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 ...

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