Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images

Abstract

Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions.superresolution microscopy | 3D reconstruction | DNA origami | multivariate statistical analysis | structural biology W ith recent advances in electron detection and image processing, electron microscopy (EM) now enables the exploration of supramolecular architectures at nanoscale resolutions (1-3). The resolution in conventional fluorescence light microscopies is limited by diffraction to a few hundred nanometers. Recently, several superresolution microscopy methods were developed to surpass the intrinsic resolution limit of conventional fluorescence microscopy (4). Uniquely, superresolution microscopies enable imaging of man-made materials and biological objects in complex environments with chemical specificity and previously unimagined detail (5-7). However, the dissection of structural heterogeneity, dynamics, and low labeling densities, most noticeable at the nanoscale, limit the full potential of current superresolution analysis methods. Recently, single-particle averaging approaches were successfully applied to superresolution optical microscopy images to unravel ultrastructural details of the nuclear pore complex (8), the endosomal sorting complexes required for transport (ESCRT) machinery at HIV assembly sites (6), and the machinery of the centrosome (9). Typically, these methods relied on the use of structural templates to seed the reconstruction process and used cylindrical or spherical symmetries to reveal information on the radial distribution of components within complexes. These approaches are thus not easily transferable to de novo systems and may lead to structures that are biased toward those of templates.Here, we present a model-free method adapted from conventional EM single-particle reconstruction (EM SPR) algorithms to extract 3D isotropic structural information from 2D singlemolecule localization microscopy (SMLM) (10) images of supramolecular structures. We demonstrate the applicability of this method by solving the 3D structures of DNA origami (11, 12) and of simulated large protein complexes. ResultsSuperresolution Imaging of Model DNA Origami. DNA origami were labeled by DNA-PAINT (13, 14). Equally spaced "docking" strands w...

Multicolor single particle reconstruction of protein complexes

“…Fluorescence-based single-molecule localization microscopy (SMLM) can help to address this challenge, as demonstrated for the nuclear pore complex using 2D averaging 7 . Extending to 3D, a recent implementation of single-particle reconstruction (SPR) from 2D SMLM images demonstrated isotropic reconstruction on DNA origami and simulated data 8 . However, multi-color particle reconstruction of actual macromolecular complexes requires generating large image libraries of multiple proteins and solving the complex problem of 3D multi-channel alignment.…”

ATP-driven separation of liquid phase condensates in bacteria

“…Super-resolved images were reconstructed and classified using our Single-Particle Analysis method (Salas et al, 2017) . 990 clusters were sorted into 50 classes ( Figure S1D): 51%…”

“…Partition complex shapes displayed heterogeneity, reflecting the different three dimensional projections and conformational states of the complex ( Figure 1B-C). Thus, we used single-particle analysis to classify them into eight major class averages that contained most partition complexes ( Figure 1C, S1D-E) and to generate 3D reconstructions with isotropic resolution ( Figure 1D) (Salas et al, 2017) .…”

About samples, giving examples: Optimized Single Molecule Localization Microscopy

“…At this scale, colocalization does not exist and is replaced by the measurement of distances between the localizations of different species (Georgieva et al, 2016;Lagache et al, 2018;Pageon et al, 2016). A recent development in SMLM analysis is the use of single particle averaging to reach molecular details beyond image resolution by averaging multiple similar objects (Broeken et al, 2015;Heydarian et al, 2018;Laine et al, 2015;Salas et al, 2017;Sieben et al, 2018a). A detailed review of the available algorithms and software is beyond the scope of the present article, but we encourage the interested reader to explore this very dynamic area that constitutes the next logical step toward leveraging SMLM possibilities.…”