Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 105-2221-E-007-MY3 and from the National Tsing Hua University under grant numbers 105N518CE1 and 106N518CE1.

1. Optical Setup
Note: The principle of the optical setup is illustrated in Figure 1.
<ol>
	<li>Set up the optical tweezer kit (see the Table of Materials) and the fluorescence module (see Table of Materials) as per their manuals. Connect a 470 nm blue light emitting diode (LED) light source to the fluorescent module.</li>
	<li>Replace the high numerical aperture (NA) (NA= 1.25, magnification 100x) oil immersion objective by a long working distance (WD) microscope objective (focal length 3.6 mm, WD=10.6 mm, NA=0.5).</li>
	<li>Remove the lens in the beam expansion section of the assembled kit to achieve loose focusing of the laser beam.</li>
	<li>Turn on the power supply and the current for the laser diode of wavelength 980 nm and use the charged coupled device (CCD) camera to make sure the laser beam is aligned properly. 
	NOTE: If the laser beam is well-aligned, the CCD camera will read a Gaussian spot.</li>
</ol>
2. Nanofabrication
<ol>
	<li>Marker Fabrication. 
	NOTE: Markers will help to position the nanoplasmonic array during the fabrication process and subsequent trapping experiment. The detailed process is illustrated in Supplementary Figure 1.
	<ol>
		<li>Deposit 40 nm indium tin oxide (ITO) film on a coverslip of thickness 0.17 mm with sputtering. 
		NOTE: The ITO film will help discharge electrons during the subsequent e-beam lithography process.</li>
		<li>Spin coat an 8 &#181;m layer of positive photoresist with spin speed 4000 rpm and time 30 s with a spin coater.</li>
		<li>Soft bake the sample at 90 &#176;C for 5 min and align the sample with the photomask for marker and expose the sample to UV light for 80 s in the mask aligner.</li>
		<li>Soak the sample in the photoresist developer for 130 s.</li>
		<li>Deposit a 2 nm layer of chromium and a 40 nm layer of gold onto the sample using thermal evaporation.18</li>
		<li>Soak the sample in acetone and place it in an ultrasonic cleaner operating at 43 kHz and 150 W for 5 min for lift off.</li>
	</ol>
	</li>
	<li>Fabrication of Nanoplasmonic Array
	<ol>
		<li>Spin coat a layer of e-beam resist PMMA 120K with spin speed 5000 rpm for 30 s on a spin coater. Bake the sample at 160 &#176;C for 3 min on a hot plate.</li>
		<li>Spin coat another layer of e-beam resist PMMA 960K with spin speed 5000 rpm for 30 s on a spin coater. Bake the sample at 160 &#176;C for 3 min on a hot plate.</li>
		<li>Use e-beam writer to expose the e-beam resist with acceleration voltage 30 kV and dosage 400 C/cm2.</li>
		<li>Deposit a 40 nm layer of gold in a thermal evaporator.</li>
		<li>Soak the sample in acetone and place it in an ultrasonic cleaner for 5 min for lift off.</li>
	</ol>
	</li>
</ol>
3. Sample cooling system and its temperature calibration
NOTE: The sample cooling stage design is shown in Supplementary Figure 2.
<ol>
	<li>Making the driver circuit for sample cooling
	<ol>
		<li>Place the resistors, bipolar junction transistors, and power metal oxide field effect transistors on the custom circuit board by following the circuit diagram in Supplementary Figure 3. Solder all these components with soldering iron.</li>
		<li>Connect wires between the control port of the circuit board and the electronic control board. Connect the wires between the output port of the circuit board and thermoelectrical cooling (TEC) element. Place the TEC element on the sample stage with heat sinking. 
		&#8203;NOTE: The TEC element has a hole in the center to allow the laser beam to go through.</li>
		<li>Connect wires from the circuit board to 5 V power supply. Use the forward-looking infrared camera to monitor the temperature to check if the thermoelectrical cooling is properly cooling down.</li>
	</ol>
	</li>
	<li>Calibration of measured temperature in the forward-looking infrared camera and resistance temperature detector (RTD) thermometer.
	<ol>
		<li>Place the RTD thermometer on a blank coverslip and apply a small amount of thermal paste onto it to ensure proper thermal contact between the RTD thermometer and coverslip.</li>
		<li>Change the output power setting of the electronic control circuit to TEC element by changing the duty cycle of pulse width modulation setting and wait for 3 min to make sure the steady state temperature is reached. Read the temperature using the RTD thermometer.</li>
		<li>Turn on the forward looking infrared camera and monitor the temperature. Repeat this at various output power settings to obtain the temperature calibration curve. A representative temperature calibration curve is shown in Supplementary Figure 4. 
		NOTE: It is crucial to do calibration between RTD thermometer and the forward looking infrared camera because the temperature reading of the forward-looking infrared camera must be accurate to ensure the correct temperature is reached.</li>
	</ol>
	</li>
</ol>
4. Trapping of Microparticles
<ol>
	<li>Dilute micro polystyrene particles of diameter 2 &#181;m in deionized water in a microcentrifugetube with proper volume ratio. 
	NOTE: The concentration of micro particles can be adjusted according to the objective of the experiment. While lower concentration allows a sample time interval between single particle trapping events, higher concentration will shorten the time for multiple particle trapping. For single particle trapping, a typical concentration is ~0.05% (w/v).</li>
	<li>Put the sample with nanoplasmonic array on the stage and turn on a 470 nm LED as the fluorescence light source and manually set the power to 5 mW for bright field imaging.</li>
	<li>Use the marker to locate the nanoplasmonic array, align the sample, and use the CCD camera to make sure the array is in the center of the region of interest on the computer screen.</li>
	<li>Dispense 10 &#181;L of the diluted micro particles of diameter 2 &#181;m on the sample with a micro pipette.</li>
	<li>Turn on the current supply to the laser diode of wavelength 980 nm to excite the plasmonic resonance of the array with a power in the range ~1 mW to 10 mW.</li>
	<li>Manually turn on the power supply to the electronic control board to cool down the sample to a steady state temperature ~4 &#176;C.</li>
	<li>In the viewer software, click the &#34;record video&#34; sequence to open the recording dialog. Click the &#34;record&#34; button to start the video recording 1.5 of the motion of the micro particles at a frame rate of 10 frames/s over the sample under the influence of laser beam using the CCD camera. Click the &#34;stop&#34; button to stop the recording. See Video 1.</li>
</ol>

<ol>
	<li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+single-beam+gradient+force+optical+trap+for+dielectric+particles.">Observation of a single-beam gradient force optical trap for dielectric particles.</a> Opt.Lett. 11, 288-290 (1986).</li><li>Grier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revolution+in+optical+manipulation.">A revolution in optical manipulation.</a> Nature. 424, 21-27 (2003).</li><li>Wright, W. H., Sonek, G. J., Berns, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+on+trapping+forces+with+optical+tweezers.">Radiation on trapping forces with optical tweezers.</a> Appl. Phys. Lett. 63, 715-717 (1993).</li><li>Kawata, S., Tani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optically+driven+Mie+particles+in+an+evanescent+field+along+a+channel+waveguide.">Optically driven Mie particles in an evanescent field along a channel waveguide.</a> Opt. Lett. 21, 1768-1770 (1996).</li><li>Yang, A. H. J., Moore, S. D., Schmit, B. S., Klug, M., Lipson, M., Erickson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Manipulation+of+nanoparticles+and+biomolecules+in+sub-wavelength+slot+waveguides.">Optical Manipulation of nanoparticles and biomolecules in sub-wavelength slot waveguides.</a> Nature. 457, 71-75 (2009).</li><li>Lin, S., Schonbrun, E., Crozier, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+manipulation+with+planar+silicon+microring+resonators.">Optical manipulation with planar silicon microring resonators.</a> Nano Lett. 10, 2408-2411 (2010).</li><li>Mandal, S., Serey, X., Erickson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomanipulation+using+silicon+photonic+crystal+resonators.">Nanomanipulation using silicon photonic crystal resonators.</a> Nano Lett. 2010 (10), 99-104 (2010).</li><li>Lin, S., Crozier, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+silicon+microrings+as+wavelength-+multiplexed+optical+traps+for+storing+and+sensing+particles.">Planar silicon microrings as wavelength- multiplexed optical traps for storing and sensing particles.</a> Lab Chip. 11, 4047-4051 (2011).</li><li>Juan, M. L., Righini, M., Quidant, M., R, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmonic+nano+optical+tweezers.">Plasmonic nano optical tweezers.</a> Nat. Photon. 5, 349-356 (2011).</li><li>Huang, J. S., Yang, Y. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+future+of+plasmonic+optical+tweezer.">The origin and future of plasmonic optical tweezer.</a> Nanomaterials. 5, 1048-1065 (2015).</li><li>Pang, Y., Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+of+single+protein.">Optical trapping of single protein.</a> Nano Lett. 12, 402-406 (2012).</li><li>Chen, K. Y., Lee, A. T., Hung, C. C., Huang, J. S., Yang, Y. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+and+trapping+in+two-dimensional+nanoscale+plasmonic+optical+lattice.">Transport and trapping in two-dimensional nanoscale plasmonic optical lattice.</a> Nano Lett. 13, 4118-4122 (2013).</li><li>Cuche, A., Stein, B., Canguier-Durand, A., Devaux, E., Genet, C., Ebbesen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brownian+motion+in+a+designer+force+field:+Dynamical+effects+of+negative+refraction+on+nanoparticles.">Brownian motion in a designer force field: Dynamical effects of negative refraction on nanoparticles.</a> Nano Lett. 12, 4329-4332 (2012).</li><li>Baffou, G., Berto, P., Urena, E. B., Quidant, R., Monnert, S. J., Polleux, J., Righeault, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+heating+of+nanoparticle+arrays.">Photoinduced heating of nanoparticle arrays.</a> ACS Nano. 8, 6478-6488 (2013).</li><li>Roxworthy, B. J., Bhuiya, A. M., Vanka, S. P., Toussant, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+controlling+plasmon-induced+convection.">Understanding and controlling plasmon-induced convection.</a> Nat. Commun. 5, (2014).</li><li>Yang, T. P., Yossifon, G., Yang, Y. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+near-field+and+convectional+transport+behavior+of+micro+and+nano+particles+in+nanoscale+plasmonic+optical+lattice.">Characterization of the near-field and convectional transport behavior of micro and nano particles in nanoscale plasmonic optical lattice.</a> Biomicrofluidics. 10, 034102 (2016).</li><li>Chen, Y. -. C., Yossifon, G., Yang, Y. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supression+of+phtothermal+convection+of+micro+particles+in+two+dimensional+nanoplamonic+optical+lattice.">Supression of phtothermal convection of micro particles in two dimensional nanoplamonic optical lattice.</a> Appl. Phys. Lett. 109, 201111 (2016).</li><li>Smith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thin film deposition: principles and practice. , (1995).</li><li>Zehtabi-Oskuie, A., Bergeron, J. G., Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+double-nanohole+optical+trapping+of+20+nm+polystyrene+nanospheres.">Flow-dependent double-nanohole optical trapping of 20 nm polystyrene nanospheres.</a> Sci. Rep. 2, 966 (2012).</li><li>Zhang, W., Huang, L., Santschi, C., Martin, O. J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+and+sensing+10+nm+metal+nanoparticles+using+plasmonic+dipole+antennas.">Trapping and sensing 10 nm metal nanoparticles using plasmonic dipole antennas.</a> Nano Lett. 10, 1006-1011 (2010).</li><li>Pang, Y., Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+of+12+nm+dielectric+spheres+using+double-nanoholes+in+a+gold+film.">Optical trapping of 12 nm dielectric spheres using double-nanoholes in a gold film.</a> Nano Lett. 2011 (11), 3763-3767 (2011).</li></ol>

The authors have nothing to disclose.

The procedure described here enables the reader to reliably reproduce trapping on a daily basis. A general empirical guideline to design a usable optical lattice is to use a comparable size for plasmonic nanoarray, interdisc distance, and trapped particle size. Compared to a single, isolated plasmonic nanostructure, the optical lattice design in conjunction with the high optical power afforded by cooling the sample to ~4 &#176;C used here greatly enhances the trapping probability. If well separated, plasmonic nanostructu...

The optical trapping of micro-scale particles was originally developed by Arthur Askin in the early 1970s. Ever since its invention, the technique has been developed as a versatile tool for micro- and nanomanipulation1,2. Conventional optical trapping based on the far-field focusing principle is inherently limited by the diffraction in its spatial confinement, wherein the trapping force decreases dramatically (following an ~a3 law for a particle of radius a)3. To overcome such diffraction limits, researchers have developed near-field optical trapping techniques based on the evanescent optical field using plasmonic metallic nanostructures and, furthermore, the trapping of nanoscale objects down to single protein molecules has been demonstrated4,5,6,7,8,9,10,11. Moreover, the plasmonic optical lattice is created from arrays of periodic plasmonic nanostructures to confer long range transport of micro- and nanoparticles and multiple particle stacking11,12. One major obstacle to disrupt trapping in an optical lattice is photothermal convection and efforts have been made to elucidate its effects by several groups14,15,16,17. Using Green&#39;s function, Baffou et al. have calculated a temperature profile by modeling each plasmonic nanostructure as a point heater and then experimentally validated their model14. Toussant&#39;s group has also measured the plasmon-induced convection with particle velocimetry15. The author&#39;s group has also characterized both near-field and convectional transport and demonstrated an engineering strategy to suppress photothermal convection16,17.
Here we present the design of an optical setup and a detailed procedure specifically for trapping experiments with plasmonic optical lattice. The optical potential was created by illuminating an array of gold nanodiscs with a loosely focused Gaussian beam. A scheme to suppress the photothermal convection by cooling down the sample to a low temperature (~4 &#176;C) for optimal trapping is also describe here17. Under Boussinesq approximation, an order of magnitude estimate for the natural convection velocity u is given by u ~L2 g&#946;&#916;T / v, where L is the length scale of the heat source and &#916;T is the temperature increase relative to the reference due to the heating. &#160;g and &#946;&#160;are the gravitational acceleration and thermal expansion coefficient, respectively. At temperatures near 4 &#176;C, the density of the water medium exhibits anomalous temperature dependence and this translates into a near-zero thermal expansion coefficient and, therefore, a vanishingly small photothermal convection.

<table><tbody><tr><td>Thermoelectric cooling element</td><td>Thorlabs</td><td>TEC 1.4-6</td><td>TEC element for sample cooling</td></tr><tr><td>RTD thermometer</td><td>Omega Engineering</td><td>RTD Thermometer 969C</td><td></td></tr><tr><td>Forward looking infrared camera</td><td>FLIR&amp;nbsp;</td><td>FLIR One</td><td>IR camera for temperature monitoring</td></tr><tr><td>light emitting diode light source</td><td>Touchbright</td><td></td><td>Light source for illumination for fluorescent imaging</td></tr><tr><td>Long working distance objective</td><td>Olympus</td><td>LMPLFLN</td><td>For illuminating the sample and imaging</td></tr><tr><td>Optical trap kit</td><td>Thorlabs</td><td>OTKB/M</td><td></td></tr><tr><td>Cover slip</td><td></td><td></td><td>thickness 0.17 mm</td></tr><tr><td>Scanning electron microscope</td><td>Hitachi</td><td>SEM-Hitachi S3400N</td><td></td></tr><tr><td>Electron beam blanker</td><td>DEBEN</td><td>PCD beam blanker</td><td>the blanker is added to the scanning electron microscope&amp;nbsp;</td></tr><tr><td>Thermal evaporator</td><td>SYSKEY Technology</td><td></td><td></td></tr><tr><td>Mask aligner</td><td>Karl Suss</td><td>MJB 3</td><td>For marker fabrication</td></tr><tr><td>Electron beam resist</td><td>Sigma Alrich</td><td>PMMA 120K</td><td>For e-beam lithography</td></tr><tr><td>Electron beam resist</td><td>Sigma Alrich</td><td>PMMA 960K</td><td>For e-beam lithography</td></tr><tr><td>Fluoresent labeled polystyrene microspheres</td><td>Polyscience</td><td></td><td>2 um diameter</td></tr><tr><td>Bipolar transistor</td><td>Mouser</td><td>2N3904</td><td>quantity 2 for TEC driver circuit</td></tr><tr><td>Bipolar transistor</td><td>Mouser</td><td>2N3906</td><td>quantity 2 for TEC driver circuit</td></tr><tr><td>MOSFET power transistor</td><td>Mouser</td><td>IRF5305</td><td>quantity 2 for TEC driver circuit</td></tr><tr><td>MOSFET power transistor</td><td>Mouser</td><td>IRF131ON</td><td>quantity 2 for TEC driver circuit</td></tr><tr><td>10 kOhm resistor</td><td>Mouser</td><td></td><td>quantity 6 for TEC driver circuit</td></tr><tr><td>910 Ohm resistor</td><td>Mouser</td><td></td><td>quantity 2 for TEC driver circuit</td></tr><tr><td>Photoresist</td><td>Microchemicals</td><td>AZ4620</td><td>For marker fabrication</td></tr><tr><td>Acetone</td><td>Sigma Alrich</td><td></td><td>For marker fabrication</td></tr><tr><td>Fluorescence Module for the OTKB/M, Metric Threads</td><td>Thorlabs</td><td>OTKB-FL/M</td><td></td></tr><tr><td>Fluorescent filter set</td><td>Thorlabs</td><td>MDF-FITC</td><td>For Fluorescein Isothiocyanate (FITC)</td></tr><tr><td>Ultrasonic cleaner</td><td>Delta</td><td>DC150H</td><td>For the lift off step</td></tr></tbody></table>

trapping of micro particles in nanoplasmonic optical lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

Single particle trajectories were recorded by a CCD camera in our experiment and the images were then processed with a custom program to extract each particle&#39;s trajectory16. Representative results are displayed in Figure 3 and Video 1 for micro-spheres with diameters of 2 &#181;m. Multiple particle trappings inside the optical lattice were observed. Successive images extracted from a representative motion video of...

Watch this Scientific Journal Video about Trapping of Micro Particles in Nanoplasmonic Optical Lattice at JoVE.com

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

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

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

trapping-of-micro-particles-in-nanoplasmonic-optical-lattice

Research

JoVE Journal

Bioengineering

6.5K Views.  National Tsing Hua University. We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

Video: Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Engineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

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.

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

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 ...

Fabrication and Operation of a Nano-Optical Conveyor Belt

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological and physical sciences over the past few decades. The progress made in this field invites further study of even smaller systems and at a larger scale, with tools that could be distributed more easily and made more widely available. Unfortunately, the fundamental laws of diffraction limit the minimum size of the focal spot of a laser beam, which makes particles smaller than a half-wavelength in diameter hard to trap and generally prevents an operator from discriminating between particles which are closer together than one half-wavelength. This precludes the optical manipulation of many closely-spaced nanoparticles and&#160;limits the&#160;resolution of optical-mechanical systems. Furthermore, manipulation using focused beams requires beam-forming or steering optics, which can be very bulky and expensive. To address these limitations in the system scalability of conventional optical trapping our lab has devised an alternative technique which utilizes near-field optics to move particles across a chip. Instead of focusing laser beams in the far-field, the optical near field of plasmonic resonators produces the necessary local optical intensity enhancement to overcome the restrictions of diffraction and manipulate particles at higher resolution. Closely-spaced resonators produce strong optical traps which can be addressed to mediate the hand-off of particles from one to the next in a conveyor-belt-like fashion. Here, we describe how to design and produce a conveyor belt using a gold surface patterned with plasmonic C-shaped resonators and how to operate it with polarized laser light to achieve super-resolution nanoparticle manipulation and transport. The nano-optical conveyor belt chip can be produced using lithography techniques and easily packaged and distributed.

The scalability and resolution of conventional optical manipulation techniques are limited by diffraction. We circumvent the diffraction limit and describe a method of optically transporting nanoparticles across a chip using a gold surface patterned with a path of closely spaced C-shaped plasmonic resonators.

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological ...

Optical Trap Loading of Dielectric Microparticles In Air

We demonstrate a method to trap a selected dielectric microparticle in air using radiation pressure from a single-beam gradient optical trap. Randomly scattered dielectric microparticles adhered to a glass substrate are momentarily detached using ultrasonic vibrations generated by a piezoelectric transducer (PZT). Then, the optical beam focused on a selected particle lifts it up to the optical trap while the vibrationally excited microparticles fall back to the substrate. A particle may be trapped at the nominal focus of the trapping beam or at a position above the focus (referred to here as the levitation position) where gravity provides the restoring force. After the measurement, the trapped particle can be placed at a desired position on the substrate in a controlled manner. 
In this protocol, an experimental procedure for selective optical trap loading in air is outlined. First, the experimental setup is briefly introduced. Second, the design and fabrication of a PZT holder and a sample enclosure are illustrated in detail. The optical trap loading of a selected microparticle is then demonstrated with step-by-step instructions including sample preparation, launching into the trap, and use of electrostatic force to excite particle motion in the trap and measure charge. Finally, we present recorded particle trajectories of Brownian and ballistic motions of a trapped microparticle in air. These trajectories can be used to measure stiffness or to verify optical alignment through time domain and frequency domain analysis. Selective trap loading enables optical tweezers to track a particle and its changes over repeated trap loadings in a reversible manner, thereby enabling studies of particle-surface interaction.

A protocol for launching and stably trapping selected dielectric microparticles in air is presented.

We demonstrate a method to trap a selected dielectric microparticle in air using radiation pressure from a single-beam gradient optical trap. Randomly ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and artificial nanomotors and may provide new routes towards single cell analysis, studies of non-equilibrium thermodynamics, and mechanical actuation of nanoscale systems. A facile way to drive rotation is to use focused circularly polarized laser light in optical tweezers. Using this approach, metallic nanoparticles can be operated as highly efficient scattering-driven rotary motors spinning at unprecedented rotation frequencies in water.
In this protocol, we outline the construction and operation of circularly-polarized optical tweezers for nanoparticle rotation and describe the instrumentation needed for recording the Brownian dynamics and Rayleigh scattering of the trapped particle. The rotational motion and the scattering spectra provides independent information on the properties of the nanoparticle and its immediate environment. The experimental platform has proven useful as a nanoscopic gauge of viscosity and local temperature, for tracking morphological changes of nanorods and molecular coatings, and as a transducer and probe of photothermal and thermodynamic processes.

Plasmonic gold nanorods can be trapped in liquids and rotated at kHz frequencies using circularly-polarized optical tweezers. Introducing tools for Brownian dynamics analysis and light scatteringspectroscopy leads to a powerful system for research and application in numerous fields of science.

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and ...

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes can alter protein biophysics and reduce the usefulness of the data acquired. Plasmonic nanotweezers is a technique that uses localized surface plasmon resonance (LSPR) on gold nanostructures to enhance the electric field within a confined hotspot region. This field enhancement allows for the use of low laser powers to trap single nanoparticles far smaller than conventional optical tweezers, down to only a few nanometers in diameter, such as single proteins. Trapping of single protein molecules within the hotspot region induces a shift in the local refractive index (nprotein &#62; nwater), altering light scattering as a product of the molecule&#39;s polarisability, which is affected by its volume, shape anisotropy, and refractive index. An avalanche photodiode (APD) collects the subsequent changes in light scattering. These alterations can then be analyzed to determine changes in the trapped molecule, including its size, global conformation, and dynamics of conformational change over time. The incorporation of microfluidics within the system allows for controlled environmental changes and real-time monitoring of their subsequent effects on the molecule. In this protocol, we demonstrate the steps to trap single protein molecules, alter their environmental solution conditions, and monitor their corresponding conformational changes using a plasmonic nanotweezers system.

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.

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric field of metallic nanostructures. Salt-induced silver nanoparticle aggregation is the most popular method for generating SERS-active substrates; however, it is limited by poor reproducibility, stability, and biocompatibility. The present protocol integrates optical manipulation and SERS detection to develop an efficient analytical platform to address this. A 1064 nm trapping laser and a 532 nm Raman probe laser are combined in a microscope to assemble silver nanoparticles, which generate plasmonic hotspots for in situ SERS measurements in aqueous environments. Without aggregating agents, this dynamic plasmonic silver nanoparticle assembly enables an approximately 50-fold enhancement of the analyte molecule signal. Moreover, it provides spatial and temporal control to form the SERS-active assembly in as low as 0.05 nM analyte-coated silver nanoparticle solution, which minimizes the potential perturbation for in vivo analysis. Hence, this optical trapping-integrated SERS platform holds great potential for efficient, reproducible, and stable molecular analyses in liquids, especially in aqueous physiological environments.

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

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Optical Trapping of Nanoparticles

Three-dimensional Optical-resolution Photoacoustic Microscopy

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

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

Fabrication and Operation of a Nano-Optical Conveyor Belt

Optical Trap Loading of Dielectric Microparticles In Air

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations