Tuneable wide-field illumination and single-molecule photoswitching with a single MEMS mirror

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Abstract

Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images. Therefore, different approaches for flat-field illumination have been introduced as alternative to the conventional Gaussian illumination. Here, we introduce a single microelectromechanical systems (MEMS) mirror as a tuneable and cost-effective device for adapting wide-field illumination in SMLM. In flat-field mode the MEMS allowed for consistent SMLM metrics across the entire field of view. Employing single-molecule photoswitching, we developed a simple yet powerful routine to benchmark different illumination schemes on the basis of local emitter brightness and ON-state lifetime. Moreover, we propose that tuning the MEMS beyond optimal flat-field conditions enables to study the kinetics of photoswitchable fluorophores within a single acquisition.
Original languageEnglish
Number of pages9
JournalACS Photonics
Early online date25 Aug 2021
DOIs
Publication statusE-pub ahead of print - 25 Aug 2021

Keywords

  • super-resolution microscopy
  • dSTORM
  • tuneable flat-field illumination
  • MEMS micromirror
  • photoswitching
  • quantitative microscopy

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