Abstract
Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.
Highlights
Diffraction The bending of light waves at the edges of an obstacle such as an aperture
The most well known of these super-resolution microscopy methods fall into three main categories: stimulated emission depletion[3], structured illumination microscopy[4,5] and single-molecule localization microscopy (SMLM)[6,7,8,9], which is the focus of this Primer
In SMLM, super-resolution images are assembled computationally and the quality of the images strongly depends on image processing
Summary
PAINT it is dependent on binding affinities and binder concentration. these parameters are usually determined manually, SMLM can be automated with control software that implements a feedback loop to tune the active fluorophore density[85,86,87]. Structural dynamics studies aim to reveal the movement of a structure composed of many molecules, such as a focal adhesion, a clathrin-coated pit or an organelle membrane[34,60,90] In these studies, time series of super-resolution images are reconstructed from the. Multicolour single-molecule localization microscopy (SMLM) using synthetic dyes can be accomplished using the classical stochastic optical reconstruction microscopy (STORM) concept and probes with both an activator and a reporter fluorophore[8]. Multicolour dSTORM is susceptible to chromatic aberrations, these can be avoided by spectral demixing In this approach, synthetic dyes are used that exhibit good photoswitching performance in the same thiol switching buffer and can be efficiently excited with the same laser wavelength, but exhibit different emission maxima. SMLM allows for the collection of orders of magnitude more molecular trajectories than single-molecule tracking without photoswitching[91,92], and enables more insights into molecular movements and the factors that control them[93]
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