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

Rowlands CJ, Ströhl F, Ramirez PPV, Scherer KM & Kaminski CF. "Flat-Field Super-Resolution Localization Microscopy with a Low-Cost Refractive Beam-Shaping Element."  Sci. Rep (2018), 8(1):5630.


 

Abstract

Super-resolution single-molecule localization microscopy, often referred to as PALM/STORM, works by ensuring that fewer than one fuorophore in a difraction-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 fuorophores are switched on; if too many fuorophores are activated, their point-spread functions overlap, which impedes efcient 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 profles that provide a uniform illumination over the whole feld of view. We present the use of a single commercially-available low-cost refractive beamshaping element that can be retroftted to almost any existing microscope; the illumination profle created by this element demonstrates a marked improvement in the power efciency of dSTORM microscopy, as well as a signifcant reduction in the propensity for reconstruction artifacts, compared to conventional Gaussian illumination.