Here we report on initial efforts to evaluate enhanced darkfield microscopy (EDFM) and light scattering Vis–NIR hyperspectral imaging (HSI) as a rapid screening tool for the offline analysis of mixed ...cellulose ester (MCE) filter media used to collect airborne nanoparticulate from work environments. For this study, the materials of interest were nanoscale titanium dioxide (TiO2) and silicon dioxide (SiO2; silica), chosen for their frequent use in consumer products. TiO2 and SiO2 nanoscale particles (NPs) were collected on MCE filter media and were imaged and analyzed via EDFM‐HSI. When visualized by EDFM, TiO2 and SiO2 NPs were readily apparent as bright spherical structures against a dark background. Moreover, TiO2 and SiO2 NPs were identified in hyperspectral images. EDFM‐HSI images and data were compared to scanning transmission electron microscopy (STEM), a NIST‐traceable technique for particle size analysis, and the current gold standard for offline analysis of filter media. As expected, STEM provided more accurate sizing and morphology data when compared to EDFM‐HSI, but is not ideal for rapid screening of the presence of NPs of interest since it is a costly, low‐throughput technique. In this study, we demonstrate the utility of EDFM‐HSI in rapidly visualizing and identifying TiO2 and SiO2 NPs on MCE filters. This screening method may prove useful in expediting time‐to‐knowledge compared to electron microscopy. Future work will expand this evaluation to other industrially relevant NPs, other filter media types, and real‐world filter samples from occupational exposure assessments.
Classified hyperspectral image of TiO2 NPs collected on MCE filter media. Hyperspectral datacube of TiO2 captured on MCE filter media (exposure concentration: 32.8 mg TiO2). Green false coloration overlay indicates TiO2(+) pixels as identified by SAM. Scale bar = 50 μm. Enhanced darkfield microscopy‐hyperspectral imaging visualizes nanoparticles and identifies samples for further intensive electron microscopy analysis. This technique is promising for rapid screening of filters from occupational exposure assessments.
Enhanced darkfield microscopy (EDFM) and hyperspectral imaging (HSI) are being evaluated as a potential rapid screening modality to reduce the time‐to‐knowledge for direct visualisation and analysis ...of filter media used to sample nanoparticulate from work environments, as compared to the current analytical gold standard of transmission electron microscopy (TEM). Here, we compare accuracy, specificity, and sensitivity of several hyperspectral classification models and data preprocessing techniques to determine how to most effectively identify multiwalled carbon nanotubes (MWCNTs) in hyperspectral images. Several classification schemes were identified that are capable of classifying pixels as MWCNT(+) or MWCNT(–) in hyperspectral images with specificity and sensitivity over 99% on the test dataset. Functional principal component analysis (FPCA) was identified as an appropriate data preprocessing technique, testing optimally when coupled with a quadratic discriminant analysis (QDA) model with forward stepwise variable selection and with a support vector machines (SVM) model. The success of these methods suggests that EDFM‐HSI may be reliably employed to assess filter media exposed to MWCNTs. Future work will evaluate the ability of EDFM‐HSI to quantify MWCNTs collected on filter media using this classification algorithm framework using the best‐performing model identified here – quadratic discriminant analysis with forward stepwise selection on functional principal component data – on an expanded sample set.
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
A significant hurdle in conducting effective health and safety hazard analysis and risk assessment for the nanotechnology workforce is the lack of a rapid method for the direct visualization and ...analysis of filter media used to sample nanomaterials from work environments that represent potential worker exposure. Current best‐known methods include transmission electron microscopy (TEM) coupled with energy dispersive x‐ray spectroscopy (EDS) for elemental identification. TEM‐EDS is considerably time‐, cost‐, and resource‐intensive, which may prevent timely health and safety recommendations and corrective actions. A rapid screening method is currently being explored using enhanced darkfield microscopy with hyperspectral imaging (EDFM‐HSI). For this approach to be effective, rapid, and easy, sample preparation that is amenable to the analytical technique is needed. Here, we compare the sample preparation steps for mixed cellulose ester (MCE) filter media specified in NIOSH Method 7400—Asbestos and Other Fibers by Phase Contrast Microscopy (PCM)—against a new method, which involves saturation of the filter media with acetone. NIOSH Method 7400 was chosen as a starting point since it is an established technique for preparing transparent MCE filters for optical microscopy. Limitations in this method led to the development and comparison of a new method. The new method was faster, easier, and rendered filters more transparent, resulting in improved visualization and analysis of nanomaterials via EDFM‐HSI. This new method is suitable for a rapid screening protocol due to its speed, ease of use, and the improvement in image acquisition and analysis.
NIOSH Method 7400 was compared to a new method for preparing mixed cellulose ester filters for hyperspectral imaging. The new method of filter saturation with liquid acetone yielded improved imaging data for nanoparticles as compared to Method 7400.
In collaboration with RTI International, the U.S. National Institute for Occupational Safety and Health (NIOSH) administered a survey to North American companies working with nanomaterials to assess ...health and safety practices. The results would contribute to understanding the impact of the efforts made by the NIOSH Nanotechnology Research Center (NTRC) in communicating occupational health and safety (OHS) considerations for workers when handling these materials. The survey, developed by RAND Corporation, was conducted online from September 2019–December 2019. Forty-five companies or organizations in the U.S. and Canada that fabricate, manufacture, handle, dispose, or otherwise use nanomaterials completed the survey. The survey was designed to answer research questions regarding the nanomaterials in use, which resources the companies have consulted for OHS guidance, and the overall OHS culture at the companies. Other questions specifically addressed whether the companies interacted with NIOSH or NIOSH resources to inform OHS policies and practices. Among participating companies, 57.8% had a maximum of 50 employees. Gold nanoparticles and polymers were most common (n = 20; 45.5% each), followed by graphene (36.4%), carbon nanotubes and nanofibers (34.1%), and zinc oxide nanoparticles (31.8%). Environmental monitoring was performed by 31.8% of the companies. While 88.9% of the companies had laminar flow cabinets, only 67.5% required it to be used with ENMs. Information and training programs were indicated by 90% of the sample, and only 29.6% performed specific health surveillance for ENM workers. Personal protective equipment primarily included gloves (100%) and eye/face protection (97.7%). More than a third (37.8%) of the respondents reported using at least one NIOSH resource to acquire information about safe handling of ENMs. The small number of companies that responded to and completed the survey is a considerable limitation to this study. However, the survey data are valuable for gauging the reach and influence of the NIOSH NTRC on nano OHS and for informing future outreach, particularly to small businesses.
Engineered nanomaterials (ENMs) are currently employed by many industries and have different physical and chemical properties from their bulk counterparts that may confer different toxicity. ...Nanoparticles used or generated in semiconductor manufacturing have the potential to enter the municipal waste stream via wastewater and their ultimate fate in the ecosystem is currently unknown. This study investigates the fate of ENMs used in chemical mechanical planarization (CMP), a polishing process repeatedly utilized in semiconductor manufacturing. Wastewater sampling was conducted throughout the wastewater treatment (WWT) process at the fabrication plant's on-site wastewater treatment facility. The goal of this study was to assess whether the WWT processes resulted in size-dependent filtration of particles in the nanoscale regime by analyzing samples using scanning electron microscopy (SEM). Statistical analysis demonstrated no significant differences in particle size between sampling points, indicating low or no selectivity of WWT methods for nanoparticles based on size. All nanoparticles appeared to be of similar morphology (near-spherical), with a high variability in particle size. EDX verified nanoparticles composition of silicon- and/or aluminum-oxide. Nanoparticle sizing data compared between sampling points, including the final sampling point before discharge from the facility, suggested that nanoparticles could be released to the municipal waste stream from industrial sources.
From 2011–2015, the National Institute for Occupational Safety and Health Nanotechnology Field Studies Team conducted 11 evaluations at worksites that either produced engineered nanomaterials (ENMs) ...via a wet process or used ENMs in a wetted, suspended, or slurry form. Wet handling or processing of ENMs reduces potential exposure compared to dry handling or processing; however, air sampling data indicated exposures may still occur. Information was gathered about each company, production processes, ENMs of interest, and control measures. Exposure assessments included air sampling using filter media, surface wipe sampling, and real-time particle counting by direct-reading instruments. Electron microscopy analysis of air filters confirmed the presence of ENMs of interest (10 of 11 sites). When a method was available, chemical analysis of filters was also used to detect the presence of ENMs (nine of 11 sites). Wipe samples were collected at four of the 11 sites, and, in each case, confirmed the presence of ENMs on surfaces. Direct-reading data showed potential nanomaterial emissions (nine of 11 sites). Engineering controls included fume hoods, cleanrooms, and enclosed processes. Personal protective equipment was required during all 11 evaluations. Recommendations to address potential exposures were provided to each company following the hierarchy of controls.
Hyperspectral imaging (HSI) and mapping are increasingly used for visualization and identification of nanoparticles (NPs) in a variety of matrices, including aqueous suspensions and biological ...samples. Reference spectral libraries (RSLs) contain hyperspectral data collected from materials of known composition and are used to detect the known materials in experimental samples through a one‐to‐one pixel “mapping” process. In some HSI studies, RSLs created from raw NPs were used to map NPs in experimental samples in a different matrix; for example, RSLs created from NPs in suspension to map NPs in biological tissue. Others have utilized RSLs created from NPs in the same matrix. However, few studies have systematically compared hyperspectral data as a function of the matrix in which the NPs are found and its impact on mapping results. The objective of this study is to compare RSLs created from metal oxide NPs in aqueous suspensions to RSLs created from the same NPs in rat tissues following in vivo inhalation exposure, and to investigate the differences in mapping that result from the use of each RSL. Results demonstrate that the spectral profiles of these NPs are matrix dependent: RSLs created from NPs in positive control tissues mapped to experimental tissues more appropriately than RSLs created from NPs in suspension. Aqueous suspension RSLs mapped 0‐602 out of 500,424 pixels per tissue image while tissue RSLs mapped 689‐18,435 pixels for the same images. This study underscores the need for appropriate positive controls for the creation of RSLs for mapping NPs in experimental samples.
This study compared hyperspectral data as a function of the matrix in which nanoparticles are found and its impact on mapping. Results show that nanoparticle spectral profiles are matrix dependent, underscoring the need for appropriate positive controls.
Inhalation exposure to engineered nanomaterials (ENMs) may result in adverse pulmonary and/or systemic health effects. In this study, enhanced darkfield microscopy (EDFM) was used as a novel approach ...to visualizing industrial metal oxide nanoparticles (NPs) (silica, ceria, or alumina) in multiple tissue types following inhalation in rats mimicking occupational exposures. Advantages of EDFM over electron microscopy (EM) include reduced cost, time, and ease of sample preparation and operation. Following 4–6 hour inhalation exposures at three concentrations (3.5–34.0 mg/m3), lungs and secondary organs were harvested at 24 hours or 7 days post‐exposure and prepared for brightfield (BF) microscopy and EDFM. NPs were visualized within the lung and associated lymphatic tissues and in major organs of excretion (liver, spleen, kidney). EDFM also revealed NPs within pulmonary blood vessels and localization within specific regions of toxicological relevance in liver and kidney, indicating pathways of excretion. Results demonstrate the utility of EDFM for rapid direct visualization of NPs in various tissue types and suggest the potential for metal oxide NPs to distribute to secondary tissues following inhalation exposure. Confirmation of the composition, distribution, and relative abundance of inhaled NPs will be pursued by combining EDFM with hyperspectral imaging (HSI) and mapping.
This study investigated the utility of enhanced darkfield microscopy (EDFM) for rapid visualization of metal oxide nanoparticles (NPs) following inhalation in rats mimicking potential occupational exposures. Silica, alumina, and ceria NPs in lung tissue, lung lymph nodes, and secondary organs (liver, kidney, spleen) were easily visualized with EDFM. Immediate next steps will combine EDFM with hyperspectral imaging (HSI) and mapping to confirm NP composition and assess NP biodistribution and abundance.
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