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Fluorescence Light Sheet Microscopy for Live 3D and 4D imaging

用于实时 3D 和 4D 成像的荧光光片显微镜

基本信息

批准号：
BB/L014947/1
负责人：
Violaine See
金额：
$ 31.5万
依托单位：
University of Liverpool
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL014947%2F1
关键词：
Fluorescence Light Sheet Microscopy Live

项目摘要

.Individual cells in a plant or an animal are exposed to changes in their environment (biochemical signals, temperature, light variations...). Cells have to interpret this information to adapt and respond appropriately. To understand the molecular mechanisms leading to a particular response (e.g., cell death, cell growth, cell migration, etc), biologists have to measure the levels and localisation of proteins in the cells. Each individual cell might respond differently from its neighbour and at a different time so it is crucial to follow the events in real time and in each individual cell. This can be achieved using live cell imaging coupled with the use of fluorescent labels. However, technological limitations have restricted the measurements to flat samples, such as cells attached to a glass coverslip. In 2004, a German group invented a new microscope called light sheet microscopy, which illuminates the sample using, as the name indicates, a sheet of light. This technology also includes rotation of the sample to acquire different views. Images can be acquired very quickly through the depth of a sample (up to 1 mm thick, which is far more than with conventional microscopes). Because of the illumination geometry, the sample does not suffer from the toxicity induced by the laser light, enabling longer term imaging of living organisms. For example, the development of a zebrafish or drosophila embryo, from a few cells up to a whole organism, with single cell resolution can be observed in real-time. The first commercial light sheet microscope was released at the end of 2012. We propose to purchase one of the first commercial light sheet instruments in the UK and to install it in the Liverpool Centre for Cell Imaging (CCI). The CCI is an open access facility, so the microscope will be accessible to groups from several universities and companies. To illustrate the breadth of the science that will be served by this equipment, we briefly present below three exemplar projects:1. Repair of muscles during ageing.As people age, their skeletal muscles starts to deteriorate. Skeletal muscle cells do not divide and hence need to have a robust mechanism in place in case of damage or loss. Repair requires a unique population of specific muscle stem cells called satellite cells. Failure in satellite cell function can lead to delayed, impaired or failed recovery after injury and such failures increase in old age. With the new microscope, we will follow for the first time, the movement, division and differentiation into muscle cells of the satellite cells, in real time on damaged muscle fibres.2. 3D cell culture models for drug testingThe reduction, replacement, and refinement of animal experiments is one of the BBSRC priorities. It requires better in vitro models to improve drug discovery and drug testing. Three dimensional multi-cellular spheroid models allow faster and less expensive screening in a 3D cellular organisation. To develop drug delivery and toxicity testing using 3D cell culture system, we need to understand how the cells survive, divide and move at different positions within the spheroid. Using Light sheet microscopy, we will image in real time cell survival, proliferation and migration of individual cells in the whole spheroid. This has attracted the attention of biotech companies developing biomaterials for 3D culture and pharmaceutical companies for drug testing.3. Uncovering cell specific changes in the plant circadian clockThis project is about understanding the regulation of intracellular signals in plants triggered by the day and light changes (circadian clock). Using 3D analysis of seedlings with the light sheet microscope, we will elucidate each cell type specific regulation of the circadian clock intracellular network. This research has important implications, because the circadian clock regulates many agronomic important processes including yield, water use efficiency, disease resistance and flowering time

植物或动物中的单个细胞暴露于环境的变化（生化信号、温度、光线变化......）。细胞必须解释这些信息才能做出适当的适应和反应。为了了解导致特定反应（例如细胞死亡、细胞生长、细胞迁移等）的分子机制，生物学家必须测量细胞中蛋白质的水平和定位。每个单独的细胞可能会在不同的时间对其邻居做出不同的反应，因此实时跟踪每个单独的细胞中的事件至关重要。这可以通过活细胞成像结合荧光标记来实现。然而，技术限制限制了对平面样品的测量，例如附着在玻璃盖玻片上的细胞。 2004 年，一个德国小组发明了一种称为光片显微镜的新型显微镜，顾名思义，它使用一片光来照亮样品。该技术还包括旋转样本以获得不同的视图。可以非常快速地获取样品深度的图像（厚度可达 1 毫米，远远超过传统显微镜）。由于照明几何形状，样品不会受到激光引起的毒性，从而能够对活体生物体进行长期成像。例如，可以以单细胞分辨率实时观察斑马鱼或果蝇胚胎的发育，从几个细胞到整个生物体。第一台商业光片显微镜于 2012 年底发布。我们建议购买英国第一批商业光片仪器之一，并将其安装在利物浦细胞成像中心 (CCI)。 CCI 是一个开放访问设施，因此来自多所大学和公司的团体都可以使用显微镜。为了说明该设备将服务的科学广度，我们简要介绍以下三个示例项目：1。衰老过程中肌肉的修复。随着人们年龄的增长，他们的骨骼肌开始退化。骨骼肌细胞不会分裂，因此需要有一个强大的机制来防止损坏或丢失。修复需要一群独特的特定肌肉干细胞，称为卫星细胞。卫星细胞功能的衰竭会导致受伤后恢复延迟、受损或失败，并且这种衰竭在老年时会增加。借助新的显微镜，我们将首次实时跟踪受损肌纤维上卫星细胞的运动、分裂和分化为肌细胞的情况。2．用于药物测试的 3D 细胞培养模型动物实验的减少、替代和细化是 BBSRC 的优先事项之一。它需要更好的体外模型来改进药物发现和药物测试。三维多细胞球体模型可以在 3D 细胞组织中进行更快、更便宜的筛选。为了使用 3D 细胞培养系统开发药物输送和毒性测试，我们需要了解细胞如何在球体内的不同位置生存、分裂和移动。使用光片显微镜，我们将实时成像整个球体中单个细胞的存活、增殖和迁移。这引起了开发用于3D培养的生物材料的生物技术公司和用于药物测试的制药公司的关注。3．揭示植物生物钟的细胞特异性变化该项目旨在了解植物中由白天和光线变化（生物钟）触发的细胞内信号的调节。利用光片显微镜对幼苗进行 3D 分析，我们将阐明每种细胞类型对生物钟细胞内网络的特异性调节。这项研究具有重要意义，因为生物钟调节许多重要的农艺过程，包括产量、水分利用效率、抗病性和开花时间