Integrated multi-dimensional molecular organ imaging

创新的光学成像技术的发展导致了光学显微镜的革命，预示着新一代显微镜的发展，有望为生物医学研究提供强大的新应用。我们相信，我们对商业可用仪器的改进、新型探针的开发和复杂生物应用中的全面测试的计划，将确保我们先进的光学显微镜“工具包”能够被利用来提供创新研究和改善健康结果。为了最大限度地促进我们对先进成像技术的创新和应用的贡献，我们专注于生命内成像，因为这个平台允许以最大可能的穿透深度详细研究活标本中亚细胞分辨率的生物事件。我们将部署最先进的活体内光学显微镜，以获取特定标记或无标记分子进入组织(S)的图像，这些组织通常是老鼠或斑马鱼。我们的创新方法包括用3D映射来补充这些图像，以便提供更完整的健康和疾病器官功能视图。有效、信息丰富的细胞标记方法在生命内成像之前仍然是将这些方法广泛应用于非转基因模型的一个重大挑战。为了扩大我们的活体成像方法的应用范围，我们将采用和评估新的荧光“智能探针”。这些智能探头已经在爱丁堡使用，通过尖端的光纤显微镜对人类肺部的关键生物和病理事件进行成像，从而提供了一条从计划中的斑马鱼和小鼠研究到生命中人类疾病器官的直接转换途径(该技术很容易应用于消化道和生殖系统)。我们的团队已经在使用广泛的模型动物方面建立了良好的记录，这些模型动物在关键目标细胞/器官中表达荧光标记的感兴趣分子。这些专业知识将保证我们开发出强大的方法学，专注于具有科学挑战性的问题，例如：巨噬细胞在乳腺癌发生中的作用是什么？神经元和神经胶质细胞如何在神经系统的发育、功能和修复中相互作用？性类固醇激素如何改变子宫中的细胞行为，以及如何对其进行调节？与脑疾病模型鼠相比，中枢神经系统蛋白在正常小鼠大脑中的分布情况如何？将活体成像应用于生物医学研究的兴奋基于其跟踪活组织中细胞及其内容的能力，为研究蛋白质分布、分子相互作用和组织组成的动态变化提供了一个新的平台。目前，这样的观察只能在组织内的单个位置进行--我们的目标是将数据采集扩展到整个器官，例如询问小鼠大脑中突触蛋白的分布。为了实现这一目标，我们将在快速共聚焦显微镜上将我们的体内技术与系统的3D图像采集相结合，从而能够扫描整个切片的器官。由此产生的3D图像系列将被存档，以确保数据保存，并将通过可搜索的基于网络的器官图书馆向更广泛的研究界提供。这些分子‘器官图’详细说明了特定标记蛋白质的位置，这将使我们能够改进后续的‘聚焦’功能体内成像，并提供高内容的3D器官图和来自活标本的功能测量。这些创新研究将为我们的翻译研究管道提供数据，并为改进诊断和治疗健康问题(包括癌症、多发性硬化症、不孕症、心血管和神经退行性疾病)提供信息。

The development of innovative optical imaging technologies has led to a revolution in light microscopy heralding the development of a generation of microscopes that promise powerful new applications for biomedical research. We believe our plans for refinement of commercially available instrumentation, development of novel probes and comprehensive testing in complex biological applications will ensure our advanced light microscopy 'toolkit' can be exploited to deliver innovative research and improved health outcomes.To maximise our contribution to the innovation and application of advanced imaging we focus on intra-vital imaging as this platform allows detailed investigation of biological events at sub-cellular resolution in living specimens at the greatest possible penetration depth. We will deploy a state-of-the-art intra-vital light microscope to acquire images of specifically labelled or label-free molecules up to one millimetre into tissue(s) of live animals typically mouse or zebrafish. Our innovative approach includes complementing these images with 3D mapping in order to provide a much more complete view of organ function in health and disease.Effective, informative, methods for labelling cells prior to intra-vital imaging remains a significant challenge in the wider application of these methods to non-transgenic models. In order to increase the reach of our intra-vital imaging methods we will employ and evaluate novel fluorescent 'Smartprobes'. These Smartprobes are already being used in Edinburgh to image key biological and pathological events in human lungs through cutting-edge fibre-optic microscopy, thus providing a direct translational pathway from planned studies in zebrafish and mice to human diseased organs in life (the technology is readily applicable to the digestive tract and the reproductive system). Our group has already established a strong track record in the use of a wide range of model animals that express fluorescently labelled molecules of interest in key target cells/organs. This expertise will guarantee we develop robust methodology focussed on scientifically challenging questions, such as: What is the involvement of macrophages in the development of breast cancer? How do neurons and glial cells interact in the development, function and repair of the nervous system? How do sex steroid hormones alter cell behaviour in the uterus and how can this be modulated? What is the distribution of central neuronal proteins in the brains of normal compared to model mice suffering from brain disorders?Excitement surrounding the application of Intra-vital imaging to biomedical research is based on its capacity to track cells and their contents within live tissues offering a novel platform for the study of dynamic changes in protein distribution, molecular interactions and tissue composition. Currently, such observations are only possible at single locations within a tissue - our goal is to expand data-acquisition to whole organs, e.g. to interrogate the distribution of synaptic proteins in a mouse brain. To achieve this, we will integrate our intra-vital techniques with systematic 3D image acquisition on a fast confocal microscope, capable of scanning whole sectioned organs. The resulting series of 3D-images will be archived to ensure data preservation and will be made available to the wider research community via searchable web-based organ libraries. These molecular 'organ maps' detailing the location of specific labelled proteins will allow us to refine subsequent 'focussed' functional intra-vital imaging and provide high-content 3D organ maps with functional measurements from live specimens.These innovative studies will provide data for our translational research pipeline and inform development of improved diagnostics and therapies for health problems including cancer, multiple sclerosis, infertility, cardiovascular and neurodegenerative diseases.