Understanding and characterizing biointerfaces via quantitative study using localization based single molecule fluorescent microscopy

Abstract

In this work, the single molecule localization microscopy (SMLM) technique was utilized to characterize biointerfaces and their functions for the first time. The power of this method is that it’s bio-friendly and its ability for visualizing individual molecules. To achieve a well-defined and well-controlled surface, indium tin oxide coated glass (ITO) was applied as the substrate. This work was carried out aiming to take molecular level measurement on molecular level fabrication.

In the first chapter, to achieve a single molecule counting approach and test the compatibility of ITO as a SMLM substrate, a quantitative study was demonstrated through imaging fluorescent-labelled BSA on glass substrate and then glass and ITO were compared as a SMLM substrate. Through optimization of dSTORM detection parameters and counting algorithm, a quantitative study of surface adsorbed BSA molecules with Alexa Fluor 647 labelling was conducted on glass surfaces. This quantitative dSTORM study (qdSTORM) established an effective approach for precisely imaging and accurately counting individual surface molecules. Then a comparison of number of surface adsorbed BSA molecules as well as photophysical property of Alexa Fluor 647 on bare and modified glass and ITO surfaces were carried out, followed by a dual-colour PALM/dSTORM imaging of cells on both glass and ITO surfaces. The overall results suggest that ITO, just like glass, is overall compatible with SMLM imaging including PALM/dSTORM.

In the second chapter, as ITO is also compatible of molecular level controlled surface fabrication, a cell-material interaction on designed ITO surface with molecular level precision was monitored using PALM/dSTORM. ITO surfaces were modified and functionalized to have a homogenous distribution of RGD ligands over a broad area and also made ITO surfaces with high to low RGD densities. The example of model interfaces for cell biology, where the density of the cell adhesive RGD peptides is shown to influence the phenotype of cells, was referred here. Then a quantitative co-localization study of RGD ligands and focal adhesions (FAs) on ITO surface was performed. Based on a nanometre level correlation analysis, it can be concluded that cells find regions of high RGD density to form FAs.

In the third chapter, a quantitative study of a typical antibody-antigen reaction was carried out. The antibody-antigen interaction is fundamental in biology however few researches have been conducted to study the reaction kinetics in molecular level precision. After immobilizing a controlled amount of Alexa Fluor 647 labelled BSA molecules on ITO surfaces, the Alexa Fluor 488 labelled anti-BSA molecules with differed concentrations were then incubated onto the surfaces, enabling a dual-colour dSTORM analysis to monitor the BSA and anti-BSA interaction. The number of surface binding BSA and anti-BSA molecules was quantified. In this study, a surface based approach was built up for getting quantitative information of the binding affinity between BSA and anti-BSA on a liquid-solid interface.