Scanning probe microscopy (SPM) techniques can be employed to measure the size, topography, composition, and structural properties of nanoparticles. Related techniques such as scanning tunneling microscopy (STM), electric field gradient microscopy (EFM), scanning thermal microscopy, and magnetic field microscopy (MFM) combined with atomic force microscopy (AFM), can be used to investigate the structural, electronic, thermal, and magnetic properties of a nanomaterial. AFM uses a nanoscale probe to detect the inter-atomic forces and interactions between the probe and the material being analyzed and is capable of determining size and shape within a spatial resolution of a few angstroms.16 Apart from the ability to measure the particle size in a dry state as well as in aqueous and physiological conditions, AFM is a useful tool to probe the interaction of nanoparticles with supported lipid bilayers. This technique has been successfully used to compare nanoparticle interactions in in vitro cell assays.17,18 The ability to image under physiological conditions makes AFM a powerful tool for the characterization of nanoparticles in a dynamic, biological context. A variant of this method, molecular recognition force microscopy (MRFM), can be employed to study the specific ligand-receptor interactions between nanoparticles and their biological targets.

Optical microscopy techniques are useful at the micron scale and are extensively used for imaging structural features. Fluorescence and confocal microscopy may be used to determine cellular binding and internalization of fluorescent-labeled nanoparticles19 or those that are inherently fluorescent, such as quantum dots. But a more precise analysis of nanomaterial size and other direct measurements of physical properties will require a more sophisticated and specialized set of microscopic and spectroscopic techniques.

Scanning electron microscopy (SEM) provides information on the size, size distribution, shape, and density of nanomaterials. Transmission electron microscopy (TEM) and high-resolution TEM are more powerful than SEM in providing details at the atomic scale and can yield information regarding the crystal structure, quality, and grain size. TEM can be coupled with other characterization tools, such as electron energy loss spectrometry (EELS) or energy dispersive x-ray spectrometry (EDS), to provide additional information on the electronic structure and elemental composition of nanomaterials. Samples for TEM are evaluated dry or in a frozen state, under high-vacuum conditions. Nanoparticles analyzed by this instrument must therefore be stable under these extreme conditions. Additionally, while considered a gold standard of microscopic characterization methods, TEM requires a great deal of skill and time to obtain good data. In principle, when establishing characterization protocols, TEM can be used to validate characterization methods that are easier to use on a routine basis. Further description of analytical technologies as they apply to the measurement of specific nanomaterial properties is provided in the following sections.

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