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

Lucas Herdly,Ralf Bauer,Paul Janin,Sebastian Van De Linde

doi:10.1021/acsphotonics.1c00843

Lucas Herdly, Ralf Bauer + Show 2 more

Open Access

https://doi.org/10.1021/acsphotonics.1c00843

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Journal: ACS photonics	Publication Date: Aug 25, 2021
Citations: 8	License type: CC BY 4.0

Affiliation: University of Strathclyde

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.

Highlights

Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images
Single-molecule localization microscopy (SMLM) techniques, such as photoactivated localization microscopy (PALM/FPALM) and stochastic optical reconstruction microscopy (STORM/dSTORM), can improve on the classical resolution limit of ∼200 nm by a factor of 10 and more.[3−6] In addition to its high-resolution capabilities, SMLM is routinely used for quantitative imaging of proteins in subcellular compartments.[7−11]
Irreversibly photoactivatable or photoconvertible dark states are employed in PALM and FPALM,[3,4] whereas reversibly photoswitchable organic dyes are used in STORM and dSTORM.[5,6]

Summary

Introduction

Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images. ON- and OFF-states are mainly dependent on the irradiation intensities[6,14] and affect the average number of localizations obtained per molecule over the course of an acquisition It is desirable, especially for quantitative SMLM, to use homogeneous illumination across the field of view (FOV). Especially for quantitative SMLM, to use homogeneous illumination across the field of view (FOV) This is per se not the case in conventional wide-field microscopy employing Gaussian illumination, in which a trade-off between homogeneous illumination and high excitation intensity exists. This leads to a confinement of laser power in the center and to a significant drop of intensity toward the edges of the FOV, affecting photoswitches nonuniformly. Fast scanning mirrors have been used to achieve flat-field illumination, called adaptable scanning for tunable excitation region (ASTER).[23]

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