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

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，是成像的理想选择；果蝇，拥有高超的基因；小鸡，有一个大的胚胎，很容易操作；而老鼠，作为一种哺乳动物，与我们人类的发展最相似。为了跟踪胚胎发育和成年生物体的动态过程，我们需要实时成像样本，使用荧光蛋白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，在检测这种荧光方面非常好，但使用高强度的激光束来照亮样品。这就产生了许多问题，限制了我们可以做的实验的类型。首先，激光聚焦在一个很小的区域，然后扫描样品以建立一个更大的图像。这很耗时，而且很难对更大的三维结构进行成像。高强度激光还能减弱或漂白荧光信号，并能加热和损伤细胞。这些因素限制了实验的时间，而且很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提供了巨大的改进，可以进行更长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，样品从侧面被一层薄薄的激光照射。不需要激光扫描来形成二维图像，因为信息是瞬间从被照亮的整个区域捕获的。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破坏。失焦光的数量被减少到最低限度，给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进行的不同项目的范围。钙信号传导就是一个例子：细胞利用钙离子对接收到的外部信息做出反应，从而影响细胞行为的变化，如运动或与其他细胞的粘附。我们使用荧光报告器，当钙信号激活时，荧光报告器会闪烁或关闭。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来成像复杂和更大的三维结构，包括用于研究干细胞的培养组织，以及活胚胎中整个发育中的器官系统，如内耳复杂的迷宫或发育中的血管。该显微镜将被安置在谢菲尔德大学光学显微镜设施内，并将提供给谢菲尔德大学和更远的地方的所有研究人员。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、动物和植物科学以及机械工程。我们的一些项目涉及国内或国际合作（学术和工业），因此这些合作伙伴也将受益。因此，我们认为显微镜将得到大量和有效的使用，而且这项建议物有所值。

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.