Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique

Abstract

Super-resolution microscopy has become an increasingly popular and robust tool across the life sciences to study minute cellular structures and processes. However, with the increasing number of available super-resolution techniques has come an increased complexity and burden of choice in planning imaging experiments. Choosing the right super-resolution technique to answer a given biological question is vital for understanding and interpreting biological relevance. This is an often-neglected and complex task that should take into account well-defined criteria (e.g., sample type, structure size, imaging requirements). Trade-offs in different imaging capabilities are inevitable; thus, many researchers still find it challenging to select the most suitable technique that will best answer their biological question. This review aims to provide an overview and clarify the concepts underlying the most commonly available super-resolution techniques as well as guide researchers through all aspects that should be considered before opting for a given technique.

Keywords: diffraction limit; fluorescence; imaging; localization; microscopy; molecular dynamics; molecular imaging; protein–protein interactions; super resolution.

Figure 1

Ensemble super-resolution microscopy techniques.A, stimulated emission depletion (STED) microscopy: (i) STED laser is aligned with excitation laser and depletes emission of fluorescent molecules in overlapping regions; (ii) depletion occurs though interruption of internal conversion process undergone by excited electrons, forcing excited fluorescent molecules to immediately return to ground state, resulting in the release of a photon with a shorter lifetime and a red-shifted wavelength that can be excluded from detection by band filtering. B, structured illumination microscopy (SIM): patterned light interferes with high-frequency sample details to produce lower-frequency Moiré fringes used to reconstruct a super-resolved image. C, pixel reassignment example—Airyscan detector: confocal pinhole substituted with 32-element detector array; each detector is equivalent to a 0.2 AU pinhole, but the array maintains light collection efficiency of a 1.25 AU pinhole. D, expansion microscopy: samples are embedded within a swellable hydrogel; fluorophores are anchored to the gel before crosslinking and digestion of cellular structures and finally expansion.

Figure 2

Single fluorophore detection–based super-resolution microscopy techniques.A, principle of SMLM. B, dSTORM uses buffers with reducing conditions to drive fluorescent molecules into short-lived (T1) or long-lived (D) dark states to cause molecules to blink. C, PALM uses photoactivatable fluorophores that need to be activated by 405 nm light before being excited. D, fluctuation-based example—SOFI: intensity fluctuations from emitters tracked through time-lapse images, then used to refine emitter localization. E, MinFlux: blinking fluorophores are combined with a torus-shaped excitation beam with a central intensity minimum; determining the point with minimum emission provides precise localization coordinates. dSTORM, direct stochastic optical reconstruction microscopy; PALM, photoactivated localization microscopy; SMLM, single-molecule localization microscopy; SOFI, super-resolution optical fluctuation imaging.

Figure 3

Simplified guide to choosing a super-resolution technique.

Figure 4

Spatiotemporal resolutions achieved by different super-resolution techniques as compared with confocal laser scanning microscopy (CLSM).A, point spread functions (PSFs) for each technique at minimal lateral (XY) and axial (Z) resolutions typically achievable on biological samples. Color coded according to temporal resolution scale in C. Adapted from Ref. (124). B, temporal resolution of each technique compared with timescales of biological processes. For single fluorophore detection techniques, such as SMLM and MINFLUX, each individual localization and the tracking of single particles can be acquired very rapidly, while imaging of gross structural changes requires numerous localizations and is relatively slow. C, ranges of lateral, axial, and temporal resolutions typically achieved using each technique on biological samples. ∗Temporal resolution is highly dependent on imaging area for laser scanning techniques. SMLM, single-molecule localization microscopy.