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

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.

This project has a wide range of potential beneficiaries over a number of time scales. In the first case, the activity will deepen the relationship between the research groups involved in the project and catalyse synergies in their future collaborative projects. Of course, outside this relationship, it is plainly evident that academics working in the field relevant to the proposal will benefit from the project outputs as they will form the basis of high impact publications in a burgeoning international research field. Apart from the PDRAs employed, research students and other PDRAs in each of the groups will also benefit from entering a truly multidisciplinary research environment.

In the medium term, the successful development of cellular probes will have impacts in cell biology and medical research, This will be relevent to the wide range of SMEs, larger medical-based companies, as well as relevant charities concerned with diagnostics, therapies and diagnostics. As a specific example: in their 2009-2014 research strategy review, Cancer Research UK state; "New opportunities in imaging could have a real impact on how cancer is treated" and has funding schemes such as its Cancer Imaging Initiative towards this goal. Clearly, this project may well lead to IP that can be exploited

In the long term the development of luminescent cell probes for biomedical research and anticancer therapeutics have potential impacts for society in general.