Light sheet microscopy for live cell imaging of photosensitive systems

海洋生物协会(MBA)的研究人员在使用显微镜研究海洋生物方面久负盛名。观察这些生物不同寻常和新颖的生理学的能力导致了许多重大发现。这项提议中要求的新显微镜设施将允许MBA研究人员以前所未有的分辨率检查细胞过程，对研究中的细胞或生物施加最小的压力，为未来对海洋生物细胞生物学的研究提供世界级的设施。MBA的研究依赖先进的荧光显微镜技术来研究海洋模型生物，使用荧光染料和蛋白质来观察细胞中特定蛋白质的位置，或直接成像细胞过程，如细胞内pH或钙的变化，这是许多信号通路的核心。MBA研究的其他方面使用模型生物来研究细胞过程是如何在更复杂的多细胞生物中进化或可能运行的。例如，我们使用藻类来研究鞭毛的结构和功能，因为鞭毛的许多方面与人体中发现的纤毛高度保守。海洋浮游植物是一种微小的藻类，代表着我们海洋食物链的基础，支持着渔业和海鲜产业，但尽管它们对地球上的生命很重要，我们对它们仍然知之甚少。许多浮游植物被由二氧化硅或碳酸钙制成的错综复杂的胞外覆盖物所包围。我们的目标是了解这些藻类如何以及为什么会产生如此复杂和华丽的细胞覆盖物，以帮助设计用于生物技术和生物医学应用的类似结构。其他研究使用在动物生命树的最底层分化的海洋生物，如海葵，来研究参与胚胎发育的复杂调控过程是如何在多细胞动物中进化的。海洋真菌代表了另一类鲜为人知的生物。最近的研究强调，真菌在海洋环境中广泛存在，这表明它们很可能在营养循环中扮演类似的角色，并作为病原体与陆地真菌类似。我们正在开发用于实验室研究的海洋真菌的模式物种，以便我们能够更多地了解它们的生理及其在环境中的潜在作用。非常小的细胞或结构的荧光显微镜需要用明亮的激发光照射对象，这对许多样品来说可能是有害的。这与光合作用生物(如海洋浮游植物)或长期研究(如生物矿化或胚胎发育的时间推移成像)特别相关。因此，MBA研究人员目前在研究他们的模型生物的重要方面的能力有限。这项提议旨在通过购买光片显微镜来解决这些限制。光片显微镜在减少激发光的损害方面非常有效，因为它们从侧面用一层非常薄的光照射样品，而不是从上面或下面直接透过样品。来自侧面的照明也具有提高显微镜分辨率的效果，因为不存在来自样品焦外区域的干扰。光片显微镜已被证明在较长时间内对光敏生物的长期延时成像特别有效，因此它是MBA研究人员目前面临的问题的理想解决方案。新设施将用于支持当前研究团队的正在进行的研究，从海洋真菌和藻类到海洋动物，以及更远的地方。因此，这些资源将支持许多不同的研究途径，因此具有极高的性价比。

Researchers at the Marine Biological Association (MBA) have a long standing reputation for excellence in the use of microscopy to study marine organisms. The ability to observe the unusual and novel physiology of these organisms has led to many significant discoveries. The new microscopy facility requested in this proposal will allow MBA researchers to examine cellular processes at unprecedented levels of resolution, with minimal stress on the cells or organisms under study, providing a world-class facility for future research into cell biology of marine organisms.Research at the MBA relies on advanced fluorescence microscopy techniques to study marine model organisms, using fluorescent dyes and proteins to view where specific proteins are found in a cell or to directly image cellular processes such as changes in intracellular pH or calcium, which are central to many signalling pathways. Other aspects of MBA research use model organisms to study how cellular processes have evolved or may operate in more complex multicellular organisms. For example, we use algae to study the structure and function of flagella, as many aspects of flagella are highly conserved with the cilia found in the human body. Marine phytoplankton are tiny algae that represent the base of the food chain in our oceans, supporting fishing and seafood industries, but despite their importance to life on our planet we still understand very little about them. Many phytoplankton surround themselves with intricate extracellular coverings made from silica or calcium carbonate. We aim to understand how and why these algae produce such intricate and ornate cell coverings to aid the design of similar structures for use in biotechnology and biomedical applications. Other research uses marine organisms from lineages that diverged at the very base of the animal tree of life, such as sea anemones, to examine how complex regulatory processes involved in embryo development evolved in multicellular animals. The marine fungi represent another group of organisms that are poorly understood. Recent research has highlighted that fungi are widespread in marine environments, suggesting that they are likely to play similar roles in nutrient recycling and as pathogens as the terrestrial fungi. We are developing model species of marine fungi for laboratory studies, so that we can understand much more about their physiology and their potential roles in the environment.Fluorescence microscopy of very small cells or structures requires that the subject is illuminated with a bright excitation light, which for many samples can be damaging. This is particularly relevant for photosynthetic organisms (such as marine phytoplankton) or for long term studies (such as time lapse imaging of biomineralisation or embryo development). MBA researchers are therefore currently limited in their ability to study important aspects of their model organisms. This proposal aims to address these limitations through the acquisition of a light sheet microscope. Light sheet microscopes are very effective at reducing damage from excitation light because they illuminate the sample with a very thin layer of light from the side, rather than passing light directly through the sample from above or below. Illumination from the side also has the effect of increasing the resolution of the microscope as there is no interference from out of focus areas of the sample. Light sheet microscopy has been shown to be particularly effective for long term time lapse imaging of photosensitive organisms for extended periods and therefore it is an ideal solution to the problems currently faced by MBA researchers.The new facility will be used to support the ongoing research of the current research teams, with interests from marine fungi and algae through to marine animals, and further afield. The resource will therefore underpin many diverse avenues of research and therefore represents excellent value for money.