus light.The light-sheet microscope offers enormous improvements over existing confocal microscope systems for longer time-lapse experiments and imaging of larger objects: we are requesting funds to purchase one of these machines to support a whole variety of research projects in the Bateson Centre and elsewhere. In a light-sheet microscope, the sample is illuminated with a thin sheet of laser light from the side. No laser scanning is required to form a two-dimensional image, as information is captured instantaneously from the entire area that is illuminated. This vastly reduces the time required to capture three-dimensional information, meaning less bleaching and less damage to the sample. The amount of out-of-focus light is reduced to a minimum, giving a beautifully sharp and clear image.A few specific examples will illustrate the range of different projects that we intend to pursue. Calcium signalling is one example: cells use calcium ions to respond to the external messages they receive, effecting changes in cell behaviour such as movement or adhesion to other cells. We use fluorescent reporters that flash on and off when calcium signalling is active. The light-sheet microscope will enable us to follow large numbers of cells over time, to identify these transient signalling events in the entire cell population. We will also use the light-sheet microscope to image complex and larger three-dimensional structures, including cultured tissues for the study of stem cells, and whole developing organ systems in the live embryo, such as the intricate labyrinth of the inner ear or the developing blood vessels. The microscope will be housed within the University of Sheffield Light Microscopy Facility, and will be made available to all researchers at the University of Sheffield and further afield. Outside the Bateson Centre, other users are drawn from a number of different academic departments, including Molecular Biology and Biotechnology, Animal and Plant Sciences, and Mechanical Engineering. Some of our projects involve national or international collaboration (academic and industrial) and so these partners will also benefit. We therefore believe that the microscope will be very heavily and efficiently used, and that the proposal is excellent value for money.

贝特森中心的研究关注的是从胚胎到成人的整个生命过程中身体是如何发育的。我们希望了解指导单个细胞-受精卵-发育成功能齐全的成熟身体的遗传和细胞机制。我们在研究中使用了许多不同的动物模型系统。其中包括斑马鱼，它有一个透明的胚胎，是理想的成像;果蝇，其高超的遗传学;小鸡，它有一个大的胚胎，很容易操纵;而老鼠，作为一种哺乳动物，与我们自身的发育有着最接近的相似之处。为了跟踪动态过程，无论是在胚胎发育期间还是在成年有机体中，我们都需要对样本进行真实的成像，使用荧光蛋白来照亮不同的细胞或组织。传统的显微镜，如激光扫描共聚焦显微镜，是非常好的检测这种荧光，但使用高强度的激光束照射样品。这造成了许多问题，限制了我们可以做的实验类型。首先，激光聚焦到一个微小的区域，并扫描样品以建立一个更大的图像。这是耗时的，并且使得难以对较大的三维结构进行成像。高强度激光还可以减弱或漂白荧光信号，并可以加热和损伤细胞。这些因素限制了实验的时间，并可能破坏我们想要了解的过程。最后，由于传统的共聚焦显微镜从上方照射样品，图像可能会因失焦而失真。光片显微镜比现有的共聚焦显微镜系统有了巨大的改进，可以进行更长时间的延时实验和更大物体的成像：我们正在申请资金购买一台这样的机器，以支持贝特森中心和其他地方的各种研究项目。在光片显微镜中，样品从侧面用一薄层激光照射。不需要激光扫描来形成二维图像，因为信息是从被照射的整个区域瞬时捕获的。这大大减少了捕获三维信息所需的时间，这意味着更少的漂白和对样品的损坏。失焦光线的数量被减少到最低限度，提供了一个美丽的锐利和清晰的图像。几个具体的例子将说明我们打算追求的不同项目的范围。钙信号传导就是一个例子：细胞使用钙离子来响应它们接收到的外部信息，影响细胞行为的变化，例如运动或粘附到其他细胞。我们使用荧光报告器，当钙信号活跃时，它会闪烁。光片显微镜将使我们能够随着时间的推移跟踪大量细胞，以识别整个细胞群中的这些瞬时信号事件。我们还将使用光片显微镜对复杂和较大的三维结构进行成像，包括用于干细胞研究的培养组织，以及活胚胎中整个发育中的器官系统，例如内耳复杂的迷路或发育中的血管。该显微镜将被安置在谢菲尔德大学的光学显微镜设施，并将提供给所有研究人员在谢菲尔德大学和更远的地方。在贝特森中心之外，其他用户来自许多不同的学术部门，包括分子生物学和生物技术，动植物科学和机械工程。我们的一些项目涉及国家或国际合作（学术和工业），因此这些合作伙伴也将受益。因此，我们相信显微镜将得到大量和有效的使用，这项建议是非常物有所值的。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Olfactory Rod Cells: A Rare Cell Type in the Larval Zebrafish Olfactory Epithelium With a Large Actin-Rich Apical Projection.

DOI：
10.3389/fphys.2021.626080
发表时间：
2021
期刊：
Frontiers in physiology
影响因子：
4
作者：
Cheung KY;Jesuthasan SJ;Baxendale S;van Hateren NJ;Marzo M;Hill CJ;Whitfield TT
通讯作者：
Whitfield TT

Olfactory rod cells: a rare cell type in the larval zebrafish olfactory epithelium with an actin-rich apical projection

嗅杆细胞：斑马鱼幼虫嗅上皮中的一种罕见细胞类型，具有富含肌动蛋白的顶端突起