Strategic and practical guidelines for successful structured illumination microscopy

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

Comprehensive mapping of the 3D epigenome by high-content super-resolution image analysis

A full understanding of the relationship between the density and packing arrangements of chromatin in interphase mammalian nuclei and epigenetic function at the nanometer size scale remains an elusive goal in the field of chromatin biology. Over the last years great technical leaps have been made through sequencing-based methods to address this question. Advancements have also allowed research to bypass long-held optical limits to resolve chromatin at a scale where its 3D topology can start to be analysed. The work in this thesis represents attempts at high-throughput data mining of 3D SIM datasets, encoding rich nanometer-scale spatial information from immunofluorescence detection of histone modifications and key epigenetic markers relative to a chromatin landscape. To do so, an automated and high-throughput image analysis workflow (ChaiN , for ChaiN analysis of the in situ Nucleome) was developed. Novel metrics for the quantitation and correlation of chromatin and the 3D epigenome reveal a chromatin network of filaments at the size scale proposed from sequencing approaches, and with segregated regions of genomic activities as a function of chromatin accessibility (its density and depth). Furthermore, ChaiN has allowed characterisation of the local and global rearrangements of chromatin when subjected to replication pressures or exogenous perturbation showing for the first time how individual genomic markers are affected at different locations throughout the chromatin network. The model hypothesised from these results tries to reconcile previous data obtained from Hi-C population ensemble studies and in silico modelling, to single cell observations at super-resolution.</p

Quantitative 3D structured illumination microscopy of nuclear structures

3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week

Stabilization of chromatin topology safeguards genome integrity

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin–CST–POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As 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 that are disrupted by DNA breakage