Name: performing spectroscopy on plasmonic nanoparticles with transmission-based nomarski-type differential interference contrast microscopy
Uploaded: 2019-06-05T13:30:00
Description: Watch this Scientific Journal Video about Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy at JoVE.com

Dr. Anthony S. Stender wishes to acknowledge technical support through the Nanoscale and Quantum Phenomena Institute (NQPI) at Ohio University. This article&#160;was made possible through start-up funding provided to Dr. Stender by Ohio University.

1. Sample preparation with standard glass microscopy slides
<ol>
	<li>Prepare glass microscope slides for sample deposition. 
	NOTE: In some circumstances, it may be more appropriate to store the glass in ultrapure water instead of ethanol. However, storing in water or air makes the glass hydrophobic over time.
	<ol>
		<li>For best results, purchase glass or quartz microscope slides and cover glass.</li>
		<li>Using a scribing pen, place a shallow and short scratch mark onto the center of each glass cover slip.</l...

<ol>
	<li>Pluta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+7:+Differential+Interference+Contrast+in.">Ch 7: Differential Interference Contrast in.</a> Advanced Light Microscopy. 2, 146-197 (1989).</li><li>Stender, A. S., Wang, G., Sun, W., Fang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Gold+Nanorod+Geometry+on+Optical+Response.">Influence of Gold Nanorod Geometry on Optical Response.</a> ACS Nano. 4 (12), 7667-7675 (2010).</li><li>Stender, A. 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=Single+Cell+Optical+Imaging+and+Spectroscopy.">Single Cell Optical Imaging and Spectroscopy.</a> Chemical Reviews. 113 (4), 2469-2527 (2013).</li><li>Mehta, S. B., Sheppard, C. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partially+coherent+image+formation+in+differential+interference+contrast+(DIC)+microscope.">Partially coherent image formation in differential interference contrast (DIC) microscope.</a> Optics Express. 16 (24), 19462-19479 (2008).</li><li>Murphy, D. B., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+1:+Fundamentals+of+Light+Microscopy.">Ch 1: Fundamentals of Light Microscopy.</a> Fundamentals of Light Microscopy and Electronic Imaging, Second edition. , 1-20 (2012).</li><li>Stender, A. S., Augspurger, A. E., Wang, G., Fang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Polarization+Setting+on+Gold+Nanorod+Signal+at+Nonplasmonic+Wavelengths+Under+Differential+Interference+Contrast+Microscopy.">Influence of Polarization Setting on Gold Nanorod Signal at Nonplasmonic Wavelengths Under Differential Interference Contrast Microscopy.</a> Analytical Chemistry. 84 (12), 5210-5215 (2012).</li><li>Wang, G., Sun, W., Luo, Y., Fang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+Rotational+Motions+of+Nano-objects+in+Engineered+Environments+and+Live+Cells+with+Gold+Nanorods+and+Differential+Interference+Contrast+Microscopy.">Resolving Rotational Motions of Nano-objects in Engineered Environments and Live Cells with Gold Nanorods and Differential Interference Contrast Microscopy.</a> Journal of the American Chemical Society. 132 (46), 16417-16422 (2010).</li><li>Kelly, K. L., Coronado, E., Zhao, L. L., Schatz, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Optical+Properties+of+Metal+Nanoparticles:+The+Influence+of+Size,+Shape,+and+Dielectric+Environment.">The Optical Properties of Metal Nanoparticles: The Influence of Size, Shape, and Dielectric Environment.</a> The Journal of Physical Chemistry B. 107 (3), 668-677 (2003).</li><li>Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+All+That's+Gold+Does+Glitter.">Not All That's Gold Does Glitter.</a> MRS Bulletin. 26 (12), 1009-1014 (2012).</li><li>Maier, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plasmonics: Fundamentals and Applications. , (2007).</li><li>Sun, W., Wang, G., Fang, N., Yeung, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength-Dependent+Differential+Interference+Contrast+Microscopy:+Selectively+Imaging+Nanoparticle+Probes+in+Live+Cells.">Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells.</a> Analytical Chemistry. 81 (22), 9203-9208 (2009).</li><li>Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A., Ligler, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+chemical+cleaning+methods+of+glass+in+preparation+for+silanization.">Comparison of chemical cleaning methods of glass in preparation for silanization.</a> Biosensors and Bioelectronics. 14 (8), 683-688 (1999).</li><li>Augspurger, A. E., Sun, X., Trewyn, B. G., Fang, N., Stender, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+Stimulated+Uncapping+Process+of+Gold-Capped+Mesoporous+Silica+Nanoparticles.">Monitoring the Stimulated Uncapping Process of Gold-Capped Mesoporous Silica Nanoparticles.</a> Analytical Chemistry. 90 (5), 3183-3188 (2018).</li><li>Murphy, D. B., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+2:+Light+and+Color.">Ch 2: Light and Color.</a> Fundamentals of Light Microscopy and Electronic Imaging, Second Edition. , 21-33 (2012).</li><li>Wayne, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+3:+The+Dependence+of+Image+Formation+on+the+Nature+of+Light.">Ch 3: The Dependence of Image Formation on the Nature of Light.</a> Light and Video Microscopy (Second Edition). , 43-78 (2014).</li><li>Stender, A. S., Wei, X., Augspurger, A. E., Fang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmonic+Behavior+of+Single+Gold+Dumbbells+and+Simple+Dumbbell+Geometries.">Plasmonic Behavior of Single Gold Dumbbells and Simple Dumbbell Geometries.</a> The Journal of Physical Chemistry C. 117 (31), 16195-16202 (2013).</li><li>Hu, 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=Dark-field+microscopy+studies+of+single+metal+nanoparticles:+understanding+the+factors+that+influence+the+linewidth+of+the+localized+surface+plasmon+resonance.">Dark-field microscopy studies of single metal nanoparticles: understanding the factors that influence the linewidth of the localized surface plasmon resonance.</a> Journal of Materials Chemistry. 18 (17), 1949-1960 (2008).</li><li>Choo, 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=Wavelength-Dependent+Differential+Interference+Contrast+Inversion+of+Anisotropic+Gold+Nanoparticles.">Wavelength-Dependent Differential Interference Contrast Inversion of Anisotropic Gold Nanoparticles.</a> The Journal of Physical Chemistry C. 122 (47), 27024-27031 (2018).</li><li>Funston, A. M., Novo, C., Davis, T. J., Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmon+Coupling+of+Gold+Nanorods+at+Short+Distances+and+in+Different+Geometries.">Plasmon Coupling of Gold Nanorods at Short Distances and in Different Geometries.</a> Nano Letters. 9 (4), 1651-1658 (2009).</li></ol>

When imaging with DIC microscopy, it is critical to optimize the optical components before collecting data. Even minor adjustments to the polarizer in the middle of an experiment can result in significant impacts to the final data6. Moreover, different materials require different polarizer settings. Although large step sizes were utilized here to demonstrate the effect of polarization angle, in an actual experiment, it is imperative to optimize the polarizer setting within 1&#176;&#8211;2&#176; of...

Since the 1980s, differential interference contrast (DIC) microscopy has largely been viewed as an important imaging method reserved for microscale objects within the biological sciences. However, during its development in the 1950s and 1960s, it was intended as a technique for materials science1. With the recent advancements in the material sciences related to plasmonic nanoparticles, an increased interest in the characterization of materials with optical microscopy has taken place.
Many optical techniques are certainly available for nanomaterial characterization (e.g., dark field, brightfield, polarized light, fluorescence, etc.). Dark field is widely popular in nanoparticle research, but it relies solely on the collection of scatter and provides limited information about complex samples2. Fluorescence can be useful, but only with samples that luminesce or that can be properly stained. DIC microscopy has several traits that make it a valuable tool for the analysis of nanoparticles. The most frequently stated advantages of DIC in comparison to other methods and in regards to plasmonic nanoparticles are: no sample staining required, no halo effects, shallow depth of field, and high lateral resolution3. DIC has additional strengths that are valuable to plasmonic nanoparticle research. First of all, two inherent and orthogonal polarization fields are present, and they can be measured simultaneously for spectroscopy purposes2. Secondly, the depolarized signal of nanoparticles is not captured in the final image2, which can be a cause for serious concern in dark field spectroscopy measurements.
The purpose of this article is to provide a clear methodology for utilizing transmitted-light Nomarski DIC microscopy to perform spectroscopy on plasmonic nanoparticles. Although DIC is a powerful technique that can be applied to highly diverse materials, it is also a technique that requires great skill and understanding to operate it properly when imaging nanoparticles. Transmission-based Nomarski DIC microscopy has a complex light path1 that will only be briefly reviewed here. The optical train of DIC is displayed in Figure 1. Light is transmitted through the microscope by first being passed through a polarizer and a beam-splitting Nomarski prism before being focused by the condenser onto the sample plane. After passing through the objective, the light encounters a beam-combining Nomarski prism and an analyzer before exiting to the detector. The two polarizers and Nomarski prisms are critical to formation of the DIC image and are responsible for producing DIC&#8217;s two orthogonal polarization fields1. For the reader interested in knowing more about the working principles and optical path of Nomarski DIC microscopes, or the differences between Nomarski DIC and other styles of DIC, please refer to other well-written accounts on these topics1,4,5,6,7.
It is equally important to understand the basic nature of plasmonic nanoparticles before attempting to perform spectroscopy on them, whether it be with Nomarski DIC, dark field, or any other microscopy technique. In the field of plasmonics, nanoparticles are defined as particulates with dimensions on the scale of 10-100 nm8,9. Nanoparticles can take on many shapes (e.g., spheres, rods, stars, dumbbells, etc.), and most of their important properties arise from interactions with light in the ultraviolet-visible-near infrared range of the electromagnetic spectrum. The term &#8220;plasmonic&#8221; is not restricted to nanoparticles10; however, when discussing nanoparticles, it is used in reference to localized surface plasmon resonance (LSPR). LSPR is a phenomenon in which the conduction electrons in a nanoparticle oscillate due to a Coulombic interaction with electromagnetic radiation of a highly specific and relatively narrow frequency band8. At these same frequencies, plasmonic nanoparticles exhibit increased absorption and scattering of light, making them observable with optical microscopy. In many cases, it is preferred to observe the nanoparticles while placing bandpass filters before the condenser2, to improve imaging contrast and to eliminate light that fails to induce the LSPR effect. Using filters also makes it possible to perform single particle spectroscopy experiments.
LSPR-related optical behavior is highly dependent on the size and shape of the nanoparticles, and it can be investigated with many optical microscopy techniques. However, in order to decipher orientation information of plasmonic nanoparticles with an anisotropic (i.e., non-spherical) shape, it is necessary to utilize polarization of the light field. By carefully rotating the polarization field or the sample substrate at small increments, it is possible to monitor the orientation-dependent spectroscopic properties of individual nanoparticles. Rotation and polarization can also aid in determining whether a spectral feature is due to a dipolar or higher order oscillation of the nanoparticle&#8217;s surface electrons. However, in the case of isotropic (i.e., spherical) nanoparticles, the spectral profile remains essentially unchanged upon rotating the sample under polarized light.
When viewed through a DIC microscope (Figure 2), nanoparticles have an airy disk with a shadow-cast white-and-black appearance against a gray background. Spherical nanoparticles will retain this appearance under rotation and with the changing of bandpass filters; however, the particles will gradually fade from view as the filter&#8217;s central wavelength becomes further separated from the sphere&#8217;s only dipolar LSPR wavelength11. The appearance of nanorods can change quite dramatically as they are rotated2. Nanorods have two LSPR bands with dipolar behavior, the location of which are based on the physical dimensions of the nanorods. When the longitudinal axis of a nanorod is oriented parallel to one of the DIC polarization fields, the airy disc will appear all white or all black if viewed with a bandpass filter associated with that LSPR wavelength. After rotating the sample 90&#176;, it will take on the opposite color. Alternatively, since the transverse axis of a nanorod is perpendicular to the longitudinal axis, the rod will take on the opposite color when switching between filters that match the LSPR wavelengths for the two axes. At other orientations and filter settings, nanorods will appear more like spheres, presenting a variety of shadow-cast airy disc patterns. For nanorods with a transverse axis &#60; 25 nm, it can be difficult to detect signal at that LSPR&#8217;s wavelength using DIC microscopy.
To perform single particle spectroscopy, it is important to use the correct optical components and to align them properly. An objective capable of DIC microscopy must be used. For single particle experiments, 80x or 100x oil objectives are ideal. Nomarski DIC prisms ordinarily come in three varieties: standard, high contrast, and high resolution. The ideal type highly depends on the purpose of the experiment and the size of the nanoparticles. Standard prisms are fine for many experiments; but when working with smaller nanoparticles (&#60; 50 nm), high contrast prisms can be beneficial, since particle contrast decreases as the particles decrease in size11. Adjusting the DIC contrast is achieved either by rotating a polarizer or by translating one of the DIC prisms, depending on the microscope brand or model6.
After setting Kohler illumination and the polarizer settings, it is critical to not readjust these settings while collecting spectroscopy data. Furthermore, a constant average background signal must be maintained at all times during data collection, even when switching between filters and angle settings. The actual ideal background value depends on the dynamic range of the scientific camera, but in general, the background should be in the range of 15%&#8211;40% of the maximum detection level of the camera. This reduces the likelihood of saturating the camera sensor while enabling optimal particle contrast. For collecting spectroscopy data, it is necessary to work with a scientific camera that captures images in black and white, as opposed to a color camera.
Sample preparation is another critical aspect of imaging plasmonic nanoparticles. It is imperative that operators of DIC microscopy have an understanding of the sample&#8217;s optical properties and of the sample&#8217;s substrate. &#8220;Pre-cleaned&#8221; microscope glass is not sufficiently prepared for imaging nanoparticles, and it must be properly re-cleaned before sample deposition to ensure unobstructed observation of the sample. Many cleaning protocols for microscope slides have been previously documented12, but it is not a step that is normally reported in experimental studies.
Finally, data analysis methods are the final component to single particle spectroscopy. The maximum and minimum intensities for each nanoparticle must be measured, as well as the local background average. Particles of interest should be located in areas with no background debris, substrate defects, or uneven illumination. One method for determining the spectral profile of a nanoparticle is by calculating particle contrast at each wavelength, using the equation below11,13,14,15:
<img alt="Equation" src="/files/ftp_upload/59411/59411eq1.jpg" />
Alternatively, a single particle&#8217;s spectrum can be split into its individual maximum and minimum signal components, which represent DIC&#8217;s two polarization fields, thereby displaying the two simultaneously-collected directionally-dependent spectra, through the two equations:
<img alt="Equation" src="/files/ftp_upload/59411/59411eq2.jpg" />
<img alt="Equation" src="/files/ftp_upload/59411/59411eq3.jpg" />

<table border="0" cellpadding="0" cellspacing="0" style="width:1117px;" width="1117">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Contrad 70</td>
			<td style="width:228px;">Decon Labs, Inc.</td>
			<td style="width:157px;">1002</td>
			<td style="width:439px;">For cleaning microscope glass, Available through many chemical suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ethanol</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:157px;">A962-4</td>
			<td>For cleaning and storing microscope glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Glass microscope cover slips</td>
			<td style="width:228px;">Ted Pella</td>
			<td style="width:157px;">260148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Glass microscope slides</td>
			<td style="width:228px;">Ted Pella</td>
			<td style="width:157px;">26007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Gold nanorods</td>
			<td style="width:228px;">Nanopartz</td>
			<td style="width:157px;">DIAM-SPR-25-650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Gold nanospheres (80 nm)</td>
			<td style="width:228px;">Sigma Aldrich</td>
			<td style="width:157px;">742023-25ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">ImageJ</td>
			<td style="width:228px;">NIH</td>
			<td style="width:157px;">N/A</td>
			<td>Free Software availabe for data analysis from NIJ</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:293px;">Nail polish</td>
			<td style="width:228px;">Electron Microscopy Sciences</td>
			<td style="width:157px;">72180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Nikon Ti-E microscope</td>
			<td style="width:228px;">Nikon</td>
			<td style="width:157px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Nitrogen gas</td>
			<td style="width:228px;">Airgas</td>
			<td style="width:157px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">ORCA Flash 4.0 V2+ digital sCMOS camera</td>
			<td style="width:228px;">Hamamatsu</td>
			<td style="width:157px;">77054098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Scribing pen</td>
			<td style="width:228px;">Amazon</td>
			<td style="width:157px;">N/A</td>
			<td>Many options available online for under $10. Not necessary to buy an expensive version.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ultrapure water</td>
			<td style="width:228px;"></td>
			<td style="width:157px;"></td>
			<td>18 megaohm</td>
		</tr>
	</tbody>
</table>

performing spectroscopy on plasmonic nanoparticles with transmission-based nomarski-type differential interference contrast microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

When working with samples that are large enough to be seen with the naked eye, placing landmarks on the glass substrate is not normally required. However, when working with nanomaterials or when rotation of the sample is required, landmarks can provide an easy method for locating, distinguishing, and tracking the orientation of the sample. Although more sophisticated techniques can be utilized for leaving landmarks on glass substrates17, scratching the glass with a...

Watch this Scientific Journal Video about Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy at JoVE.com

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using ...

DIC microscopy is superior to dark field at imaging plasmonic nanoparticles in complex environments. However, DIC also requires greater knowledge and skill to obtain reproducible results. DIC microscopy provides both high lateral resolution and shallow depth of field.Both of these traits are extremely important when monitoring nanoparticles within cells or in complex environments. To begin, use a scribing pen to place a shallow and short scratch mark onto the center of each glass coverslip, and then clean the glass as described in the accompanying text protocol. Next, use a micropipette to remove 100 microliters of a 05-milligram-per-milliliter gold nanoparticle solution from its original storage container, and eject the solution into a 1.5-milliliter centrifuge tube.Centrifuge the sample for 10 minutes at 6, 000 times gravity. When finished, remove the supernatant with a micropipette to get rid of the excess surfactant from the nanoparticle solution. Next, add 100 microliters of ultra-pure water to the centrifuge tube.Briefly vortex the sample to break up the pellet, and then sonicate it for 20 minutes immediately afterwards to fully resuspend the nanoparticle aggregates. Using a micropipette, dropcast six microliters of nanoparticle solution onto the cleaned and scratched glass coverslip. Then carefully flip the coverslip and place it on top of a second larger piece of microscope glass.The drop should quickly spread out evenly between the two pieces of glass. Take care to avoid getting air bubbles trapped between the two pieces of glass. Use a narrow line of nail polish to seal off the edges of the coverslip to prevent evaporation of the liquid.Place the sample onto the microscope stage, and align the objective and condenser of the microscope. Adjust the focus to find the focal plane with the sample on it and locate the scratch mark created earlier. Focus on it and fine-tune the focus until the nanoparticles come into view.To determine the accurate placement of the condenser, utilize the Kohler illumination method by starting with the 20x objective, and then moving to high magnifications such as 80 or 100x. Next, select a region of interest within the sample for imaging. Center the region in the camera's field of view, and adjust the focus as necessary.If the microscope has the de Senarmont design, start with the polarizer set near to maximum background extinction and gradually rotate the polarizer towards decreasing background extinction. The background intensity will gradually increase. The ideal setting is achieved when the nanoparticles reach their greatest intensity difference compared to the local background average.For plasmonic nanoparticles, greatest contrast is typically achieved with a relatively dark background at or near the maximum background extinction. Turn off room lighting to prevent stray illumination from interacting with the process. Put into place a 10-nanometer full-width at half-maximum band-pass filter with its central wavelength co-located with the main localized surface plasmon resonance wavelength in order to view the region of interest.Next, adjust the lamp intensity or exposure time until the background level is at 5%to 40%of the camera's maximum capacity level. No objects within the region of interest should exhibit signal intensities that exceed 90%of the camera's maximum intensity level. Image the sample with a series of band-pass filters that each has a full width at half-maximum of 10 nanometers, and that as a whole enable imaging across the entire wavelength range of interest.Ensure that the background intensity in the images remains within 5%of one another by adjusting the exposure time and not the lamp power. After switching filters, refocus the sample before capturing images. Save the images as uncompressed TIFF files and/or in the software's native file format in order to preserve all relevant information.To begin analysis, open the image with ImageJ. Use the rectangle tool to draw a rectangle around the main region of interest. Then on the toolbar, select image, go to zoom, and select to selection.The imaging window will zoom in on the selected area. Next, select image, go to adjust, and select brightness/contrast. To improve the view of the sample region, adjust the minimum, maximum, brightness, and contrast of the image.These adjustments do not alter the scientific data, they merely enable better visibility of the sample region. Now, use the rectangle tool again and draw a box around the first nanoparticle to be measured. The box should be only slightly larger than the nanoparticle's airy disc.Once selected, go to analyze, and select measure. A new window appears that reports the minimum, maximum, and mean intensities for the pixels located inside of the selected box. Retaining the original size of the box, drag the box to an area immediately adjacent to the particle where the background contrast is relatively even, and no particles or contaminants are present.Use the measure tool again to determine the mean intensity for the background area. Repeat this process for each particle in all of the images in the series. Then export the data to a spreadsheet to calculate the contrast or intensity of each particle across all wavelengths and angles.Enter in the following equation to calculate each particle's contrast. Finally, graph the spectral profile at a given nanoparticle position by plotting data with the wavelength along the X-axis and the contrast or intensity along the Y-axis. To determine the optimal imaging setting, these gold nanospheres were imaged at several polarizer settings.At zero degrees, the particles appear mostly white with a dark stripe running across their midsection. This is indicative of cross-polarization for nanosphere samples. When the polarizer is rotated to different angles, the particles cast dark shadows towards one corner.However, the particle contrast values change dramatically. For this sample, the optimal imaging setting is with a polarizer shift of 10 degrees. Here the wavelength is plotted against contrast of gold nanospheres.Each data point represents an average of 20 nanospheres for each particle diameter. The peak contrast for each particle shifts slightly depending on its size. The intensity and rotation is even more important with anisotropic shapes such as this gold nanorod.Here the wavelength is plotted against intensity at two different polarizer settings. In each case, the bright side's intensity is much stronger than the dark side's. The intensity profile of a single gold nanorod at its localized surface plasmon resonance wavelength during rotation of the stage shows a large intensity difference between the dark and bright sides as the sample is rotated through 180 degrees.Sample imaging is extremely important. It requires both diligence and patience on the part of the microscopist. If you are performing an experiment that requires reimaging the sample over a series of days, then landmarks such as the scratch marks are critical in relocating your region of interest.DIC microscopy is growing in interest by researchers who study nanoparticles, especially in dynamic and cellular environments. Because DIC offers optimal spatial resolution, especially in complex sampling environments.

performing-spectroscopy-on-plasmonic-nanoparticles-with-transmission

Research

JoVE Journal

Chemistry

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal structure and probing the hydrogen-bond (H-bond) dynamics of the water molecules adsorbed on solid surfaces are fundamental issues of water science, which remains a great challenge owing to the light mass and small size of hydrogen. Scanning tunneling microscopy (STM) is a promising tool for attacking these problems, thanks to its capabilities of sub-&#197;ngstr&#246;m spatial resolution, single-bond vibrational sensitivity, and atomic/molecular manipulation. The designed experimental system consists of a Cl-terminated tip and a sample fabricated by dosing water molecules in situ onto the Au(111)-supported NaCl(001) surfaces. The insulating NaCl films electronically decouple the water from the metal substrates, so the intrinsic frontier orbitals of water molecules are preserved. The Cl-tip facilitates the manipulation of the single water molecules, as well as gating the orbitals of water to the proximity of Fermi level (EF) via tip-water coupling. This paper outlines the detailed methods of submolecular resolution imaging, molecular/atomic manipulation, and single-bond vibrational spectroscopy of interfacial water. These studies open up a new route for investigating the H-bonded systems at the atomic scale.

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced Raman spectroscopy (SERS) with a low detection limit of ~0.2 &#956;M for multiple characteristic peaks in the fingerprint region, using a modular spectrometer. This biosensing scheme is mediated by the host-guest complexation between a macrocycle, cucurbit[7]uril (CB7), and UA, and the subsequent formation of precise plasmonic nanojunctions within the self-assembled Au NP: CB7 nanoaggregates. A facile Au NP synthesis of desirable sizes for SERS substrates has also been performed based on the classical citrate-reduction approach with an option to be facilitated using a lab-built automated synthesizer. This protocol can be readily extended to multiplexed detection of biomarkers in body fluids for clinical applications.

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced ...

Preparation of Silver-Palladium Alloyed Nanoparticles for Plasmonic Catalysis under Visible-Light Illumination

Localized surface plasmon resonance (LSPR) in plasmonic nanoparticles (NPs) can accelerate and control the selectivity of a variety of molecular transformations. This opens possibilities for the use of visible or near-IR light as a sustainable input to drive and control reactions when plasmonic nanoparticles supporting LSPR excitation in these ranges are employed as catalysts. Unfortunately, this is not the case for several catalytic metals such as palladium (Pd). One strategy to overcome this limitation is to employ bimetallic NPs containing plasmonic and catalytic metals. In this case, the LSPR excitation in the plasmonic metal can contribute to accelerate and control transformations driven by the catalytic component. The method reported herein focuses on the synthesis of bimetallic silver-palladium (Ag-Pd) NPs supported on ZrO2 (Ag-Pd/ZrO2) that acts as a plasmonic-catalytic system. The NPs were prepared by co-impregnation of corresponding metal precursors on the ZrO2 support followed by simultaneous reduction leading to the formation of bimetallic NPs directly on the ZrO2 support. The Ag-Pd/ZrO2 NPs were then used as plasmonic catalysts for the reduction of nitrobenzene under 425 nm illumination by LED lamps. Using gas chromatography (GC), the conversion and selectivity of the reduction reaction under the dark and light irradiation conditions can be monitored, demonstrating the enhanced catalytic performance and control over selectivity under LSPR excitation after alloying non-plasmonic Pd with plasmonic metal Ag. This technique can be adapted to a wide range of molecular transformations and NPs compositions, making it useful for the characterization of the plasmonic catalytic activity of different types of catalysis in terms of conversion and selectivity.

Presented here is a protocol for the synthesis of silver-palladium (Ag-Pd) alloy nanoparticles (NPs) supported on ZrO2 (Ag-Pd/ZrO2). This system allows for harvesting energy from visible light irradiation to accelerate and control molecular transformations. This is illustrated by nitrobenzene reduction under light irradiation catalyzed by Ag-Pd/ZrO2 NPs.

Localized surface plasmon resonance (LSPR) in plasmonic nanoparticles (NPs) can accelerate and control the selectivity of a variety of molecular ...

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron microscopy. In this paper, we describe how to prepare an in situ heating experiment for studying the effect of temperature on the formation of gold nanoparticles driven by radiolysis in water. The protocol of the experiment is fairly simple involving a special liquid cell with uniform heating capabilities up to 100 &#176;C, a liquid-cell TEM holder with flow capabilities and an integrated interface for controlling the temperature. We show that the nucleation and growth mechanisms of gold nanoparticles are drastically impacted by the temperature in liquid cell. Using STEM imaging and nanodiffraction, the evolution of the density, size, shape and atomic structure of the growing nanoparticles are revealed in real time. Automated image processing algorithms are exploited to extract useful quantitative data from video sequences, such as the nucleation and growth rates of nanoparticles. This approach provides new inputs for understanding the complex physico-chemical processes at play during the liquid-phase synthesis of nanomaterials.

Temperature control during liquid-phase electron microscopy experiments opens up new perspectives of studying the dynamic of nanoparticles in liquid environments mimicking their formation or application media. Using recently developed heating liquid cells, we directly observed the influence of temperature on the nucleation and growth processes of gold nanoparticles in water.

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron ...

Synthesis of Metal Nanoparticles Supported on Carbon Nanotube with Doped Co and N Atoms and its Catalytic Applications in Hydrogen Production

A method for facile synthesis of nanostructured catalysts supported on carbon nanotubes with atomically dispersed cobalt and nitrogen dopant is presented herein. The novel strategy is based on a facile one-pot pyrolysis treatment of cobalt (II) acetylacetonate and nitrogen-rich organic precursors under Ar atmosphere at 800 &#176;C, resulting in the formation of Co- and N- co-doped carbon nanotube with earthworm-like morphology. The obtained catalyst was found to have a high density of defect sites, as confirmed by Raman spectroscopy. Here, cobalt (II) nanoparticles were stabilized on the atomically dispersed cobalt- and nitrogen-doped carbon nanotubes. The catalyst was confirmed to be effective in the catalytic hydrolysis of ammonia borane, in which the turnover frequency was 5.87 mol H2&#183;molCo-1&#183;min-1, and the specific hydrogen generation rate was determined to be 2447 mL H2&#183;gCo-1&#183;min-1. A synergistic function between the Co nanoparticle and the doped carbon nanotubes was proposed for the first time in the catalytic hydrolysis of ammonia borane reaction under a mild condition. The resulting hydrogen production with its high energy density and minimal refueling time could be suitable for future development as energy sources for mobile and stationary applications such as road trucks and forklifts in transport and logistics.

Here, we present a protocol to synthesize Co nanoparticles supported on carbon nanotubes with Co- and N- dopants for hydrogen productions.

A method for facile synthesis of nanostructured catalysts supported on carbon nanotubes with atomically dispersed cobalt and nitrogen dopant is presented ...

Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy

With the ever-increasing use of Li-ion batteries, especially due to their adoption in electric vehicles, their safety is in prime focus. Thus, the all-solid-state batteries (ASSBs) that use solid electrolytes instead of liquid electrolytes, which reduce the risk of flammability, have been the center stage of battery research for the last few years. However, in the ASSB, the ion transportation through the solid-solid electrolyte-electrode interface poses a challenge due to contact and chemical/electrochemical stability issues. Applying a suitable coating around the electrode and/or electrolyte particles offers a convenient solution, leading to better performance. For this, researchers are screening potential electronic/ionic conductive and nonconductive coatings to find the best coatings with suitable thickness for long-term chemical, electrochemical, and mechanical stability. Operando transmission electron microscopy (TEM) couples high spatial resolution with high temporal resolution to allow visualization of dynamic processes, and thus is an ideal tool to evaluate electrode/electrolyte coatings via studying (de)lithiation at a single particle level in real-time. However, the accumulated electron dose during a typical high-resolution in situ work may affect the electrochemical pathways, evaluation of which can be time-consuming. The current protocol presents an alternative procedure in which the potential coatings are applied on Si nanoparticles and are subjected to (de)lithiation during operando TEM experiments. The high volume changes of Si nanoparticles during (de)lithiation allow monitoring of the coating behavior at a relatively low magnification. Thus, the whole process is very electron-dose efficient and offers quick screening of potential coatings.

Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy

Utilizing the volume change of Si nanoparticles during (de)lithiation, the present protocol describes a screening method of potential coatings for all-solid-state batteries using in situ transmission electron microscopy.