Avoiding drying-artifacts in transmission electron microscopy: Characterizing the size and colloidal state of nanoparticles

Abstract

Standard transmission electron microscopy nanoparticle sample preparation generally requires the complete removal of the suspending liquid. Drying often introduces artifacts, which can obscure the state of the dispersion prior to drying and preclude automated image analysis typically used to obtain number-weighted particle size distribution. Here we present a straightforward protocol for prevention of the onset of drying artifacts, thereby allowing the preservation of in-situ colloidal features of nanoparticles during TEM sample preparation. This is achieved by adding a suitable macromolecular agent to the suspension. Both research- and economically-relevant particles with high polydispersity and/or shape anisotropy are easily characterized following our approach (http://bsa.bionanomaterials.ch), which allows for rapid and quantitative classification in terms of dimensionality and size: features that are major targets of European Union recommendations and legislation.

Figure 1. TEM micrographs of drop-cast samples of suspended Au NPs.

TEM micrographs of drop-cast samples of suspended Au NPs without BSA (a), with BSA but well below the optimal concentration (b), and with BSA at the optimal concentration (c). The magnified view (middle) - the right side of which is bi-leveled for high contrast - shows aggregation due to van der Waals forces (a), aggregation also due to protein bridging (b), and stability as single particles owing to the presence of an intact BSA ‘shield’ (c).

Figure 2. TEM analysis of in situ aggregates of Au NPs.

Panel (a) and (b) depict representative TEM micrographs (width: 2.68 μm) and close-up views of typical in situ aggregates of Au NPs. Panel c shows the result of counting 2200 single and pre-aggregated particles. Particles were classified in terms of long axis (2a) and aspect ratio (δ), following the same approach as for Figure 1(a–c). Panel (d) depicts the result summarized into a two-dimensional histogram quantifying the occurrence of existing combinations of aspect ratio and size.

Figure 3. In situ characterization of Au NPs.

UV-Vis extinction spectra (left) and dynamic depolarized light scattering results (right, empty circles) of single and in situ aggregated NPs. The dashed lines are correlation functions reconstructed from TEM analysis of BSA-prepared samples (Supplementary Information, SI 2).

Figure 4. Micrographs of various suspended nanopowders deposited onto the TEM grid without and with BSA.

The result of image analysis is summarized by the histograms. The Zeta potential, relevant for describing the effective surface charge, increases from top to bottom.

Figure 5. Illustration of an aqueous drop and the dominant processes influencing the deposition pattern of suspended particles onto a hydrophobic surface.

Red arrows indicate effective particle flow due to the coffee ring effect. Yellow lines show counter flow introduced by the Marangoni flow.