Single-protein optical holography

Light scattering by nanoscale objects is a fundamental physical property defined by their scattering cross-section and thus polarisability. Over the past decade, a number of studies have demonstrated single-molecule sensitivity by imaging the interference between coherent scattering from the object of interest and a reference field. This approach has enabled mass measurement of single biomolecules in solution owing to the linear scaling of the image contrast with the molecular polarisability. Nevertheless, all implementations to date are based on a common-path interferometer and cannot separate and independently tune the reference and scattered light field, thus prohibiting access to the rich toolbox available to holographic imaging. Here, we demonstrate comparable sensitivity using a non-common path geometry based on a dark-field scattering microscope, similar to a Mach-Zehnder interferometer. We separate the scattering and reference light into four parallel, inherently phase stable detection channels, delivering a five orders of magnitude boost in sensitivity in terms of scattering cross-section over state-of-the-art holographic methods. We demonstrate the detection, resolution and mass measurement of single proteins with mass below 100 kDa. Separate amplitude and phase measurement also yields direct information on sample identity and experimental determination of the polarisability of single biomolecules