Imaging nanoscale nuclear structures with expansion microscopy

Abstract

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, reducing the numbers of three-dimensional, nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy solves many of these limitations but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120-130 nm. Using DNA damage response proteins, BRCA1, 53BP1 and RAD51 as exemplars we quantitatively describe the three-dimensional nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin regulated events and show the simultaneous assessment of four elements. This study thus provides the means by which expansion microscopy can contribute to the investigation of nanoscale nuclear processes.

Chromatin expansion microscopy reveals nanoscale organization of transcription and chromatin

“…2). ChromExM provides markedly higher resolution than previous ExM applications for chromatin imaging (~3 to 15 nm versus ~65 nm) ( 31 ). Other methods to visualize chromatin, such as ChromEMT ( 10 ), lack multimodal labeling, and the resolution of single-molecule localization microscopy is limited by the size of the fluorescent labels (~20 nm for primary and secondary antibody), which becomes negligible in ChromExM given that labels are applied after expansion ( 11 ).…”

“…Previous studies have reported conflicting results regarding the isotropy of ~4× to 8× expansion of chromatin ( 31 , 32 ). To address whether chromatin structure is perturbed by physical expansion, we developed an assay in which a pattern of parallel stripes is photocleaved into biotin-labeled DNA before expansion and visualized after expansion (Fig.…”

Current Progress in Expansion Microscopy: Chemical Strategies and Applications

“…To date, various strategies has been applied to quantify the distortions of expansion at the macroscopic and nanoscopic levels. Macroscopic expansion is commonly evaluated by comparing the distance variation between two identical sites of the hydrogel pre- and postexpansion. ,,, To assess expansion isotropy at the nanoscale, the most commonly used method is to compare the postexpansion confocal image with the super-resolution image of the same features in unexpanded states (e.g., SIM or d STORM). ,,,, Following a nonrigid registration process between two images, the amount of distortions after expansion can be measured. Expansion distortions can also be evaluated by applying ExM to quantify well-characterized biological targets, e.g., centrioles and nuclear pore complexes, , or well-defined structures, e.g., DNA origami and nanopillars .…”

“…54,58,100,101 To assess expansion isotropy at the nanoscale, the most commonly used method is to compare the postexpansion confocal image with the super-resolution image of the same features in unexpanded states (e.g., SIM or dSTORM). 17,19,54,59,101 Following a nonrigid registration process between two images, the amount of distortions after expansion can be measured. Expansion distortions can also be evaluated by applying ExM to quantify well-characterized biological targets, e.g., centrioles 30 and nuclear pore complexes, 54,100 or well-defined structures, e.g., DNA origami 71 and nanopillars.…”

“…Macroscopic expansion is commonly evaluated by comparing the distance variation between two identical sites of the hydrogel pre-and postexpansion. 54,58,100,101 To assess expansion isotropy at the nanoscale, the most commonly used method is to compare the postexpansion confocal image with the super-resolution image of the same features in unexpanded states (e.g., SIM or dSTORM). 17,19,54,59,101 Following a nonrigid registration process between two images, the amount of distortions after expansion can be measured.…”