Benchtop, turnkey super-resolution microscopy for biology, biophysics and biotechnology

Much of modern biology aims to understand processes of life, health and disease in terms of the molecular-scale building blocks that make up our body and living organisms in general. A major obstacle to such research is that it is hard to "see" these molecular building blocks, for the simple reason that they are so small. Major breakthroughs in this direction have been the use of X-rays and of electrons to probe living systems, but these methods require the samples under investigation to be fixed and/or frozen and put in a vacuum chamber. To see these molecular building blocks at work, we need to image them in salty water, and - ideally - in or on living cells. In part, this can be achieved by attaching fluorescent labels to the molecules of interest, precisely locating them and/or tracking their behaviour. Thanks to a set of methods collectively named "super-resolution microscopy", there has been major progress (awarded a Nobel Prize in 2014) in the accuracy by which this can be done. Yet such methods are not trivial to use: the operation of such microscopes remains complex and this complexity is a barrier to its wider use in various fields of research.

We aim to purchase a super-resolution microscope that has been developed to address this barrier. It does not require any particularly stable environment nor involves any risk of user exposure to high-power laser (it is in the same risk class as low-power laser pointers), and can operate on a normal bench (or kitchen table, for that matter). Moreover, it includes a way ('microfluidics') to handle small volumes of biological samples in a more convenient matter, further facilitating experiments.

We will make this microscope widely accessible to academic and - where appropriate - also commercial users. To start with, we have identified various research projects that will benefit from this microscope, to address questions including how viruses can infect cells, how our DNA is packed and when needed unpacked in the nucleus, and how to make better reagents to kill bacteria and/or cancer cells.

Most current biological research focusses on understanding biological and biotechnological processes in terms of the underlying molecular structures, recognition events and dynamics. In this context, super-resolution fluorescence microscopy remains unique by providing the combination of:
- biochemically specific information;
- a spatial resolution that can be as good as ~10 nm; and
- a temporal resolution that can be in the range of few milliseconds.
Yet, super-resolution microscopy depends on instrument operation and data analysis that largely remain the realm of specialists. This still hampers its wider use, in particular for its advanced modes of operation to probe single-molecule dynamics. In addition, its wider use is limited by the relatively slow throughput of such experiments. We have identified a range of experiments and potential users of super-resolution techniques, for which throughput and easy access and operation are now key requirements. To meet these requirements, we propose to purchase a benchtop, turnkey super-resolution microscope with integrated microfluidic sample handling, to make high-end super-resolution imaging available and practical for use by a wide, non-expert scientific community that includes structural, molecular and cell biologists as well as (biochemical) engineers and physical scientists.

The proposed equipment will benefit research activities across disciplines and also across length scales, ranging from cellular length scales to single-molecule assays and ranging from basic bioscience, via biophysics, to (bio)chemical engineering. These activities include projects on viral infection, on cell polarity, on motor proteins, on immune proteins that target bacteria and cancer cells, on DNA structure, packing, unpacking and remodelling in chromatin, on chemically engineering membrane nanopores for new antibiotics and biosensing, and on bioprocessed T-cells and the signalling that determines their efficacy.

The here proposed research ranges from basic biology to bioprocessing and (bio)chemical engineering. As a consequence, the economical and societal impact will cover a wide range of areas. By research projects on viral infection, immune effectors, and on various DNA-protein interactions of relevance in cancer, it will generate basic biological understanding to underpin health and healthcare applications. More towards applications, it will contribute to the chemical engineering of antimicrobial nanopores, to address the rising challenge of antimicrobial resistance, and strengthen an on-going, EPSRC-funded "Future Targeted Healthcare Manufacturing Hub", which is directed to processing immune cells to target disease, notably by engineering T-cells for cancer immunotherapies. We therefore anticipate societal and economic impact to lie in the development of novel therapeutic approaches based on the research outcomes of this proposal.

By providing graduate students low-threshold access to super-resolution microscopy methods, this proposal will directly contribute to the training of life and physical scientists as well as engineers on advanced characterisation methods. For example, UCL is the lead organisation of the BBSRC-funded London interdisciplinary doctoral training program (LIDo), one of the BBSRC's largest DTPs that encompasses students across 8 London HEIs including, Birkbeck, King's, QMUL. Following BBSRC strategic priorities, use and development of new imaging methods is a strong theme among LIDo's PhD projects. Most of the investigators on this grant are active in offering PhD and rotation projects on this program with numerous LIDo students having trained in their labs over the past 5 years. UCL has also just been awarded a major Wellcome Trust PhD programme on Optical Biology (with co-I Henriques being one of the directors), and hosts numerous other programmes, e.g., an MRC Doctoral Training Programme at the Institute of Structural and Molecular Biology, and EPSRC funded Centres for Doctoral Training on Bioprocess Engineering Leadership, on Advanced Characterisation of Materials (joint with Imperial College) and on Transformative Pharmaceutical Technologies (with Nottingham University), for which several of our investigators are project supervisors.

Finally, capacity-permitting, we anticipate to open up the equipment for use by existing and future commercial clients. At the atomic force microscopy user facility at the London Centre for Nanotechnology, we have a similar approach, with 5 current industrial clients making regular use of microscopy facilities. We expect the proposed equipment to facilitate materials characterisation for biotechnology, pharmaceutical and photonic industries.