Surface passivation and functionalization for interferometric scattering microscopy

Abstract Interferometric scattering (iSCAT) microscopy enables the label-free observation of biomolecule interactions with surfaces. Consequently, single-particle imaging and tracking with iSCAT is a growing area of study. However, establishing reliable cover glass passivation and functionalization methods are crucial to reduce non-specific binding and prepare surfaces for in vitro single-molecule binding experiments. Existing protocols for fluorescence microscopy can contain strongly scattering or mobile components, which make them impractical for iSCAT-based microscopy. In this study, we characterize several different surface coatings using iSCAT. We present approaches for cover glass passivation using 3-aminopropyltriethoxysilane (APTES) and polyethylene glycol (PEG, 2k) along with functionalization via a maleimide-thiol linker. These coatings are compatible with water or salt buffers, and show low background scattering; thus, we are able to measure proteins as small as 60 kDa. In this technical note, we offer a surface preparation suitable for in vitro experiments with iSCAT.