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.
