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.

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.

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.

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.

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.

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.

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.

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.