Interferometric scattering (iSCAT) microscopy enables the label-free observation of biomolecules. Consequently, single-particle imaging and tracking with the iSCAT-based method known as mass photometry (MP) is a growing area of study. However, establishing reliable cover glass passivation and functionalisation methods is crucial to reduce nonspecific 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 MP-based microscopy. In this study, we characterise several different surface coatings using MP. We present approaches for cover glass passivation using 3-aminopropyltriethoxysilane (APTES) and polyethylene glycol (PEG, 2k) along with functionalisation 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 MP.
Keywords: interferometric scattering; label‐free microscopy; mass photometry; surface functionalisation; surface immobilisation; surface passivation.
© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.