NanoProbes; Development of novel probes for biological submicroscopic multicolour imaging

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.

The approach of this proposal to develop Fluorescent Electron Microscopy unites the work of three distinctly separate research centres in a close collaboration that will operate throughout the project with results being fed between the three centres to drive the research forwards. Brunel will develop novel nanocrystallite phosphors (probes). These will be optimised for their CL performance in the size range 2-50nm. The result will be a range of new luminescent probe particles prepared with narrow emission bands at various different wavelengths (in the visible region). These probes will be composed of chemically inert metal oxide lattices, designed not to be cytotoxic even if their protective coatings (where used) were to become detached. This will allow several types of probe to be utilised and allow multicolour FEM. The probes will be coated (using organic materials such as block copolymers) to further increase biocompatibility and allow them to be biologically targeted. York and NIMR (MRC Mill Hill) will concentrate on the application of the probes in biological samples. A new cold FEGSEM with a CL detector will be acquired for the project. Uncoated probes will be characterised on this FEGSEM as well as by other techniques e.g confocal microscopy, in order to assess their suitability and performance. The probes will be modified to bind to specific biological targets. Our first probe tag will be to an anti-GFP antibody for immediate applicability to a diverse range of biological samples and can be exploited by many members of our assembled SAG. Techniques will then be developed specifically to facilitate their use. The suitability of probes and coating materials will be explored in collaboration between the centres so that the most useful materials in terms of application towards biological specimen labelling will be developed. The work as planned will facilitate new avenues of research for all three centres, enhancing and supporting their current activities.