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WIREs Nanomed Nanobiotechnol
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Cryo‐electron microscopy and cryo‐electron tomography of nanoparticles

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Cryo‐transmission electron microscopy (cryo‐TEM or cryo‐EM) and cryo‐electron tomography (cryo‐ET) offer robust and powerful ways to visualize nanoparticles. These techniques involve imaging of the sample in a frozen‐hydrated state, allowing visualization of nanoparticles essentially as they exist in solution. Cryo‐TEM grid preparation can be performed with the sample in aqueous solvents or in various organic and ionic solvents. Two‐dimensional (2D) cryo‐TEM provides a direct way to visualize the polydispersity within a nanoparticle preparation. Fourier transforms of cryo‐TEM images can confirm the structural periodicity within a sample. While measurement of specimen parameters can be performed with 2D TEM images, determination of a three‐dimensional (3D) structure often facilitates more spatially accurate quantization. 3D structures can be determined in one of two ways. If the nanoparticle has a homogeneous structure, then 2D projection images of different particles can be averaged using a computational process referred to as single particle reconstruction. Alternatively, if the nanoparticle has a heterogeneous structure, then a structure can be generated by cryo‐ET. This involves collecting a tilt‐series of 2D projection images for a defined region of the grid, which can be used to generate a 3D tomogram. Occasionally it is advantageous to calculate both a single particle reconstruction, to reveal the regular portions of a nanoparticle structure, and a cryo‐electron tomogram, to reveal the irregular features. A sampling of 2D cryo‐TEM images and 3D structures are presented for protein based, DNA based, lipid based, and polymer based nanoparticles. WIREs Nanomed Nanobiotechnol 2017, 9:e1417. doi: 10.1002/wnan.1417 This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Examples of cryo‐TEM images of protein‐RNA based, lipid based, polymeric, and metallic nanoparticles. (a) Protein‐RNA nanoparticles (T = 1 polyomavirus SV40 VP1 virus‐like particles) 22 nm in diameters (arrows) and larger assemblies, scale bar, 50 nm. (Reprinted with permission from Ref . Copyright 2013 American Chemical Society) (b) Lipid‐based hexosome nanoparticles, scale bar, 50 nm. The lower panels show a magnified area and a Fourier transform of this area. (Reprinted with permission from Ref . Copyright 2005 American Chemical Society) (c) Spherical polyelectrolyte brushes with added cesium ions and bovine serum albumin to enhance the image contrast of the brush layer, scale bar, 200 nm. (Reprinted with permission from Ref . Copyright 2005 American Chemical Society) (d) Iron oxide polymeric nanoparticles, 40 weight percent iron oxide clustered within the amphiphilic diblock copolymer, 5 K‐poly(ethylene oxide)‐b‐10 K‐poly(d,l‐lactide), scale bar, 50 nm. (Reprinted with permission from Ref . Copyright 2014 American Chemical Society)
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Cryo‐TEM analysis of mixed PAA/PS polymer brush‐grafted silica nanoparticles in organic and aqueous solvents. (a) One tilt image from a tilt‐series of the nanoparticles in DMF. (b) Central slice from a cryo‐electron tomogram of the nanoparticles in DMF. (c) Segmented representation of one nanoparticle from the tomogram in panel b, with the silica core shown in red and PAA domains in blue. (d) One tilt image from a tilt‐series of the nanoparticles in water. (e) Central slice from a cryo‐electron tomogram of the nanoparticles in water. (f) Segmented representation of one nanoparticle from the tomogram in panel e, with the silica core shown in gold and PAA domains in green. The PAA chains were positively stained with uranyl acetate and have a darker contrast. Scale bars, 100 nm in panels (a) and (b), and 50 nm in panels (c–f). (Reprinted with permission from Ref . Copyright 2015 American Chemical Society).
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Cryo‐TEM analysis of a multicompartment micelle formed with a triblock terpolymer and interpolyelectrolyte complex (IPEC) formation with a diblock copolymer. (a) Cryo‐TEM image. (b) Overview of the cryo‐electron tomogram. (c) Isosurface representation of the entire micelle. (d) Side and tilt views of top slice (red). (e) Top view of middle slice (blue). (f) Isosurface of cropped micelle. Scale bars, 50 nm. (Reprinted with permission from Ref . Copyright 2014 American Chemical Society).
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Cryo‐electron tomogram of liposomes with incorporated clusters of superparamagnetic iron oxide nanoparticles (purple). Scale bar, 50 nm. (Reprinted with permission from Ref . Copyright 2014 American Chemical Society).
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Cryo‐TEM analysis of Doxil, a pegylated liposome that encapsulates doxorubicin. (a) Cryo‐TEM image. (b) Central slice (0.5 nm thick) from a cryo‐electron tomogram. (c) Segmented representation of a cryo‐electron tomogram, with liposome density shown in purple and doxorubicin density in pink. Scale bars, 100 nm. (Reprinted by permission from Ref . Copyright 2008 Medicine Ltd).
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Cryo‐TEM single particle reconstructions of gold nanoparticles built using DNA origami frames. (a) A P6 DNA octahedron with all sticky ends encoded to coordinate 7 nm nanoparticles into a symmetric six‐nanoparticle cluster. Scale bar, 7 nm. (b) A P4(1234) cluster with 10 nm gold nanoparticles at vertices 1‐2‐3‐4 of the octahedral frame. Scale bar, 10 nm. The structures in panel (a) and (b) are both composites of a reconstruction of the DNA portion of the origami frame together with a reconstruction of the gold nanoparticle portion of the assembly. The composite structures are viewed along the octahedral 4‐fold, 3‐fold, and 2‐fold symmetry axes (left to right) with the gold nanoparticles shown in gold and the DNA density radially color coded from red to blue representing the inner to outer radii. (Reprinted by permission from Ref . Copyright 2015 Macmillan Publishers Ltd: Nature Nanotechnology).
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Cryo‐TEM structure of the vault‐nanodisk complex. The structure is a composite of a cryo‐TEM single particle reconstruction of the vault (green) and a cryo‐electron tomogram of the internal phospholipid bilayer nanodisk (red). The overall dimensions of the vault are 71 nm × 42 nm × 42 nm. (Reprinted with permission from Ref . Copyright 2011 John Wiley and Sons).
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Cryo‐TEM single particle reconstruction of a protein‐RNA nanoparticle with icosahedral symmetry (T = 1 polyomavirus SV40 VP1 virus‐like particles). Both full (left) and cropped views (right) are shown in radially colored surface representations. The internal RNA shell (blue) appears disconnected from the outer protein shell (red). Oval, triangle, and pentagon symbols indicate the locations of icosahedral 2‐fold, 3‐fold, and 5‐fold axes, respectively. Scale bar, 5 nm. (Reprinted with permission from Ref . Copyright 2013 American Chemical Society).
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Examples of nanoparticle cryo‐TEM images in different solvents. (a) Cryo‐TEM image of mixed PAA/PS brush‐grafted silica nanoparticles in DMF. The electron dense spots in the center of the image are 10 nm fiducial nanogold markers. (b) One enlarged nanoparticle in panel (a). (c) Cryo‐TEM image of mixed PAA/PS brush‐grafted silica nanoparticles in water. (d) One enlarged nanoparticle in panel C. The PAA chains were positively stained with uranyl acetate. Scale bars, 100 nm. (Reprinted with permission from Ref . Copyright 2015 American Chemical Society).
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Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
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