Developing cell identification methods using atomic force microscopy

• Main Author: Pulleine, Ellie Mui Mui
• Published: University of Glasgow 2017
• Subjects: 616.02; Q Science (General)
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.716879

This body of work describes the development of a non-invasive and label-free method for characterization of cell surface markers. The motivation for such a method is the ability to measure cells whilst maintaining function, minimizing contamination and disturbance but enabling downstream applications. The technique would impact on life sciences applications including; phenotype identification of both individual and populations of cells, dynamic measurement of cellular response and monitoring cell-microenvironment interactions. The method described centers on molecular recognition interactions which are associated with specific binding forces. These specific forces can be measured in a highly sensitive manner using force instruments. In this study atomic force microscopy (AFM) was employed because of its powerful capability of highly sensitive force measurement at a nanoscale spatial resolution. The objective to develop a force based method for characterization of cell surface molecules may be considered in more specific aims; the development of a functional AFM probe for identification of specific molecules and establishment of quantitative measurement of surface markers. The probe developed has a colloidal geometry which encourages multivalent binding due to greater contact areas, which can reveal presence on cells in just few measurements. On non-deformable surfaces few interactions occur and regular force increments and probability of unbinding indicate presence of target molecules. With multivalent interactions on deformable samples other variables of adhesion indicate identification of interactions; namely distance of total separation, total peaks of unbinding and energy for total separation. With these variables, the identity of HeLa and HFF1 cells was indicated by cluster of differentiation markers 24, 44 and 98 in a semi-quantitative manner. Additionally individual mesenchymal stems cells are identified by the presence of cluster of differentiation marker 90 and dynamic measurement of Human Leukocyte Antigen. Single-cell force spectroscopy was employed to investigate cellular binding to cancerous matrices to gain greater understanding of tumour angiogenesis. Total internal reflection fluorescence microscopy was employed to inform the experimental setting of contact area and sampling density. The method developed illustrates the potential of force based measurement for label-free, non-invasive measurements on cells. Further development and automation may allow the dynamic measurement of multiple markers. This would allow for a number of applications; the identification of true stem cell clones which is of great importance for stem cells therapies, for monitoring of differentiation, where both short and long term activations could be investigated.