Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Abstract

Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50–60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25–35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.

Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy

“…ExM not only provides a high spatial resolution (∼75 nm or better when using a standard confocal microscope with ∼4× expanding gels), but also enables antibody labels to be covalently tethered to the hydrogel scaffold, such that DNA FISH can subsequently be performed without loss of antibody fluorescence. ExM has previously been combined with DNA FISH to either visualize the HER2 gene in tissue ( 41 ), or to visualize repetitive centromere regions in plants ( 42 ). However, this combination has not yet been used to determine the density of a protein structure, such as histone mark clusters, at specific genomic regions.…”

Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy

“…ExM has previously been combined with DNA FISH to either visualize the HER2 gene in tissue, 40 or to visualize repetitive centromere regions in plants. 41 However this combination has not yet been used to determine the density of a protein structure, such as histone mark clusters, at specific genomic regions. We refer to this new methodology as Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE), as a tool to quantify the fluorescence signal of immunolabeled histone marks or proteins structures at individual FISH-labeled genomic loci within individual cells ( fig.…”

Nanoscale fluorescence imaging of biological ultrastructure via molecular anchoring and physical expansion

“…Indirect anchoring of genomic DNA via retention of the histone proteins has been used for DNA FISH-based detection of genomic regions, such as in expansion pathology (ExPath) [ 74 ], U-ExM [ 58 ], and TRanslational Epigenetic Encoding (SCEPTRE) [ 90 ]. Furthermore, physical entanglement of the genomic DNA by the hydrogel polymer network has also been explored for non-covalent anchoring.…”