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Tunable Wide-Field Illumination and Single-Molecule Photoswitching with a Single MEMS Mirror.

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 tunable 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.

SUBMITTER: Herdly L

PROVIDER: S-EPMC8447260 | biostudies-literature |

REPOSITORIES: biostudies-literature

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