Liverpool BioAFM: an integrated optical and atomic force microscope for research across the life sciences

Individual cells in a plant or an animal are exposed to changes in their environment (mechanical forces, biochemical signals, temperature, light variations...). Cells have to interpret this information to adapt and respond appropriately. To understand the molecular mechanisms leading to a particular response (e.g. cell death, differentiation, adhesion, changes in topology etc), biologists need means of manipulating the environment in a carefully controlled manner and measuring the effects, for instance on the levels and localisation of proteins inside cells or on the structural properties of the cell surface. Each individual cell might respond differently from its neighbour and at a different time so it is crucial to follow the events in real time and in each individual cell.

This can be achieved using BioAFM imaging, an emerging technology, which combines Atomic Force Microscopy (AFM) with advanced optical imaging (e.g. confocal, TIRF). AFM is one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale. Information is gathered by "feeling" the surface with a mechanical probe. However, this technique does not provide information about the events inside cells, which can only be imaged using advanced optical imaging using fluorescently labelled molecules. Although BioAFM techniques have existed for over a decade in specialist centres, technological limitations restricted the ability to readily combine measurements from these complementary approaches. Only now that the technology has advanced and been refined through the development of user-friendly routines to capture and overlay information has it reached the point where it can be made available more widely to the life science community.

We propose to purchase one of the first BioAFM microscopes in the UK and to install it in the Liverpool Centre for Cell Imaging (CCI). The CCI is an open access facility, so the microscope will be accessible to groups from several universities and companies. To illustrate the breadth of the science that will be served by this equipment, we briefly present below three of our exemplar projects:

1. Novel surfaces for anti-microbial resistance
Repeated warnings from the WHO and the UK Government's Chief Medical Officer emphasise the serious global threat of increasing antimicrobial resistance. Microbial activity and biofilms on surfaces cost UK industry billions of pounds each year due to product contamination, energy losses and equipment damage. Infection control, via advanced anti-microbial surfaces, is a key strategy to combat resistance. Uniquely, the BioAFM will enable us to learn how harmful bacteria attach to and respond to surfaces, and develop materials engineered at the nanoscale that can combat infection.

2. Developing water-efficient biofuel crops
There is an urgent and pressing need to improve the ability of biofuel plants to grow productively and sustainably on marginal land that is unsuitable for major food crops. Using the new microscope, we will better understand the mechanisms used by drought-adapted desert plants to conserve water by opening their stomatal pores at night and closing them during the hot, dry light period. These principles will then be applied to generate biofuel crops with these properties.

3. Repair of joints during ageing
Regeneration of cartilage, which acts as a flexible cushion between joints, depends on the ability of chondrocytes to produce and maintain the cartilaginous material. Failure in chondrocyte cell function is observed in old age and is associated with osteoarthritis, the most common type of arthritis in the UK, which afflicts around 1 million people every year. With the new microscope, we will follow for the first time, the effects of ageing on the ability of chondrocytes to respond to compression forces at the molecular level and begin to dissect the key pathways that trigger cartilage production.

Multiparameter quantitative imaging in live cells provides important insights into how cells communicate with each other and interact effectively with their environment. However, a critical gap in our knowledge is how cells integrate and generate mechanical cues. BioAFM is an emerging technology combining atomic force microscopy (AFM) with advanced optical imaging (confocal and TIRF), which addresses directly this gap, because it provides precise measurements of mechanical signalling and its integration with effector signalling inside cells. Recent improvements in both software and hardware mean that BioAFM systems are no longer just at the disposal of one or two centres capable of integrating these technologies. However, because developments for correlative force and optical measurements are so recent, there is no open access BioAFM facility in the UK. We propose to install a BioAFM in the Liverpool Centre for Cell Imaging to serve a range of projects within the BBSRC remit for users from the University of Liverpool, and external academic and industrial partners. The proposed projects include understanding: the rules of attachment and responses of biofilm-forming bacteria to nanostructured surfaces; inside-out signalling to regulate the mechanical and physical properties of guard cells in CAM plants; structural arrangement of photosynthetic elements in the chloroplast during circadian regulation and respiratory domains in bioenergetic membranes; outside-in signalling to regulate extracellular matrix production in chondrocytes etc. Liverpool is uniquely placed to implement this step-changing technology, by having an open access facility with expertise in the requisite imaging techniques and a multidisciplinary team of developmental, cell and plant biologists as well as a physicist and chemist for optimal exploitation of the quantitative data generated.

Where and who is our user pool?

A key beneficiary of this investment in BioAFM microscopy will be our user pool. The Centre for Cell Imaging (CCI) is setup specifically for live cell imaging in control environmental conditions with capacity to image live cell or tissue cultures from a range of model organisms, including plants and animals. Therefore our primary user base will be academics interested in the quantitative measurement of real-time bio-mechanical events in a variety of experimental systems. With recent investment from the Optical Microscopy cross-council (MR/K015931/1) and BBSRC Alert13 (BB/L014947/1) initiatives we have been able to significantly extend our user base. We current host an average of around 70 users/pa from across the University of Liverpool, UK and the rest of the world, including industry. Moreover, we have been recommended to become a Euro-Bioimaging Node. While our facility is well established and supports projects using AFM and confocal microscopy, there is a growing demand for BioAFM (combined AFM-confocal-TIRF) capacity, which will enable correlative studies.

1. The biotechnology Industry has interests in developing materials with the ability to interact with and modify the behaviour of biological materials, cultures and biofilms in specific ways (e.g. for directed stem cell differentiation and use as antimicrobials, see letters of support). The agro-biotechnology industry will also be interested in BioAFM imaging of plants for example to understand plant development and function.

2. The pharmaceutical industry will be interested in using BioAFM to develop and validate model systems of tissues and to use those for drug testing, for instance by correlating the distribution of cell types to their metabolic activity following chemical or physical perturbation in living specimens over time, and mapping these changes to variation in biomarkers of human disease (e.g. see project P1, Case for Support).

3. Scientific Software Industry. Data generated by the BioAFM microscopes will present opportunities for software manufacturers to enhance data analysis software for correlative studies.

4. Scientific community. The major impact will come through access to the BioAFM to a wide user base (see letters of support). We will ensure the academic impact of this work through timely seminars, workshops and publications.

5. Outreach. The PIs and Co-Is have active collaborations with the Liverpool World Museum and local schools. The team will use these links to host events showcasing the applicability of the imaging techniques to "grand challenges" in biology and develop teaching resources.

6. A next generation of Scientists. The CCI will provide strong training of young scientists in cutting-edge technologies, assisting in their career progression. The CCI annually trains approximately 8 undergraduate and 20 postgraduate/postdoctoral scientists per annum, who will have access to the new BioAFM facility. More senior researchers who are trying to establish themselves as independent investigators will be supported via the Technology Directorate voucher scheme (see letter from TD). The CCI has had 6 projects funded by this programme. The Centre for Cell Imaging is also active in working with schools. For instance, sixth form projects are run within the CCI.
BioAFM is set to become an important microscopy technique; consequently access to this equipment will be accompanied by training and workshops. On top of individual hands-on training, we plan to host a series of events and workshops on BioAFM explaining its application as well as specific and specialist training on imaging techniques from instrument makers (Zeiss and JPK) and external speakers. We will also include training on image analysis. Workshops will be available to new and existing CCI users in the UK, industry and across Europe via the Eurobioimaging network.