Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d.

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

To safeguard genome integrity in response to DNA double-strand breaks (DSB), mammalian cells mobilize the neighboring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes 1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-Polα complex 2. How this pathway reflects and impacts on the three-dimensional (3D) nuclear architecture is not known. Here, we applied super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes 3D chromatin topology at sites of DNA breakage. This is initiated by 53BP1 accrual at compact chromatin regions colocalizing with topology-associated domain (TAD) sequences and followed by RIF1 recruitment to boundaries between such domains. The alternating 53BP1 and RIF1 distribution stabilizes several neighboring TAD-sized structures at a single DBS site to an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement, leading to decompaction of DSBflanking chromatin, reduction of interchromatin space, aberrant spreading of DNA repair proteins, and DNA-end hyper-resection. Similar topological distortions are triggered by depletion of cohesin, suggesting that maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape 3D nuclear organization. Since topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci disrupted by DNA breakage. quantitative nanoscopy texture (QUANTEX) analysis tool, which indeed revealed a significant increase in 53BP1-MD Mean breadth and Principal axis length (Fig. 1e, f; Extended Data Fig. 3a-c). It was further reproduced by silencing RIF1 with multiple siRNAs, by replacing endogenous 53BP1 with a mutant unable to promote RIF1 recruitment 10 , and in several cancer-derived as well as non-cancerous cells (Extended Data Fig. 4a-e). Together, these data indicate that 53BP1 and RIF1 form an autonomous module where RIF1 is required to stabilize 53BP1-NDs into ordered, circular chromatin architecture (Extended Data Fig. 4f). In support of this, knockdown of 53BP1 or RIF1 phenocopied each other by disrupting γH2AX-marked chromatin into disordered and elongated shapes (Extended Data Fig. 4g-i). To study how 53BP1 and RIF1 cooperate to stabilize chromatin topology, we set out to determine RIF localization with respect to 53BP1. While conventional microscopy only generally indicates 53BP1 and RIF1 proximity at DSB-sites, 3D-SIM and STED revealed that RIF1 localized to the chromatin boundaries between neighboring 53BP1-NDs (Fig. 2a). To understand the purpose of this alternating localization, we tracked 53BP1 dynamics from pre-to post-damaged state using live-cell 3D-SIM (live-SIM; Extended Data Fig. 5a). The fir...