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WIREs Nanomed Nanobiotechnol
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Hyperspectral microscopy as an analytical tool for nanomaterials

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Hyperspectral microscopy is an advanced visualization technique that combines hyperspectral imaging with state‐of‐the‐art optics and computer software to enable the rapid identification of materials at the micro‐ and nanoscales. Achieving this level of resolution has traditionally required time‐consuming and costly electron microscopy techniques. While hyperspectral microscopy has already been applied to the analysis of bulk materials and biologicals, it shows extraordinary promise as an analytical tool to locate individual nanoparticles and aggregates in complex samples through rapid optical and spectroscopic identification. This technique can be used to not only screen for the presence of nanomaterials, but also to locate, identify, and characterize them. It could also be used to identify a subset of samples that would then move on for further analysis via other advanced metrology. This review will describe the science and origins of hyperspectral microscopy, examine current and emerging applications in life science, and examine potential applications of this technology that could improve research efficiency or lead to novel discoveries. WIREs Nanomed Nanobiotechnol 2015, 7:565–579. doi: 10.1002/wnan.1330 This article is categorized under: Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
Example of CytoViva® application for visualization of nanoparticle internalization into the cells of Caenorhabditis elegans. AgNPs are ingested and internalized into the cells of C. elegans. (a) CIT10 AgNPs are taken up along with food by C. elegans. (b) Some CIT10 AgNPs are also taken up into the cells of the nematodes, and are transferred to the offspring. AgNP identity was confirmed by hyperspectral analysis. (c) Hyperspectral image (HSI) showing the presences of PVPS AgNPs inside and outside C. elegans after exposure; colored rectangles correspond to the pixel areas [regions of interest (ROI)] in HSI where spectral profiles are collected. (d) The spectral profiles of AgNPs and hypodermis region of C. elegans; green, red, and dark blue represent internal AgNPs clusters, yellow, cyan, and maroon external AgNP clusters, and magenta background. (e and f) Very little signal is detected in nonexposed nematodes; note that y‐axis values are much lower in (f) than (d); higher contrast was used in (e) than (c) for visualization. Images taken using CytoViva hyperspectral imaging technology with dark‐field microscopy at 100× (panels a, b and e) or 40× (panel c) total magnification. The color of spectral profile corresponds to color of the rectangle in the image and each profile represents the average of all the pixels present in each square. (Reprinted with permission from Ref . Copyright 2010 Elsevier)
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Hyperspectral imaging of nanoparticles in complex waters. Hyperspectral image of Ag‐PVP55 nanoparticles in mesocosm (a), 600× zoom in of a portion of the sample image (a), (c) n‐dimensional visualization analysis of endmembers, and (d) endmembers of Ag‐PVP55 nanoparticles of different sizes the sample. Images were acquired using 100× objective/1.3 oil iris. Scale: 2 cm = 200 µm. (Reprinted with permission from Ref . Copyright 2012 Elsevier)
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Comparison of nanoparticle binding via light and enhanced dark field microscopies. (a) Silver NP (MIC of 31.25 ppm) viewed via light microscopy (bound NP not detectable). (c) Silver NP viewed via dark‐field microscopy (NP easily detected). (b) Cobalt NPs (MIC of 0.24 ppm) viewed via light microscopy (encrustation of cobalt NP visible even via light microscopy). (d) Binding of cobalt NP viewed via enhanced dark‐field microscopy. Inset shows detail of highlighted area, magnified 2.25×. (e) Copper NP (MIC of 15.63 ppm) demonstrating ‘bejeweled’ appearance. Inset shows detail of highlighted area, magnified 2.5×. (f) ‘Bejeweled’ appearance of copper–silver alloy NP (MIC of 250 ppm). Scale bars for all panels 5 µm. (Reprinted with permission from Ref . Copyright 2009 Elsevier)
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Dark‐field imaging of nanospheres in lung homogenates. (a–c) Polystyrene spheres in lung homogenates (1 day post‐exposure) imaged by dark‐field illumination (CytoViva). A sample of homogenate was allowed to dry on a slide. The lung tissue had a dull and spotty reflective property while the spheres generate very bright, spherical images. There is a clear difference between the 1000 nm spheres (c) versus the 100 nm (b) and 20 nm (a) spheres. The inserts show magnified areas of the spheres in tissue; some agglomeration of 20 nm and 100 nm spheres can be seen but the 1000 nm spheres appear to be monodisperse. Owing to the resolution of the microscope there is no difference in the image of the 20 nm spheres versus the 100 nm spheres. Arrows point to the spheres. (Reprinted with permission from Ref . Copyright 2009 Elsevier)
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Visualization of intra‐ and extracellular clusters of nanoparticles. Internalization of silver nanoparticles in U937 cells was examined with CytoViva URI system 2 h after silver nanoparticles treatment (2.5 µg/mL). Silver nanoparticles of 100 nm were detected inside the cells and presented as multiple red spots. With 5 nm silver nanoparticles treatment, several red spots suggested internalization of 5 nm silver nanoparticles, which was not detected in the control. (Reprinted with permission from Ref . Copyright 2012 Elsevier)
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Cerium oxide particles in lung tissue and pulmonary fibroblasts isolated from CeO2‐exposed rats, at 28 days post‐exposure. Cerium oxide particles in lung tissue and pulmonary fibroblasts isolated from CeO2 (a single intratracheal dose of 7 mg/kg)‐exposed rats, at 28 days post‐exposure. Control lung tissues exhibit no particles under high resolution, dark‐field illumination (a). Illuminated CeO2 particles, using dark‐field‐based illumination, were clearly detected in macrophages (MAC), the interstitium (arrow), in acellular surfactant clumps (arrow head), in the airspace as free particles (b). (c) Representative intensity versus wavelength spectra of points (pixels) of CeO2 particles in the cerium oxide‐exposed tissue section (upper panel) and spectra of control tissue. Each different colored curve represents a different point. Small arrow: MAC; big arrow: interstitium; arrow head: acellular mass of surfactant‐cerium oxide in the air space. (Reprinted with permission from Ref . Copyright 2012 Elsevier)
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Identification of nano‐TiO2 particles in lung tissues. Lung tissues from mice harvested 1 and 28 days following a single intratracheal instillation of low (18 µg) and high (162 µg) doses of nano‐TiO2 were subjected to VNIR hyperspectral imaging to identify particle retention in these tissues. (a) Reference spectral library from nano‐TiO2 exposed tissue. (b) Reference spectral library from control tissue. (c) Dark‐field images from nano‐TiO2 exposed tissues (upper panel). Dark‐field hyperspectral images from nano‐TiO2 exposed tissues identifying these nanoparticles, which appeared as aggregates of white inclusions (middle panel). Hyperspectral mapping of nano‐TiO2 in these tissues appeared as red dots or aggregates (bottom panel). (Reprinted with permission from Ref . Copyright 2013 Elsevier)
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Visualization of nanoparticles in suspension. Light scattering images of the nanoparticle suspensions obtained by Olympus microscope BX51 with the CytoViva Adapter (100x oil objective): (a) 5% glucose, (b) PLGA, (c) PEI/DNA, and (d) complex PLGA/PEI/DNA. Bar = 5 µm. (Reprinted with permission from Ref . Copyright 2008 Elsevier)
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Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Diagnostic Tools > In Vitro Nanoparticle-Based Sensing

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