Optical imaging of individual biomolecules in densely packed clusters

Abstract

Recent advances in fluorescence super-resolution microscopy have allowed sub-cellular features and synthetic nanostructures down to ~15 nm in size to be imaged. However, direct optical observation of individual molecular targets (~5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography) - a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridisation - on synthetic DNA nanostructures. We examined the effects of high photon count, high blinking statistics, and appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves <1 nm residual drift (r.m.s.) over hours. This allowed us to image a densely packed triangular lattice pattern with ~5 nm point-to-point distance, and analyse DNA origami structural offset with angstrom-level precision (2 Å) from single-molecule studies. By combining the approach with multiplexed Exchange-PAINT imaging, we further demonstrated an optical nano-display with 5×5 nm pixel size and three distinct colours, and with <1 nm cross-channel registration accuracy. This method opens up possibilities for direct and quantitative optical observation of individual biomolecular features in crowded environments.

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

“…This description of possible errors is not exhaustive, and other errors such as those induced by sample drift over the course of acquisition(106, 160–162) are also necessary to correct in quantitative single-molecule microscopy. (In the case of drift, correction can be achieved using fiducial markers, interferometry, or cross-correlation with post-processing or active feedback.)…”

DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging

“…However, the binding of diffusing ligands to their targets is achieved by electrostatic or hydrophobic interactions and is thus hard to program for different target species in a single cell, thus preventing easy-to-implement multiplexed detection. DNA-PAINT, 12–17 a variation of PAINT, achieves stochastic switching of fluorescence signals between the ON- and OFF-states by the repetitive, transient binding of short fluorescently labeled oligonucleotides (“imager” strands) to complementary “docking” strands that are conjugated to targets (Fig. 1a).…”

In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT

“…Below, we provide brief descriptions of Oligopaints, OligoSTORM, and OligoDNA-PAINT, followed by protocols for generating Oligopaint probes from a library of oligonucleotides (oligos) and preparing samples for imaging by OligoSTORM or OligoDNA-PAINT. As for the actual steps of image acquisition, we refer the reader to published literature for STORM and OligoSTORM [5, 6, 9, 10, 11, 15] and DNA-PAINT and OligoDNA-PAINT [5, 13, 14, 16, 17]. …”

“…Imager strands, which are continuously replenished from the buffer, are designed to be short (e.g., 9-10 bases) in order to promote the transient nature of their binding. Additionally, as the concentration of imager strands and thermodynamic stability of the hybridized duplex control the rates of, respectively, binding and dissociation, users can tune the conditions of the imaging buffer to attain a desired blinking rate [13, 14, 16, 17]. Moreover, users can achieve numerous pseudocolors using only one spectral channel via the assignment of orthogonal docking strand sequences to distinct targets and, using a version of DNA-PAINT, called Exchange-PAINT, enable multiple targets to be distinguished via serial imaging [14].…”