Cellular Force Microscope.

Over the past decade the field of mechanobiology has expanded exponentially due to a growing realisation that the coupling between mechanical properties and biochemistry plays a central role in biology and potentially in a number of diseases. This expansion has been enabled by the adoption of a number of techniques for measuring mechanical properties at a cellular and sub-cellular scale, arguably the most widely used of which is atomic force microscopy (AFM). By indenting a sharp probe attached to a force sensing cantilever into a sample surface, a curve of indentation against force can be measured, and from this the sample's elastic modulus can be calculated. AFM instrument manufacturers have made the process relatively straightforward, automated the analysis, and have spawned the growth of an active scientific community. The numbers obtained are now used to inform models and improve understanding of fundamental biological processes such as cellular motility, endocytosis and tissue development, as well as to drive translational aspects such as tissue repair and intervention. Unfortunately there is a fundamental problem in that the measurements of modulus obtained contain systematic errors typically of 10s to 100s of percent - the force sensor, the AFM cantilever, is simply not sensitive enough to accurately measure the small forces asked of it.

To address this problem we will use a new generation of small and soft AFM cantilevers that have the required sensitivity, we will build a detection system that enables accurate measurement of their stiffness and deflection, removing another major source of measurement error, and house it within a custom designed instrument able to measure indentation accurately over 10s of micrometres. The resultant Cellular Force Microscope (CFM) will have the sensitivity and accuracy necessary to measure the properties of soft cells and tissue.

We will use the instrument to look at two example systems in which we have considerable experience. Nerves in the peripheral nervous system (PNS) contain a number of different cell types, including neurones and Schwann cells, both of which are exceptionally soft even for animal cells. Traumatic damage to the PNS is common and can result in loss of feeling and function. Nerve repair remains a considerable challenge, and better understanding of nerve mechanical properties has an important role to play in the selection of suitable tissue engineering scaffolds for nerve regeneration. We will use our new instrument to obtain preliminary data on the mechanical properties of cells from the PNS, with the aim of informing the selection of mechanical property matching materials. The second system we will study is the mechanics of the soft tissue in the interior of bone. This is tissue that is mechanically heterogeneous but contains regions that are very soft and as such is an excellent example of the kind of advanced, more in vivo, mechanobiology study that AFM is starting to be used for. As part of a project on breast cancer metastasis we are currently characterising the properties of bone using conventional AFM so it provides an excellent tissue model for applying the new technology, using surplus bone tissue. Our new data will contribute to that work on understanding whether the mechanics of the bone influences the secondary spread of cancer to this site.

AFM is a standard tool for measuring the mechanical properties of cells and tissue with sub-cellular resolution. It is widely used to obtain the elastic modulus of cells and to look for variations across different cell types, between different disease states, or due to external perturbation. Our understanding of cell mechanics is increasingly reliant on the capabilities of AFM. However, there is a fundamental issue with measurements as they are currently made, particularly when applied to softer cell types and tissue. Current AFMs are simply not sensitive enough to force or accurate enough in their measurements to perform many of the experiments they are used for, resulting in errors of up to 100s of percent. These are systematic errors for most ways of analysing data, so they do not result in broad distributions, and experiments are repeatable when the same measurement conditions are used, but the actual numbers obtained for modulus are incorrect. The solution is to use much more sensitive cantilevers, meaning both softer and smaller. Such small soft cantilevers are commercially available for a different application, and will be adapted for use in probing cell mechanics. We will address the issue that cantilever deflection is not accurately measured by incorporating a traceable interferometric detection system, providing several advantages for making mechanical measurements of this type. The resulting Cellular Force Microscope will enable accurate measurement of Young's Modulus with errors less than 5% even for the low moduli found in soft animal cells and tissue.

We will combine instrument development with application to two challenging systems: measuring the mechanical properties of the very soft cells found in the peripheral nervous system (PNS) namely Schwann cells and neurons, and measuring how the elastic modulus varies across the interior of a bone, a model tissue and important in understanding what controls the site of breast cancer metastasis in bone.

One of the reasons for the rapid expansion of the field of mechanobiology is the growing realisation that mechanical, as opposed to purely biochemical, properties play a central role in how many biological systems develop and function. This is true for a fundamental understanding of how life works, but also has broad translational aspects in understanding and tackling disease, aging and physical trauma. The initial exemplar projects that we will study in the life-time of this proposal, targeted to the areas of nerve growth and regeneration and cancer metastasis, acknowledge this wider societal importance. We will develop the technology to be capable of addressing such systems, so that it can be directly applied to medically relevant problems.

In the short term we expect to collect preliminary data on nerve cell mechanics, enabling a follow-up grant on nerve growth in the context of regenerative medicine. Nerve damage and ensuing morbidity affects 65,000 people annually in the UK, with consequences such as common brachial plexus injuries equitable to spinal cord injury in socio-economic terms - improving outcomes in this area will have wide impact both on individuals and on their ability to be economically productive. Similarly our work on the mechanics of bone will feed into our project on cancer metastasis and the influence of various drugs on rates of bone metastasis. In the UK more than 10,000 women die from breast cancer each year, and improving outcomes will have considerable impact. These are both long term potential impacts, but are indicative of the sorts of areas where an improved ability to measure cell mechanics would be beneficial. Our research is already actively translational, for instance in the development of new nerve guidance conduits for nerve regeneration (Haycock), and we are very well placed to optimise any potential in these areas.

Cell mechanics have been shown to play a key role in developmental biology, and are implicated in a number of different diseases. Better capability for accurate measurement may have future impact in these areas, as improved understanding leads to the development of new drugs and treatment regimes.

We will develop a new microscope with novel capabilities. This will be of interest to the AFM industry, as a large and growing market is in the life sciences and mechanobiology. We are exceptionally well linked with existing AFM manufacturers, both in the UK and internationally. Hobbs also has experience of spinning out a company in this area (Infinitesima Ltd) which is now successful in the silicon fabrication inspection market. We are well placed to ensure that the technology, once developed, can be moved as rapidly as is feasible towards the market. To this end, we will protect intellectual property where appropriate (Hobbs looked after Infinitesima's patent portfolio during the company's initial stages, and is well experienced in this area), helping to provide security to enable future exploitation.

As well as ensuring that the new technology is well exploited in the areas that we have developed it for, we will actively search for other potential applications and partnerships. We have found in the past that technology can have its most powerful application in unexpected areas - the VideoAFM technology that lies behind Infinitesima's inspection business was originally developed for following dynamic processes in polymers, very far from its ultimate market measuring defects in silicon fabrication masks.