Development of high-speed SICM for biological applications.

Scanning ion conductance microscopy(SICM) has been pioneered in the UK to image the surface of live cells , under solution, at high resolution to visualize structures that are so small that they are only detectable using an electron microscope, when the cells are fixed. It can be used to follow changes in cell dynamics during important processes at the cell membrane such as the entry or exit of molecules or viruses and the reorganisation of the cell membrane during signalling. However the application of SICM has been limited due to the time it takes to obtain an image. If it was possible to image faster then it would be possible to follow, for the first time, some key biological processes and observe many more details of other important biological processes. To do this we aim to redesign the instrument taking advantage of improvements that have been made to atomic force microscopy to obtain high speed images and advances in high speed computer boards to control the instrument. The redsigned instrument will be optimised on test samples and then finally demonstrated on live cells.

Scanning ion conductance microscopy (SICM) is a form of scanning probe microscopy that allows nanoscale imaging of live cells without contact between the nanopipette probe and the soft cell surface. We propose to redesign the SICM to obtain a a step function change in the imaging speed, exploiting methods developed for high speed AFM and working in collaboration with Professor Miles at Bristol. This will firstly improve the quality of all SICM images, since sample drift and cellular movement will be significantly reduced. Secondly it will allow us to follow many important biological processes occuring at the cell surface including clathrin mediated endocytosis and virus entry. The improvements are based on using a fast FPGA board for feedback control and seperating the distance feedback control into two parts: using a slow piezo to follow the predetermined overall surface topography and fast piezo to determine the accurate position of the surface and then combining these measurements to determine the exact surface position. Fast modulation of the pipette position is used for the feedback signal that is used for the distance feedback control. In combination the imaging speed will be improved by at least a factor of 20 allowing 1um x1um region to be imaged in 100 ms with 32x 32 pixels, which should allow many important membrane processes to be directly imaged. In the first year the instrument will be built and programmed and then tested on fixed and then live samples which undergo significant dynamics. In the final year the instrument will be used to follow the neuronal growth cone and obtain high speed images of clathrin mediated endocytosis. This high speed SICM should have widespread application in both the biological and physical sciences allowing nanoscale imaging of dynamic processes.

Once this instrument is designed and built it is important to get the instrument into the hands of the users in the physical and biological communities as quickly as possible.

An important aspect of ensuring maximum impact of this technology is the involvement of the spin-out company Ionscope. Under suitable commercial agreements the company is well placed to sell high speed SICM instruments making the technology readily available to potential users and present the technology at workshops, meetings as well as performing demos and sending out literature. It should also be possible for scientists to perform feasibility experiments to see how well the technology works before deciding to purchase an instrument. The company has no rights to any IPR generated by this project but owns key patents on SICM imaging, so this may be an excellent and fast route for exploitation.