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

Summary

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

Abstract

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'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.

Introduction

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 ecosystem^1-3. 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 habitat^4,5. 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 animal^7-10. 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 µm)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 range^12-14. 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.

Protocol

1. Experimental Setup

1. 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.
2. 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).
3. 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.
4. Use a notch filter to prevent the laser from entering the camera.
5. Use a power meter to measure the laser power after the objective.

2. Sample Preparation

1. Pipette a water droplet (180 µl) on a microscope glass slide and position the sample on the dark field microscope.
2. Pipette a Nauplius from a small water tank to the water droplet.
3. Use a 10X air objective to observe the movement of the Nauplius in the solution and record a video stream.
4. 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.

3. Particle Tracking Experiment

1. 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.
2. Turn off the laser of the optical tweezer and release the gold nanoparticle from the trap.
3. 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.
4. 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:
   1. Drag the video file to the open program ‘Video Spot Tracker’.
   2. Mouse click on the particle seen in the first picture of the video stream and a circular region of interest appears.
   3. Choose “symmetric” and “optimize” in the top command prompt window to optimize the tracking of the particle.
   4. 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.
   5. Mouse click “play video” on the left command prompt window of the tracking program and wait until all frames of the video are analyzed.
   6. Close the program and open the saved data spreadsheet with ‘Origin’. Set the column values as “y₁” and “y₂”.
   7. Set time steps for each video frame as “x” in the ‘Origin’ data spreadsheet.
   8. 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.
   9. Plot the amplitudes of the calculated FFT signal in x- and y-direction versus the frequency.

4. Numerical Simulation

1. Calculate the polarizability α of the 60 nm gold particle by using the computer program ‘Mathematica’.
   1. Use equation (1) to calculate the polarizability according to Kuwata et al.¹⁶:
      (1)
   2. 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.
2. 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:
   (2)
   1. Use equations (3)-(6) from Agayan et al.¹⁷ to calculate both, the gradient and scattering forces acting on the particle:
      (3)
      (4)
      (5)
      (6)
   2. In the program code, define the parameters for the laser power, the numerical aperture of the objective, and the complex polarizability of the nanoparticle.
   3. Sum up the gradient force and the scattering force to calculate the total optical force acting on the gold particle in an optical trap.
3. Run the simulation by simultaneously pressing “Control” and “Enter”.

Results

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 particle^12,14. 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...

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microscope Zeiss Axio Scope.A1	Carl Zeiss	490035-0012-000	dark field illumination
Water objective Achroplan	Carl Zeiss	440087	100X magnification, NA = 1.0
Air objective Epiplan	Carl Zeiss	442934	10X magnification, NA = 0.2
Dark field oil condenser	Carl Zeiss	445323	NA = 1.2
Cobolt Rumba CW 1,064 nm DPSSL	Cobolt	1064-05-01-2000-500	1,064 nm, CW, λ = 1,064 nm, 2 Watt, TEM00
Beam expander	Edmund Optics		Part no. 1064 2-8X 64414
High Speed Camera Dimax HD	PCO. Germany
Color Camera Canon EOS 500 D	Canon		FAQ-ID: 8201395700
Notch filter StopLine 532/1064	Semrock	A11149-711265	Part no. NF01-532U
Water
Nauplius Artemia salina
Gold colloid	BBInternational	Batch 13741	Diameter 60 nm
MQMie Version 3.2	Dr. Michael Quinten
Mathematica 8.0	Wolfram
Comsol Multiphysics 4.0	COMSOL, Inc.