Abstract text (incl. figure legends and references)
Introduction

DNA origami is a versatile and flexibly adaptable molecular template to control self-assembly of nanoparticles (NPs) and other nanoscopic building blocks [1]. Particle-particle and particle-substrate interactions can be controlled effectively by choosing appropriate functionalization of the NPs. As DNA-based processes require liquid environments, high-resolution microscopy applicable to liquids is needed to characterize, understand, and optimize the underlying physico-chemical mechanisms. Here, we use in-situ (scanning) transmission electron microscopy ((S)TEM) to study Au NPs, which are site-specificly bound to DNA origami structures directly in solution.

Objectives

The study focuses on imaging individual Au NPs adhered to DNA origami, determining the precision, to which particle sizes and positions can be measured in solution, and the characterization of beam-induced artefacts, which may impede the microscopic view and alter the solution chemistry and assembly.

Materials & methods

Au NPs (52±1 nm in diameter) were synthesized by seed-mediated growth and covered with DNA oligomers using a freeze-thaw approach [2]. 2 x 40 x 150 nm3 large quasi 2D DNA origami pads were designed and synthesized by scaffolded self-assembly [1,3]: Long ssDNA (M13mp18) is folded by oligonucleotides, some of which were extended by ssDNA, whose sequence is comple­mentary to that on the Au NP surface. Two different sequences were used for positioning of Au NP dimers with defined spacing on the pad.

Ex-situ (S)TEM and in-situ liquid phase (S)TEM (LP-(S)TEM) were conducted on a JEOL F-200 micro­scope equipped with a Gatan OneView camera and a HAADF detector at 200 kV. For LP-(S)TEM investigations, a Protochips Poseidon Select holder was used to keep the samples in a liquid environment that was sealed with SiN membranes against the high vacuum of the line tube.

Results

While ex-situ (S)TEM allows to clearly image Au NPs dried from solution and the individual DNA origami attached to them (despite large differences in contrast, see insert of Fig. 1), resolution and contrast of the LP-STEM images are reduced by beam-induced crystallization (predominantly of salts from solution). Nonetheless, upon optimizing the scanning parameters, a precision measurement of particle positions and distances is also possible in solution and first measurements indicate a preferred inter-particle distance as intended by the specifically "programmed" DNA pads (cf. Fig. 2). Indications are that complementary bright and dark field imaging may allow for a better resolution in STEM than in TEM.

Conclusion

LPEM bears a great potential for the quantitative characterization of NPs in solution. However, resolution-limiting artefacts and side effects render the continuous conduction of ex-situ control experiments indispensable.

Financial support by DFG through RTG 2767 and EU"s Horizon 2020 Programme (grant #964248) is gratefully acknowledged. We thank Gunnar Klös for nanoparticle synthesis and functionalization.

[1] J. Zessin et al., Nano Lett. 17 (2017) 5163.

[2] G. Klös et al., Zenodo (2022), https://doi.org/10.5281/zenodo.6516945.

[3] P.W.K. Rothemund, Nature 440 (2006) 297.