Application for a TRI-SPIM fluorescence lightsheet microscope

生命科学研究的一个重要目标是研究和理解构成生命系统各层次组织基础的关键过程的复杂时空动态。研究各种分子如何相互作用形成细胞膜、细胞骨架、细胞核、高尔基体、溶酶体和产生能量的线粒体等细胞器是生物化学、细胞生物学和分子遗传学的主要目标。了解细胞如何执行不同的复杂功能，如自我复制、形状变化、运动和分化，以形成组织、器官甚至生物体，是细胞和发育生物学和遗传学的另一个关键目标，对我们理解健康和疾病中的生物体具有许多重要意义。这些技术的前沿是在最高可能的时空分辨率下研究生命系统中从分子、细胞、组织到有机体尺度的复杂动力学的全谱。光学显微镜成像是一种关键的方法，提供了在生命系统中进行分子、细胞和组织动力学定量测量的能力。荧光显微镜是研究分子、细胞、组织甚至小生物尺度过程的一种关键融合技术。基于荧光的成像是非常强大的，因为它允许检测非常特定的标记成分，具有高特异性和对比度。此外，用荧光蛋白在体内特异性标记许多分子已成为可能。一般来说，有必要尽量减少光照射，因为暴露在许多高能光子下会对待研究的分子和系统造成损害。理想情况下，使用的光强度不应超过相当于天空中一个太阳的光强度。最近开发的一种称为光片显微镜的技术在实现这些目标方面走了很长一段路，在活体样品成像期间具有出色的时空分辨率，同时由于仅对正在成像的部分样品进行选择性照明而大大减少了辐射负荷。这是通过将照明路径与成像路径分开来实现的，这两个路径通常彼此成90度。光片产生非常薄的光片，将标本一分为二，第二个物镜用于在高灵敏度和分辨率的相机上对这个被照亮的部分进行成像。我们在这个项目中要获得的仪器，一个triSPIM光片显微镜，通过使用两个相反方向的照明和同时从三面收集光来改进这项技术。这导致了荧光信号的最佳收集和分辨率的提高。这些仪器将使我们的实时成像能力处于目前技术可行的最前沿。一组经验丰富的研究人员将利用这一方法来研究细胞复制的机制，包括有丝分裂纺锤体的形成、分裂细胞中期板上染色体的排列、染色体分离。进一步的研究旨在了解脊髓和大脑形成过程中细胞极化和神经前体细胞不对称分裂的机制，以及干细胞在肠道功能中的作用，以及在胚胎发育和免疫系统功能中重要的细胞形状变化和细胞运动的作用。这些和其他未来要解决的研究项目将对我们对健康和疾病的理解产生重要影响。一旦这个仪器得到很好的建立，毫无疑问，它将被用于许多其他尖端的研究项目。目前，这将是英国首台此类显微镜。最后，我们计划以该仪器为基础，推动这种基于光片显微镜的生命系统关键过程成像技术的发展

An important goal in Life Science research is to study and understand the complex spatio-temporal dynamics of key processes that underlie living systems at all levels of organisation. Studying how various molecules interact to form organelles such as membranes, the cytoskeleton, the nucleus, the Golgi apparatus, lysosomes and energy producing mitochondria is a major goal of biochemistry, cell biology and molecular genetics. Understanding how cells perform different complex functions such a self-replication, shape change, movement and differentiation to form tissues, organs and even organisms are other key goals of cell and developmental biology and genetics and have many important implications for our understanding of organisms in health and disease. The frontier of these techniques is to study the full spectrum of complex dynamics from the molecular, cellular, tissue to organism scale in living systems at the highest possible spatio-temporal resolution. Light microscopy based imaging is a key methodology provides the ability to perform quantitative measurements molecular, cellular and tissue dynamics in living systems. Fluorescence light microscopy is a key merging technique of choice to study processes at the molecular, cellular, tissue and even small organism scale. Fluorescence based imaging is very powerful since it allows, detection of very specific labelled components with high specificity and contrast. Furthermore it has become possible to label many molecules specifically in-vivo with fluorescent proteins. In general there is a need to minimise the irradiation with light, since exposure to many high energy photons results in damage to the molecules and systems to be studied. Ideally the light intensity used should not exceed more than that of the equivalent of one sun in the sky. A recently developed technique known as light sheet microscopy goes a long way in reaching these goals, excellent spatiotemporal resolution during imaging of live samples while greatly reducing the radiation load due to selective illumination of only the part of the sample that is being imaged. This is achieved by separating the illumination path from the imaging path, which are typically at 90 degrees to each other. The lightsheet generates a very thin sheet of light that bisects a specimen and a second objective is used to image this illuminated section on a high sensitivity and resolution camera.The instrument, a triSPIM lightsheet microscope that we aim to acquire in this project improves this technology by using illumination from two opposing directions and simultaneous light collection from three sides. This results in optimal collection of the fluorescence signals and an improved resolution. This instruments will put our live imaging capabilities at the forefront of what is presently technically possible. A group experienced researchers will use this to study the mechanism underlying cell replication, including formation of the mitotic spindle, line up of chromosomes on the metaphase plate in dividing cells, chromosome separation. Further studies are aimed at understanding the mechanisms of cell polarisation and asymmetric division of neural precursor cells during formation of the spinal cord and brain as well as the role of stem cells in the function of the gut and the role of cell shape changes and cell motility, important during embryonic development and the function of the immune system. These and other future research projects to be tackled will have important consequences for our understanding of health and disease. As soon as this instrument is well established it will without doubt be used in many other cutting edge research projects. At present this will be the first microscope of its kind in the UK. Finally we plan to use this instrument as a basis to drive forward the development of this type of lightsheet microscopy based imaging technology of critical processes in living systems