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生命系统各级组织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器，如膜，细胞骨架，细胞核，高尔基体，溶酶体和能量产生线粒体是生物化学，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其他关键目标，并对我们了解健康和疾病的生物体具有许多重要意义。这些技术的前沿是以尽可能高的时空分辨率研究生命系统中从分子、细胞、组织到生物体尺度的复杂动力学的全谱。基于光学显微镜的成像是一种关键方法，提供了定量测量生命系统中分子、细胞和组织动力学的能力。荧光显微镜是研究分子、细胞、组织甚至小生物体尺度过程的关键融合技术。基于荧光的成像非常强大，因为它允许以高特异性和对比度检测非常特异的标记组分。此外，已经可以用荧光蛋白在体内特异性地标记许多分子。一般来说，需要尽量减少光的照射，因为暴露于许多高能光子会导致待研究的分子和系统受损。理想情况下，所使用的光强度不应超过天空中一个太阳的当量。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的路，在活体样品成像期间具有出色的时空分辨率，同时由于仅对被成像的样品的一部分进行选择性照明而大大降低了辐射负荷。这是通过将照明路径与成像路径分开来实现的，照明路径与成像路径通常彼此成90度。光片产生一个非常薄的光片，平分一个标本和第二个物镜是用来成像这个照明部分上的高灵敏度和分辨率camera.The仪器，一个triSPIM光片显微镜，我们的目标是在这个项目中获得改进，通过使用从两个相反的方向照明和同时从三个方面的光收集这一技术。这导致荧光信号的最佳收集和提高的分辨率。这些仪器将使我们的实时成像能力处于目前技术上可能的最前沿。一组有经验的研究人员将利用这一点来研究细胞复制的机制，包括有丝分裂纺锤体的形成，分裂细胞中染色体在中期板上的排列，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神经前体细胞的细胞极化和不对称分裂机制，以及干细胞在肠道功能中的作用，以及细胞形状变化和细胞运动的作用，这在胚胎发育和免疫系统功能中很重要。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。一旦这种仪器得到很好的建立，它无疑将被用于许多其他尖端的研究项目。目前，这将是英国同类显微镜中的第一台。最后，我们计划使用该仪器作为基础，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