The DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution fluorescence microscopy technique that achieves single-molecule 'blinking' by transient DNA hybridization. Despite blinking kinetics being largely independent of fluorescent dye choice, the dye employed substantially affects measurement quality. Thus far, there has been no systematic overview of dye performance for DNA-PAINT.

Quantification of absolute labeling efficiency at the single-protein level.

State-of-the-art super-resolution microscopy allows researchers to spatially resolve single proteins in dense clusters. However, accurate quantification of protein organization and stoichiometries requires a general method to evaluate absolute binder labeling efficiency, which is currently unavailable. Here we introduce a universally applicable approach that uses a reference tag fused to a target protein of interest.

Spatial proteomics in neurons at single-protein resolution.

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution.

Ångström-resolution fluorescence microscopy.

Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions.

Quantitative Imaging With DNA-PAINT for Applications in Synaptic Neuroscience.

Super-resolution (SR) microscopy techniques have been advancing the understanding of neuronal protein networks and interactions. Unraveling the arrangement of proteins with molecular resolution provided novel insights into neuron cytoskeleton structure and actin polymerization dynamics in synaptic spines. Recent improvements in quantitative SR imaging have been applied to synaptic protein clusters and with improved multiplexing technology, the interplay of multiple protein partners in synaptic active zones has been elucidated.

DNA-Barcoded Fluorescence Microscopy for Spatial Omics.

Innovation in genomics, transcriptomics, and proteomics research has created a plethora of state-of-the-art techniques such as nucleic acid sequencing and mass-spectrometry-based proteomics with paramount impact in the life sciences. While current approaches yield quantitative abundance analysis of biomolecules on an almost routine basis, coupling this high content to spatial information in a single cell and tissue context is challenging. Here, current implementations of spatial omics are discussed and recent developments in the field of DNA-barcoded fluorescence microscopy are reviewed.

Calcium stabilizes the strongest protein fold.

Staphylococcal pathogens adhere to their human targets with exceptional resilience to mechanical stress, some propagating force to the bacterium via small, Ig-like folds called B domains. We examine the mechanical stability of these folds using atomic force microscopy-based single-molecule force spectroscopy. The force required to unfold a single B domain is larger than 2 nN - the highest mechanostability of a protein to date by a large margin.