Dinuclear ruthenium light-switches as multi-output sub-cellular imaging probes within live cells and tissues

正如原子是物质的基本单位一样，细胞是生命的基本单位。维持健康生物体所需的所有功能最终都可以追溯到细胞内发生的分子过程。当这些过程出现故障或被破坏时，包括癌症在内的疾病状态就会出现。为了更深入地了解细胞组分的复杂结构和功能，细胞生物学家希望在分子水平上探测细胞。然而，由于细胞大多是透明无色的，因此必须使用彩色或发光染色剂来标记和可视化特定的细胞成分。在之前的工作中，托马斯小组已经确定了一种化合物，它是细胞核内DNA（遗传“蓝图”分子）的发光探针。这种探针特别令人兴奋，因为与商业细胞不同，它的发射是由低能量光照射引起的，它可以穿透细胞层进入组织，并且对活样本无害。此外，一旦被“点亮”，结合探针的发射寿命也是DNA的独特标记。这是很重要的，因为这个生命周期标记可以作为“指纹”，即使细胞内其他自然发生的分子正在发射。在这个项目中，这些令人兴奋的结果将进一步发展。我们在原始研究中使用的探针包含两个未被分解的手性金属中心（不可重叠的“镜像”），因此它是产物的混合物。由于许多生物分子也是手性的，分子之间的结合可能高度依赖于每个组分的手性，在本项目中，我们将对原始探针的手性纯示例进行研究，以研究单个立体异构体是否被摄取并结合/成像不同的细胞靶标。我们还将制作一系列相关的探针来结合不同的细胞靶标。虽然光学显微镜是一种有吸引力的动态成像技术，但它依赖于探针在绑定到目标时发出光，这并不总是如此。此外，其他技术——如电子显微镜——可能提供更高分辨率的（静态）成像。因此，新化合物作为多功能探针的使用也将被研究。特别是它们作为透射电子显微镜和拉曼显微镜技术探针的使用也将继续进行。这种探针将是有用的，因为使用相同的探针可以在一定范围内成像，并且有可能通过不同的模式产生可以对不同结构进行成像的系统。我们发现的化合物不是被动地扩散到细胞中，而是被主动吸收。非常有趣的是，我们发现，尽管在生物和医学研究中使用的大多数常用细胞系都使用探针，但并不是所有细胞系都使用探针。为了利用这一惊人的结果，在一项概念验证研究中，我们将在一个3-D皮肤肿瘤模型中研究“肿瘤发生”。这项研究的成功结果将有助于了解肿瘤的发展过程，并可能导致新的诊断技术。

Just as atoms are the basic unit of matter, cells are the basic unit of life. All the functions required to maintain a healthy organism can ultimately traced back to molecular processes occurring within cells. When there is a malfunction of these processes or they are disrupted, disease states, including cancer, can arise. To understand the complex structures and functions of cellular components in more depth, cell biologists wish to probe cells at a molecular level. However, since cell are mostly transparent and colourless, coloured or luminescent stains must be used to mark and visulise specific cellular components. In previous work the Thomas group has identified a compound that is a luminescent probe for DNA (the genetic "blueprint" molecule) within the nucleus of cells. This probe is particularly exciting as, unlike commercial cells, its emission is induced by illumination with low energy light, which can penetrate into tissue through layers of cells and is not deleterious to live samples. Furthermore once "lit up" the bound probe emission lifetime is also a distinctive marker for DNA. This is significant as this lifetime marker can be used as a "fingerprint" , even if emission from other naturally occurring molecules within the cell is occurring,nIn this project these exciting results will be further developed.The probe we used in our original studies contains two chiral metal centres (non-superimposible "mirror images") that were not resolved, consequently it is a mixture of products. Since many biomolecules are also chiral, and binding between molecules can be highly dependent on the chirality of each component, in this project we will carry out studies on chirally pure examples of the original probes to investigate whether the individual stereoisomers are taken up and bind/image different cellular targets. We will also make a series of related probes designed to bind to different in cellulo targets. While optical microscopy is an attractive tecnique for dynamic imaging, it relies on a probe emitting light when bound to a target, which is not always the case. Furthermore other techniques - such as electron microscopy - can potentially provide (static) imaging at a higher resolution. Consequently, the use of the new compounds as multifunctional probes will also be investigated. In particular their use as probes for Transmission Electron Microscopy and Raman Microscopy techniques will also be pursued. Such probes will be useful as imaging at a range of scales using the same probe will be possible and, potentially, systems that can image separate structures through different modalities will be produced.The compounds we have identified do not passively diffuse into cells, but are actively taken up. Very interestingly, we have found that although they are taken up by most of the commonly used cell lines used in biological and medical research, not all lines take up the probes. To exploit this striking result, in a proof-of-concept study we will investigate "tumour genesis" in a 3-D skin tumour model. A successful outcome in this study will help understand the process of tumour development and may lead to new diagnostic technologies.