FastScan atomic force microscope for rapid imaging and property measurement of biological systems under natural conditions.

Atomic force microscopy (AFM) is now starting to deliver its potential for biology, allowing us to follow biological dynamics at the molecular scale in their natural (liquid) environment, to monitor the forces that drive biological processes and locally measure mechanical properties from the sub-protein level to entire tissues. Over recent years in Sheffield we have pioneered the use of this technology to answer questions from protein organisation in photosynthetic membranes, to the architecture of bacterial cell walls and we are expanding into entirely new applications such as the measurement of local mechanical properties in the walls of individual plant cells that are important in enabling their function. However, a new generation of AFMs is now commercially available that provides capabilities that are vital for understanding many of the most pressing problems. Processes at molecular length scales are inherently fast, while AFM, because each pixel of an image is collected consecutively, has traditionally had limited imaging rate. The new fast scanning systems have tackled this problem and are able to follow processes with sub-second image rates. Similarly, single proteins typically operate with very small forces, at the picoNewton level, and the properties of structures such as bacterial cell walls vary subtly over tiny, nanometre, length scales in a way that is important for their function. The new AFMs are able to use smaller cantilevers that are inherently more sensitive to force and less susceptible to noise than those used in conventional microscopes, allowing us to really start to unravel the molecular scale properties of living systems.

The purpose of this project is to purchase a fast scanning AFM system and to use it to tackle a diverse range of questions including biological solar energy harvesting, bacterial cell division, dynamic changes as plant epidermal cells respond to environmental changes, and membrane protein function. As well as these initial applications, the Principal Investigator has an excellent track record of building successful collaborations using AFM to tackle biological problems, and the new machine will be vital in further expanding the questions that can be broached. Beyond this, the new microscope will form a vital component of a new centre at the University of Sheffield that will bring together state-of-the-art techniques for AFM, super-resolution optics and cryo-electron microscopy, in combination with internationally leading biology, to really understand living systems from individual molecules to entire cells and tissue. This ambition will occur within an extensive portfolio of BBSRC funded research providing an underpinning facility, vital for our future success.

Atomic force microscopy (AFM) provides a unique set of capabilities for biology, including an ability to image with nanometre resolution under physiological conditions, to measure forces with the sensitivity necessary for examining conformational changes in individual proteins, and to examine properties and structures across length scales from the sub-molecular scale to entire cells and even tissue in a living system. In Sheffield we have pioneered the application of AFM in areas such as the cell-wall architecture of bacteria, and protein organisation in photosynthetic membranes, and are currently expanding into entirely new areas such as the relationship between plant cell wall properties and form and function. However, we have found that our current systems, due to limited scanning speed and force sensitivity, are making it impossible for us to tackle some of the most pressing problems. The new generation of fast scanning AFMs fills this capability gap. They can image at the sub-second imaging speeds necessary to follow molecular processes, can obtain exceptionally large data sets rapidly enough to make correlative microscopy at the molecular scale between TEM and AFM, and can utilise the newly available small cantilevers that have inherent advantages in signal-to-noise and hence force sensitivity.

Without the new machine our BBSRC funded research runs the risk of losing our cutting edge in many important areas. Highlights include: how are the photosystem I and cytochrome b6f complexes organised in the periphery of grana membranes in plant cells? Understanding the basis of photosynthesis is crucial in developing enhanced methodologies to ensure future food security. How do the mechanical properties of the cell wall of S. aureus change during the division cycle? This fundamental research is revealing how bacteria are able to maintain their viability, grow and divide, with important implications for the understanding of how antibiotics kill bacteria.

The aim of this proposal is to purchase a new microscope. Here we will detail primarily the Impact of the research that we propose to carry out with that microscope in the immediate future. This is a flavour of the total impact that the new system will have, as it is our consistent experience that the number of users, projects, and biological questions we are addressing with a machine expands well beyond that initially envisaged as the full potential of new capabilities are realised.

A focus of the work to be carried out with the microscope will be the fundamental underpinning biology of the important human pathogens S. aureus, C. difficile and N. meningitidis, as well as food pathogens such as B. cereus. Between them these kill thousands of people in the UK every year, and the basic understanding that will be enabled by the new microscope will contribute towards future developments such as new antibiotic targets. Antibiotic resistance is an immense problem leading to a renewed emphasis on the development of novel drugs. The projects are fundamental underpinning science in this extremely important area of public concern and will have long-term impact. Foster has extensive experience in both interaction with pharmaceutical companies and has launched a university spin-out in this area (Absynth Biologics), so we are well positioned to ensure impact from work in this domain. Where ever appropriate IP will be secured to facilitate income generation in the long-term, helped by FusionIP, our partner commercialization company.

A second focus is photosynthetic membranes in plants and bacteria. Understanding photosynthesis is of fundamental importance as the basis of life on Earth, but also has the potential to impact upon the search for a sustainable energy future. There are many scientists in academia and industry trying to learn from how plants harvest solar energy, yet in reality there is much at a fundamental level that we simply don't understand. We expect the new findings that the microscope will enable to have a substantial impact in this area.

A third focus is the mechanics of stomata, small pores in plants crucial for the control of water loss. Understanding processes involved in improving crop water use efficiency is central to the themes of Food Security and Living with Environmental Change. The new insights that this machine will provide will allow us to pursue strategies to breed crops with improved water use, a key present and future constraint in agriculture worldwide. Fleming and associates have excellent links with the plant biotech and breeding industry (e.g. Syngenta, RAGT seeds, SAB Millar, Heineken), and are well positioned to deliver impact in this area.

The new microscope will facilitate training in cutting edge microscopy and inter-disciplinary science. As part of the Imagine: Imaging Life initiative it will form an integral component of a suite of super-resolution techniques, complementing optics and electron microscopy, providing a unique training environment for the next generation of biologists. We will provide highly skilled students and RAs capable of working in this inter-disciplinary environment throughout the UK. The new instrument will contribute to the establishment of Sheffield as a hub for interdisciplinary biophysics research, the new system attracting users from other Universities and Industry. These users will benefit from the excellent training environment as well as from the integrated approach being taken.