Stretching super-resolution microscopy limits


Rebecca Pool

Thursday, July 7, 2016 - 13:00
Wyss Institute at Harvard University researchers, US, claim the highest resolution possible in optical imaging [Wyss Institute]
Researchers have developed super resolution fluorescence microscopy to image individual molecular targets only 5 nm apart, within a densely packed biomolecular cluster.
Using DNA tags to achieve discrete molecular imaging, Professor Peng Yin from Harvard's Wyss Institute for Biologically Inspired Engineering and colleagues visualised single protein-sized particles in synthetic DNA nanostructures and now plan to image biological complexes.
While advances in fluorescence super-resolution microscopy have allowed researchers to visualise closely positioned molecules only 10 to 20 nm in size, so-called DNA-PAINT pushes the boundaries of imaging resolution back even further.
As Yin points out, DNA-PAINT - points accumulation for imaging in nanoscale topography - exploits programmable transient oligonucleotide hybridization on synthetic DNA nanostructures.
Densely packed individual targets that are just 5 nm apart from each other in DNA origami structures (left). Top right image on shows clear pattern of individual signals. Bottom right three different target species within the same origami structure have been visualised [Wyss Institute at Harvard University].
Developed a few years ago, the method creates 'imager strands' by tagging small pieces of DNA with fluorescent dye.
Each of these fluorescently labelled oligonucleotides then transiently, and repeatedly, binds to a matching DNA strand attached to the target molecule, so the target appears to 'blink'.
This allows researchers to obtain sub-diffraction resolution single molecule images of structures.
"In [standard] reconstruction microscopy, most molecules are switched to a fluorescent dark, OFF state, and only a few emit fluorescence - the ON state. Each molecule is then localised... by fitting its emissions to a 2D Gaussian function," explains Yin.
"In DNA-PAINT, the 'switching' between the ON- and OFF-states is facilitated by repetitive, transient binding of the imager strands to complementary 'docking strands'," he adds.
But while the method has already been used to study myriad processes at a molecular level, researchers have now honed the experimental set-up to boost resolution further.
The researchers first examined the effects of a high photon count and blinking cycles on imaging quality; several factors have been known to limit performance, including limited control over blinking kinetics  and unsatisfactory fluorophore imaging efficiency.
The researchers went onto develop a framework for discrete molecular imaging, including developing a software-based drift correction method that compensated for disruptive movements in the microscope stage and reduced residual drift to less than 1 nm.
As Yin highlights: "This allowed us to image a densely-packed triangular lattice pattern with around 5 nm point-to-point distance and to analyse the DNA origami [structure] with angstrom-level precision from single-molecule studies."
The researchers went onto combine this approach with so-called Exchange-PAINT - a multiplexing approach for sequentially imaging many targets - and imaged three different target species within the DNA origami structure.
The researchers now hope to study diverse cellular systems including cell membrane receptor clusters and neuronal synapses as well as the molecular states of individual protein components within macromolecules.
Research is published in Nature Nanotechnology.
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