Letter pubs.acs.org/NanoLett
DNA Origami Nanopillars as Standards for Three-Dimensional Superresolution Microscopy Jürgen J. Schmied,†,∥ Carsten Forthmann,†,∥ Enrico Pibiri,† Birka Lalkens,† Philipp Nickels,‡ Tim Liedl,‡,§ and Philip Tinnefeld*,†,§ †
Institut für Physikalische und Theoretische Chemie, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany ‡ Fakultät für Physik, Ludwig Maximilians Universität, Geschwister-Scholl-Platz, 80539 München, Germany § Center for NanoScience, Ludwig Maximilians Universität, Schellingstraße 4, 80799 München, Germany S Supporting Information *
ABSTRACT: Nanopillars are promising nanostructures composed of various materials that bring new functionalities for applications ranging from photovoltaics to analytics. We developed DNA nanopillars with a height of 220 nm and a diameter of ∼14 nm using the DNA origami technique. Modifying the base of the nanopillars with biotins allowed selective, upright, and rigid immobilization on solid substrates. With the help of site-selective dye labels, we visualized the structure and determined the orientation of the nanopillars by three-dimensional fluorescence superresolution microscopy. Because of their rigidity and nanometer-precise addressability, DNA origami nanopillars qualify as scaffold for the assembly of plasmonic devices as well as for threedimensional superresolution standards. KEYWORDS: DNA origami, superresolution, single molecules, nanotechnology, fluorescence, biophysics
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for dsDNA and an average interhelix distance of 0.7 nm,16,17 Figure 1a). At the bottom, the DNA origami nanopillars have a broader base of ∼25 nm diameter for surface attachment. DNA nanopillars can conveniently be fabricated in paralleled production of trillions of identical structures by self-assembly in solution. The selectivity of DNA hybridization and the incorporation of reactive groups furthermore allow stoichiometric control with nanometer accuracy over large distance enabling the site specific modification of DNA origami, e.g., with biomolecules,13,18 organic dyes,19−21 and semiconductor or metallic nanoparticles.22−26 Very recently, the attachment of gold nanoparticles to a DNA nanopillar that created a plasmonic hotspot for fluorescence enhancement has been presented.27 In this and many other examples of nanopillar applications, the functionality of the nanopillar is strongly coupled to its orientation on the surface. In contrast to lithographically produced nanopillars, the DNA origami nanopillars have to be placed on the surface after production and their orientated immobilization has to be ensured. In order to determine the structure and orientation of the DNA origami nanopillars, we attached a fluorescent dye to the top and to the bottom of the structure and carried out three-
aterials reduced to nanoscopic dimensions often show distinct properties that are different than those of the macroscopic counterparts. In many cases, the altered characteristics are related to confinement effects and to changes of the surface-to-volume ratio. In addition, the material type as well as the shape strongly influences the properties of the nanostructures. Nanopillars, which commonly are nanoscale extensions from a bulk material, are an emerging class of nanostructures. Nanopillars can create nanoscale functionality in different ways, e.g., by increasing surface areas which can lead to enhanced photovoltaics,1,2 lighting, and solar hydrogen production.3 Altered electronic properties of nanopillars can also be used for plasmonics and surface-enhanced spectroscopy.4−7 Arrays of nanopillars are used in (DNA) separation technologies,8,9 and their mechanical sensitivity is exploited for force mapping of cells.10 Individual nanopillars offer interesting properties caused by their small size allowing the recording of action potentials11 and localized fluorescence imaging.12 Depending on the application and properties aimed for, nanopillars are made of a variety of different materials such as glass, elastomers, semiconductors, metals, and metal oxides and are commonly produced lithographically. Here, we report on self-assembled DNA nanopillars based on the DNA origami technique.13,14 The pillars are made of a 12helix bundle15 adopting a shape with an overall length of ∼220 nm and a diameter of ∼14 nm (assuming a diameter of 2.0 nm © 2013 American Chemical Society
Received: December 5, 2012 Revised: January 15, 2013 Published: January 31, 2013 781
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pillar base consists of 30 helices and is 20 nm high whereas the central 12 helices extend to the top of the pillar (see cross sectional schemes in Figure 1b). Successful folding of the DNA nanopillar was ensured by atomic force microscopy (Figure 1c), electron microscopy (Figure 1d) and, as described in the following, by superresolution fluorescence microscopy. To study the effective end-to-end distance of the DNA origami nanopillar and to check for correct folding by fluorescence microscopy, we first immobilized the DNA nanopillar horizontally. Therefore, one biotin at the bottom and one biotin at the top bound the DNA nanopillar on BSA− biotin−neutravidin-coated coverslips (see inset in Figure 2a).
Figure 2. (a) Superresolution image of DNA nanopillars immobilized horizontally on a BSA−biotin−neutravidin surface via two biotin molecules as shown in the inset. Scale bar is 500 nm. (b) Histogram of measured distances with a mean value of 184 ± 14 nm (standard deviation).
Figure 1. (a) Sketch of the DNA origami nanopillar with one Alexa647 dye close to the bottom and one Alexa647 dye close to the top. Θ is defined as the angle between nanopillar axis and surface. The inset highlights the surface immobilization via 15 biotin-modified staple strands that bind mediated by neutravidin to the BSA−biotincoated coverslip. (b) Sketches of nanopillar cross sections. (i) Cross section of helices for the base of the nanopillar. Biotin-labeled helices are shown in green. (ii) Cross section of helices of the pillar. (c) Atomic force micrograph of nanopillars adsorbed on a mica surface. Scale bar = 500 nm. (d) Transmission electron micrograph of the nanopillar.
We used dSTORM imaging35 to measure the distance of the two dyes that were placed at a contour length distance of 200 nm. For dSTORM, oxygen was removed enzymatically and 140 mM β-mercaptoethanol was added (see Supporting Information for experimental details). Using simultaneous 532 nm (1.5 kW/cm2) and 644 nm (10 kW/cm2) excitation, the dyes reversibly enter an OFF state of several seconds so that the individual dyes on the nanopillar are imaged and localized successively.36,37 By plotting each localization as a Gaussian peak with the width of the localization standard deviation, superresolved images as in Figure 2a are obtained.38 From such superresolution images, we extracted the interdye distance of 184 ± 14 nm (Figure 2b). This value is slightly smaller than the contour length which might indicate a small degree of bending. Smaller than designed distances were also observed for other biotin-immobilized DNA origami.21,39,40 After the DNA nanopillar was proven intact and rigid in 2D experiments, we attached 15 biotin molecules to the base of the DNA origami to place the nanopillar upright (see Figure 1a). To localize single molecules in 3D, we exploited optical astigmatism induced by a cylindrical lens inserted in the detection pathway.28 The cylindrical lens yields elliptical spots and the degree of ellipticity reports on the z-position of the dye (see Supporting Information for experimental details). dSTORM images of individual nanopillars are shown as 3D representations in Figure 3 with point clouds for the localizations of the dyes at the top and at the bottom of the nanopillar as well as their projections onto the xy-, xz-, and yzplanes. While some nanopillars stand almost vertical (Figure 3a), other subpopulations exhibit an intermediate angle with respect to the surface or lie horizontally on the coverslip (Figure 3b,c). To correctly describe the distribution of nanopillar orientations without bias induced by manually picking spots for analysis, we developed an algorithm that detects fluorescent
dimensional fluorescence superresolution microscopy in solution. In recent years, superresolution imaging has been extended to the third dimension, e.g., by localizing single molecules in x, y, and z. This can be achieved by different approaches including optical astigmatism by a cylindrical lens,28 biplane imaging,29,30 and interferometric imaging31 or by other means of engineering the point-spread function.32,33 Commonly, new superresolution methods are demonstrated, and microscopes are validated by imaging cellular filaments of the cytoskeleton. However, such samples do not provide easily reproduced distances and fluorophore numbers. Using two-dimensional DNA origami, we presented validation standards for various microscopic techniques with a defined number of fluorescent dye molecules per mark on nanorulers.21,34 By placing the DNA origami nanopillars selectively on the small nanopillar base, calibration standards for three-dimensional superresolution microscopy with a dynamic range of 0−200 nm can be created. The DNA origami nanopillars were folded from one 8634 nucleotides long scaffold strand and 199 short staple strands. (see scheme in Figure 1 and Supporting Information for materials and methods and sequences).15 The nanopillars carry one Alexa647 fluorophore at the top and one close to the bottom. The inset in Figure 1a shows the immobilization scheme to a BSA−biotin−neutravidin surface via 15 biotin molecules attached below the bottom of the pillar. The broader 782
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Figure 3. Three-dimensional superresolution representations of individual nanopillars with different orientations with respect to the surface: (a) perpendicular, (b) at Θ = 35°, and (c) lying almost horizontally.
Figure 4. (a) Correlation between measured pillar length and pillar angle with respect to the surface. Because of the refractive index mismatch, the nanopillar length appears to correlate with the z-component. The fit describes a theoretical relation assuming a constant magnification factor for the z-direction. (b) For z-magnification corrected distribution. (c) Histogram of measured distances between the two dyes on the nanopillar corrected for the refractive index mismatch between glass and solution. (d) Angular distribution of DNA origami nanopillars presented as solid angle density (occurrence normalized by 1/cos(Θ)). (e) Solid angle distribution of the nanopillar tips with the bottom of the nanopillar placed in the center of the sphere. The scale is normalized to the density of a uniform distribution. The subpopulation of upright orientated nanopillars is clearly visible.
(1.35) and a numerical aperture of 1.4 assuming isotropic emission.28,41 The deviation to the measured value might be related to the close proximity to the surface where a lower value is expected, and the assumption of isotropic emission is not strictly valid. The fact that the data can be well fitted with the equation underlines the applicability of the model that is based on a rigid and immobile nanopillar and a simple correction factor for the z-component. The corrected pillar length does not show the angle dependence (Figure 4b), and these values were used for further analysis. The standing nanopillar shows a homogeneous length distribution with no sign of fluctuations within the accuracy of our measurement (Figure 4c). The orientational distribution of the nanopillar is depicted in Figure 4d. The solid angle density is obtained by normalizing the frequency of angles with the geometric probability for the upper dye being on the surface of a sphere (i.e., by normalizing with 1/cos(Θ), where Θ defines the angle between the nanopillar axis and the surface). The solid angle density directly visualized on a hemisphere is shown in Figure 4e. Obviously, the biotin immobilization promotes a nanopillar orientation upstanding on the surface. This finding is corroborated by comparing the solid angle density distribution of the standing nanopillar to that of the lying nanopillar that was immobilized by one biotin close to the top and one biotin close to the bottom (Figure 4d). The difference of the distributions for the two immobilization schemes indicates the specific immobilization of the standing nanopillar on the small
spots representing nanopillars and measures their length (distance between the two marker dyes) as well as their angle Θ with respect to the surface. This algorithm assigns each localization to one of the two dyes on the nanopillar, determines the location of the individual dyes from all localizations originating from one nanopillar, filters the data, and finally provides the required parameters such as length and angle. It also supplies parameters characterizing the localizations of the top and bottom dye, respectively (see Supporting Information for a full description of the algorithm). Interestingly, we found that the length of the nanopillar seems to correlate with the angle to the surface from about 190 nm for flat lying pillars up to ∼300 nm for the pillars standing up vertically (Figure 4a). This effect reflects a magnification induced by the diffractive index mismatch of the coverslip and the buffer.28 It requires a correction factor that depends on the z-component of the nanopillar length. We fitted the data in Figure 4a to the equation Lm = L
1 + tan 2(θm) 1 + f 2 tan 2(θm)
(1)
and obtained a nanopillar length of 190 ± 2 nm (standard error, N = 236) and a correction factor of the z-component of f = 0.66 (see Supporting Information for derivation of eq 1). The theoretical correction value of 0.79 is approximately a constant for the first few micrometers above the interface.41 It was calculated for the diffractive indices of glass (1.52) and buffer 783
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(13N11461), the German Research Foundation (DFG Ti329/ 6-1 and LI1743/2-1), and the Center for NanoScience, München. We thank A. Gietl, P. Holzmeister, D. Grohmann, and A. Tiefnig for help with experiments and proofreading.
base (Figure 4d). Control measurements without neutravidin indicate that the contribution of small angles with respect to the surface for the standing nanopillar sample can be attributed to some degree of unspecific binding of the pillar to the surface (data not shown). Importantly, most nanopillars stand still for the duration of the measurements. Localizations and point clouds are of similar quality for the bottom and the top dye (see Supporting Information Figure S1 and Table S1 for more information). In a few exceptions, however, movement of the nanopillar tip with respect to a fixed base occurred on a time scale of a few seconds (see Supporting Information Figure S2). In these examples, the tip of the nanopillar moves on the surface of a sphere and the bottom of the nanopillar remains fixed. In summary, we constructed 220 nm DNA nanopillars from single scaffolds. The data show that the DNA nanopillars are straight and rigid within the accuracy of our measurements. They are selectively placed on the small nanopillar base which substantiates their applicability for functional plasmonic devices27 and qualifies them as standards for three-dimensional superresolution microscopy with a dynamic range of 0−200 nm. As calibration standards they could be used to compare superresolution methods, to distinguish between microscope and sample specific error sources, and to characterize the magnification induced by the diffraction index mismatch at the glass−sample interface. For applications with more stringent requirements on the homogeneity of nanopillar orientations, a flatter surface could be used and the attachment chemistry could be further developed. Alternatively, the area of the base could be increased at the cost of a smaller, more flexible or more complex nanopillar. The option for site-specific modifications of DNA origami opens new routes for nanopillars with elaborated functionalities. DNA origami nanopillars could, for example, serve as building blocks for higher orders of organization forming complex networks.
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ASSOCIATED CONTENT
* Supporting Information S
Materials and methods, Figures S1−S5, and Tables S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Author Contributions ∥
REFERENCES
These authors contributed equally.
Notes
The authors declare the following competing financial interest(s): Braunschweig University of Technology has filed a provisional patent application on the described method of DNA origami based standards for three-dimensional microscopy.
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ACKNOWLEDGMENTS This work was supported by a starting grant (SiMBA) of the European Research Council, the Biophotonics IV program of the Federal Ministry of Education and Research (BMBF, VDI) 784
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