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DNA-PAINT shows sub-20 nm resolution

Challenge

DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) is a method for overcoming the diffraction limit, for super-resolution imaging, using a simple reagent-based approach. Molecules of interest in the cell are labeled with antibodies that are attached to single DNA strands. Complementary DNA strands are labeled with fluorophores and introduced in solution; they transiently bind to their complementary strand on the antibody according to the binding kinetics. In this way, the binding and unbinding of labeled DNA strands induces the same blinking effect that occurs stochastically from the dyes in traditional dSTORM.

Solution with nanoimager

DNA-PAINT works in fixed cells, and offers the advantage of simple multi-color imaging, by adding and washing away orthogonal DNA complementary strands which could be labeled with the same fluorophore. It also negates the effect of photobleaching, as there is an abundance of labeled DNA strands in solution. Complex buffers for controlling blinking are also no longer required, as the blinking is performed by the binding and release of labeled DNA strands. As such, this approach simplifies many experimental considerations when compared to dSTORM.

A convenient way of characterizing super-resolution instruments and demonstrating their achievable resolution takes advantage of DNA-PAINT in the form of nanorulers, from GATTAquant GmbH. DNA origami nanorulers are attached to a surface, and labeled DNA strands in a sealed reusable slide transiently bind to the nanorulers at binding sites that are a prescribed distance apart. In the image, we show GATTA-PAINT 40RG nanorulers with 3 binding sites, each binding site is 40 nm apart and there are two fluorophores (red and green) that can bind at each site. The results demonstrate the high mapping accuracy between the two channels in the Nanoimager.

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Case study

In this figure, we demonstrate the ability of the Nanoimager to easily obtain 20 nm resolution, by imaging binding sites on GATTA-PAINT nanorulers that are separated by 20 nm. Nanorulers with all three binding sites localized are indicated in gray. (Those with two out of three sites localized are in yellow, probably caused by no binding at this site during the acquisition period.) Imaging at this resolution requires drift correction, a feature comprehensively supported by the Nanoimager.