Financial support by the ERC through the Advanced Investigator Grant HYMEM, by the DFG through the Nanosystems Initiative Munich (NIM) and through the Sonderforschungsbereich (SFB1032), project A8 is gratefully acknowledged. We are thankful to Dr. Alexander Ohlinger, Dr. Sol Carretero-Palacios and Spas Nedev for support and fruitful discussions.

1. Experimental Setup
<ol>
	<li>Use an up-right microscope and a dark field oil condenser with a numerical aperture (NA) = 1.2 for dark field illumination. Use a water immersion objective with 100X magnification and a NA = 1.0 for particle observations and trapping. Use an air objective with 10X magnification and a NA = 0.2 to follow the motion of the Nauplius.</li>
	<li>Use an optical tweezers setup with a 1,064 nm continuous wave laser coupled into the up-right microscope. Set the laser power of the optical trap to 100 mW (measured with a power meter after the objective).</li>
	<li>Use a CMOS high-speed camera or a digital single lens reflex (DSLR) camera to detect and image the gold particle movement in the optical trap and the motion of the Nauplius.</li>
	<li>Use a notch filter to prevent the laser from entering the camera.</li>
	<li>Use a power meter to measure the laser power after the objective.</li>
</ol>
2. Sample Preparation
<ol>
	<li>Pipette a water droplet (180 &#181;l) on a microscope glass slide and position the sample on the dark field microscope.</li>
	<li>Pipette a Nauplius from a small water tank to the water droplet.</li>
	<li>Use a 10X air objective to observe the movement of the Nauplius in the solution and record a video stream.</li>
	<li>Use a gold nanoparticle with a diameter of 60 nm as a detector to observe the fluid motion generated by the Nauplius. Therefore, add 5 &#181;l of a highly diluted particle solution into the water droplet, so that approximately one particle can be seen in the field of view with a 100X water immersion objective.</li>
</ol>
3. Particle Tracking Experiment
<ol>
	<li>Trap one gold nanoparticle with the optical tweezer. Therefore, bring the 1,064 nm trapping laser close to a gold nanoparticle that is diffusing in solution by moving the microscope stage. The attractive optical forces pull the gold nanoparticle towards the focal point of the laser beam. The trapped particle is not diffusing anymore and rather keeps its position. Take a video stream of the trapped nanoparticle with the DSLR camera at a frame rate of 50 Hz for 30 sec.</li>
	<li>Turn off the laser of the optical tweezer and release the gold nanoparticle from the trap.</li>
	<li>Use a particle tracking program to readout the position of the optically trapped gold particle at each frame of the video stream. A fast Fourier transformation (FFT) of the particle&#39;s x-y-position over time reveals a frequency spectrum. 
	NOTE: Here, a self-written &#8216;IGOR PRO&#8217; computer program code was used to analyze the particle center position in the x-y-plane over time and for FFT analysis.</li>
	<li>As an alternative to a self-written IGOR code use the freely available &#8216;Video Spot Tracker&#8217; program for tracking the particle in the video. Use the commercial software &#8216;Origin&#8217; to perform the Fourier transformation of the tracking data:
	<ol>
		<li>Drag the video file to the open program &#8216;Video Spot Tracker&#8217;.</li>
		<li>Mouse click on the particle seen in the first picture of the video stream and a circular region of interest appears.</li>
		<li>Choose &#8220;symmetric&#8221; and &#8220;optimize&#8221; in the top command prompt window to optimize the tracking of the particle.</li>
		<li>Mouse click &#8220;logging&#8221; in the top command prompt window and choose a folder to save the data. The tracking data will be saved as a data spreadsheet.</li>
		<li>Mouse click &#8220;play video&#8221; on the left command prompt window of the tracking program and wait until all frames of the video are analyzed.</li>
		<li>Close the program and open the saved data spreadsheet with &#8216;Origin&#8217;. Set the column values as &#8220;y1&#8221; and &#8220;y2&#8221;.</li>
		<li>Set time steps for each video frame as &#8220;x&#8221; in the &#8216;Origin&#8217; data spreadsheet.</li>
		<li>Mark the x-position column and perform a FFT by choosing &#8220;Data Analysis&#8221; and &#8220;FFT&#8221; in the top command prompt window. Repeat the step for the y-position column.</li>
		<li>Plot the amplitudes of the calculated FFT signal in x- and y-direction versus the frequency.</li>
	</ol>
	</li>
</ol>
4. Numerical Simulation
<ol>
	<li>Calculate the polarizability &#945; of the 60 nm gold particle by using the computer program &#8216;Mathematica&#8217;.
	<ol>
		<li>Use equation (1) to calculate the polarizability according to Kuwata et al.16: 
		<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/51502/51502eq1.jpg" width="300" /> (1)</li>
		<li>Define the following three parameters in the program code: the wavelength-dependent complex dielectric function of the gold particle, the nanoparticle radius, and the refractive index of the surrounding medium.</li>
	</ol>
	</li>
	<li>Use the description of the electric field distribution of a focused Gaussian beam according to Agayan et al.17 to calculate the optical forces acting on a 60 nm gold particle: 
	<img alt="Equation 2" fo:content-width="4in" src="/files/ftp_upload/51502/51502eq2.jpg" width="500" /> (2)
	<ol>
		<li>Use equations (3)-(6) from Agayan et al.17 to calculate both, the gradient and scattering forces acting on the particle: 
		<img alt="Equation 3" fo:content-width="3.5in" src="/files/ftp_upload/51502/51502eq3.jpg" width="350" /> (3) 
		<img alt="Equation 4" fo:content-width="3in" src="/files/ftp_upload/51502/51502eq4.jpg" width="300" /> (4) 
		<img alt="Equation 5" fo:content-width="5in" src="/files/ftp_upload/51502/51502eq5.jpg" width="500" /> (5) 
		<img alt="Equation 6" fo:content-width="3in" src="/files/ftp_upload/51502/51502eq6.jpg" width="300" /> (6)</li>
		<li>In the program code, define the parameters for the laser power, the numerical aperture of the objective, and the complex polarizability of the nanoparticle.</li>
		<li>Sum up the gradient force and the scattering force to calculate the total optical force acting on the gold particle in an optical trap.</li>
	</ol>
	</li>
	<li>Run the simulation by simultaneously pressing &#8220;Control&#8221; and &#8220;Enter&#8221;.</li>
</ol>

<ol>
	<li>Hellawell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological indicators of freshwater pollution and environmental management. , (1986).</li><li>Diamond, J. M., Barbour, M. T., Stribling, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+comparing+bioassessment+approaches+and+their+results:+A+perspective.">Characterizing and comparing bioassessment approaches and their results: A perspective.</a> Journal of the North American Benthological Society. 15, 713-727 (1996).</li><li>Carlisle, D. 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=The+quality+of+our+Nation&#8217;s+waters&#8212;Ecological+health+in+the+Nation&#8217;s+streams,+1993&#8211;2005.">The quality of our Nation&#8217;s waters&#8212;Ecological health in the Nation&#8217;s streams, 1993&#8211;2005.</a> U.S. Geological Survey Circular. 1391, (2013).</li><li>Boxhall, G. A., Defaye, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+diversity+of+copepods+(Crustacea+Copepoda)+in+freshwater.">Global diversity of copepods (Crustacea Copepoda) in freshwater.</a> Hydrobiologia. 595 (1), 195-207 (2008).</li><li>Ferdous, Z., Muktadir, A. K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review:+Potentiality+of+Zooplankton+as+Bioindicator.">A Review: Potentiality of Zooplankton as Bioindicator.</a> American Journal of Applied Sciences. 6 (10), 1815-1819 (2009).</li><li>Andersen Borg, C. M., Bruno, E., Ki&#248;rboe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kinematics+of+Swimming+and+Relocation+Jumps+in+Copepod+Nauplii.">The Kinematics of Swimming and Relocation Jumps in Copepod Nauplii.</a> PLoS ONE. 7 (10), (2012).</li><li>Gilchrist, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+Form+of+the+Brine+Shrimp+Artemia+Salina.">Growth and Form of the Brine Shrimp Artemia Salina.</a> Journal of Zoology. 134 (2), 221-235 (1960).</li><li>Boone, E., Baas-Becking, L. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+Effects+on+Egga+and+Nauplii+of+Artemia+Salina+L.">Salt Effects on Egga and Nauplii of Artemia Salina L.</a> Journal of General Physiology. 14 (6), 453-763 (1931).</li><li>Drewes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+investigations+of+hatching+in+brine+shrimp+cysts.">Quantitative investigations of hatching in brine shrimp cysts.</a> Association for Biology Laboratory Education. 27, 299-312 (2006).</li><li>Williams, T. 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+model+of+rowing+propulsion+and+the+ontogeny+of+locomotion+in+Artemia+larvae.">A model of rowing propulsion and the ontogeny of locomotion in Artemia larvae.</a> Biological Bulletin. 187, 164-173 (1994).</li><li>Croghan, P. 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+Mechanism+of+Osmotic+Regulation+in+the+Artemia+Salina+(L.):+The+Physiology+of+the+Branchiae.">The Mechanism of Osmotic Regulation in the Artemia Salina (L.): The Physiology of the Branchiae.</a> Journal of Experimental Biology. 35, 234-242 (1958).</li><li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+single-beam+gradient+force+optical+trap+for+dielectric+particles.">Observation of a single-beam gradient force optical trap for dielectric particles.</a> Optics Letters. 11 (5), 288-290 (1986).</li><li>Svoboda, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+of+metallic+Rayleigh+particles.">Optical trapping of metallic Rayleigh particles.</a> Optics Letters. 19 (13), 930-932 (1994).</li><li>Hansen, P. M., Bhatia, V. K., Harrit, N., Oddershede, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+Optical+Trapping+Range+of+Gold+Nanoparticles.">Expanding the Optical Trapping Range of Gold Nanoparticles.</a> Nano Letters. 5 (10), 1937-1942 (2005).</li><li>Ohlinger, A., Deak, A., Lutich, A. A., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optically+Trapped+Gold+Nanoparticle+Enables+Listening+at+the+Microscale.">Optically Trapped Gold Nanoparticle Enables Listening at the Microscale.</a> Physical Review Letters. 108 (1), (2012).</li><li>Kuwata, H., Tamaru, H., Esumi, K., Miyano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resonant+light+scattering+particles:+Practical+analysis+beyond+Rayleigh+approximation.">Resonant light scattering particles: Practical analysis beyond Rayleigh approximation.</a> Applied Physics Letters. 83 (22), 4625-4628 (2003).</li><li>Agayan, R. R., Gittes, F., Kopelman, R., Schmidt, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+near+resonance+absorption.">Optical trapping near resonance absorption.</a> Applied Optics. 41 (12), 2318-2327 (2002).</li><li>Klar, T., Perner, M., Grosse, S., von Plessen, G., Spirkl, W., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-Plasmon+Resonances+in+Single+Metallic+Nanoparticles.">Surface-Plasmon Resonances in Single Metallic Nanoparticles.</a> Physical Review Letters. 80, 4249-4252 (1998).</li><li>Ohlinger, A., Nedev, S., Lutich, A. A., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optothermal+Escape+of+Plasmonically+Coupled+Silver+Nanoparticles+from+a+Three-Dimensional+Optical+Trap.">Optothermal Escape of Plasmonically Coupled Silver Nanoparticles from a Three-Dimensional Optical Trap.</a> Nano Letters. 11 (4), 1770-1774 (2011).</li><li>Urban, A. S., Lutich, A. A., Stefani, F. D., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Printing+Single+Gold+Nanoparticles.+Nano+Letters.">Laser Printing Single Gold Nanoparticles. Nano Letters.</a> Nano Letters. 10 (12), 4794-4798 (2010).</li><li>Urban, A. S., Fedoru, K. M., Nedev, S., Lutich, A., Lohmueller, T., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shrink-to-fit+Plasmonic+Nanostructures.">Shrink-to-fit Plasmonic Nanostructures.</a> Advanced Optical Materials. 1 (2), 123-127 (2013).</li></ol>

The authors declare that they have no competing financial interests.

Dark field microscopy is a powerful tool for visualizing gold nanoparticles with dimensions below the optical diffraction limit, since the scattering cross section of the metal nanoparticles exceeds their geometric cross section (cp. Figure 2A)18. In a tweezer setup, this approach even allows to distinguish if only a single or multiple gold nanoparticles are trapped by the laser beam because plasmonic coupling between the particles causes a red-shift of the plasmon resonance frequency15</...

Water quality assessment based on chemical and biological indicators is of fundamental importance to gain insight on the state and environmental conditions of an aquatic ecosystem1-3. Classical methods for chemical water analysis are based on organoleptic properties or the determination of physicochemical parameters. Biological indicators, on the other hand, are animal species whose presence and viability provide insight on the environmental conditions and the effect of pollutants for an ecosystem that they occur in. Typical examples for bioindicators are Copepods, a group of small water crustaceans, which can be found in nearly any water habitat4,5. Observing the activity and viability of these species from a water sample can thus be used to obtain information on the overall conditions of an ecosystem5. The larvae of Copepods, which are called Nauplii, use rhythmic strokes of their antennae (each larva has three pairs of appendages at their head region) to swim in water6. The frequency and intensity of these strokes is thereby a direct indicator of the age, fitness, and environmental conditions of the animal7-10. Any investigations on these specimens are usually done with a microscope by observing and counting the antenna strokes of the Nauplii directly. Due to their size (~100-500 &#181;m)11, this often requires to do measurements either one by one or to fix a single Nauplius to a substrate.
Here, we demonstrate a new approach to observe the activity of Copepod larvae in water samples by using an optically trapped gold nanoparticle as an ultra-sensitive detector. Optical tweezers are typically used by many groups as a fine experimental tool to apply or measure forces between molecules down to the piconewton range12-14. More recently, the range of applications for optical tweezers has been expanded to observe acoustic vibrations and solvent fluctuations in liquid media by monitoring the motion of nano- and microparticles that are confined in an optical trap15. Particles that are immersed in a liquid are subjected to Brownian motion. Inside an optical trap, however, this motion is partially damped by a strong, laser induced, gradient force. Therefore, the stiffness of the optical trap and the localization of the particle within the focus of the laser beam can be tuned by the laser power. At the same time, it is possible to reveal characteristics about the trapping potential and to analyze interactions of molecules with the particle by monitoring the time-dependent particle motion in the trap. This approach renders it possible to pick up the frequency, intensity, and the direction of the fluidic motion that is generated by a moving object in its liquid environment. We demonstrate how this general idea can be applied to obtain a frequency spectrum of the motion of an individual Nauplius without the requirement to directly interfere with the specimen. This experimental approach introduces a new general concept for the observation of the motile behavior of aquatic specimens in a very sensitive way. For observations on bioindicator species, this could expand the current methodology for water analysis and could be applied to gain information about the health and the integrity of aquatic ecosystems.

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Microscope Zeiss Axio Scope.A1</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:201px;">490035-0012-000</td>
			<td style="width:303px;">dark field illumination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Water objective Achroplan</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:201px;">440087</td>
			<td>100x magnification, NA=1.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Air objective Epiplan</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:201px;">442934</td>
			<td>10x magnification, NA=0.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Dark field oil condenser</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:201px;">445323</td>
			<td>NA=1.2</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:244px;">Cobolt Rumba CW 1064 nm DPSSL</td>
			<td style="width:127px;">Cobolt&#160;</td>
			<td style="width:201px;">1064-05-01-2000-500</td>
			<td>1064nm, CW, &#955;=1064nm, 2 Watt, TEM00</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:244px;">Beam expander</td>
			<td style="width:127px;">Edmund Optics</td>
			<td style="width:201px;"></td>
			<td>Part no. 1064 2-8X 64414</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">High Speed Camera Dimax HD</td>
			<td style="width:127px;">PCO. Germany</td>
			<td style="width:201px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Color Camera Canon EOS 500 D&#160;</td>
			<td style="width:127px;">Canon</td>
			<td></td>
			<td>FAQ-ID: 8201395700</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Notch filter StopLine 532/1064</td>
			<td style="width:127px;">Semrock</td>
			<td style="width:201px;">A11149-711265</td>
			<td>Part no. NF01-532U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Water&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:201px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Nauplius Artemia Salina</td>
			<td style="width:127px;"></td>
			<td style="width:201px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Gold colloid</td>
			<td style="width:127px;">BBInternational</td>
			<td style="width:201px;">Batch 13741&#160;</td>
			<td>Diameter 60nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MQMie Version 3.2&#160;</td>
			<td style="width:127px;">r. Michael Quinten</td>
			<td style="width:201px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mathematica 8.0</td>
			<td style="width:127px;">Wolfram</td>
			<td style="width:201px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Comsol Multiphysics 4.0&#160;</td>
			<td style="width:127px;">COMSOL, Inc.</td>
			<td style="width:201px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the movement of the nauplius 'artemia salina' by optical tracking of plasmonic nanoparticles

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A single gold nanoparticle held by an optical tweezer is used as a sensor to quantify the rhythmic motion of a Nauplius larva (Artemia salina) in a water sample. This is achieved by monitoring the time dependent displacement of the trapped nanoparticle as a consequence of the Nauplius activity. A Fourier analysis of the nanoparticle&#39;s position then yields a frequency spectrum that is characteristic to the motion of the observed species. This experiment demonstrates the capability of this method to measure and characterize the activity of small aquatic larvae without the requirement to observe them directly and to gain information about the position of the larvae with respect to the trapped particle. Overall, this approach could give an insight on the vitality of certain species found in an aquatic ecosystem and could expand the range of conventional methods for analyzing water samples.

A schematic illustration of the experimental setup is shown in Figure 1A. A dark field configuration is necessary to optically detect the displacement of a 60 nm gold particle in an optical trap15. The wavelength of 1,064 nm for the trapping laser is chosen to guarantee a stable confinement of the detector gold particle12,14. A beam splitter in the microscope is used to focus the trapping beam through the objective and a notch filter prevents the trapping laser from entering the det...

Watch this Scientific Journal Video about Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles at JoVE.com

Analyzing the Movement of the Nauplius &#039;Artemia salina&#039; by Optical Tracking of Plasmonic Nanoparticles

We use optical tracking of plasmonic nanoparticles to probe and characterize the frequency movements of aquatic organisms.

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A ...

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10.4K Aufrufe.  Ludwig-Maximilians-Universität.  Wir verwenden optische Tracking plasmonischer Nanopartikel zu untersuchen und zu charakterisieren, die Frequenz Bewegungen von Wasserorganismen. 

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Fabricating Metamaterials Using the Fiber Drawing Method

Metamaterials are man-made composite materials, fabricated by assembling components much smaller than the wavelength at which they operate 1. They owe their electromagnetic properties to the structure of their constituents, instead of the atoms that compose them. For example, sub-wavelength metal wires can be arranged to possess an effective electric permittivity that is either positive or negative at a given frequency, in contrast to the metals themselves 2. This unprecedented control over the behaviour of light can potentially lead to a number of novel devices, such as invisibility cloaks 3, negative refractive index materials 4, and lenses that resolve objects below the diffraction limit 5. However, metamaterials operating at optical, mid-infrared and terahertz frequencies are conventionally made using nano- and micro-fabrication techniques that are expensive and produce samples that are at most a few centimetres in size 6-7. Here we present a fabrication method to produce hundreds of meters of metal wire metamaterials in fiber form, which exhibit a terahertz plasmonic response 8. We combine the stack-and-draw technique used to produce microstructured polymer optical fiber 9 with the Taylor-wire process 10, using indium wires inside polymethylmethacrylate (PMMA) tubes. PMMA is chosen because it is an easy to handle, drawable dielectric with suitable optical properties in the terahertz region; indium because it has a melting temperature of 156.6 &deg;C which is appropriate for codrawing with PMMA. We include an indium wire of 1 mm diameter and 99.99% purity in a PMMA tube with 1 mm inner diameter (ID) and 12 mm outside diameter (OD) which is sealed at one end. The tube is evacuated and drawn down to an outer diameter of 1.2 mm. The resulting fiber is then cut into smaller pieces, and stacked into a larger PMMA tube. This stack is sealed at one end and fed into a furnace while being rapidly drawn, reducing the diameter of the structure by a factor of 10, and increasing the length by a factor of 100. Such fibers possess features on the micro- and nano- scale, are inherently flexible, mass-producible, and can be woven to exhibit electromagnetic properties that are not found in nature. They represent a promising platform for a number of novel devices from terahertz to optical frequencies, such as invisible fibers, woven negative refractive index cloths, and super-resolving lenses.

Metamaterials at terahertz frequencies offer unique opportunities, but are challenging to fabricate in bulk. We adapt the fabrication procedure for microstructured polymer optical fibers to inexpensively fabricate metamaterials potentially on an industrial scale. We produce polymethylmethacrylate fibers containing ~10 &mu;m diameter indium wires separated by ~100 &mu;m, which exhibit a terahertz plasmonic response.

Metamaterials are man-made composite materials, fabricated by assembling components much smaller than the wavelength at which they operate 1. They owe ...

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Evaluating Plasmonic Transport in Current-carrying Silver Nanowires

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information support1,2,3. In this context, metal nanowires are especially desirable for realizing dense routing networks4. A prerequisite to operate such shared nanowire-based platform relies on our ability to electrically contact individual metal nanowires and efficiently excite surface plasmon polaritons5 in this information support. In this article, we describe a protocol to bring electrical terminals to chemically-synthesized silver nanowires6 randomly distributed on a glass substrate7. The positions of the nanowire ends with respect to predefined landmarks are precisely located using standard optical transmission microscopy before encapsulation in an electron-sensitive resist. Trenches representing the electrode layout are subsequently designed by electron-beam lithography. Metal electrodes are then fabricated by thermally evaporating a Cr/Au layer followed by a chemical lift-off. The contacted silver nanowires are finally transferred to a leakage radiation microscope for surface plasmon excitation and characterization8,9. Surface plasmons are launched in the nanowires by focusing a near infrared laser beam on a diffraction-limited spot overlapping one nanowire extremity5,9. For sufficiently large nanowires, the surface plasmon mode leaks into the glass substrate9,10. This leakage radiation is readily detected, imaged, and analyzed in the different conjugate planes in leakage radiation microscopy9,11. The electrical terminals do not affect the plasmon propagation. However, a current-induced morphological deterioration of the nanowire drastically degrades the flow of surface plasmons. The combination of surface plasmon leakage radiation microscopy with a simultaneous analysis of the nanowire electrical transport characteristics reveals the intrinsic limitations of such plasmonic circuitry.

Silver nanowires can simultaneously transport electrons and optical information in the form of surface plasmons. A procedure is described here to realize such a shared circuitry and the limitations at propagating both information carriers are evaluated.

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information ...

Patterning via Optical Saturable Transitions - Fabrication and Characterization

This protocol describes the fabrication and characterization of nanostructures using a novel nanolithographic technique called Patterning via Optical Saturable Transitions (POST). In this technique the chemical properties of organic photochromic molecules that undergo single-photon reactions are exploited, enabling rapid top-down nanopatterning over large areas at low light intensities, thereby, allowing for the circumvention of the far-field diffraction barrier.4 Simple, cost-effective, high throughput and resolution alternatives to nanopatterning are being explored, such as, two-photon polymerization5,6, beam pen lithography (BPL)7, scanning electron beam lithography (SEBL), and focused ion beam (FIB) patterning. However, multi-photon approaches require high light intensities, which limit their potential for high throughput and offer low image contrast. Although, electron and ion beam lithographic processes offer increased resolution, the serial nature of the process is limited to slow writing speeds, which also prevents patterning of features over large areas. Beam-pen lithography is an approach towards parallel near-field optical lithography. However, the gap between the source of the beam and the surface of the photoresist needs to be controlled extremely precisely for good pattern uniformity and this is very challenging to accomplish for large arrays of beams. Patterning via Optical Saturable Transitions (POST) is an alternative optical nanopatterning technique for patterning sub-wavelength features1-3. Since this technique uses single photons instead of electrons, it is extremely fast and does not require high light intensities1-3, opening the door to massive parallelization.

We report that the diffraction limit of conventional optical lithography can be overcome by exploiting the transitions of organic photochromic derivatives induced by their photoisomerization at low light intensities.1-3 This paper outlines our fabrication technique and two locking mechanisms, namely: dissolution of one photoisomer and electrochemical oxidation.

This protocol describes the fabrication and characterization of nanostructures using a novel nanolithographic technique called Patterning via Optical ...

Experiments on Ultrasonic Lubrication Using a Piezoelectrically-assisted Tribometer and Optical Profilometer

Friction and wear are detrimental to engineered systems. Ultrasonic lubrication is achieved when the interface between two sliding surfaces is vibrated at a frequency above the acoustic range (20 kHz). As a solid-state technology, ultrasonic lubrication can be used where conventional lubricants are unfeasible or undesirable. Further, ultrasonic lubrication allows for electrical modulation of the effective friction coefficient between two sliding surfaces. This property enables adaptive systems that modify their frictional state and associated dynamic response as the operating conditions change. Surface wear can also be reduced through ultrasonic lubrication. We developed a protocol to investigate the dependence of friction force reduction and wear reduction on the linear sliding velocity between ultrasonically lubricated surfaces. A pin-on-disc tribometer was built which differs from commercial units in that a piezoelectric stack is used to vibrate the pin at 22 kHz normal to the rotating disc surface. Friction and wear metrics including effective friction force, volume loss, and surface roughness are measured without and with ultrasonic vibrations at a constant pressure of 1 to 4 MPa and three different sliding velocities: 20.3, 40.6, and 87 mm/sec. An optical profilometer is utilized to characterize the wear surfaces. The effective friction force is reduced by 62% at 20.3 mm/sec. Consistently with existing theories for ultrasonic lubrication, the percent reduction in friction force diminishes with increasing speed, down to 29% friction force reduction at 87 mm/sec. Wear reduction remains essentially constant (49%) at the three speeds considered.

We present a protocol for using a piezoelectrically-assisted tribometer and optical profilometer to investigate the dependence of ultrasonic wear and friction reduction on linear velocity, contact pressure, and surface properties.

Friction and wear are detrimental to engineered systems. Ultrasonic lubrication is achieved when the interface between two sliding surfaces is vibrated at ...

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to neutral gold Au(0) by reducing agents, such as sodium citrate or sodium borohydride. The extension of this method to decorate iron oxide or similar nanoparticles with gold nanoparticles to create multifunctional hybrid Fe2O3-Au nanoparticles is straightforward. This approach yields fairly good control over Au nanoparticle dimensions and loading onto Fe2O3. Additionally, the Au metal size, shape, and loading can easily be tuned by changing experimental parameters (e.g., reactant concentrations, reducing agents, surfactants, etc.). An advantage of this procedure is that the reaction can be done in air or water, and, in principle, is amenable to scaling up. The use of such optically tunable Fe2O3-Au nanoparticles for hyperthermia studies is an attractive option as it capitalizes on plasmonic heating of gold nanoparticles tuned to absorb light strongly in the VIS-NIR region. In addition to its plasmonic effects, nanoscale Au provides a unique surface for interesting chemistries and catalysis. The Fe2O3 material provides additional functionality due to its magnetic property. For example, an external magnetic field could be used to collect and recycle the hybrid Fe2O3-Au nanoparticles after a catalytic experiment, or alternatively, the magnetic Fe2O3 can be used for hyperthermia studies through magnetic heat induction. The photothermal experiment described in this report measures bulk temperature change and nanoparticle solution mass loss as functions of time using infrared thermocouples and a balance, respectively. The ease of sample preparation and the use of readily available equipment are distinct advantages of this technique. A caveat is that these photothermal measurements assess the bulk solution temperature and not the surface of the nanoparticle where the heat is transduced and the temperature is likely to be higher.

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

We describe the synthesis and properties of multifunctional Fe2O3-Au nanoparticles produced by a wet chemical approach and investigate their photothermal properties using laser irradiation. The composite Fe2O3-Au nanoparticles retain the properties of both materials, creating a multifunctional structure with excellent magnetic and plasmonic properties.

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to ...

Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step process that finishes with Negishi coupling. Negishi coupling is a cross-coupling process in which a chemical precursor is first lithiated, followed by transmetallation with ZnCl2. The resultant organozinc compound can be coupled to a dibrominated aromatic precursor to give the conjugated monomer. Polymer films can be prepared via electropolymerization of the monomer and characterized using cyclic voltammetry and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy. Nanoparticles (NPs) are prepared via emulsion polymerization of the monomer using a two-surfactant system to yield an aqueous dispersion of the polymer NPs. The NPs are characterized using dynamic light scattering, electron microscopy, and UV-Vis-NIR-spectroscopy. Cytocompatibility of NPs is investigated using the cell viability assay. Finally, the NP suspensions are irradiated with a NIR laser to determine their effectiveness as potential materials for photothermal therapy (PTT).

A protocol is presented for the synthesis and preparation of nanoparticles consisting of electroactive polymers.

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step ...

Use of Sacrificial Nanoparticles to Remove the Effects of Shot-noise in Contact Holes Fabricated by E-beam Lithography

Nano-patterns fabricated with extreme ultraviolet (EUV) or electron-beam (E-beam) lithography exhibit unexpected variations in size. This variation has been attributed to statistical fluctuations in the number of photons/electrons arriving at a given nano-region arising from shot-noise (SN). The SN varies inversely to the square root of a number of photons/electrons. For a fixed dosage, the SN is larger in EUV and E-beam lithographies than for traditional (193 nm) optical lithography. Bottom-up and top-down patterning approaches are combined to minimize the effects of shot noise in nano-hole patterning. Specifically, an amino-silane surfactant self-assembles on a silicon wafer that is subsequently spin-coated with a 100 nm film of a PMMA-based E-beam photoresist. Exposure to the E-beam and the subsequent development uncover the underlying surfactant film at the bottoms of the holes. Dipping the wafer in a suspension of negatively charged, citrate-capped, 20 nm gold nanoparticles (GNP) deposits one particle per hole. The exposed positively charged surfactant film in the hole electrostatically funnels the negatively charged nanoparticle to the center of an exposed hole, which permanently fixes the positional registry. Next, by heating near the glass transition temperature of the photoresist polymer, the photoresist film reflows and engulfs the nanoparticles. This process erases the holes affected by SN but leaves the deposited GNPs locked in place by strong electrostatic binding. Treatment with oxygen plasma exposes the GNPs by etching a thin layer of the photoresist. Wet-etching the exposed GNPs with a solution of I2/KI yields uniform holes located at the center of indentations patterned by E-beam lithography. The experiments presented show that the approach reduces the variation in the size of the holes caused by SN from 35% to below 10%. The method extends the patterning limits of transistor contact holes to below 20 nm.

Uniformly sized nanoparticles can remove fluctuations in contact hole dimensions patterned in poly(methyl methacrylate) (PMMA) photoresist films by electron beam (E-beam) lithography. The process involves electrostatic funneling to center and deposit nanoparticles in contact holes, followed by photoresist reflow and plasma- and wet-etching steps.

Nano-patterns fabricated with extreme ultraviolet (EUV) or electron-beam (E-beam) lithography exhibit unexpected variations in size. This variation has ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...