38 www.microscopy-today.com฀฀•฀฀2011 September doi:10.1017/S1551929511001179 Simplifying Electron Diffraction Pattern Identification of Mixed-Material Nanoparticles Jacopo Samson, 1 Patrick C. Nahirney, 2 Charles Michael Drain, 1 and Irene Piscopo 3 * 1 Department of Chemistry, Hunter College, 695 Park Avenue, New York, NY 10065 2 Division of Medical Sciences, University of Victoria, Victoria, BC V8W 2Y2, Canada 3 EM Consulting, 57 Soundview Drive, Huntington, NY 11743 * irene.piscopo@gmail.com Introduction Metallic and non-metallic nanoparticles (NPs), ranging in size from 1–200 nm, have unique functional properties that difer from their bulk materials and their component atoms or molecules [1]. Tese unique properties have driven the demand for nano-sized materials and new methods to synthesize NPs, which are used in drug delivery systems [2], bio-imaging agents [3], catalysts [4], photonics, and optical devices [5]. Inorganic NPs can be synthesized with a variety of methods that impart size, shape, and other structural properties. Cobalt-based NPs, for instance, display unique size and shape-dependent magnetic properties [6], while the band gap, UV blocking properties and stability of zinc oxide (ZnO) NPs enable new applications in products ranging from cosmetics [7] to solar cell power [8]. Approaches to NP synthesis include solvothermal, biological, and other templates [9], as well as ligands to seed NP growth and molding strategies [10]. Our approach for synthesizing metal NPs involves using toroidal topologies of plasmid DNA as sacrifcial molds and varying conditions to fabricate size-tunable gold, nickel, and cobalt NPs [9]. Plasmid DNA provides a relatively inexpensive monodispersed template that can be engineered to form in a range of sizes and exploits the well-established high afnity for metal cations. Tis strategy is generally a greener approach to NP synthesis because the solvent is water and the template is biodegradable. We have characterized these NPs by atomic force microscopy (AMF) and transmission electron microscopy (TEM). For example, a pcDNA3.1 (+) plasmid can be used as a sacrifcial mold to yield disc-shaped gold and nickel NPs in the range of 28 ± 3 nm × 8 ± 1 nm and 52 ± 5 nm × 13 ± 1 nm, respectively. Columnar-shaped ZnO NPs were synthesized using a pH gradient and imaged to reveal a bimodal distribution in the range of 70 ± 10 nm × 50 ± 10 nm and 135 ± 15 nm × 80 ± 10 nm. In order to confrm the nature of these NPs, which were composed of both metals and non-metallic materials, we compared their electron microdifraction (μD) patterns to known standards [11–12]. Tere are two methods for obtaining electron difraction (ED) patterns [13]. Te selected area difraction (SAD) method uses an aperture to select the area producing the ED pattern, while μD and convergent beam electron difraction (CBED) techniques use the beam to select the area producing the pattern. Te minimum area that can be selected on a 100 kV TEM by the SAD method is 1 μ [12]. Because μD uses the beam to select the area, the minimum size in the TEM mode is limited by the electron source. Te sharp difracted beams of μD, as opposed to the discs of CBED, are produced by using a small (20–30 μ) second condenser aperture [14]. Because the size of the NPs under examination was less than 200 nm, μD was the method of choice. Microdifraction (μD) is a reliable method of verifying the identity of individual NPs when there is not enough sample for powder X-ray difraction (XRD) analysis. In the use of plasmid molds, the resulting materials could be the starting metal ion salts, the metal oxides, the target metal NPs, or combinations of these (for example, nickel metal, NiO, Ni 2 O 3 , NiX 2 ). Similar analytical criteria are needed for the formation of inorganic materials such as ZnO and TiO 2 . Morphology alone cannot diferentiate these NPs because the metals in the NPs sometimes exist in more than one oxidation state. In other cases, similar morphologies proved to be two diferent materials. Identifcation of the NPs necessitated the indexing of individual difraction patterns, a very time consuming and tedious procedure. To simplify the identifcation of materials, when one has an idea what the material might be (that is, NiO or Ni 2 O 3 ) and standards with which to compare them, we present two easily applied and straightforward methods for comparing electron difraction (ED) patterns. Identifying total unknowns will still require indexing individual difraction patterns. Te example shown in Figure 1 illustrates that this technique can be applied to inclusions in tissue samples as well as to particulate materials. Materials and Methods Materials. Me 3 PAuCl and Co(II)Cl 2 . 6H 2 O were pur- chased from Sigma Aldrich, and Ni(II)Cl 2 . 6H 2 O was purchased from Fisher Scientifc. Te pcDNA3.1(+) plasmid was obtained from Invitrogen, amplifed with Qiagen kit to a mother stock suspension of 1 mg/mL and diluted when mixed with the cationic-containing solutions. Te 12 mM stock solutions of metal chlorides were prepared in nanopure water. Te gold solution was prepared by adding an equal portion of deionized water to 100 mL of a 24 mM stock solution of Me 3 PAuCl dissolved in acetone. Zn(NO 3 ) 2 . 6H 2 O (Sigma Aldrich) was dissolved in deionized water at a concentration of 50 mM. Tris (Sigma Aldrich) was prepared in deionized water at a concen- tration of 100 mM. Tris-EDTA (TE) bufer (10 mM Tris, 1 mM EDTA, pH 8) was included in the Qiagen kit.  Instrumentation. Samples for TEM observation and μD were dispersed onto carbon-coated copper grids (Electron Microscopy Sciences). Te samples were imaged and the μD patterns collected at 120 kV using a Tecnai G2 Biotwin (FEI). All images and μD patterns were collected with an AMT 2K CCD camera. We obtained μD patterns of known standards and then, under the same conditions (kV and camera length), we obtained μD patterns of the unknowns. For both methods to https://doi.org/10.1017/S1551929511001179 Downloaded from https:/www.cambridge.org/core. IP address: 54.191.40.80, on 09 Jul 2017 at 04:10:16, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.