Name: identification of metal oxide nanoparticles in histological samples by enhanced darkfield microscopy and hyperspectral mapping
Uploaded: 2015-12-08T14:00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping at JoVE.com

The authors thank G&#252;nter Oberd&#246;rster, DVM, PhD and Alison Elder, PhD (University of Rochester) and Mary Frame, PhD (Stony Brook University) for animal research collaborations resulting in tissue samples for analysis. Additionally, the authors thank: Christina Rotondi (Albany Medical College Histology Core); Rani Sellers, DVM, PhD and Barbara Cannella, PhD (Albert Einstein College of Medicine Histology and Comparative Pathology Facility); Leonardo Bezerra and Ahlam Abuawad (Brenner research team members); and Leslie Krauss, Byron Cheatham and Elyse Johnson (CytoViva). This work was supported in part by CDC-NIOSH grant OH-009990-01A1 and the NanoHealth and Safety Center, New York State, awarded to S.B.

<ol>
	<li>Sun, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+of+a+single+mesoporous+silica+nanoparticle+into+a+human+lung+cancer+cell+observed+by+differential+interference+contrast+microscopy.">Endocytosis of a single mesoporous silica nanoparticle into a human lung cancer cell observed by differential interference contrast microscopy.</a> Anal Bioanal Che. 391 (6), 2119-2125 (2008).</li><li>Anselme, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+cells+and+bacteria+with+surfaces+structured+at+the+nanometre+scale.">The interaction of cells and bacteria with surfaces structured at the nanometre scale.</a> Acta Biomate. 6 (10), 3824-3846 (2010).</li><li>Anshup, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+gold+nanoparticles+in+human+cells.">Growth of gold nanoparticles in human cells.</a> Langmui. 21 (25), 11562-11567 (2005).</li><li>Weinkauf, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+dark+field+microscopy+for+rapid+artifact+free+detection+of+nanoparticle+binding+to+Candida+albicans+cells+and+hyphae.">Enhanced dark field microscopy for rapid artifact free detection of nanoparticle binding to Candida albicans cells and hyphae.</a> Biotechnol. 4 (6), 871-879 (2009).</li><li>Sondi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticles+as+antimicrobial+agent:+a+case+study+on+E+coli+as+a+model+for+Gram+negative+bacteria.">Silver nanoparticles as antimicrobial agent: a case study on E coli as a model for Gram negative bacteria.</a> J Colloid Interface Sc. 275 (1), 177-182 (2004).</li><li>Berry, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+exploitation+of+magnetic+nanoparticle+cell+interaction+for+biomedical+applications.">Possible exploitation of magnetic nanoparticle cell interaction for biomedical applications.</a> J Mater Chem. 15 (5), 543-547 (2005).</li><li>Chithrani, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+the+mechanism+of+cellular+uptake+and+removal+of+protein+coated+gold+nanoparticles+of+different+sizes+and+shapes.">Elucidating the mechanism of cellular uptake and removal of protein coated gold nanoparticles of different sizes and shapes.</a> Nano Lett. 7 (6), 1542-1550 (2007).</li><li>Chithrani, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+size+and+shape+dependence+of+gold+nanoparticle+uptake+into+mammalian+cells.">Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells.</a> Nano Lett. 6 (4), 662-668 (2006).</li><li>Elechiguerra, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+silver+nanoparticles+with+HIV+1.">Interaction of silver nanoparticles with HIV 1.</a> J Nanobiotechnol. 3 (6), 1-10 (2005).</li><li>Carlson, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+cellular+interaction+of+silver+nanoparticles+size+dependent+generation+of+reactive+oxygen+species.">Unique cellular interaction of silver nanoparticles size dependent generation of reactive oxygen species.</a> J Phys Chem. 112 (43), 13608-13619 (2008).</li><li>Roth, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+microscopy+as+an+analytical+tool+for+nanomaterials.">Hyperspectral microscopy as an analytical tool for nanomaterials.</a> WIREs Nanomed Nanobiotechnol In Press. , (2015).</li><li>Williams, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+kernel+hardness+classification+by+near+infrared+(NIR)+hyperspectral+imaging+and+multivariate+data+analysis.">Maize kernel hardness classification by near infrared (NIR) hyperspectral imaging and multivariate data analysis.</a> Anal Chim Act. 653 (2), 121-130 (2009).</li><li>Ziph Schatzberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+imaging+enables+industrial+applications.">Hyperspectral imaging enables industrial applications.</a> Industrial Photonic. , (2014).</li><li>Sun, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hyperspectral imaging for food quality analysis and contro. , (2010).</li><li>ElMasry, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+imaging+for+nondestructive+determination+of+some+quality+attributes+for+strawberry.">Hyperspectral imaging for nondestructive determination of some quality attributes for strawberry.</a> J Food En. 81 (1), 98-107 (2007).</li><li>Mortimer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+hyperspectral+imaging+microscopy+for+semi+quantitative+analysis+of+nanoparticle+uptake+by+protozoa.">Potential of hyperspectral imaging microscopy for semi quantitative analysis of nanoparticle uptake by protozoa.</a> Environ Sci Techno. 48, 8760-8767 (2014).</li><li>Sarlo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+20+nm+100+nm+and+1000+nm+fluorescent+polystyrene+latex+nanospheres+following+acute+systemic+or+acute+and+repeat+airway+exposure+in+the+rat.">Tissue distribution of 20 nm 100 nm and 1000 nm fluorescent polystyrene latex nanospheres following acute systemic or acute and repeat airway exposure in the rat.</a> Toxico. 263, 117-126 (2009).</li><li>Husain, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+instillation+of+low+doses+of+titanium+dioxide+nanoparticles+in+mice+leads+to+particle+retention+and+gene+expression+changes+in+the+absence+of+inflammation.">Pulmonary instillation of low doses of titanium dioxide nanoparticles in mice leads to particle retention and gene expression changes in the absence of inflammation.</a> Toxicol Appl Pharmacol. 269, 250-262 (2013).</li><li>Meyer, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+uptake+and+associated+toxicity+of+silver+nanoparticles+in+Caenorhabditis+elegan.">Intracellular uptake and associated toxicity of silver nanoparticles in Caenorhabditis elegan.</a> Aquatic Toxicol. 100, 140-150 (2010).</li><li>Badireddy, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=characterization+and+abundance+of+engineered+nanoparticles+in+complex+waters+by+hyperspectral+imagery+with+enhanced+darkfield+microscopy.">characterization and abundance of engineered nanoparticles in complex waters by hyperspectral imagery with enhanced darkfield microscopy.</a> Environ Sci Technol. 46 (18), 10081-10088 (2012).</li><li>Dettmeyer, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Staining techniques and microscopy Forensic histopathology fundamentals and perspectives. , 370 (2011).</li><li>Titford, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+development+of+microscopical+techniques+for+diagnostic+pathology.">Progress in the development of microscopical techniques for diagnostic pathology.</a> J Histotechnol. 32 (1), 9-19 (2009).</li><li>Kumar, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Special stains and H&amp;E. , (2014).</li><li>Manolakis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+image+processing+for+automatic+target+detection+applications.">Hyperspectral image processing for automatic target detection applications.</a> Lincoln Laboratory Journal. 14 (1), 79-116 (2003).</li><li>Mercer, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+fibrotic+response+to+aspiration+of+multi+walled+carbon+nanotubes.">Pulmonary fibrotic response to aspiration of multi walled carbon nanotubes.</a> Part Fibre Toxicol. 8 (1), 21 (2011).</li><li>Kim, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+reflectance+and+fluorescence+imaging+system+for+food+quality+and+safety.">Hyperspectral reflectance and fluorescence imaging system for food quality and safety.</a> T Am Soc Ag En. 44 (3), 721-730 (2001).</li><li>Mercer, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrapulmonary+transport+of+MWCNT+following+inhalation+exposure.">Extrapulmonary transport of MWCNT following inhalation exposure.</a> Part Fibre Toxico. 10 (1), 38 (2013).</li></ol>

The authors have nothing to disclose.

For tissue samples that have undergone conventional histological staining, the identification and analysis of metal oxide nanoparticles may be achieved through a combination of EDFM, HSI mapping, and image analysis techniques. While there is flexibility in sample preparation for histology or immunohistochemistry (e.g., using fixed or frozen tissues; type of stain), it is important that samples are sectioned to a thickness of 5-10 &#181;m for optimal visualization. Samples used here were formalin-fixed and paraffin-embedded prior to sectioning with a rotary microtome to 6 &#181;m thickness, mounted on glass microscope slides, stained with hematoxylin and eosin and coverslipped. Porcine skin tissues from an ex vivo cutaneous penetration toxicology collaboration were used for this study. The tissues were exposed to metal oxide nanoparticles (alumina, silica, ceria) in aqueous suspensions. Detection of the region(s) of interest (high contrast elements) with EDFM is a critical first step that facilitates subsequent HSI mapping and analysis. Positive and negative control samples must be imaged and analyzed first in order to create a spectral library for reference. The collected spectra from the positive control are exported to a positive control spectral library. Then, all the spectra from the negative control images are subtracted from the positive control&#8217;s spectral library in order to improve specificity (reduce false positives). The resulting filtered spectral library is considered the RSL that serves for the analysis of materials of interest. All tissue samples undergo the same imaging process and are mapped against the RSL. The resulting image will contain only areas with elements of interest over a black background. This image could then be analyzed with ImageJ using its threshold and particle analysis functions to obtain the area of mapped particles per field of view. Numerical data obtained from ImageJ can be exported to a spreadsheet for further analysis.
It is important to consider that as biological samples are inherently different from one another, and staining methods may affect visualization through EDFM and HSI, the exposure settings should be determined in accordance to what produces the best high contrast image for a specific type of sample. Although reduction of false positives can be achieved through the filtration of spectral libraries, it is crucial to obtain reliable negative controls that have avoided contamination with the element of interest, as the spectra corresponding to this contamination could potentially be filtered from the positive control&#8217;s spectral library, increasing false negative rates. Also, the spectrum intensity range that is detectable with the hyperspectral imaging software cannot surpass the limit of the particular software (for this study, that is 16,000 units): areas with a high number of accumulated particles that produce spectral intensities above the intensity limit are left out of the spectral library, due to the risk of increasing the number of false negatives.
While the HSI system confers many advantages over traditional methods, there are some drawbacks and limitations to consider. One is that the large amount of optical data collected may require substantial computing power. Another is that HSI can be time-intensive, particularly at the beginning stages when reference spectral libraries are being created. Also, imaging time may require several minutes per image capture, making it slower than simple darkfield imaging; however, it is still faster than performing sampling preparation and visualization by electron microscopy. Additionally, complex systems may result in multiple characteristic spectra, which require the development of highly specialized controls and make the establishment of standardized, universal reference libraries difficult26. Finally, the technique results in lower resolution than probe- or electron-microscopy techniques, such as atomic force microscopy or transmission electron microscopy, which can resolve individual atoms. This technique&#8217;s resolution is limited due to its photonic nature, which prevents it from being a high-precision tool for measuring particle sizes at the nanometer level or for precisely locating materials at a sub-micron level. While the technique may be able to pinpoint particles within certain tissue compartments or cellular organelles greater than 1 &#181;m (such as cell nuclei), smaller organelles or features are challenging to visualize accurately with this method. Also of note, given its spatial resolution, this method cannot differentiate between single nanoparticles and agglomerates11.
Other considerations include: certain materials (such as noble metals) have much higher reflectance and distinct spectral profiles, which can make them easier to analyze and spectrally map with this tool. Others, such as the semi-metallic oxides investigated in this study and carbon-based nanomaterials24, 27, may be more challenging due to their elemental composition, shape, and depending on the matrix. In two murine inhalation studies by Mercer et al., a similar system to the one employed in this study was used in order to locate carbon nanotubes in the lung and in secondary organs based on their remarkably high contrast with surrounding tissues. However, hyperspectral mapping was not demonstrated in either study, likely because the unique shape of carbon fibers was a sufficient trait for identification. Another consideration pertains specifically to tissues: since depositing nanoparticles of interest to specific organs through normal biophysical processes is unpredictable (and often itself the subject of study), determination of an applicable positive control can be difficult and requires consideration of how generation of the control might affect the state of a material of interest. For example, if a spectral library is created from pristine nanoparticles of interest, it may be difficult to map the library to those same nanoparticles in tissues or cells due to changes in the spectra resulting from alteration of the particle (e.g., due to change in pH, dissolution, agglomeration, protein binding) and the overall microenvironment or matrix. Finally, the technique is limited in its semi-quantitative nature: it can only be as quantitative as other two-dimensional microscopy techniques of similar resolution, which means it cannot be easily used to perform tasks such as characterizing total organ burden of a material.
Overall, EDFM and HSI provide several advantages over conventional nanomaterial imaging and characterization techniques, such as TEM, HAADF and DIC. EDFM/HSI allows for faster image acquisition and analysis, which saves time and cost in comparison to more intensive conventional techniques. Additionally, sample preparation for EDFM/HSI is typically both minimal and non-destructive, which saves time and also allows for more flexible analysis of a given sample since it may then be used for other techniques. Furthermore, HSI is versatile, allowing for analysis of nanoscale materials of many compositions and in a variety of matrices. The research team is working to refine the method described here for other materials and sample types, including an in-depth assessment of the specificity of the technology. A critical next step under investigation by the research team is validation of these techniques against traditional gold standards (e.g., Raman, TEM, SEM) for the materials and tissue types of interest.

As nanomaterials are increasingly used in a variety of industries and applications, there is a need for nanoscale imaging and characterization methods that are more rapid, affordable, and convenient than traditional modalities, such as electron microscopy. To visualize nanoparticle (NP) interactions with cells, tissues, and living systems, many optical techniques have been employed, including differential interference contrast (DIC) microscopy1 and evanescent field-based approaches, such as total internal reflection (TIR) or near-field scanning optical microscopy (NSOM)2,3. However, these are high-end analytical approaches, out of reach of most non-specialist labs4. Electron microscopy, including transmission electron microscopy (TEM), has also been used to study NP interaction with cells5,6,7,8. High angle annular darkfield (HAADF) scanning transmission electron microscopy has been used to study the interaction of NPs with viruses9. Confocal microscopy is another popular technique used to study NP-cell interactions10.
In recent years, darkfield-based hyperspectral imaging (HSI) techniques have been used as a promising analytical tool for studying NPs in biological matrices11. HSI systems generate a three-dimensional representation of spatial and spectral data, known as a hypercube or datacube12. The spatial map of spectral variation is used for material identification. Spectral profiles of known materials can be generated and used as reference libraries for comparison to unknown samples. One of the major advantages with HSI systems is its ability to combine imaging with spectroscopy, thereby establishing the location and distribution of unknown NPs in vivo or ex vivo as well as linking them to another reference particle of known, similar composition.
There are several advantages of using HSI systems over conventional imaging techniques: minimal sample preparation is required; sample preparation is typically non-destructive in nature; image acquisition and analysis is faster; the technique is cost-effective13; and the spatial distribution and analysis of compounds of mixed composition and/or in complex matrices is more easily accomplished14.
For nanomaterials research involving precious samples, one of the most important considerations is the availability of a non-destructive imaging method, which allows for the potential to repeatedly examine samples by one or more methods. Repeated or multiple analyses may be desired to develop comprehensive datasets that would not be available from a single method. To this regard, studying its optical properties is the safest way to analyze the sample. By using an enhanced darkfield microscope (EDFM) and HSI system to study the optical response of the sample &#8211; namely reflectance, but also absorbance and transmittance &#8211; feature identification and characterization can be performed15. Potential characterization endpoints include an assessment of relative size and shape of nanoparticles or agglomerates and distribution of nanoparticles within a sample.
In this paper, we describe mapping methods specifically for metal oxide nanoparticles in post-mortem tissue using a HSI system based on a pixel-spectral match algorithm referred to as a spectral angle mapper (SAM). We chose this particular application because it has the potential to complement current and future in vivo nanotoxicology research, wherein animal models are used to evaluate the health implications of exposure to engineered nanomaterials. Application of this method could also inform nanoscale drug delivery research that utilizes tissue or animal models. In particular, nanoparticle absorption, distribution, metabolism, and excretion throughout organs and tissues could be investigated with this system. A wide variety of applications are being investigated for use in biomedical research11.
This method could be utilized for assessment of different biological samples (such as various tissues types, bronchoalveolar lavage samples, and blood smears) that have been exposed to nanoparticles of a variety of elemental compositions16-19. Furthermore, this method is useful for studying nanoparticle biodistribution in vivo and in vitro, which is pertinent for nanoscale drug delivery studies11. Beyond biological samples, EDFM and HSI can be used to evaluate nanoparticles in environmental samples, such as wastewater20. Occupational exposure assessment can be facilitated by the use of this technique as well, since it may be used to evaluate the efficacy of personal protective equipment in preventing nanoparticle penetration. Furthermore, the research team is currently developing a similar EDFM and HSI protocol for the evaluation of filter media samples of nanoparticles collected from occupational exposure assessments. While preparation of these different sample types for EDFM and HSI may vary, it is important that they are prepared in such a way that they may be easily visualized by the optical system. Typically, the sample should be prepared as if it would be visualized via traditional brightfield microscopy. There are several hyperspectral imaging systems commercially available11.

<table border="0">
	<tbody>
		<tr>
			<td>CytoViva 150 Unit (condenser)</td>
			<td>CytoViva (Auburn, AL)</td>
			<td></td>
			<td>mounted to Olympus BX43 microscope</td>
		</tr>
		<tr>
			<td>Olympus BX43 Microscope - Analyzer Slot - HSI with 10x and 40x air objectives and 100x oil immersion objective</td>
			<td>obtained through CytoViva (Auburn, AL)</td>
			<td></td>
			<td>for use with CytoViva 150 Unit condenser</td>
		</tr>
		<tr>
			<td>Dagexcel-M Digital Firewire Camera - Cooled; includes Exponent 7 software</td>
			<td>obtained through CytoViva (Auburn, AL)</td>
			<td></td>
			<td>enhanced darkfield camera and software</td>
		</tr>
		<tr>
			<td>CytoViva Hyperspectral Imaging System 1.4; includes Pixelfly hyperspectral camera, XY stage controller, ENVI hyperspectral imaging software</td>
			<td>obtained through CytoViva (Auburn, AL)</td>
			<td></td>
			<td>hyperspectral camera and software</td>
		</tr>
		<tr>
			<td>cleanroom cleaned glass microscope slides (glass B slides)</td>
			<td>Schott NEXTERION</td>
			<td>1025087</td>
			<td>reduced debris and artifacts compared to conventional glass microscope slides for optimal imaging</td>
		</tr>
		<tr>
			<td>cleanroom cleaned glass microscope coverslips (#1.0; 22mm x 22mm x&#160; 1.45mm)</td>
			<td>Schott NEXTERION</td>
			<td>custom</td>
			<td>reduced debris and artifacts compared to conventional glass coverslips for optimal imaging</td>
		</tr>
		<tr>
			<td>type A microscopy immersion oil</td>
			<td>Fisher Scientific</td>
			<td>12368B</td>
			<td>multiple suppliers</td>
		</tr>
		<tr>
			<td>70% isopropanol in water</td>
			<td></td>
			<td></td>
			<td>multiple suppliers</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health (NIH)</td>
			<td></td>
			<td>free open-source software online download</td>
		</tr>
		<tr>
			<td>metal oxide nanoparticles</td>
			<td>supplied to the research team by industrial partners</td>
			<td></td>
			<td>alumina, silica, and ceria nanoparticles in aqueous suspensions. Due to a Non-Disclosure Agreement between the authors and industry partners, further product information cannot be disclosed.</td>
		</tr>
	</tbody>
</table>

identification of metal oxide nanoparticles in histological samples by enhanced darkfield microscopy and hyperspectral mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Hyperspectral microscopy is useful for its ability to identify materials in a manner similar to spectrophotometry. As indicated in Figure 1, each material has several characteristic spectra and an overall shape of its reflectance, which is unique. Furthermore, Figure 1 illustrates the matrix-dependent nature of the spectral profiles: the spectral profiles for each of the three metal oxides in histological tissue samples (top panel) are different from the spectral profiles for each of the three metal oxides in aqueous suspension (bottom panel). By mapping the characteristic spectral profiles to unknown samples in the same matrix, the technique is useful for determining presence or absence of a material, and can also semi-quantitatively compare relative amounts of material in samples.
The reference spectral libraries created from positive control skin samples are appropriate for mapping to experimental skin samples. As seen in Figure 2, the reference spectral libraries map well to the corresponding positive controls and do not map to the corresponding negative controls.
The most significant advantage of the technique is its ability to detect low levels of materials of interest in samples, as well as to differentiate them from contaminants. For example, individual nanotubes can be observed in lungs during inhalation of doses as low as 40 &#181;g in a 20-30 g rat25. Figure 3 demonstrates this in histological skin samples: while some particles are readily obvious in the darkfield optical image (column 2), others are detected using hyperspectral imaging (column 3) that might have been missed using brightfield microscopy (column 1) or other methods. The use of darkfield HSI also serves to improve specificity over simple brightfield microscopy. These images can then be subject to more quantitative forms of analysis, notably particle analysis via ImageJ.
Enhanced darkfield microscopy has several different applications, even in the absence of hyperspectral imaging. The first is its ability to generate high quality, high-contrast images. This is best demonstrated by Figure 3, in which particles of interest are readily identifiable in comparison to their brightfield counterparts. These images can also be subject to quantitative particle analysis, though in the absence of hyperspectral mapping, caution is necessary to avoid confusing a contaminant particle from a particle of interest.
<img alt="Figure 1" src="/files/ftp_upload/53317/53317fig1.jpg" /> 
Figure 1.&#160;Reference spectral libraries (tissue matrix) compared to spectral libraries (nanoparticles in suspension). This figure demonstrates the importance of generating a reference spectral library from nanomaterials of interest in the same matrix as experimental samples. The first row shows the reference spectral library (RSL) for each material in this study (alumina, silica and ceria). <a href="https://www.jove.com/files/ftp_upload/53317/53317fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This RSL was created by the method described in this protocol, where positive control tissue samples were filtered against negative control tissue samples. The second row shows spectral libraries (SL) created from the nanoparticles of interest in liquid suspension on a glass slide. For alumina, a bimodal peak at wavelengths of 500 and 650 was found in the RSL, whereas a more curved and less distinctive peak was seen in the alumina NP suspension SL at around 600. For silica, a peak at a wavelength of around 650 was present in the RSL, whereas a more curved and less distinctive peak was seen in the silica NP suspension SL at around 600. For ceria, a bimodal peak was found at wavelengths of 520 and 620 in the RSL, whereas a less distinctive peak was seen in the ceria NP suspension SL at around 560. This suggests that the matrix in which the NPs are embedded creates a shift in spectra that can be considerable when selecting the controls to create the SL for as specific experiment.
<img alt="Figure 2" src="/files/ftp_upload/53317/53317fig2.jpg" /> 
Figure 2. Reference spectral libraries (tissue matrix) mapped on positive control and negative control porcine skin tissue. This figure serves as confirmation of the RSLs as appropriate for mapping to experimental samples, in that each RSL (left column) maps well to its corresponding positive control (middle column) and does not map to its corresponding negative control (right column). <a href="https://www.jove.com/files/ftp_upload/53317/53317fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53317/53317fig3.jpg" /> 
Figure 3. Hyperspectral mapping of experimental porcine skin tissue exposed to metal oxide nanoparticles. Rows from top to bottom correspond to different layers of the skin, from superficial to deepest layer: epidermis, dermis, and subcutaneous tissue, respectively. The first row shows the stratum corneum of the epidermis from a skin sample that was exposed to alumina NPs in suspension; the second row was exposed to silica NPs in suspension; and the third row to ceria NPs in suspension. Each column depicts the same area of the skin imaged with different techniques. The first column corresponds to brightfield microscopy (40X mag.; scale bar = 50 &#181;m), where the area enclosed in a red square was magnified (100X mag; scale bar = 10 &#181;m) with enhanced darkfield microscopy (EDFM) in the second column, where white arrows are pointing to the high contrast NPs . The third column was obtained with a hyperspectral camera (HSI), showing the same high contrast NPs (white arrows). EDFM and HSI images were taken at 100X magnification; scale bar = 10 &#181;m. The fourth column shows the HSI image mapped against the respective RSL for each exposure group, where alumina NPs are shown in green, silica NPs in red, and ceria NPs in yellow, respectively. <a href="https://www.jove.com/files/ftp_upload/53317/53317fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping at JoVE.com

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

identification-metal-oxide-nanoparticles-histological-samples

Research

JoVE Journal

Bioengineering

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning

Surgical margin analysis (SMA), an essential procedure to confirm the complete excision of cancerous tissue in tumor resection surgery, requires intraoperative diagnostic tools to avoid repeated surgeries due to a positive surgical margin. Recently, by taking the advantage of the high intrinsic optical absorption of DNA/RNA at 266 nm wavelength, ultraviolet photoacoustic microscopy (UV-PAM) has been developed to provide high-resolution histological images without labeling, showing great promise as an intraoperative tool for SMA. To enable the development of UV-PAM for SMA, here, a high-speed and open-top UV-PAM system is presented, which can be operated similarly to conventional optical microscopies. The UV-PAM system provides a high lateral resolution of 1.2 &#181;m, and a high imaging speed of 55 kHz A-line rate with one-axis galvanometer mirror scanning. Moreover, to ensure UV-PAM images can be easily interpreted by pathologists without additional training, the original grayscale UV-PAM images are virtually stained by a deep-learning algorithm to mimic the standard hematoxylin- and eosin-stained images, enabling training-free histological analysis. Mouse brain slice imaging is performed to demonstrate the high performance of the open-top UV-PAM system, illustrating its great potential for SMA applications.

A high-speed and open-top ultraviolet photoacoustic microscope that can provide histological images intraoperatively for surgical margin analysis is demonstrated, including the system configuration, optical alignment, sample preparation, and experimental procedures.