Home
This Title All WIREs
WIREs RSS Feed
How to cite this WIREs title:
WIREs Nanomed Nanobiotechnol
Impact Factor: 6.14

Nanoscale imaging in DNA nanotechnology

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Abstract DNA nanotechnology has developed powerful techniques for the construction of precisely defined molecular structures and machines, and nanoscale imaging methods have always been crucial for their experimental characterization. While initially atomic force microscopy (AFM) was the most widely employed imaging method for DNA‐based molecular structures, in recent years a variety of other techniques were adopted by researchers in the field, namely electron microscopy (EM), super‐resolution fluorescence microscopy, and high‐speed AFM. EM is now typically applied for the characterization of compact nanoobjects and three‐dimensional (3D) origami structures, as it offers better resolution than AFM and can be used for 3D reconstruction from single‐particle analysis. While the small size of DNA nanostructures had previously precluded the application of fluorescence microscopic methods, the development of super‐resolution microscopy now facilities the application of fast and powerful optical methods also in DNA nanotechnology. In particular, the observation of dynamical processes associated with DNA nanoassemblies—e.g., molecular walkers and machines—requires imaging techniques that are both fast and allow observation under native conditions. Here single‐molecule fluorescence techniques and high‐speed AFM are beginning to play an increasingly important role. WIREs Nanomed Nanobiotechnol 2012, 4:66–81. doi: 10.1002/wnan.173 This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

This WIREs title offers downloadable PowerPoint presentations of figures for non-profit, educational use, provided the content is not modified and full credit is given to the author and publication.

Download a PowerPoint presentation of all images


Stochastic readout principle for super‐resolution fluorescence microscopy. The localization of a single fluorophore in a diffraction‐limited area is possible with high precision by fitting a two‐dimensional (2D) Gaussian. If the point‐spread function (PSF) of two or more emitters within a diffraction‐limited area overlap, localization is not possible. Using temporal separation of fluorescence emission by stochastically switching fluorescence between ON and OFF states, the locations of the molecules can be reconstructed in a super‐resolved image.

[ Normal View | Magnified View ]

DNA walkers and assembly lines. (a) A DNA spider walks on a prescriptive track on a planar DNA origami structure. High‐resolution fluorescence microscopy is used to monitor the movement of the walker (top). Atomic force microscopy (AFM) allows the observation of specific actions at the start, on the track and at the stop site (bottom, sites marked with the green arrow respectively). (Reprinted with permission from Ref 72. Copyright 2010 Macmillan Publishers Ltd) (b) A DNA walker proceeds by toehold‐mediated strand displacement on a linear DNA track (top). Kymographs constructed from AFM images at different times allow analysis of the progress of the walker. The histogram obtained from the height profiles reveals the walker's stepping motion (bottom). (Reprinted with permission from Ref 74. Copyright 2011 Macmillan Publishers Ltd) (c) Molecular assembly line. A transporter made from DNA can take up cargo from three loading sites. The loading sites consist of two‐state DNA machines that can either present or hide their cargo. The cargos themselves are combinations of differently sized gold nanoparticles (left). AFM imaging in air shows the uptake of all three cargos by the transporter (right), whereas transmission electron microscopy (TEM) micrographs show the results for all eight possible combinations of the cargo (bottom). (Reprinted with permission from Ref 75. Copyright 2010 Macmillan Publishers Ltd)

[ Normal View | Magnified View ]

Three‐dimensional (3D) origami. (a) A 3D structure based on a honeycomb lattice is constructed by stacking of two‐dimensional (2D) DNA origami slices (top). Transmission electron microscopy (TEM) micrographs confirm the successful formation of origami structures (bottom). (Reprinted with permission from Ref 59. Copyright 2009 Macmillan Publishers Ltd) (b) An alternative approach to form 3D origami structures based on a square lattice cross section (top), and corresponding TEM images (bottom). (Reprinted with permission from Ref 60. Copyright 2009 American Chemical Society) (c) A nanoscale box made from 2D DNA origami with a controllable lid (top). Atomic force microscopy (AFM) images of the box in liquid show unfolded, lid‐closed, and lid‐opened states (middle). Single‐particle analysis of cryo‐EM images allows for a 3D reconstruction of the DNA box. The theoretical model on the left agrees well with the surface representation of the cryo‐EM map (bottom). (Reprinted with permission from Ref 61. Copyright 2009 Macmillan Publishers Ltd)

[ Normal View | Magnified View ]

Electron microscopy (EM) of a three‐dimensional (3D) DNA object. (a) Molecular model of a DNA tetrahedron. (b) 3D density maps obtained by single‐particle reconstruction from cryo‐EM images at 20 Å resolution, and (c) at 12 Å resolution—the length of the scale bar is 5 nm. (Reprinted with permission from Ref 47. Copyright 2009 American Chemical Society)

[ Normal View | Magnified View ]

DNA‐point accumulation for imaging in nanoscale topography (PAINT) concept. (a,b) DNA origami structures can easily be modified with single‐stranded extensions of staple strands. A complementary imager strand carrying a fluorophore can transiently bind from solution to these extension strands on the surface‐immobilized origami, thus producing an apparent blinking as shown in the intensity versus time trace. Apart from utilizing the directly obtained fluorescence ON and OFF times for the analysis of hybridization kinetics and dynamic processes on the single‐molecule level, the precise tunability of these times allows for localization‐based reconstruction microscopy. (c) The positions of three extended staple strands at a distance of approximately 130 nm on an origami structure cannot be resolved with diffraction‐limited total internal reflection (TIRF) microscopy. Using DNA‐PAINT and localization‐based reconstruction, the staple positions can be well resolved showing the designed distance.

[ Normal View | Magnified View ]

Super‐resolution microscopy with DNA origami. (a) Total internal reflection fluorescence (TIRF) image of surface‐immobilized DNA origami containing two ATTO655‐labeled staple strands. The positions of the single fluorophores cannot be determined because of their overlapping point‐spread functions (PSFs). The positions of the fluorophores on the origami nanostructure are shown in the scheme at the bottom, their distance is 89.5 nm. (b) Using blink microscopy (BM) super‐resolution microscopy, the positions of the two fluorophores can be well resolved. The image shows an overlay of the diffraction‐limited TIRF image with a super‐resolved reconstruction. The fluorescence time trace obtained from one origami molecule is shown below the micrograph, highlighting the blinking of the fluorophores. Two magnified views of several structures from (b) reveal two single fluorophores. The distance distribution shown at the bottom yields (88.2 ± 9.5 nm).

[ Normal View | Magnified View ]

Design and imaging of single‐layer DNA origami structures. (a) Formation of rectangular DNA origami by thermal annealing. (Reprinted with permission from Ref 41. Copyright 2010 Macmillan Publishers Ltd) (b) Schematic representations and atomic force microscopy (AFM) images in liquid from DNA origami structures. In the rightmost structure, single‐staple strands are highlighted using dumbbell‐shaped hairpin extensions to represent the map of the Americas on an origami scaffold. (Reprinted with permission from Ref 40. Copyright 2006 Macmillan Publishers Ltd)

[ Normal View | Magnified View ]

A DNA lattice formed from double‐crossover (DX) tiles. (a) Architecture of a DX tile: two DNA double helices are connected at two crossover points. (b) DX tiles can be connected by ‘sticky‐end hybridization’ to form extended molecular lattices in two dimensions (2D). (c,d) Atomic force microscopy (AFM) images in liquid of a 2D DX crystal, scale bars: 300 nm. (Reprinted with permission from Ref 2. Copyright 1998 Macmillan Publishers Ltd)

[ Normal View | Magnified View ]

Related Articles

Super‐resolution microscopy for nanosensing
Atomic force microscopy of biological samples
Potential role of atomic force microscopy in systems biology (WIREs Systems Biology and Medicine)
Unraveling DNA dynamics using atomic force microscopy
Novel DNA materials and their applications
Controlling forces and pathways in self‐assembly using viruses and DNA
Fluorescence in Nanodiagnostics

Browse by Topic

Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts