124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics.

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.

Correlation analysis framework for localization-based superresolution microscopy.

Superresolution images reconstructed from single-molecule localizations can reveal cellular structures close to the macromolecular scale and are now being used routinely in many biomedical research applications. However, because of their coordinate-based representation, a widely applicable and unified analysis platform that can extract a quantitative description and biophysical parameters from these images is yet to be established. Here, we propose a conceptual framework for correlation analysis of coordinate-based superresolution images using distance histograms. We demonstrate the application of this concept in multiple scenarios, including image alignment, tracking of diffusing molecules, as well as for quantification of colocalization, showing its superior performance over existing approaches.

Super-resolution microscopy with DNA-PAINT.

Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.

Universal Super-Resolution Multiplexing by DNA Exchange.

Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time-consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super-resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.

Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging.

Photoactivatable fluorescent proteins (PA-FPs) are widely used in live single-molecule super-resolution imaging but emit substantially fewer photons than organic dyes do. Herein, we show that in heavy water (D2O) instead of H2O, common PA-FPs emit 26-54% more photons, effectively improving the localization precision in super-resolution imaging.

Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm.

Highly accurate sample drift correction is essential in super-resolution localization microscopy to guarantee a high spatial resolution, especially when the technique is used to visualize small cell organelle. Here we present a localization events-based drift correction method using a redundant cross-correlation algorithm originally developed to correct beam-induced motion in cryo-electron microscopy. With simulated, synthesized as well as experimental data, we have demonstrated its superior precision compared to previously published localization events-based drift correction methods. The major advantage of this method is the robustness when the number of localization events is low, either because a short correction time step is required or because the imaged structure is small and sparse. This method has allowed us to improve the effective resolution when imaging Golgi apparatus in mammalian cells.

A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.

Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy.

Single-molecule switching based super-resolution microscopy techniques have been extended into three dimensions through various 3D single-molecule localization methods. However, the localization accuracy in z can be severely degraded by the presence of aberrations, particularly the spherical aberration introduced by the refractive index mismatch when imaging into an aqueous sample with an oil immersion objective. This aberration confines the imaging depth in most experiments to regions close to the coverslip. Here we show a method to obtain accurate, depth-dependent z calibrations by measuring the point spread function (PSF) at the coverslip surface, calculating the microscope pupil function through phase retrieval, and then computing the depth-dependent PSF with the addition of spherical aberrations. We demonstrate experimentally that this method can maintain z localization accuracy over a large range of imaging depths. Our super-resolution images of a mammalian cell nucleus acquired between 0 and 2.5 µm past the coverslip show that this method produces accurate z localizations even in the deepest focal plane.

Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.

The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.