NanoProbes; Development of novel probes for biological submicroscopic multicolour imaging

基因组学和蛋白质组学计划日益推动我们对复杂生物系统的理解；蛋白质科学的进步使我们能够越来越详细地了解蛋白质的形态和功能，而纳米技术研究项目正在开发工具来研究和操纵1-100nm长度范围内的系统。目前的“盲点”是我们无法将基因组和蛋白质组学数据与生命系统中分子机制和生化途径的理解结合起来。例如，我们可能知道蛋白质的原子结构，了解其蛋白质或配体的相互作用，以及它如何以及在何处组装成细胞内的多组分结构。然而，我们无法以必要的分辨率直接在活细胞中描绘出这些过程中的任何一个，从而对事物的工作原理给出一个完整而令人满意的理解。最近在欧洲和英国领先的显微镜家论坛都强调了这个问题，也迫切需要实现更高分辨率的多色活细胞显微镜。虽然新的光学技术不断发展，但在使用电子源和基于光学的方法之间仍然存在重大差距。为了应对这一挑战，我们提出了我们的新型探针的发展，最终将导致活细胞，多色/组分成像在电子显微镜下，使表观荧光电子显微镜（FEM）。通过将光学和电子成像方面的最新技术进步与我们新型发光探针的开发相结合，我们相信这种方法将创造一种远远超过目前正在开发的任何其他已知技术的技术，并提供显微镜所需的阶跃变化，从而在很少的限制下开始真正的多色亚光分辨率成像，并解决生物学中的这一主要限制。这将是生物成像领域的一个突破性进展。我们认识到，任何试图实现亚光/衍射限制纳米分辨率成像的新技术都受到当前可用的荧光/发光探针的限制，这些探针都是为光致发光成像而设计的。我们的方法是封装或化学钝化，特别设计的纳米尺寸（在10nm区域）阴极发光材料，如用于P43（彩色电视屏幕荧光粉）的材料，用于细胞标记。这些探针将具有额外的优势，它们也将适用于标准光子激发，并且由于其高电子束和光稳定性，非常窄的发射峰和惰性性质，与大多数其他荧光染料相比，它们表现出更好的性能。利用传统光学显微镜的优势，使用不同的发光探针来研究生活环境中的多种细胞成分，以及使用扫描电子束可以实现的分辨率，我们将消除目前研究生命系统的“盲点”。这将对生物和医学研究产生深远的影响，阐明精细的粒子图和研究受体组织和相互作用的能力。众所周知，这种相互作用在细胞信号传导、识别和其他对健康细胞功能和疾病至关重要的重要细胞功能中起着关键作用，但由于我们目前无法可视化活样本，因此对这些相互作用知之甚少。除了在生物领域的应用外，我们相信我们的新型发光探针技术将在聚合物研究、表面科学、微纳米技术等许多其他领域产生广泛的影响。因此，我们的探针和FEM将在许多学术和工业学科中得到最广泛的应用。

Genomic and proteomic programmes increasingly drive our understanding of complex biological systems; advances in protein science allow us to understand protein form and function in ever increasing detail whilst nanotechnology research programmes are developing tools to study and manipulate systems on the length scale of 1-100nm. The current 'blind spot' is our inability to combine genomic and proteomic data with understandings of molecular mechanism and biochemical pathways in living systems. For instance, we may know the atomic structure of a protein, understand its protein or ligand interactions, how and where it assembles into multi-component structures within the cell. However, we are unable to image any of these processes directly in living cells with the necessary resolution to give a complete and satisfactory understanding of how things work. Recent forums of leading microscopists both in Europe and the UK have highlighted this issue and also the pressing needs to achieve higher resolution multicolour live cell microscopy. While new optical techniques are constantly evolving there still remains a critical gap between what is possible using electron sources and optically based methods. To meet this challenge we propose the development of our novel probes that will eventually result in a live cell, multicolour/component imaging within an Electron Microscope, making an apparent Fluorescence electron microscope (FEM). By combining recent technological advances in both optical and electron imaging with the development of our novel luminescent probes, we believe that this approach will create a technology that will far surpass any other known technique currently being developed and provide the step change required in microscopy to start true multicolour sub-light resolution imaging with few constraints and address this major limitation in biology. This would be a ground-breaking advance in biological imaging. We recognised that any new technology trying to enable sub-light/diffraction limited nanometre resolution imaging is limited by currently available fluorescent/luminescent probes that have all been designed for photoluminescent imaging. Our approach is to encapsulate, or chemically passivate, specially engineered nano-sized (in the region of 10nm) cathodoluminescent materials such as the material used for P43 (in colour TV screen phosphors) for cell labelling. These probes will have the added advantage that they will also be suitable for standard photon excitation and exhibit far better properties compared to most other fluorochromes due to their high electron beam and photo-stability, very narrow emission peaks and inert nature. Taking the advantage of conventional optical microscopy and the use of different luminescent probes to study multiple cellular components in a live environment and the resolution that can be achieved using a scanning electron beam, we will remove the current 'blind spot' in studying living systems. This will have a far-reaching impact on biological and medical research with the elucidation of fine detailed particle maps and the ability to study receptor organisation and interactions. Such interactions are known to play key roles in cell signalling, recognition and other vital cellular functions that are critical for healthy cell function and disease, yet little is understood due to our current inability to visualise live samples. As well as the biological applications, we believe our new luminescent probe technology will impact widely on many other fields, such as polymer research, surface science, micro and nanotechnology. Our probes and FEM will therefore have the widest possible application across many academic and industrial disciplines.