MICA: A super resolution optical microscopy facility at the MRC Laboratory of Molecular Biology.

Light microscopy plays an indispensable role in modern biomedical research. By providing the ability to visualise specific processes within healthy and diseased cells, often in real-time, this technique has been a driving force in increasing our knowledge of biological processes. This knowledge is, of course, essential for developing new strategies for the diagnosis and treatment of specific human diseases. Until recently, light microscopy has been hampered by the so-called "diffraction limit". This is a theoretical limit to how far two objects need to be apart before they can be resolved by the microscope, and is typically in the region of half the wavelength of visible light (~ 250 nanometers (nm), or 1/4000th of a millimetre). Many important biological structures within our cells are smaller than 250nm and therefore remain poorly characterised. These structures include the "organelles" that perform specific cellular functions and the membrane-bound carriers and filaments along which trafficking occurs, as well as infectious viruses. Several microscopy techniques have been developed in the last few years that allow the diffraction limit to be bypassed, thereby providing access to previously unappreciated processes within cells. These 'super resolution" (SR) methods-some of which allow specific molecules to be localised with a precision of up to ten times greater than conventional microscopy-promise to revolutionise biomedical research, particularly when combined with other techniques in which the UK has pre-established expertise.

Two MRC-funded research organisations in Cambridge-the Laboratory of Molecular Biology (LMB) and Mitochondrial Biology Unit (MBU)-are proposing to jointly establish a multi-user centre of innovation in applied super resolution (SR) optical microscopy. Scientists at LMB and MBU have made, and continue to make, major discoveries into fundamental biological processes and have frequently translated them into commercial and therapeutic successes. There is a remarkable track record of studying biological processes at the scale of the structure of individual proteins and protein complexes, as well as at the cellular level. SR platforms would close the gap between these molecular and cellular studies, allowing multi-scale analysis of cellular processes and the relationship to disease in a world-renowned research environment. The critical importance of light microscopy to LMB and MBU is demonstrated by the presence of > 20 highly used confocal or wide-field microscopes, until recently the highest quality optical microscopes available. Ready access to SR microscopy will make a major impact on the productivity of the organisations. Indeed, 26 groups within the LMB and MBU have projects that require specific SR platforms. Their needs can only be fulfilled by providing three different, leading SR systems: structured illumination, single molecule localisation microscopy (e.g. PALM/STORM) and stimulated emission depletion (STED) microscopy. These systems have non-overlapping strengths and different projects require different systems.

Substantial added value will be provided by (i) capitalising on the highly successful research programmes and complementary technological expertise within the partner organisations, (ii) building on pioneering biotechnology developed at LMB to develop superior labelling methods for SR techniques, (iii) strong collaborations with industrial partners and (iv) provision of access to other, local users to facilitate their research and foster collaborations with LMB and MBU.

The project is sustainable. It builds on well-established infrastructure for management of microscopy resources and user training, and includes early access to new developments through the establishment of industrial partnerships and a significant financial commitment from LMB and MBU. There is also a strong component of cultivating the next generation of scientists by providing training in SR techniques.

The aim of this proposal is to equip a super resolution microscopy facility serving research groups in the Laboratory of Molecular Biology and the Mitochondrial Biology Unit on the Cambridge Biomedical Campus at Addenbrooke's, as well as local collaborators. The availability of such a facility would have a major impact on a wide-range of scientifically tractable projects where super resolution imaging methods will clearly yield important new insights. Given the number of groups and the scope of their projects we have asked for three different systems each yielding super resolution images through application of a different technique. Each has its own strengths and areas of application. These are as follows:

- A structured illumination microscope that is capable of a twofold improvement in resolution in all three dimensions and is compatible with standard fluorescence labelling methods.
- A gated STED (stimulated emission depletion) microscope capable of 50nm lateral resolution in two channels. This method relies on the use of specific fluorescent labels with well defined properties and emission wavelength.
- A single molecule localisation microscope capable of resolution in the 25nm range dependent upon sufficient labelling density. Here a super resolution image is generated by capturing the location at high precision of a sparse subset of fluorescently labelled molecules and repeating for many subsets to build a high resolution composite image. This relies on the use of fluorescent labels with switchable fluorescence properties.

We also seek to develop novel labeling methods building on pioneering work at LMB on site-specific, bioorthogonal labelling of proteins with unnatural amino acids. These microscopes will be installed in a purpose built super resolution microscopy facility managed by a team of microscopy specialists to facilitate their efficient and productive use.

Research work originating from the LMB and the MBU has had a profound impact in the area of public health. Many drugs and even whole fields of therapeutic intervention such as the use of engineered antibodies to treat disease, can trace their origins to work carried out in these labs and its collaborators. The super resolution microscopy facility will be a central resource to a large number of research groups working in a very diverse range of fields in cell and structural biology. It will therefore contribute to work that, in time may lead to the discovery of new drug-able targets and the invention of new therapeutic agents. Whilst such benefits may take a decade or more to reach this stage, it is the general public at large who will benefit through improved public health.

The general public at large will also benefit through the LMB's, the MBU's and on a wider scale the MRC's activities in fostering the public understanding of science. Scientific imaging particularly microscopy in its various forms is often used as a tool to start or illustrate discussions in this field. Super resolution microscopy as a newly emerging research tool is well placed to feature in such discussions. It has a "gee whiz" factor that can grab attention which in turn leads to better engagement with the intended audience. Examples of this include participation by LMB and MBU scientists at the Cambridge science festival and the Royal Society's summer science exhibition.

The academic output of researchers in the LMB and the MBU is for the most part, put straight into the public domain. Articles intended for the popular press can draw on this resource for ideas and factual information. Super resolution microscopy images will contribute to this pool.

The fraction of academic output that is not immediately put into the public domain usually contains commercially valuable intellectual property. It is likely that some discoveries made with the help of super resolution imaging will fall into this category. The MRC has a strong track record in capitalising on such intellectual property, eventually generating income for the UK government and creating business activity in the biotech sector.