The overall goal of this procedure is to trap micron sized biological objects with plasmonic and photonic crystal tweezers. This is accomplished by first fabricating the plasmonic substrate or photonic crystal substrate. Next, the biological sample is prepared by adding BSA to the cell solution and providing adequate spacing for the cell solution beneath the top cover slip.
Then the laser is aligned to the microscope, which has been fitted with the correct filter scheme. The microscope is now focused on the sample and trapping is performed. Finally, micro or nano-sized particles can be trapped with reduced incident intensity through the aid of plasmonic or photonic crystal nano structures.
The main advantage of this technique over existing methods like conventional optical tweezers, is that conversion of the optical fields through plasmonic and photonic crystal nano structures reduces the intensity required to trap small particles like cells, and it also doesn't require a tightly focused beam, which reduces the risk of cell photo damage from exposure to radiation. This method can help answer key questions in many biological fields, such as cell to cell interaction and the study of various forces in biology. It can also be a useful tool for fabrication in micro and nanotechnology by providing precise manipulation of micro and nanoparticles To form a gold nanoparticles array using an e-beam evaporator and chromium as the adhesion layer evaporate gold on a glass cover slip to a thickness of 20 nanometers.
Clean the sample using oxygen plasma for about one minute to remove organic impurities on the surface. After inducing the polystyrene monolayer to self-assemble, drop, cast the liquid onto the sample, incubate it for about one hour, then use a large volume of water to wash away the non adsorb spheres air dry. The monolayer finally evaporate a second 20 nanometer layer of gold onto the latex sphere, monolayer to form a random gold nanoparticles array.
The nanoparticle array is now ready for the biological sample. Place the prepared nanoparticle slide on the stage of a fluorescent microscope. The next step is to prepare mouse cell nuclei for optical trapping.
Begin with mouse cell nuclei tagged with aine orange to prevent the nuclei from sticking to the substrate. Place the cell nuclei in a small vial and add 10%BSA in PBS at a concentration of one to 10 BSA mixed by vortexing. Then pipette five microliters of the nuclei BSA solution onto the prepared slide.
To provide spacing between the nuclei and a cover slip, place a stack of two spacer cover slips around the droplet, and then place another cover slip over the top. For trapping and simultaneous imaging, use a customized fluorescence microscope configuration, including a bypassed excitation filter and two custom dichroic beam splitters. The optical tweezers are constructed by sending a 35 milliwatt helium neon laser through a zes axio imager D one M microscope, equipped with a GFP 17 filter set, which is modified to allow for 633 nanometer laser radiation to reach the sample.
The microscope will focus the laser beam on the sample, and then the beam will be diffracted by either the plasmonic nanoparticle substrate or the diffraction gradating. To image the cell nuclei, which are about five microns in diameter, use a Zeiss LDEC epi plan with a Neola 50 x objective. Begin by placing the sample on the objective and focusing on the plane with the most nuclei as they're sitting on the surface of the substrate.
Next, focus the microscope on a nucleus to trap by finding free floating cell nuclei above the layers of cells on the surface. Then position the laser trap spot over the particle. At this point, it should be trapped and should maintain its position in the laser spot even when the stage is moved.
The mouse nuclei that are at the focal plane of the microscope will likely be drifting with a slight microfluidic current caused by sample heating and evaporation, but the nuclei in the trap will stay in one position. This figure shows a scanning electron micrograph of self-assembled gold nanoparticles that each have a diameter of approximately 450 nanometers. Here is a nearfield scanning optical microscopy image of the plasmonic substrate where the nanoparticles distribution is sparse, showing the nearfield radiation.
The wavelength of the excitation laser is 633 nanometers, a scattering efficiency spectrum of the plasmonic substrate showing the peak at 624 nanometers is shown here. An absorption efficiency spectrum of the plasmonic substrate shows the peak at 668 nanometers. Gold nanospheres randomly distributed on a 2D domain of one by one square.
Micrometer are shown here. Each blue circle represents the center of the nanosphere. Shown in these panels are scattering field distributions on observation planes, which are parallel to the random nanosphere array.
These plots show the minimum laser intensities to maintain the trap as a function of flow rate of the surrounding fluid. Utilizing plasmonic trapping, all of the optical intensities are measured at the sample plane under the microscope objective. Each panel shows the measurement results for single polystyrene beads with different diameters.
The insets show the corresponding microscopic images of particles. The scale bars in all images represent five micrometers in length. Plotted here is the slope of the fitted line through the origin in the previous plots versus particle size.
For plasmonic trapping. The error bars show the standard deviations of the linear fits. The results show that larger particles require greater laser intensity to maintain adequate trapping stability.
This schematic shows the enhanced optical trapping utilizing 1D periodic nanostructures. The incident beam is defracted by the periodic nanostructure at far field. Shown here is the intensity distribution of light with two orthogonal polarizations at the surface of an aluminum grading.
With a 417 nanometer period obtained using finite difference time domain simulations, the distribution is normalized to the intensity on a flat aluminum surface. These figures show the trapping potential for particles directly above the grading surface versus location of the particle. For CA 350 nanometer polystyrene bead and DA one micrometer polystyrene bead, the white circles illustrate the sizes of particles.
Insets show the trapping potential above a flat aluminum surface. For the same particle size as comparisons. The values are normalized for each particle size.
For all FDTD simulation figures, the field of view is 10 by eight micrometers squared. Here is the trap efficiency and minimum trapping intensity measured for polystyrene beads of various sizes with the beam polarization perpendicular to the grading lines. The inset shows trap asymmetry in trapping efficiency for translating a 3.87 micrometer, polystyrene bead, perpendicular, and parallel to the rules of the grading.
Here is a schematic representation of the translational angle of the particle. This inset shows the enhancement versus translation direction for different polarizations and particle sizes. The solid line large asymmetry is obtained with incident light polarized perpendicular to the grading and the dash line.
Small asymmetry is obtained with incident light polarized parallel to the grading. These panels represent a trapping demonstration of a fluorescent 590 nanometer polystyrene bead. The red circle indicates the position of the laser spot as the laser light was too dim to be seen at first.
The particle is trapped within the spot at higher power. As the power is lowered, the brownian motion of the particle overcomes the trapping force, allowing the particle to escape. Here, a fluorescent ovarian cancer cell nucleus trapped the minimum intensity required to initiate trapping was 16 micro watts per micrometer squared obtained using a 20 x objective lens.
After watching this video, you should have a good understanding of how to create a low intensity optical trap for the non-invasive manipulation of micro and nano-sized biological specimens using plasmonic and photonic crystal nano structured substrates.
