Name: trapping of micro particles in nanoplasmonic optical lattice
Uploaded: 2017-09-05T12:30:00
Description: Watch this Scientific Journal Video about Trapping of Micro Particles in Nanoplasmonic Optical Lattice at JoVE.com

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 l...

<ol>
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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. 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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. 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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.

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	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Thermoelectric cooling element</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">TEC 1.4-6</td>
			<td class="xl70" style="width:170pt" width="227">TEC element for sample cooling</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">RTD thermometer</td>
			<td class="xl69" style="width:131pt" width="174">Omega Engineering</td>
			<td class="xl69" style="width:122pt" width="162">RTD Thermometer 969C</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Forward looking infrared camera</td>
			<td class="xl69" style="width:131pt" width="174">FLIR&#160;</td>
			<td class="xl69" style="width:122pt" width="162">FLIR One</td>
			<td class="xl70">IR camera for temperature monitoring</td>
		</tr>
		<tr height="46" style="height:34.5pt">
			<td class="xl69" height="46" style="width:207pt;height:34.5pt" width="276">light emitting diode light source</td>
			<td class="xl69" style="width:131pt" width="174">Touchbright</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">Light source for illumination for fluorescent imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Long working distance objective</td>
			<td class="xl69" style="width:131pt" width="174">Olympus</td>
			<td class="xl69" style="width:122pt" width="162">LMPLFLN</td>
			<td class="xl70">For illuminating the sample and imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Optical trap kit</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB/M</td>
			<td class="xl70"></td>
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			<td class="xl71" height="21" style="height:15.75pt">Cover slip</td>
			<td class="xl69" style="width:131pt" width="174"></td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">thickness 0.17 mm</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Scanning electron microscope</td>
			<td class="xl69" style="width:131pt" width="174">Hitachi</td>
			<td class="xl69" style="width:122pt" width="162">SEM-Hitachi S3400N</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="44" style="height:33.0pt">
			<td class="xl71" height="44" style="height:33.0pt">Electron beam blanker</td>
			<td class="xl69" style="width:131pt" width="174">DEBEN</td>
			<td class="xl69" style="width:122pt" width="162">PCD beam blanker</td>
			<td class="xl69" style="width:170pt" width="227">the blanker is added to the scanning electron microscope&#160;</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Thermal evaporator</td>
			<td class="xl69" style="width:131pt" width="174">SYSKEY Technology</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Mask aligner</td>
			<td class="xl69" style="width:131pt" width="174">Karl Suss</td>
			<td class="xl69" style="width:122pt" width="162">MJB 3</td>
			<td class="xl69" style="width:170pt" width="227">For marker fabrication</td>
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		<tr height="24" style="height:18.0pt">
			<td class="xl69" height="24" style="width:207pt;height:18.0pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 120K</td>
			<td class="xl69" style="width:170pt" width="227">For e-beam lithography</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 960K</td>
			<td class="xl70">For e-beam lithography</td>
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		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">Fluoresent labeled polystyrene microspheres</td>
			<td class="xl69" style="width:131pt" width="174">Polyscience</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">2 um diameter</td>
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			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3904</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
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		<tr height="28" style="height:21.0pt">
			<td class="xl69" height="28" style="width:207pt;height:21.0pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3906</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF5305</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF131ON</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">10 kOhm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 6 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">910 Ohm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Photoresist</td>
			<td class="xl69" style="width:131pt" width="174">Microchemicals</td>
			<td class="xl69" style="width:122pt" width="162">AZ4620</td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Acetone</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl69" height="41" style="width:207pt;height:30.75pt" width="276">Fluorescence Module for the OTKB/M, Metric Threads</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB-FL/M</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
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			<td class="xl69" height="49" style="width:207pt;height:36.75pt" width="276">Fluorescent filter set</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">MDF-FITC</td>
			<td class="xl70">For Fluorescein Isothiocyanate (FITC)</td>
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			<td class="xl70" height="21" style="height:15.75pt">Ultrasonic cleaner</td>
			<td class="xl69" style="width:131pt" width="174">Delta</td>
			<td class="xl70">DC150H</td>
			<td class="xl70">For the lift off step</td>
		</tr>
	</tbody>
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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 ...

The overall goal of this experiment is to demonstrate how to trap microparticles with plasmonic optical lattices with a technique that suppresses photothermal convection. The main feature of this technique is that we use the array of plasmonic nanostructures to enhance the trapping efficiency. Also we use a unique brewing property of water, 0%thermal expansion coefficient at low temperature to suppress the photothermal convection.Demonstrating this procedure will be Dinesh Bhalothia, a grad student from my laboratory. The setup for the experiment builds on a optical tweezer kit. This modified optical tweezer kit with a florescence module is ready on a breadboard.An LED and a diode laser provide light for florescence and manipulation. Mirrors direct light through an objective lens. The objective focuses the light on a sample stage which also serves as a heat sink.A CCD camera captures images from the sample. These elements are more evident in this schematic. A 470 nanometer blue LED is the light source for the florescence module.A 980 nanometer laser diode provides laser light in loose focus for manipulation. The lens is a long working distance microscope objective. Activity on the sample stage is recorded by a CCD camera.Turn on the power supply and current for the 980 nanometer laser diode. Use the CCD camera to check the laser beam alignment. If the beam is well aligned, the camera image will be a Gaussian spot.Turn off the laser for the next steps. An important aspect of the setup is the cooling system. The sample stage is a heat sink designed to accommodate a thermoelectric cooler.Work with the system electronics to prepare to add the thermoelectric cooler. There is a custom driver circuit for this experiment. Make the connections between the driver circuit and the electronic control board.Next, get a thermoelectric cooling element that will fit in the sample stage and with a hole in it to allow the laser beam through. Connect the output of the driver circuit to the thermoelectric cooling element. Move the cooling element to the sample stage.Before continuing, connect the driver circuit to a five volt power supply. To monitor the temperature, use a forward-looking infrared camera, and check that the system is cooling properly before proceeding. For this, use a resistance thermometer unit and its sensor.Begin with the sensor on a glass cover slip. Transfer the sensor and cover slip to the sample stage. Apply a small amount of thermal paste to ensure thermal contact.There, put the the assembly in contact with the stage. At the controls, adjust the power to the thermoelectric cooling element. After three minutes, read the temperature using the resistance temperature detector thermometer.In addition, record the temperature with the forward-looking infrared camera. Repeat these two measurements at various output power settings to obtain a temperature calibration curve similar to this one. Calibration is essential.Before proceeding, shut off the power to the thermoelectric cooler. The final element of the setup is the nanoplasmonic array. The slide cover has a custom fabricated array and is ready to mount in the experiment.This scanning electron microscope image of the array provides more detail. It is an array of about 16 micrometers square of 22 by 22 gold nanodiscs. Each nanodisc has a thickness of 40 nanometers and a diameter of 550 nanometers.The center to center distance between discs is 750 nanometers. Put the slide cover with the nanoplasmonic array on the sample stage. It should be in contact with the thermoelectric cooler.Next, set up the light source. Turn on the florescent light source, and set the power to five milliwatts for bright field imaging. Monitor the CCD image while manipulating the slide.Use the marker on the slide to locate and align the array. Make sure the array is in the center of the region of interest on the computer screen. Now, turn to the sample for the experiment.It is a mixture of two micrometer diameter polystyrene particles in deionized water. Use a micropipette to dispense 10 microliters onto the nanoarray slide. Move to the current supply of the 980 nanometer laser diode.Turn it on to excite the plasmonic resonance of the array. Then work with the power supply for the cooling system to achieve a temperature of four degrees Celsius. Finally, start recording video of the microparticles with the CCD camera.This is an example of the video recorded during an experiment with two micrometer polystyrene spheres. The optical power of the 980 nanometer wavelength laser used for plasmonic resonance excitation is five milliwatts. Note the particles cluster in a hexagonal, close-packed structure.These still images are of the accumulated trapped microparticles over time. Again, the hexagonal close-pack structure is clear. Images such as these can provide data to produce a plot of the number of trapped microparticles as a function of time.These five colored curves are examples of microparticle trajectories that can be extracted from the recorded video using image processing techniques, and the centroid algothrithm. The scale bar represents two micrometers. The procedure reported here enables a researcher to reproduce trapping on a daily basis.While attempting this procedure, it's important to remember to use the infrared camera to monitor the sample temperature to avoid the sample breakage. After its development, this technique pave the way for researchers in the field of optical trapping to study a larger class of transport phenomena in plasmonic optical lattice. After watching this video, you should have a good understanding of how to do the optical trapping with the plasmonic optical lattice,

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

Research

JoVE Journal

Bioengineering

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 ...

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...