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 1...

<ol>
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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. 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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. 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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 ビュー.  Ludwig-Maximilians-Universität. 私たちは、水生生物の周波数の動きを調査し、特徴づけるためにプラズモニックナノ粒子の光学式トラッキングを使用しています。 