A Method to Resolve Atomic Force Microscopy Feature Definition Issues for Cells Cultured on Nanofibrillar Scaffolds

Atomic force microscopy is emerging as a new standard technique for nanobiomedical investigation. Even so, AFM remains an under-utilized technique and more importantly an under-developed enabler of significant new biomedical discoveries due to a general problem with inconsistent feature definition. In this work, astrocyte neural cells cultured on nanofibrillar tissue scaffolds that have shown promise for brain and spinal cord injury repair were investigated by AFM. It was inherently not possible to distinguish potentially important cell-scaffold interactions within the dynamic range of conventional AFM height, deflection, amplitude or phase images because the cell processes and edges were on the same spatial order as the background nanofibers, ∼100 to 200 nm. We developed a diagnostic method based on analysis of standard AFM section measurements that provided clear guidance for the selection of a combination of image processing techniques to extract boundary information actually contained within the AFM images all along. The diagnostic conclusions were that the combination of dynamic range enhancement with low frequency component suppression would enhance feature definition of cellular edges and process relative to nanofibrillar tissue scaffolds. Implementation of this combination resulted in clear images of cellular processes and edges on the nanofibrillar surfaces. The clear images revealed previously unrecognized cell-cell interactions and provided new information for ongoing investigations of why cells cultured on nanofibrillar surfaces seem more biomimetic. The same diagnostic method was successfully applied to cerebellar granular neurons cultured on nanofibrillar surfaces, and would be useful in similar investigations, e.g., cardiomyocytes on nanofibrillar surfaces. The methods developed here can therefore extend the usefulness of AFM nanoscale imaging in regenerative medicine.