Flat-Field Super-Resolution Localization Microscopy with a Low-Cost Refractive Beam-Shaping Element

Abstract

Super-resolution single-molecule localization microscopy, often referred to as PALM/STORM, works by ensuring that fewer than one fluorophore in a diffraction-limited volume is emitting at any one time, allowing the observer to infer that the emitter is located at the center of the point-spread function. This requires careful control over the incident light intensity in order to control the rate at which fluorophores are switched on; if too many fluorophores are activated, their point-spread functions overlap, which impedes efficient localization. If too few are activated, the imaging time is impractically long. There is therefore considerable recent interest in constructing so-called 'top-hat' illumination profiles that provide a uniform illumination over the whole field of view. We present the use of a single commercially-available low-cost refractive beamshaping element that can be retrofitted to almost any existing microscope; the illumination profile created by this element demonstrates a marked improvement in the power efficiency of dSTORM microscopy, as well as a significant reduction in the propensity for reconstruction artifacts, compared to conventional Gaussian illumination.