Combined light sheet and scanning ion conductance microscopy : a new tool to perform single molecule biology in live cells

A cell has many different molecules that interact which each other to respond to external signals and carry out functions that are essential for life and in many diseases these processes go awry. Techniques have recently been developed that allow one to follow individual molecules, so that one can directly watch molecules perform their function in live cells.

The aim of this proposal is to make a major advance in the types of biological processes that it is possible to follow. We will exploit a method that we have developed to deliver (down to) a single molecule to a defined location on the cell surface or within a cell, track the spatial position of that molecule and then any subsequent changes in the cell behaviour with time. Our method is based on a nanopipette, which is less than 100 nm in diameter, to deliver the molecules to the cell and we plan to generate a thin sheet of light to then image where these molecules go, recording a video of their fluorescence. Since we can synchronise when we deliver the molecules with when the light sheet is turned on, we can make the same measurements repetitively while minimally perturbing the cell. The idea is to use the cell as a 'test tube' to perform these types of experiments and automate the measurements so we can study many cells.

The project will start by adapting our existing instrument, building the light sheet illumination and then writing software to control the measurements. We will then perform a series of proof-of-concept experiments to optimise the instrument and method of making measurements. This will be used to stain a small number of molecules on the cell surface, trigger a key receptor associated with the immune response and then add damaging aggregates of the protein alpha synuclein into cells. This new method should then be applicable to a wide range of important biological and biomedical problems since in many cases one or a few molecules can perform key functions or cause disease. There are also many fundamental biological processes that take place in mammalian cells such as the copying and repairing of DNA that could then be studied using this new method in future work.

SICM combined with light sheet imaging will allow one to follow imaging of many key biological processes and will have many potential applications in biology and biomedicine. This would include the molecular basis of neurodegenerative diseases such as Alzheimer's and Parkinson's disease and studying DNA replication and repair in mammalian cells, processes associated with cancer. The technical developments described in this proposal are likely to lead to new intellectual property which will be patented to protect these inventions. In addition it important to get this new technology to the scientific community as quickly as possible, once it has been developed.

Intellectual property and patents will be exploited by the University Technology transfer office. One exploitation plan may be to license to the technology to a large SPM or microscope company, This would also get the technology rapidly to the end-users and may have advantages in terms of the amount of money and resources available.

The main group that will benefit will be those performing fundamental biomedical and biological research but instrument manufacturers will want to incorporate these advances into their microscopes since they should open up a range of new experiments.