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.</li>
		<li>Clean all microscope glass, even if it is purchased &#8220;pre-cleaned&#8221;, to remove glass shards, dust, powder, organic residue, and any other contaminants that affect imaging quality or sample deposition. 
		NOTE: This cleaning method below works well for the types of samples described here and avoids the use of harsh chemicals. Harsher chemicals can etch the glass and require more care in handling and disposal.
		<ol>
			<li>Place microscope glass onto storage racks and then into a beaker, or into a staining jar. Do not place microscope glass at the bottom of beakers and other lab glassware without racking, because each piece and surface of microscope glass should be fully exposed to the cleaning agents.</li>
			<li>Pour ~1 mL of liquid detergent (Table of Materials) into the container and top off the container with water. Sonicate for 30 min. 
			NOTE: Once the cleaning process begins, only handle the glass while wearing gloves, to avoid leaving fingerprint residue on the glass.</li>
			<li>Pour out the liquid contents of the cleaning container into a sink. Rinse container several times with ultrapure water to remove all appearance of detergent. Refill the container with ultrapure water. Sonicate the container with microscope glass for another 30 min.</li>
			<li>Repeat the previous step at least once more. Perform additional rounds of sonication in water until it is obvious that all traces of the detergent have been removed.</li>
			<li>Pour out the contents of the cleaning container. Rinse the container with ultrapure water. Refill the container with ethanol. Sonicate microscope glass for 30 min.</li>
			<li>Pour out the contents of the cleaning container into a waste container. Refill with ethanol. Cover the container to prevent loss of ethanol through evaporation. Store the microscope glass in this container until time of experiment. Slides remain clean and usable as long as they remain submerged in ethanol inside a covered container.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of nanoparticle solution
	<ol>
		<li>Using a micropipette, remove a 100 &#181;L aliquot of 0.05 mg/mL gold nanoparticle solution from its original storage container and eject the solution into a 1.5 mL centrifuge tube.</li>
		<li>Centrifuge the sample for 10 min at 6,000 x g.</li>
		<li>Remove the supernatant with a micropipette, in order to remove excess surfactant.</li>
		<li>Using a micropipette, place 100 &#181;L of ultrapure water into the centrifuge tube. 
		NOTE: If not all of the supernatant can be removed on the first attempt, repeat the centrifugation and resuspension steps.</li>
		<li>Briefly vortex the sample to resuspend the pellet. Sonicate immediately afterwards for 20 min to fully resuspend and break up nanoparticle aggregates. 
		NOTE: If the sample is not used immediately, it should be sonicated again for 20 min before depositing solution onto microscope glass.</li>
	</ol>
	</li>
	<li>Sample deposition
	<ol>
		<li>Remove cleaned cover slips and microscope slides from their storage containers. Blow dry the glass with pressurized nitrogen or argon.</li>
		<li>Using a micropipette, drop-cast 6 &#181;L of nanoparticle solution from step 1.2.5 onto the cover slip. To spread out the droplet evenly, carefully place a second, larger piece of microscope glass on top of the cover slip, such as a second cover slip or a microscope slide. Avoid getting air bubbles trapped between the two pieces of glass.
		<ol>
			<li>Turn the sample substrate over, and seal off the edges of the cover slip with a narrow line of nail polish in order to prevent evaporation of the medium solution.</li>
			<li>Alternatively, to image the sample &#8220;dry&#8221;, allow the solution to stand for 5&#8211;15 min on the cover slip, before removing the unwanted piece of glass. Gently blow the cover slip dry with pressurized nitrogen or argon.</li>
		</ol>
		</li>
		<li>If possible, image samples immediately after preparation. If that is not possible, store samples in a covered container, such as a Petri dish until imaging.</li>
	</ol>
	</li>
</ol>
2. DIC imaging
<ol>
	<li>Align objective and condenser.
	<ol>
		<li>After placing the sample onto the microscope, find the focal plane with the sample on it. First locate and focus on the scratch mark created earlier. Then fine tune the focus until nanoparticles come into view.</li>
		<li>To determine the accurate placement of the condenser, utilize the Kohler Illumination method.5 Kohler Illumination at high magnification (80x, 100x) is more easily achieved by first setting the Kohler Illumination at a lower magnification, such as 20x. 
		NOTE: Normally, Kohler Illumination does not need to be re-adjusted during imaging of a single sample. However, it is good practice to verify that Kohler Illumination is properly set when switching to a new microscope slide.</li>
	</ol>
	</li>
	<li>Optimize contrast settings.
	<ol>
		<li>Select a region of interest within the sample for imaging. Center the region in the camera&#8217;s field of view and adjust focus as necessary.
		<ol>
			<li>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.</li>
			<li>If the microscope does not have a de Senarmont design, start with the optical train set at maximum background extinction. In this case, gradually adjust the objective prism position towards decreasing background extinction. 
			NOTE: The ideal setting is achieved when the nanoparticles reach their greatest intensity difference (i.e., contrast) from the averaged local background value. For plasmonic nanoparticles, optimal contrast is normally achieved with a relatively dark background, thus at settings near maximum background extinction.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Image the sample.
	<ol>
		<li>Turn off room lighting to prevent stray illumination from interacting with process.</li>
		<li>While viewing the nanoparticles with a scientific imaging camera, determine the optimal background level. Using a 10 nm full width at half maximum (FWHM) bandpass filter with its central wavelength co-located with the main LSPR wavelength, view the region of interest. Adjust the lamp intensity or exposure time until the background level is in the range of 15%&#8211;40% of the camera&#8217;s maximum capacity level and no objects within the region of interest exhibit signal intensities that exceed 90% of the camera&#8217;s maximum intensity level. 
		NOTE: The goal of step 2.3.2 is to prevent saturating the sensor when switching between filters. The ideal background level will vary between samples and cameras. Once this step is completed, exposure time can be adjusted but not the lamp intensity.</li>
		<li>Image the sample with a series of bandpass filters that each has a FWHM of 10 nm and that as a whole enable imaging across the entire wavelength range of interest. Ensure that the background intensity remains consistent from image to image (within ~5% of one another) by adjusting the exposure time. After switching filters, re-focus the sample before image capture.</li>
		<li>Save the Images as uncompressed TIFF files and/or in the software&#8217;s native file format, in order to preserve all information.</li>
	</ol>
	</li>
	<li>Rotate the sample.
	<ol>
		<li>After collecting images of the sample at its original position, the sample can now be rotated and imaged at additional orientations in the light path. Perform rotation at regular intervals (e.g., 10&#176; or 15&#176;) across either a 180&#176; or 360&#176; range. 
		NOTE: Rotation requires a rotatable sample stage.</li>
		<li>As in sections 2.1-2.3, adjust camera settings to provide a consistent background level from image to image. 
		NOTE: No adjustment to Kohler Illumination should be made.</li>
	</ol>
	</li>
</ol>
3. Data analysis using ImageJ
NOTE: The following calculations can be performed in a variety of software packages, and sometimes in the native program used to collect the images. ImageJ is a freely available software from the National Institutes of Health (NIH).
<ol>
	<li>Calculate particle contrast or intensity.
	<ol>
		<li>Open the image with ImageJ.</li>
		<li>Select the Rectangle tool and draw a rectangle around the main region of interest.</li>
		<li>On the Tool bar, select Image, then Zoom, then To Selection. The imaging window will zoom in on the selected area.</li>
		<li>On the Tool bar, select Image, then Adjust, then Brightness/Contrast. A new window appears. To enable better viewing of the sample region, adjust the four settings: Minimum, Maximum, Brightness, and Contrast. These adjustments do not alter the scientific data, they merely enable better visibility of the sample region. 
		NOTE: Steps 3.1.3 and 3.1.4 may be performed multiple times and in reverse order.</li>
		<li>Using the rectangle tool again, draw a box around the first nanoparticle to be measured. The box should be only slightly larger than the nanoparticle&#8217;s airy disc.</li>
		<li>On the Tool bar, select Analyze, then Measure. A new window appears that reports the Minimum, Maximum, and Mean Intensities for the pixels located inside of the selected box.</li>
		<li>Drag the box used to measure the nanoparticle to an area immediately adjacent to the particle, where the background contrast is relatively even and no particles or contaminants are present. Retain the original size of the box.</li>
		<li>Use the Measure tool to determine the Mean Intensity for the background area.</li>
		<li>Measure the remaining particles and an adjacent background area for each.</li>
		<li>Repeat the process for each particle in all of the images in the series.</li>
		<li>Export the data to a spreadsheet to calculate the contrast or intensity of each particle, across all wavelengths and angles.</li>
		<li>Calculate each particle&#8217;s contrast, using the following equation13,14,15: 
		<img alt="Equation" src="/files/ftp_upload/59411/59411eq1.jpg" /> 
		NOTE: Using this equation, particle contrast should always be &#62; 0.</li>
		<li>Calculate the particle&#8217;s background-adjusted maximum value by dividing the measured maximum particle intensity by the background mean: 
		<img alt="Equation" src="/files/ftp_upload/59411/59411eq2.jpg" /></li>
		<li>Likewise, calculate the background-adjusted minimum value by dividing the measured minimum particle intensity by the background mean: 
		<img alt="Equation" src="/files/ftp_upload/59411/59411eq3.jpg" /> 
		NOTE: As calculated, the maximum should have a value greater than one, while the minimum will be less than one. It is acceptable to subtract each value by &#8220;1&#8221;, so that the average background is essentially zero, the maximum is represented as a positive value, and the minimum value is assigned a negative value16. This latter approach allows the analyst to separately consider what is occurring along each of the polarization fields, which is useful when studying anisotropic particles.</li>
		<li>To graph the spectral profile at a given nanoparticle position, plot data with the wavelength along the x-axis and the contrast or intensity along the y-axis.</li>
		<li>To graph the rotational profile at a given wavelength, plot the rotation angle along the x-axis and the contrast or intensity along the y-axis.</li>
	</ol>
	</li>
</ol>

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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. 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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. 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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>

The author has nothing to disclose.

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 ...

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

Research

JoVE Journal

Chemistry

7.5K Views.  Ohio University. 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.

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

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) ...

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

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 ...

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.