Expansion stimulated emission depletion microscopy (ExSTED)

Abstract

Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ~10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ~4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ~50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy

“…2C, Left), the signal was not distributed uniformly along the length of the CC, and appeared primarily in clusters at irregular spacings along the long axis. A similar pattern for Arl13B was also observed via superresolution imaging in other primary cilia (31,32). This observation of clustering has important implications for Arl13B function in the CC, suggesting its association with large sparse complexes along the ciliary axis.…”

“…In addition to the value of our findings in their own right, they demonstrate the power of the methodology we present and the cellular/molecular map we have produced. Emerging technologies in both static and dynamic imaging (27,31,49,60,61) will likely continue to enhance both the resolution and the functional content of such maps.…”

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy

“…In expansion microscopy of cultured cells, correctly estimating the dimensions of cell structures depends on precisely estimating the expansion factor at which these structures were pulled apart. The swelling of the gel block is commonly used as a prompt to estimate the expansion factor of the label and thus the "real" -pre-expansiondimension of the protein structures under study 15,16 . However, since the cell is a dense aggregate of proteins and these are cross-linked to the acrylamide polymer that is made within the cell, we speculated that the swelling ratios of this ionic polymer and those of the ionic polymer densely cross-linked to cellular proteins would differ.…”

“…In ExM, to extract quantitative information from expanded samples, it is imperative to have a correct estimation of the expansion factor at which the cellular components were pulled apart. So far, expansion factor estimations have been performed either by measuring the gel block before and after water dialysis-induced swelling 15,16 , by measuring the very same location before and after expansion 17,18 or by studying structures whose dimensions were already known by other techniques, such as fluorescence nanoscopy or electron microscopy. The values reported by these approaches vary significantly, with macroscopic measurement of the gel block seeming to underestimate the actual expansion of cellular proteins.…”

Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy

“…Higher expansion ratios up to 10-to 20-fold have been achieved by using different gel recipes (41) or multiple rounds of expansion (42). Combinations of expansion microscopy with other superresolution microscopes, such as SIM (ExSIM) (19,43), STED (ExSTED) (44)(45)(46), and lattice light sheet microscopy (ExLLSM) (47) have also been reported to boost the resolution and throughput of imaging. Meanwhile, with expansion ratio of 3 to 5, the homogeneity of expansion has been confirmed by imaging DNA origami nanorulers (48) and nuclear pore complexes (49).…”