High-speed High-throughput AFM For Cell And Developmental Biology

Atomic force microscopy is a form of microscopy in which a tiny needle-like tip is scanned over a surface, thus feeling its contours in a manner similar to that used to read Braille. This technique also functions for work on surfaces immersed in water, which is of great relevance for biology as salty water is the medium that surrounds the minute machinery that makes up living cells, and thus also the human body.

The use of Atomic force microscopy is wide-spread in the Physical sciences. However, its use for biology research has been hampered by a number of drawbacks. The aim of this proposal is to create a new-generation atomic force microscopy system that will be uniquely suited to research in cell and developmental biology. The new system will have greatly improved sensitivity and be capable of imaging biological cells and tissues within seconds, compared to minutes for conventional instruments. Importantly, it can go from atomic-resolution imaging of small surfaces to indenting and stretching cells over many microns to determine their mechanical properties. Finally, it can be combined with optical microscopy, which facilitates the detection of fluorescently labelled proteins that be used to report on other phenomena occurring in the cell as it is being probed by the atomic force microscope.

These properties make the new-generation atomic force microscopy system highly suitable for addressing a wide range of scientific questions. With our investigators/ collaborators, we have identified five topics for which the application of this system will be most productive:
(i) Understand the changes in mechanics that take place during the formation of the spinal chord. Perturbations in the mechanical forces exerted by tissues are at the root of spinal malformations such as spina bifida.
(ii) Investigate the mechanical changes that occur during cell division. The answer to this fundamental question has potential implications for future cancer therapies, as tumours are characterised by uncontrolled cell divisions.
(iii) Understand how the signals that orchestrate the mechanical changes that occur during cell division.
(iv) Determine the physical forces that allow cancer cells to leave a primary tumour and establish secondary tumours in the body.
(v) Investigate how pores are formed in the membranes of bacteria and virus infected cells during key functions of the immune response.

The instrument we plan to purchase will be located in a user facility at UCL that is widely-used by researchers from all major research-intensive London universities. This will ensure that the expertise gained by the co-investigators will get passed on to the wider AFM user community and that it will benefit biosciences in London at large.

Mechanical forces play an important role in normal physiology and disease. As a consequence, researchers have been turning to physical characterisation techniques such as Atomic Force Microscopy to probe the role of mechanics and forces in Biology. AFM is a unique tool for visualising functional biological samples at (sub)nanometre resolution, as well as for characterising the mechanical properties of molecules, cells and tissues. On intact cells, however, the spatial resolution is too poor to distinguish macromolecular assemblies, partly because scan rates are slow (minutes per frame) compared to the time-scale of fluctuations of the cell membrane. Moreover, typical scan ranges are small compared to the dimensions of cultured cells, tissues, and embryos. These limitations currently prevent widespread use of AFM for biomedical research on living cells and tissues.
These limitations can be overcome by a new generation of AFM instruments that benefits from components with order-of-magnitude enhanced resonance frequencies. Such instruments feature an improved temporal resolution allowing images to be acquired in seconds rather minutes, as well as a force resolution of a few picoNewtons, all while maintaining a high spatial resolution (~1 nm). Within this project, we will purchase an AFM system that combines this enhanced performance with inverted optical microscopy (allowing acquisition of complementary information by fluorescence), while also enabling advanced mechanical measurements necessitating topographical height changes of up to 100 um and multi-positioning possibilities for high-throughput experimentation.
This combination of abilities makes it possible to address a whole range of important biological questions on cellular- and tissue-scale responses to mechanical forces, such as: how forces exerted by monolayers participate in development or what determines cellular morphogenesis in mitosis, extending to the effects of nm-scale domain formation in membranes.

A direct non-academic impact will be via the availability of this new atomic force microscopy system to industrial users in the UK. Despite their proven pharmaceutical and biomedical applications, atomic force microscopes still remain relatively uncommon in the biomedical and pharmaceutical industry, especially within small to medium firms. Over the years, the LCN has succeeded in engaging with these industries allowing them to incorporate atomic force microscopy within their research strategies via fee-for-usage access to the AFM user facility.

Examples of such impact are: (i) Syngenta, who already makes use of the atomic force microscopes at the London Centre for Nanotechnology to test samples in new crop design, and will benefit from the higher throughput and more sensitive measurements enabled by the new instrument; (ii) MedImmune, a branch of AstraZeneca, who has a long standing collaboration with PI Hoogenboom and will benefit from the new system for imaging structural changes in cell surface receptors on exposure to various drugs in development, as well as for determining drug affinities directly on the cell surface. Such use of the equipment will thus directly benefit UK industry.

It will also benefit medical research, in particular via greater mechanistic understanding of the working of next-generation antibiotics on bacterial membranes and of the immune response which can be exploited to design drugs that suppress the immune response following bone marrow transplantation. This will be helped by engagement with researchers from clinical units such as the Institute of Child Health which is attached to Great Ormond street hospital.

In summary, we expect direct, short-term benefits for UK biotechnology industry and longer-term benefits for healthcare.