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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.

SUBMITTER: Rowlands CJ

PROVIDER: S-EPMC5884788 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Owing to the development of X-ray focusing optics during the past decades, synchrotron-based X-ray microscopy techniques allow the study of specimens with unprecedented spatial resolution, down to 10 nm, using soft and medium X-ray photon energies, though at the expense of the field of view (FOV). One of the approaches to increase the FOV to square millimetres is raster-scanning of the specimen using a single nanoprobe; however, this results in a long data acquisition time. This work employs an array of inclined biconcave parabolic refractive multi-lenses (RMLs), fabricated by deep X-ray lithography and electroplating to generate a large number of long X-ray foci. Since the FOV is limited by the pattern height if a single RML is used by impinging X-rays parallel to the substrate, many RMLs at regular intervals in the orthogonal direction were fabricated by tilted exposure. By inclining the substrate correspondingly to the tilted exposure, 378000 X-ray line foci were generated with a length in the centimetre range and constant intervals in the sub-micrometre range. The capability of this new X-ray focusing device was first confirmed using ray-tracing simulations and then using synchrotron radiation at BL20B2 of SPring-8, Japan. Taking account of the fact that the refractive lens is effective for focusing high-energy X-rays, the experiment was performed with 35 keV X-rays. Next, by scanning a specimen through the line foci, this device was used to perform large FOV pixel super-resolution scanning transmission hard X-ray microscopy (PSR-STHXM) with a 780 ± 40 nm spatial resolution within an FOV of 1.64 cm × 1.64 cm (limited by the detector area) and a total scanning time of 4 min. Biomedical implant abutments fabricated via selective laser melting using Ti-6Al-4V medical alloy were measured by PSR-STHXM, suggesting its unique potential for studying extended and thick specimens. Although the super-resolution function was realized in one dimension in this study, it can be expanded to two dimensions by aligning a pair of presented devices orthogonally.

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

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