Measuring expansion from macro‐ to nanoscale using NPC as intrinsic reporter

Abstract

Expansion microscopy is a super‐resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. One of the main concerns in this approach regards the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well‐known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale to measure subcellular features with an accuracy from 10 to 5 nm. Therefore, it is an excellent method for structural studies of macromolecular complexes.

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

“…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). Here, by integrating expansion microscopy with STORM, we also confirm that our sample expansion is mostly homogeneous at 50 nm length scales.…”

Optical nanoscopy

“…Since the quantitative goal is related to understand the real chance of deciphering organizational motifs at the nanoscale in a crowded environment a further step is related to the modulation of key parameters like radius and pitch of DNA clusters taking advantage of the development in expansion microscopy. The combination of such expansion microscopy expansion techniques with optical nanoscopy methods, including STED [152], STORM [182] and SIM [183], demonstrated the access to finer details of several biological structures. So far, since when the sample buffer is expanded, the density of the chiral structures is reduced, the coupling between expansion and CIDS microscopy produces a global improvement in terms of spatial resolution and imaging contrast [153].…”

“…The final resolution is about 20nm. The field of view is 7 µm been possible to identify a nanoscale ruler to address the quantitative question related to the strength of the expansion process from millimetres down to the nanoscale by such a coupling [152]. Recently, expanded specimens have been utilized to improve the resolution and imaging contrast of label-free CIDS [82] scanning microscopy .…”

High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy

“…In addition to the improved signal-to-background ratio, expansion microscopy improves the effective resolution that can be achieved with any given objective. The resolution improvement is achieved equally in both the lateral and axial direction as the volumetric expansion of samples has been shown to be homogenous even at the nanoscale (Pesce et al, 2019). As the resolution of a given microscope is typically far worse in the axial direction, cell structures are more difficult to segment if they are in close proximity and oriented axial to the detection objective.…”

“…The expansion microscopy protocols used here have been shown to provide isotropic expansion on protein structures down to 10 nm (Pesce et al, 2019). Although most antibodies worked in our hands, some epitopes which are heat-sensitive may be denatured during the protocol.…”