The work was supported in part by the Breast Cancer Research Foundation under grant BCRF-20-043.

1. Perfluorocarbon nanodroplet formulation
<ol>
	<li>Rinse out a 10 mL round-bottom flask with chloroform and wash out a 10 &#181;L and 1 mL gas tight glass syringe with chloroform by repeatedly aspirating the full syringe volume and expelling it for a total of three times. 
	CAUTION: Chloroform is volatile and can be toxic if inhaled. All work with this solvent should be performed in a fume hood.</li>
	<li>Using the syringes, add 200 &#181;L of DSPE-mPEG2000 (25 mg/mL), 6.3 &#181;L of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, 25 mg/mL) and 1 mL of IR 1048 (1 mg/mL in chloroform) into the round-bottom flask. Remember to clean out the syringes between lipids/dye to prevent contamination of the stock. 
	NOTE: Infrared dyes are light sensitive, and work should be done in dim conditions or flasks should be covered in aluminum foil.</li>
	<li>Remove the solvent utilizing a rotary evaporator. Ensure that the vacuum is slowly adjusted to 332 mbar to prevent bumping. After 5 min, reduce the pressure to 42 mbar to remove any water that may have entered the solution. 
	NOTE: The lipid cake can be stored overnight in a round bottom flask covered with parafilm at 4 &#176;C.</li>
	<li>Suspend the lipid cake in 1 mL of phosphate buffered saline (PBS) and sonicate or vortex at room temperature for 5 min or until all of the lipid cake has been suspended and dissolved in the solution. Sonicate for an additional 2 min to homogenize the solution.</li>
	<li>Transfer the solution to a 7 mL glass vial and place the vial in a glass dish filled with ice to allow the solution to cool down for 5 min before adding 50 &#181;L of perfluorohexane using a gas tight glass syringe. Remember to rinse out the syringe with perfluorohexane before dispensing it into the vial.</li>
	<li>Place the glass vial containing the lipids and ice bath in the probe sonicator enclosure and submerge the probe tip below the miniscus. Ensure that the sides of the sonicator probe does not touch the lip of the glass vial.</li>
	<li>Probe sonicate the mixture with the following settings: Amplitude 1, Process time: 20 s, Pulse-On: 1s, Pulse-off: 5s. Then sonicate at the following settings: Amplitude: 50, Process time: 5 s, Pulse-on: 1 s, Pulse-off: 10 s.</li>
	<li>Transfer the nanodroplet solution to a 1.5 mL centrifuge tube and centrifuge at 300 x g for 3 min to separate out the larger droplets (&#62;1 &#181;m) from smaller droplets.</li>
	<li>Discard the pellet and transfer the supernatant to another 1.5 mL centrifuge tube. Wash the supernatant by centrifuging at 3000 x g for 5 min to pellet all the droplets in solution. Resuspend the PFCnDs in 1 mL of PBS by pipetting the pellet up and down and then sonicate in a bath sonicator for 1 min.</li>
	<li>Measure the size of the droplets using dynamic light scattering (DLS). Dilute the stock PFCnDs by 100-fold (10 &#181;L of PFCnD stock in 990 &#181;L of PBS) and bath sonicate to disperse the PFCnDs before measuring. Representative results are shown in Figure 1.</li>
	<li>Determine the concentration of the PFCnDs utilizing the nanoparticle tracking analyzer (see Table of Materials). Dilute the PFCnDs by 100-1000-fold to ensure accurate measurement of the concentration. The protocol typically yields droplets at a concentration on the order of 1010 particles/mL.</li>
	<li>Prepare 10 mL of ultrasound coupling gel in a 50 mL centrifuge tube and add 1% (v/v) or 100 &#181;L of PFCnDs to make solution of ~108 particles/mL. Vortex the solution to mix. Centrifuge the mixture at 4000 x g for 3 min to remove bubbles.</li>
</ol>
2. Polyacrylamide phantom preparation
<ol>
	<li>Degas water by filling a 500 mL vacuum flask with 400 mL of deionized water, seal with a rubber cork, and connect the flask to the vacuum line. Open the vacuum line and submerge the bottom of the flask in the bath sonicator. Sonicate for 5 min or until no gas bubble formation is visible.</li>
	<li>Prepare 10% ammonium persulfate (APS) solution by dissolving 500 mg in 5 mL of degassed water. Gently swirl the solution if the ammonium persulfate does not fully dissolve.</li>
	<li>In a 400 mL beaker with a stir bar on a stir plate, add 150 mL of degassed water and 50 mL of 40% (w/v) acrylamide-bisacrylamide solution to form 200 mL of 10% acrylamide-bisacrylamide solution. Stir the mixture at 200 rpm to allow for the proper mixing without introducing bubbles. 
	CAUTION: Acrylamide is a carcinogen, and all work should be done in a fume hood with gloves, especially if working with acrylamide in powder form.</li>
	<li>Weigh out 400 mg of silica to and add it to the 10% acrylamide-bisacrylamide solution from step 2.3 to form a 0.2 %(w/v) of silica and acrylamide solution. 
	CAUTION: Silica when inhaled can be a carcinogen. All work including weighing should be performed in a fume hood.</li>
	<li>Prepare a 58 mm x 58 mm x 78 mm square mold with a cylindrical inclusion by cutting off the tips of from a plastic transfer pipette and supporting it in the mold with lab tape. See Figure 2.</li>
	<li>Add 2 mL of 10 % APS solution to the beaker to make a final concentration of 0.1% APS and add 250 &#181;L of Tetramethylethylenediamine (TEMED) to the phantom solution. Allow the solution to stir briefly (less than a min).</li>
	<li>Quickly pour the solution into the mold, while being careful not to introduce air bubbles into the solution. The solution should polymerize within 10 min. Remove the phantom by running the flat end of a lab spatula around the edge of the mold and inverting the mold. 
	&#8203;NOTE: These phantoms can be reused multiple times and should be submerged in water and stored at 4&#176;C.</li>
</ol>
3. Perfluorocarbon nanodroplet imaging
<ol>
	<li>Turn on and warm up the pulsed laser system for ~20 min following manufacturer instructions. Ensure that the fiber optical bundle is properly connected to the laser output and the two legs are properly placed within the fiber bundle holder.</li>
	<li>Turn on the ultrasound imaging system, connect array imaging transducer (L11-4v) to the system and fix the transducer within the holder to align its imaging plane with laser cross section.</li>
	<li>Set the pulse repetition frequency of the laser system to 10Hz and place a powermeter at the end of the fiber bundle to measure energy. Tune the q-switch delay until the estimated fluence is 70 mJ/cm2. 
	CAUTION: Appropriate eyewear must be worn when firing the laser and laser curtains must enclose the space.</li>
	<li>Backfill one of the channels in the polyacrylamide phantom with the ultrasound gel/PFCnD mixture using a 1 mL plastic slip tip syringe. Liberally cover the top of the channel with ultrasound gel and remove any bubbles with a 1 mL plastic slip tip syringe. Place the polyacrylamide phantom underneath the transducer and fiber bundle as shown in Figure 3.</li>
	<li>Use the combined laser ultrasound and elasticity (CLUE) imaging platform based on the software20 to image PFCnD synchronized with optical activation. Change the general user-defined parameters in Param structure for imaging: set start/end depth to 0/40mm, center frequency to 6.9MHz, and transducer name to &#39;L11-4v&#39;.</li>
	<li>Define a new RunCase and design a module sequence for repeated optical activation/recondensation and US imaging of PFHnDs. This is done by listing predefined modules such as ultrafast imaging (mUF), external laser (mExtLaser) and idle(mIdle).
	<ol>
		<li>Repeat the sequence set mExtLaser-mIdle-mUF-mExtLaser-mUF twice to acquire both n-pulse and p-pulse imaging data. 
		NOTE: The first mExtLaser module in each sequence is set as a sham laser by setting ExtLaser.Enable to 0 and the &#39;mIdle&#39; is included to minimize time between background US images and the n/p-pulse US images after laser activation.</li>
	</ol>
	</li>
	<li>Set module parameters for each module placed in the current run case&#39;s module sequence. Access each module parameter by index corresponding to its order in module sequence. Modules will execute pre-defined operations with module parameters user set here.
	<ol>
		<li>Set ExtLaser.QSdelay in external laser modules to the value of laser Q-switch delay tuned in step 3.3, in microseconds. This module waits for laser system&#39;s flashlamp trigger out and generates Q-switch trigger after the delay specified in QSdelay.</li>
		<li>In ultrafast imaging module, set Resource.numFrame to 100, set SeqControl.PRI to 200 (&#181;s), and set TW.polarity to 1 for P-pulse and -1 for N-pulse (see Figure 4 for corresponding pulse shape). This module will transmit ultrafast 0-degree planar wave with pulse type specified in TW.polarity.
		<ol>
			<li>Acquire full aperture imaging window of 38.8 mm wide for number of frames in Resource.numFrame, pulse repetition interval of SeqControl.PRI, then save data for off-line processing.</li>
		</ol>
		</li>
		<li>Set SeqControl.lastPRI_Module in idle module to the length of time between laser pulses (100 ms) subtracted by Q-switch delay, imaging data acquisition time (20 ms), and a 20 &#181;s margin for the signal to travel. This module keeps the system under &#39;no operation&#39; state for the time in SeqControl.lastPRI_Module to fill the time gap between the end of imaging data acquisition and next laser pulse excitation.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Probe sonication is a relatively simple and easy to learn method to fabricate PFCnDs. There are a few steps where care must be taken. When handling chloroform, it is imperative that a positive displacement pipette or glass syringes is used, as it is volatile and will &#34;leak&#34; from standard air displacement pipettes. Furthermore, if using a positive displacement, ensure that an appropriate tip is used as chloroform will dissolve most plastic tips, which can introduce contaminants into the solution. A positive displa...

Microbubbles are the most ubiquitous ultrasound contrast agent owing to their biocompatibility and excellent echogenicity in comparison to soft tissues. This makes them valuable tools for visualizing blood flow, organ delineation, and other applications1. However, their size (1-10 &#181;m), which makes them exceptional for imaging based on their resonant frequency, restricts their applications to the vasculature2.
This limitation has led to the development of PFCnDs, which are nano-emulsions composed of a surfactant encased around a liquid perfluorocarbon core. These nanoparticles can be synthesized at sizes as small as 200 nm and are designed to take advantage of &#34;leaky&#34; vasculature or pores and open fenestrations found in tumor vasculature. While these disruptions are tumor dependent, this permeability allows for extravasation of nanoparticles from ~200 nm - 1.2 &#181;m depending on the tumor3,4. In their initial form, these particles produce little to no ultrasound contrast. Upon vaporization - induced acoustically or optically - the core phase changes from liquid to gas, inducing a two and half to five-fold increase in diameter5,6,7 and generating photoacoustic and ultrasound contrast. While acoustic vaporization is the most common activation method, this approach creates acoustic artifacts that limits the imaging of the vaporization. Additionally, most perfluorocarbons require focused ultrasound with a mechanical index beyond the safety threshold to vaporize8. This has led to the development of lower boiling point PFCnDs, which can be synthesized by condensing microbubbles into nanodroplets9. However, these droplets are more volatile and subject to spontaneous vaporization10.
Optical droplet vaporization (ODV), on the other hand, requires the addition of an optical trigger such as nanoparticles11,12,13 or dye6,14,15 and can vaporize higher boiling point perfluorocarbons using fluences within the ANSI safety limit11. PFCnDs synthesized with higher boiling point cores are more stable and will recondense after vaporization, allowing for background free imaging16, multiplexing17, and super-resolution18. One of the major limitations of these techniques is the fact that high boiling point PFCnDs are echogenic after vaporization for only a short timeframe, on the scale of milliseconds19, and are relatively faint. While this issue can be mitigated through repeated vaporizations and averaging, detection and separation of droplet signal remains a challenge.
Taking inspiration from pulse inversion, the duration and contrast can be enhanced by modifying the phase of the ultrasound imaging pulse19. By starting the ultrasound imaging pulse with a rarefaction phase (n-pulse), both the duration and contrast of the vaporized PFCnDs increases. In contrast, starting the ultrasound imaging pulse with a compression phase (p-pulse), results in reduced contrast and shorter in duration. This article will describe how to synthesize optically triggerable perfluorocarbon nanodroplets, polyacrylamide phantoms commonly used in imaging, and demonstrate contrast enhancement and improved signal longevity through acoustic modulation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>VWR</td>
			<td>97064-592</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850365C</td>
			<td>Lipids, these can be purchased suspended in chloroform or in powder form. For long term storage, powder form is the best but chloroform is more practical.</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG)</td>
			<td>Avanti Polar Lipids</td>
			<td>880120C</td>
			<td>Lipids, these can be purchased suspended in chloroform or in powder form. For long term storage, powder form is the best but chloroform is more practical.</td>
		</tr>
		<tr>
			<td>Acrylamide : Bisacrylamide solution (19:1) 40% (w/v), OmniPur&#174;</td>
			<td>VWR</td>
			<td>EM-1300</td>
			<td>acrylamide solution, lower concentration/ powder</td>
		</tr>
		<tr>
			<td>IR-1048</td>
			<td>Sigma</td>
			<td>405175</td>
			<td>Infrared dye</td>
		</tr>
		<tr>
			<td>L11-4v</td>
			<td>Verasonics</td>
			<td>-</td>
			<td>ultrasound linear array transducer</td>
		</tr>
		<tr>
			<td>Microtip 1/8&#34;</td>
			<td>Qsonica LLC</td>
			<td>4418</td>
			<td>microtip for probe sonicator</td>
		</tr>
		<tr>
			<td>N, N, N&#8242;, N&#8242; -Tetramethylethylenediamine (TEMED)</td>
			<td>VWR</td>
			<td>97064-902</td>
			<td>Used to polymerize polyacrylamide by forming free radicals in the presence of ammonium persulfate</td>
		</tr>
		<tr>
			<td>Nova II</td>
			<td>Ophir-Spiricon</td>
			<td>7Z01550</td>
			<td>laser power meter</td>
		</tr>
		<tr>
			<td>Perfluorohexane</td>
			<td>Fluoromed</td>
			<td>APF-60M</td>
			<td>perfluorocarbon liquid</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) tablets</td>
			<td>VWR</td>
			<td>97062-732</td>
			<td>Tablets used to make PBS</td>
		</tr>
		<tr>
			<td>Q500</td>
			<td>Qsonica LLC</td>
			<td>Q500-110</td>
			<td>Probe sonicator</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Sigma-Aldrich</td>
			<td>288500</td>
			<td>2-25 &#956;m particle size</td>
		</tr>
		<tr>
			<td>Tempest 30</td>
			<td>New wave research</td>
			<td>-</td>
			<td>Pulsed laser system</td>
		</tr>
		<tr>
			<td>Vantage 128</td>
			<td>Verasonics</td>
			<td>-</td>
			<td>research ultrasound imaging system</td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>Malvern Instruments Ltd</td>
			<td>-</td>
			<td>Makes size measurements based on dynamic light scattering</td>
		</tr>
	</tbody>
</table>

formulation and acoustic modulation of optically vaporized perfluorocarbon nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Successful formulation and centrifugal separation of the PFCnDs should yield droplets around the size of 200-300 nm in diameter (Figure 1A). Improperly separated droplets may show small peaks around 1 &#181;m. These solutions can be further bath sonicated to break up the larger droplets. The size of the droplets will increase over time due to coalescing and/or diffusion in a process known as Ostwald ripening21,22 (<strong class="xfig...

Watch this Scientific Journal Video about Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets at JoVE.com

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

formulation-acoustic-modulation-optically-vaporized-perfluorocarbon

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2.0K Views.  Georgia Institute of Technology and Emory University. Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Video: Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different techniques and varies in price, materials, and purpose. Many of these fabrication methods result in polydisperse populations with non-uniform activation thresholds. Additionally, controlling the droplet sizes typically requires stable perfluorocarbon liquids with high activation thresholds that are not practical in vivo. Producing uniform droplet sizes using low-boiling point gases would be beneficial for in vivo imaging and therapy experiments. This article describes a simple and economical method for the formation of size-filtered lipid-stabilized phase-shift nanodroplets with low-boiling point decafluorobutane (DFB). A common method of generating lipid microbubbles is described, in addition to a novel method of condensing them with high-pressure extrusion in a single step. This method is designed to save time, maximize efficiency, and generate larger volumes of microbubble and nanodroplet solutions for a wide variety of applications using common laboratory equipment found in many biological laboratories.

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Scalable Generation of Uniform Cell Spheroids Using a 3D Acoustic Assembly Device

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...