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，细胞也是生命的基本单位。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中断时，可能会出现包括癌症在内的疾病状态。为了更深入地了解细胞成分的复杂结构和功能，细胞生物学家希望在分子水平上探索细胞。然而，由于细胞大多是透明和无色的，必须使用有色或发光染料来标记和观察特定的细胞成分。在之前的工作中，Thomas团队已经确定了一种化合物，它是细胞核内DNA(基因蓝图分子)的发光探针。这种探测器特别令人兴奋，因为与商业细胞不同，它的发射是由低能量光照射引起的，可以透过细胞层穿透组织，对活体样本没有伤害。此外，一旦被“点亮”，结合的探测器发射寿命也是DNA的一个独特的标志。这一点很有意义，因为这个寿命标记可以用作“指纹”，即使细胞内其他自然产生的分子正在发射，在这个项目中，这些令人兴奋的结果将得到进一步的发展。我们在最初的研究中使用的探针包含两个手性金属中心(非重叠的“镜像”)，但尚未分辨，因此它是一种混合产品。由于许多生物分子也是手性的，分子之间的结合高度依赖于每个组分的手性，在这个项目中，我们将对原始探针的手性纯样本进行研究，以调查单个立体异构体是否被占据并结合/成像不同的细胞靶标。我们还将制作一系列相关的探针，旨在结合不同的纤维素靶。虽然光学显微镜是一种有吸引力的动态成像技术，但它依赖于探针在与目标结合时发光，但情况并不总是如此。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用途也将得到研究。特别是，还将继续将它们用作传输电子显微镜和拉曼显微镜技术的探针。这种探针将是有用的，因为使用同一探针在一系列尺度上进行成像将是可能的，并且潜在地将产生能够通过不同模式对不同结构进行成像的系统。我们已经识别的化合物不会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，尽管它们被大多数用于生物和医学研究的常用细胞系所吸收，但并不是所有的细胞系都采用探针。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3D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理解肿瘤的发展过程，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.