| Résumé: | <p>Interferometric scattering microscopy (iSCAT) has emerged in recent years as a highly sensitive imaging technique, capable of detecting the scattering signal even from single unlabelled proteins in suitably designed in vitro single molecule assays where the scattering background is kept to a minimum. Here we present numerical aperture filtering in an iSCAT microscope for enhanced contrast of point-like scatterers, and show that it can lead to improved sensitivity through enabling the use of higher incident light intensities.</p>
<p>We apply this development to measuring the scattering contrast of proteins at the single molecule level. This scattering contrast shows the expected linear dependence on molecular weight, allowing the extraction of mass distributions built up from the quantification of one molecule at a time, an approach we refer to as mass photometry. The mass measured in this way shows ∼2% mass accuracy and ∼2% precision. Distinct species are resolved with a full width at half maximum as low as 20 kDa, although this increases with mass, sufficient for low-order homo-oligomeric resolution. These capabilities are exemplified with measurements of the equilibrium constants for the BanLec- induced aggregation of the HIV protein Env, and dynamic imaging of the early stages of α-synuclein aggregation on supported lipid bilayers. We conclude with a detailed discussion of considerations relevant to the acquisition and analysis of high quality, reproducible mass photometry data.</p>
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