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Optical imaging of individual biomolecules in densely packed clusters.

ABSTRACT: Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and synthetic nanostructures down to 10-20 nm in size to be imaged. However, the 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 hybridization-on synthetic DNA nanostructures. We examined the effects of a high photon count, high blinking statistics and an appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves <1?nm residual drift (root mean squared) over hours. This allowed us to image a densely packed triangular lattice pattern with ?5?nm point-to-point distance and to analyse the DNA origami structural offset with ångström-level precision (2?Å) from single-molecule studies. By combining the approach with multiplexed exchange-PAINT imaging, we further demonstrated an optical nanodisplay with 5?×?5?nm pixel size and three distinct colours with <1?nm cross-channel registration accuracy.

SUBMITTER: Dai M

PROVIDER: S-EPMC5014615 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Optical imaging of individual biomolecules in densely packed clusters.

Dai Mingjie M Jungmann Ralf R Yin Peng P

Nature nanotechnology 20160704 9

Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and synthetic nanostructures down to 10-20 nm in size to be imaged. However, the 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 e ...[more]

PMID: 27376244

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Project description:BackgroundThe detailed interpretation of mass phenomena such as human escape panic or swarm behaviour in birds, fish and insects requires detailed analysis of the 3D movements of individual participants. Here, we describe the adaptation of a 3D stereoscopic imaging method to measure the positional coordinates of individual agents in densely packed clusters. The method was applied to study behavioural aspects of shimmering in Giant honeybees, a collective defence behaviour that deters predatory wasps by visual cues, whereby individual bees flip their abdomen upwards in a split second, producing Mexican wave-like patterns.ResultsStereoscopic imaging provided non-invasive, automated, simultaneous, in-situ 3D measurements of hundreds of bees on the nest surface regarding their thoracic position and orientation of the body length axis. Segmentation was the basis for the stereo matching, which defined correspondences of individual bees in pairs of stereo images. Stereo-matched "agent bees" were re-identified in subsequent frames by the tracking procedure and triangulated into real-world coordinates. These algorithms were required to calculate the three spatial motion components (dx: horizontal, dy: vertical and dz: towards and from the comb) of individual bees over time.ConclusionsThe method enables the assessment of the 3D positions of individual Giant honeybees, which is not possible with single-view cameras. The method can be applied to distinguish at the individual bee level active movements of the thoraces produced by abdominal flipping from passive motions generated by the moving bee curtain. The data provide evidence that the z-deflections of thoraces are potential cues for colony-intrinsic communication. The method helps to understand the phenomenon of collective decision-making through mechanoceptive synchronization and to associate shimmering with the principles of wave propagation. With further, minor modifications, the method could be used to study aspects of other mass phenomena that involve active and passive movements of individual agents in densely packed clusters.

Dataset Information

Optical imaging of individual biomolecules in densely packed clusters.

Publications

Optical imaging of individual biomolecules in densely packed clusters.

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