Scanning ion conductance microscopy : modelling and approaches to studying peptide secretion

Scanning ion conductance microscopy (SICM) offers a number of benefits for both imaging and recording from biological samples. However, several fundamental issues concerning this technique, including the image resolution, remain to be clearly understood. In this thesis a model of SICM has been developed to address these issues. This model is quite general and can accommodate arbitrary geometries for both the probe and the sample surface. Model simulations using simple geometries are in good agreement with known analytical solutions and experimental data. The simulations have therefore been extended to examine a number of imaging issues, including the lateral resolution of SICM in response to a surface 'step' and to a pair of adjacent objects. The probe’s interactions with features that are smaller than, or comparable to, the size of the probe tip have also been investigated. Further, the scanning of sloped objects has also been examined. The results of these simulations suggest some important considerations for scanning. The potential for extending the biological uses of SICM has also been examined in the area of protein/peptide secretion. It is demonstrated that SICM can be used to image secretory events from the Weibel-Palade bodies in endothelial cells. Further, to explore similar issues, on a smaller scale, the use of SICM to study neuropeptide Y (NPY) secretion has been examined. To this end a new preprohormone construct of NPY, which allows visualisation of the location of secretory events, has been tested. This construct contains a region coding for the pH-sensitive ecliptic pHluorin, and is shown to be capable of reporting fusion events, without major disruption of the kinetics of secretion. Experiments using this construct in conjunction with SICM are described and the potential for using SICM to provide insight into peptide secretion is discussed.