Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy

Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction-limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approximately T–1/2, where T is the transmissivity of the mask. Numerical-aperture-based differentiation of background from scattered light represents a general approach to increasing extinction contrast and enables routine label-free imaging down to the single-molecule level.