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

Herdly, Lucas and Janin, Paul and Bauer, Ralf and van de Linde, Sebastian (2021) Tunable wide-field illumination and single-molecule photoswitching with a single MEMS mirror. ACS Photonics, 8 (9). 2728–2736. ISSN 2330-4022 (https://doi.org/10.1021/acsphotonics.1c00843)

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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 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.

ORCID iDs

Herdly, Lucas, Janin, Paul, Bauer, Ralf ORCID logoORCID: https://orcid.org/0000-0001-7927-9435 and van de Linde, Sebastian;
CORE (COnnecting REpositories)