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 fluorescent 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 practicable 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.
Linear two- or three-dimensional structured illumination microscopy (SIM or 3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with sub-diffraction 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 days.
Genomic regulation is achieved at the linear DNA scale, and through higher order chromatin folding. However, chromatin topology at diffraction limiting scales (<200 nm) is poorly understood. Super-resolution 3D-SIM (3D structured illumination microscopy) resolves a 200-500nm wide 3D network of chromatin permeated by a DNA-free network (interchromatin) leading to nuclear pores . We propose that accessibility to different chromatin densities is functionally significant at this scale.
Recent advances in super-resolution microscopy enable the study of subchromosomal chromatin organization in single cells with unprecedented detail. Here we describe refi ned methods for pulse-chase replication labeling of individual chromosome territories (CTs) and replication domain units in mammalian cell nuclei, with specifi c focus on their application to three-dimensional structured illumination microscopy (3D- SIM). We provide detailed protocols for highly effi cient electroporation-based delivery or scratch loading of cell impermeable fl uorescent nucleotides for live cell studies. Furthermore we describe the application of (2′S)-2′-deoxy-2′-fl uoro-5-ethynyluridine (F-ara-EdU) for the in situ detection of segregated chromosome territories with minimized cytotoxic side effects.
Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy technique that can provide both increased resolution, beyond the diffraction limit of light, and excellent optical sectioning. Several commercial super-resolution SIM systems are available, and are accessible to users at many academic imaging facilities: DeltaVision OMX (by GE/Applied Precision), ELYRA (by Zeiss), and N-SIM (by Nikon). These systems provide many of the advantages of the traditional, widefield, light microscope. Fluorescently labeled fixed or living biological specimens can be imaged with visible light, mounted on a microscope slide under a coverslip or in a glass-bottom cell culture dish. In terms of light dose, SIM is mild on the sample compared to other superresolution techniques. Standard, linear SIM achieves a factor of two resolution improvement, producing images with around ~100 nm lateral and ~300 nm axial resolution. Even finer details can be resolved when linear SIM is combined with TIRF. Using a non-linear SIM approach, true super-resolution imaging with theoretically unlimited resolution is possible. There is no hard limit to what resolution can be obtained, but the signal-to-noise ratio (SNR) of the data in practice determines what resolution can be reached (Heintzmann et al. 2002, Gustafsson 2005). The highest resolution non-linear SIM data reported in biological imaging to this date is ~50 nm (Rego et al. 2012).
A guide to super-resolution fluorscence microscopy
Schermelleh et al. 190 (2): 165
Abstract: For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.
The Limits of Light
Optical microscopy is one of the most versatile tools in a biologist’s arsenal, since it allows the observation of the interior of 3D preserved fixed or living cell with minimal perturbation. However, from its invention in the 17th century until a few decades ago its spatial resolution was constrained by a seemingly impenetrable barrier: the diffraction limit. Simply put, the minimal distance at which two adjacent objects can still be discerned equals the wavelength used to observe them divided by twice the numerical aperture (NA) of the microscope (the NA roughly expresses the focusing strength of the microscope’s objective lens). With visible light, the smallest practical wavelength is around 450 nm (blue emission), and the largest NA possible is around 1.4
Even with advances such as confocal microscopy and deconvolution algorithms, only biological structures separated by more than about 200 nm in the lateral dimension (x and y axis) could be discerned. Resolution along the axial dimension (z axis) is even lower, being limited to roughly the wavelength of the emitted light, or 500 nm at best. Further, these values are only theoretical limits under optimal optical conditions. The effective resolution for typical biological samples is significantly decreased due to distortions from the sample itself such as light scattering, out-of-focus blur, spherical aberration, and poor signal-to-noise ratios.
Uncovering the motifs of a higher order nuclear architecture and its implications on nuclear function has raised increasing interest in the past decade. The nucleus of higher eukaryotes is considered to display a highly dynamic interaction of DNA and protein factors. There is an emerging view that there are hierarchical levels of gene regulation, reaching from epigenetic modifications at the DNA- and histone level to a higher order functional nuclear topology, in the context of which gene-activating and -repressing processes influence the gene expression profile of an individual cell beyond the sequence information of the DNA. The present work focuses on the analysis of the dynamic aspects of higher order nuclear architecture in living cells. As a prerequisite, an in vivo replication labeling strategy was developed, that enabled the simultaneous visualization of early and mid-to-late replicating chromatin as well as single chromosome territories on the basis of a labeling/segregation approach. The presented scratch replication labeling protocol combines a high labeling efficiency with reduced “damaging” effects and can be successfully applied to a number of adherently growing cell lines, including primary human fibroblasts. In addition, a live cell observation system was developed that facilitates time-lapse confocal (4D) microscopy over elongated time periods which made it possible to follow a complete cell cycle or more.