Name: Analyzing the Movement of the Nauplius Artemia salina by Optical Tracking of Plasmonic Nanoparticles (Video) | JoVE Journal
Uploaded: 2014-07-15T14:00:00.000+00:00
Duration: 5 min 52 s
Description: Learn optical tracking of plasmonic nanoparticles to analyze Artemia salina movement frequencies using optical tweezers. This method enables precise biophysical measurements of aquatic microorganism behavior.

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5 minutes

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 µl) 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 µl 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's x-y-position over time reveals a frequency spectrum. 
	NOTE: Here, a self-written ‘IGOR PRO’ 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 ‘Video Spot Tracker’ program for tracking the particle in the video. Use the commercial software ‘Origin’ to perform the Fourier transformation of the tracking data:
	<ol>
		<li>Drag the video file to the open program ‘Video Spot Tracker’.</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 “symmetric” and “optimize” in the top command prompt window to optimize the tracking of the particle.</li>
		<li>Mouse click “logging” 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 “play video” 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 ‘Origin’. Set the column values as “y1” and “y2”.</li>
		<li>Set time steps for each video frame as “x” in the ‘Origin’ data spreadsheet.</li>
		<li>Mark the x-position column and perform a FFT by choosing “Data Analysis” and “FFT” 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 α of the 60 nm gold particle by using the computer program ‘Mathematica’.
	<ol>
		<li>Use equation (1) to calculate the polarizability according to Kuwata et al.16: 
		<img alt="Alpha equation for optical absorption analysis, formula image used in photonics research." fo:content-width="3in" src="/files/ftp_upload/51502/51502eq1.jpg" width="300" data-altupdatedat="1787630408000" data-alt="Equation 1"> (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="Gaussian beam radius equation, \( E(r) = E_0 \sqrt{\frac{2}{\pi} \frac{\omega_0}{\omega(z)}} \), formula." fo:content-width="4in" src="/files/ftp_upload/51502/51502eq2.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Equation 2"> (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="Optical force equation, \(F_{grad_z}\), formula, laser trapping, photonics, scientific analysis." fo:content-width="3.5in" src="/files/ftp_upload/51502/51502eq3.jpg" width="350" data-altupdatedat="1787630408000" data-alt="Equation 3"> (3) 
		<img alt="Optical force equation; diagram; describes gradient force calculation in photonics context." fo:content-width="3in" src="/files/ftp_upload/51502/51502eq4.jpg" width="300" data-altupdatedat="1787630408000" data-alt="Equation 4"> (4) 
		<img alt="Fscatt_z equation in light-matter interaction study; mathematical formula for scattering force." fo:content-width="5in" src="/files/ftp_upload/51502/51502eq5.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Equation 5"> (5) 
		<img alt="Scattering force formula in optics; equation for electromagnetic scattering; Gaussian beam parameters." fo:content-width="3in" src="/files/ftp_upload/51502/51502eq6.jpg" width="300" data-altupdatedat="1787630408000" data-alt="Equation 6"> (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 “Control” and “Enter”.</li>
</ol>

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<li>Klar, T., Perner, M., Grosse, S., von Plessen, G., Spirkl, W., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface-Plasmon+Resonances+in+Single+Metallic+Nanoparticles%5BTitle%5D)+AND+%22Physical+Review+Letters%22%5BJournal%5D)">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/pubmed?term=((Optothermal+Escape+of+Plasmonically+Coupled+Silver+Nanoparticles+from+a+Three-Dimensional+Optical+Trap%5BTitle%5D)+AND+%22Nano+Letters%22%5BJournal%5D)">Optothermal Escape of Plasmonically Coupled Silver Nanoparticles from a Three-Dimensional Optical Trap.</a> Nano Letters. 11 (4), 1770-1774 (2011).</li>
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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><tbody><tr><td>Microscope Zeiss Axio Scope.A1</td><td>Carl Zeiss</td><td>490035-0012-000</td><td>dark field illumination</td></tr><tr><td>Water objective Achroplan</td><td>Carl Zeiss</td><td>440087</td><td>100X magnification, NA = 1.0</td></tr><tr><td>Air objective Epiplan</td><td>Carl Zeiss</td><td>442934</td><td>10X magnification, NA = 0.2</td></tr><tr><td>Dark field oil condenser</td><td>Carl Zeiss</td><td>445323</td><td>NA = 1.2</td></tr><tr><td>Cobolt Rumba CW 1,064 nm DPSSL</td><td>Cobolt</td><td>1064-05-01-2000-500</td><td>1,064 nm, CW, &amp;lambda; = 1,064 nm, 2 Watt, TEM00</td></tr><tr><td>Beam expander</td><td>Edmund Optics</td><td></td><td>Part no. 1064 2-8X 64414</td></tr><tr><td>High Speed Camera Dimax HD</td><td>PCO. Germany</td><td></td><td></td></tr><tr><td>Color Camera Canon EOS 500 D&amp;nbsp;</td><td>Canon</td><td></td><td>FAQ-ID: 8201395700</td></tr><tr><td>Notch filter StopLine 532/1064</td><td>Semrock</td><td>A11149-711265</td><td>Part no. NF01-532U</td></tr><tr><td>Water&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Nauplius &lt;em&gt;Artemia salina&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Gold colloid</td><td>BBInternational</td><td>Batch 13741&amp;nbsp;</td><td>Diameter 60 nm</td></tr><tr><td>MQMie Version 3.2&amp;nbsp;</td><td>Dr. Michael Quinten</td><td></td><td></td></tr><tr><td>Mathematica 8.0</td><td>Wolfram</td><td></td><td></td></tr><tr><td>Comsol Multiphysics 4.0&amp;nbsp;</td><td>COMSOL, Inc.</td><td></td><td></td></tr></tbody></table>

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

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...

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

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

The overall goal of this procedure is to analyze the frequencies of the movement of an aquatic microorganism using an optically trapped plasmonic nanoparticle. This is accomplished by first observing OPIS larvae in water under a microscope, equipped with an optical tweezer and a camera. The second step is to add gold nanoparticles of 16 nanometer diameter to the OPIS environment.Next, the optical tweezer is used to trap a single gold nanoparticle near the NOIs. The final step is to observe the diffusion of the trapped nanoparticle as it is influenced by the motion of the Nous. Video data is captured and analyzed by particle tracking software.Ultimately, the time dependent nanoparticle position is transformed into Fourier space to extract the movement frequencies of the nous. The main advantage of this technique over existing methods like common microscopy, is that fluidic vibrations are analyzed, and therefore this method is not restricted to any optical resolution. This method can help to answer key questions in environmentalism, such as water analysis of Socratic ecosystems.Begin by preparing an upright microscope equipped for dark field illumination. Provide the microscope with both a water immersion and an air objective. In addition, couple a 1064 nanometer continuous wave optical tweezer to the microscope.Next, mount a camera with a notch filter at 1064 nanometers to detect and image gold particle and opus motion. Use a power meter after the objective to set the laser power to 1000 milliwatts. Turn off the laser until needed.Sample preparation begins with pipetting a water droplet of 180 microliters on a microscope glass slide. Position the sample on the dark field microscope at this point, pipet OPIS from a water tank. Transfer it to the water droplet.Select the 10 x air objective. Observe the movement of the OPIS in the solution and record a ten second video stream at 25 frames per second. When this is done, prepare for the next step.Dilute one part stock solution of 60 nanometer diameter gold nano particles in 100 parts water. Return to the microscope and measure out five microliters of the solution. Add this to the water droplet with opus.When ready, change to a 100 x water immersion objective. To view the water droplet proceed when approximately one gold nanoparticle can be seen in the field of view. To trap a particle, turn on the laser of the optical trap and observe the droplet.Move the microscope stage to bring the laser beam close to a gold nanoparticle. The particle will be attracted towards the focal point of the laser beam and will stop diffusing. Take a video of the trapped nanoparticle at 50 frames per second for 30 seconds, turn off the laser of the optical tweezer to release the particle from the trap.The next step is to analyze the video on a computer. Use particle tracking software to determine the particle's XY position as a function of time, and find the frequency spectrum with a fast Fourier transform of this data. This plot shows the XY displacement of a trapped gold nanoparticle, which is only undergoing brownie in motion.The distribution is Gaussian. After adding an OIS to the water, the XY displacement of the particle changes due to fluidic vibrations. The microfluidic flow generated by the animal causes a frequency dependent distortion in the Y direction.These plots show the frequency spectra in the x and y directions in black, a reference spectra for an optically trapped particle that is not in the presence of opis. The red curves show the spectra of a trapped particle with a swimming opis. The spectrum in the X direction does not show a strong signal due to the position of the OPIS relative to the trapped particle.The flow is primarily in the Y direction as indicated in the inset. The spectrum in the Y direction taken with the swimming knobs shows a response. The broad frequency response in the measurement is consistent with the organism motility, or example, the movement of the main antenna or other body parts.Frequency maxima. Across all measurements were found to be between 3.0 and 7.2 hertz in good accordance with directly observed frequencies. Once mastered, this technique can be done in less than 30 minutes if it is performed properly.While attempting this procedure, it's important to remember to have a stable three-dimensional trapping of the gold nanoparticle using optical forces.

analyzing-movement-nauplius-artemia-salina-optical-tracking-plasmonic

Research

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We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

A Protocol for Real-time 3D Single Particle Tracking

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Plasmonic nanotweezers use localized surface plasmon resonance in gold nanostructures to trap single nanoparticles, including proteins, within a nanometer-scale optical field. Changes in the scattered signal reveal protein presence and conformational dynamics, enabling monitoring without fluorophore modifications or surface tethering.

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Learn optical tracking of plasmonic nanoparticles to analyze Artemia salina movement frequencies using optical tweezers. This method enables precise biophysical measurements of aquatic microorganism behavior.

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

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