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

Abstract

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.

Single molecule mass photometry of nucleic acids

“…The experiment involves placing a small droplet of solution on top of microscope coverglass, to which molecules bind non-specifically, although in the case of DNA appropriate charging of the glass surface is advantageous to achieve tight binding (see Methods). We then visualise individual binding events on top of the static imaging background caused by residual substrate roughness by computing the differences between batches of averaged reflectivity images, which leads to the appearance and disappearance of single molecule signals from irreversible binding events in a continuous recording (Supplementary Movie 1) (12,15). By determining the point in time when each individual molecule binds, we can then quantify the associated reflectivity change, yielding highly accurate, precise and resolved contrast distributions.…”

Quantifying the heterogeneity of macromolecular machines by mass photometry

“…We then quantify individual binding events by illuminating the interface between the sample and cover glass and interferometrically recording reflectivity changes caused by a modification of local refractive index when an adhering biomolecule replaces water. Continuous recording of these events results in a movie of individual proteins binding to the cover glass surface (Supplementary Movie 1), with species appearing and disappearing in time as a consequence of the data analysis procedure 18,19 . Optimization of the image contrast 18 then enables very accurate quantification of the reflectivity change caused by single molecule events, ultimately resulting in exceptional mass accuracy, resolution and precision 16,18 ( Supplementary Fig.…”

Quantifying protein-protein interactions by molecular counting with mass photometry

“…Microscope coverslip cleaning and assembly was performed as described previously [26] . The experimental mass photometry setup has been described elsewhere [25,26] . Briefly, a 525 nm laser diode was used for illumination with the following instrument parameters: acquisition camera frame rate = 955 Hz, pixel binning = 4x4, 5-fold time-averaging.…”

“…MP detects single biomolecules by their light scattering as they bind non-specifically to a microscope cover glass surface. Each binding event leads to a change in refractive index at the glass/water interface, which effectively alters the local reflectivity and can be detected with high accuracy by taking advantage of optimized interference between scattered and reflected light (Figure 1a) [25] . The magnitude of the reflectivity change can be converted into a molecular mass, for polypeptides with ~2% mass accuracy and up to 20 kDa mass resolution by calibration with biomolecules of known mass [26] .…”