Publication
Label-free microscopy is a rapidly advancing field that allows for imaging and tracking of objects without the need for (fluorescent) labels. One technique is interferometric scattering (iSCAT) microscopy, which allows the detection and quantification of single biomolecules based on their refractive index and size. It provides a high sampling rate, great localization precision, and exceptional capability for imaging surface interactions. Since its introduction in 2004, iSCAT microscopy has witnessed expanding applications in both the life sciences and material sciences, although its use remains limited. In this thesis, we investigate the interactions of single molecules with various surfaces through the application of mass photometry. First, we use mass photometry (MP), an iSCAT-derived method, to study the interactions between long double-stranded DNA (dsDNA) molecules and 2D materials, specifically hexagonal boron nitride (hBN). Compared to traditional methods, MP allows to image the native dynamics of the DNA molecules in an aqueous environment and without labels. Through millisecond temporal resolution, we capture transient binding events and demonstrate that DNA molecules exhibit enhanced binding affinity at engineered defects and edges of long lanes on the hBN surface. This approach showcases the potential of MP in studying biomolecular interactions with 2D materials, providing valuable insights for biosensor development. Second, we address the critical aspect of surface modification for MP. The chapter presents a comprehensive evaluation of various passivation and functionalization methods for cover glass to minimize nonspecific binding and optimize conditions for in vitro single-molecule experiments. An established protocol using 3-aminopropyltriethoxysilane (APTES) and polyethylene glycol (PEG, 2k) for passivation, and functionalization with maleimide-thiol linkers, enables low background scattering and reliable measurement of proteins as small as 60 kDa. Finally, we focus on enhancing measurement precision by addressing the noise limitations of iSCAT microscopy. We demonstrate that using a camera with a high well capacity of 2M photoelectrons significantly improves contrast precision, providing a five-fold enhancement over a camera with 32k photoelectrons. Altogether, this work combines iSCAT microscopy, surface engineering strategies, and technical optimizations to advance the study of surface interactions with single molecules.