Super-resolution fluorescence atomic force (SURFACE) microscopy

Bacteria are microscopic organisms of immense importance to humans. Given their diminutive size much of our understanding of how they grow and divide has been determined by a variety of microscopy approaches. Since the earliest microscopes many important discoveries have been made and continued technological developments have permitted new and exciting revelations. Bacterial cell biology and the ability to determine the subcellular localization of components have been largely driven by fluorescence microscopy, whereby individual components are labelled with fluorescent markers. However the limit of detection, coupled with the small dimensions of bacteria has limited the true potential of this approach. Very recently new techniques have allowed fluorescence microscopy to traverse this barrier to attain a level of resolution approaching single molecules. However due to the technical difficulties, lack of commercial availability and expense of some of these developments they have not as yet made a great impact. We have been using a complementary approach called atomic force microscopy, which gives very high resolution of surfaces. We have applied this to the study of bacterial cell walls (the site of action of important antibiotics such as penicillin). This has revealed many important new insights. The aim of the project is to build a new type of microscope coupling super resolution fluorescence and atomic force. We will use our existing microscopy set up as a scaffold to rapidly and efficiently develop the new machine, given the interdisciplinary expertise of the investigator team. The new machine will be tested and further developed within our current bacterial cell wall architecture and dynamics research area. This will provide an ideal framework to develop a new microscope, which can then be utilized and duplicated more widely to allow the rapid uptake of a novel and exciting approach.

Peptidoglycan (PG) is the major structural component of the bacterial cell wall and the target of important antibiotics (e.g. penicillin and vancomycin). We study PG architecture using Atomic Force Microscopy (AFM) which yields images at nanometre resolution. We also use fluorescence microscopy to localise chemically distinct regions of peptidoglycan (e.g. vancomycin for nascent material). However, the resolution of fluorescence microscopy is limited to about 200nm and we need to resolve this spatio-chemical order on the same scale as AFM, to connect PG architectural features to chemistry and thus growth, synthesis and cell cycle. Very recently exciting new developments have allowed fluorescent molecules to be located to within about 1nm (e.g. Stochastic Optical Reconstruction Microscopy, STORM). The focus of this application is to develop the first AFM with STORM capability permitting single fluorescent molecule localization coupled with high resolution AFM. We will design, build and test the new Super-resolution fluorescence atomic force (SURFACE) microscope, specifically within the context of our ongoing PG research. We will use a simplified optical set up, exploiting commercially available fluorescent makers, with a drift correction system to facilitate integration with the AFM and acquisition of data. We will use fluorescent vancomycin to give molecular level resolution of the sites of nascent PG, within the context of the observed architecture, and fluorescent lectins to explore glycan chain length. The SURFACE microscope will also be tested for its ability to localize the PG biosynthetic machinery using immunofluorescence with a range of antibodies available in the laboratory. The project will develop a new combined microscope with wide potential application across the biosciences. We will endeavour to promulgate the use of the new technology both internally and more widely by the free availability of the design details and software source code.

The proposed project will build a new type of microscope and apply this to our current research area on how bacteria are able to maintain their viability, grow and divide. There will be a variety of impacts over a range of timescales and in different arenas. The Scientific Community - Development and extension of microscopy approaches. The project will develop a first in class novel microscope combining super resolution fluorescence and atomic force microscopy. - Establishment of Sheffield as a hub for interdisciplinary biophysical research. The project will enhance our standing in the area and lead to further inward investment. - BBSRC priorities. The project addresses the BBSRC research priority of Basic Bioscience Underpinning Health. - Antibiotics. Peptidoglycan biosynthesis is the target of penicillin etc. and the project will have a long-term impact in this area. - Interdisciplinary research. The project is an excellent example of a cutting edge interdisciplinary collaboration, developing a new microscope to address an important biological research area, which absolutely requires the increased resolution to attain its goals. - Publications. We will produce high quality data that will be published in leading international journals to provide maximum access to user communities. Our recent manuscripts have been published in PNAS and Nature Communications - International collaborations in the field. Wider interactions will develop the area of microscopy and cell wall research in the UK to maintain and enhance our international prestige in the area. - Oral communications. We will participate in national and international meetings and conferences to publicise the work to a diverse audience. Industry, Policy, the Public and UK-PLC - Intellectual property. Where appropriate IP will be secured to facilitate income generation in the long-term. We have a strong history in patent protection of our scientific findings. - New microscopy. The project will develop a new microscopy approach, that may well be of interest to instrument manufacturers (see letter of support from Asylum). JKH has strong links with the microscopy industry as well as a successful history of commercialising microscopy instrumentation through a spin-out company (Infinitesima Ltd.). - New antibiotic targets. The project will provide fundamental data to inform the development of new antibiotic targets. I (SJF) have many links with the UK and international pharmaceutical industry for potential exploitation of results. - Public Engagement. I (SJF) have participated in several radio and newspaper interviews over the years and hosted school parties to inform the public concerning our research and other pertinent issues of concern. Training - Training of project staff in interdisciplinary approaches to biology. Specifically the RA will become an expert in a diverse range of skills across the disciplines. - The next generation of scientists. The RA will be actively involved in transfer of their skills and knowledge to PhD students, MSc students and undergraduates who will be on projects in the laboratories of the investigators. This will provide a very fertile atmosphere for the generation of a new breed of researcher able to span biology to physics. - Dissemination of skills and expertise. Visitors from other laboratories in the UK and internationally will be trained in the new technologies. - Teaching. The project not only develops a new microscopy approach but will also be crucial to the development of new models to explain the fundamental principles of bacterial growth and division. Thus the research will have implications at the level of teaching of microbiology and microscopy to undergraduates. In particular the project will also have direct relevance to the teaching of a new, BBSRC funded, Masters course in 'Mechanistic Biology', organised and run by the Krebs Institute.