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享有盛誉。观察这些生物体的不寻常和新颖的生理学的能力导致了许多重大发现。该项目所要求的新的显微镜设备将使MBA研究人员能够以前所未有的分辨率检查细胞过程，对所研究的细胞或生物体的压力最小，为未来研究海洋生物的细胞生物学提供世界一流的设施。MBA的研究依靠先进的荧光显微镜技术来研究海洋模式生物，使用荧光染料和蛋白质观察细胞中发现的特定蛋白质或直接成像细胞过程，如细胞内pH值或钙的变化，这是许多信号传导途径的核心。MBA研究的其他方面使用模式生物来研究细胞过程如何进化或可能在更复杂的多细胞生物中运作。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。海洋浮游植物是代表我们海洋食物链基础的微小藻类，支持渔业和海鲜产业，但尽管它们对我们星球上的生命很重要，我们仍然对它们知之甚少。许多浮游植物被由二氧化硅或碳酸钙组成的复杂的细胞外覆盖物包围着。我们的目标是了解这些藻类如何以及为什么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。其他研究使用来自动物生命树最底层分支的海洋生物，如海葵，来研究涉及胚胎发育的复杂调控过程如何在多细胞动物中进化。海洋真菌代表了另一组知之甚少的生物。最近的研究强调，真菌在海洋环境中广泛存在，这表明它们可能在营养物再循环中发挥类似的作用，并作为陆地真菌的病原体。我们正在开发用于实验室研究的海洋真菌模式物种，以便我们能够更多地了解它们的生理学及其在环境中的潜在作用。对非常小的细胞或结构进行荧光显微镜检查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。这对于光合生物（如海洋浮游植物）或长期研究（如生物矿化或胚胎发育的延时成像）特别相关。因此，MBA研究人员目前在研究其模式生物的重要方面的能力有限。该提案旨在通过获得光片显微镜来解决这些限制。光片显微镜在减少来自激发光的损伤方面非常有效，因为它们从侧面用非常薄的光层照射样品，而不是从上方或下方直接通过样品。从侧面照明也具有增加显微镜分辨率的效果，因为没有来自样品焦点外区域的干扰。光片显微镜已被证明是特别有效的长时间的时间推移成像的光敏生物的延长期，因此它是一个理想的解决方案，目前所面临的问题的MBA研究人员。新的设施将被用来支持目前的研究团队正在进行的研究，从海洋真菌和藻类通过海洋动物的利益，以及更远的地方。因此，该资源将支持许多不同的研究途径，因此是非常物有所值的。

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.