We would like to acknowledge the use of the clean room facility SMIF at Duke University. We also want to thank Hao Qiang for his assistance in measuring the jet velocity. The authors thank Todd Rumbaugh of Hadland Imaging for providing the Shimadzu HPV-X camera used in this study.The work was funded in part by NIH through grants 5R03EB017886-02 and 4R37DK052985-20.

1. Microfabrication
NOTE: All the microfabrication procedures are performed in a cleanroom. A chrome mask is designed prior to the microfabrication, see Figure 2.
<img alt="Figure 2" src="/files/ftp_upload/55106/55106fig2.jpg" /> 
Figure 2: Schematic of the channel design in the microfluidic chip and the dimensions of the working units. a) Mask design of the aligned PDMS microchannels (green) and the patterns on the glass substrate (blue and red). b) Enlarged image of the mask design in a representative section of the working unit arrays. c) Schematic of one working unit showing a cell pattern (blue) and a pair of gold dots (red). All the length scales are shown on the bottom right. <a href="http://cloudfront.jove.com/files/ftp_upload/55106/55106fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Gold dot patterning 
	NOTE: The area of each gold dot is designed to be within 25-30 &#181;m2, so that it is large enough to absorb laser energy for bubble generation, but small enough to avoid individual cells adhering to it. A schematic diagram for the gold dot fabrication is shown in Figure 3a.
	<ol>
		<li>Glass slide cleaning (in a chemical hood)
		<ol>
			<li>Flush the glass slide with acetone followed by isopropyl alcohol (IPA), and then dry it with N2 flow.</li>
			<li>Soak the slide in piranha solution (H2SO4:H2O2 = 4:1) for 10 min.</li>
			<li>Take out the slide, rinse it with DI water, and then dry it with N2 flow.</li>
			<li>Bake the glass slide on a hotplate at 120 &#176;C for 10 min.</li>
			<li>Treat the slide in a plasma asher at 100 W for 90 s.</li>
		</ol>
		</li>
		<li>Spin-coating (spin-coating hood)
		<ol>
			<li>Turn on a hotplate and wait until the temperature is stable at 95 &#176;C.</li>
			<li>Follow the coating procedure: 
			1,000 rpm for 5 s with a 500-rpm/s ramp; 
			then 3,000 rpm for 30 s with a 1,000-rpm/s ramp.</li>
			<li>Fix the cleaned glass slide onto the spin coater by applying a vacuum.</li>
			<li>Cover the slide surface with P-20 and start the coating recipe.</li>
			<li>Repeat the coating process for NFR-negative photoresist.</li>
			<li>Bake the slide at 95 &#176;C for 60 s and wait until it cools down to room temperature.</li>
		</ol>
		</li>
		<li>Photolithography
		<ol>
			<li>Mount the chrome mask onto the mask aligner and make sure that the pattern side is facing down towards the slide.</li>
			<li>Set the photolithography recipe to hard exposure mode with 9 s of UV exposure and quickly align the glass substrate with the mask.</li>
			<li>After carrying out the UV exposure, bake the slide at 95 &#176;C for 1 min, and then let it cool down to room temperature.</li>
		</ol>
		</li>
		<li>Development
		<ol>
			<li>Develop the pattern on the slide in developer solution for 60 s.</li>
			<li>Remove the slide from the developer, flush it with DI water, and dry it with N2 flow.</li>
			<li>Check the pattern geometry under a microscope, and measure and record the feature size.</li>
		</ol>
		</li>
		<li>Gold deposition (E-beam evaporation) and lift-off
		<ol>
			<li>Bake the slide at 120 &#176;C for 5 min, and let it cool down to room temperature.</li>
			<li>Clean the slide with plasma in the reactive ion etching (RIE) machine for 90 s at 500 Torr and 100 W.</li>
			<li>Deposit 5 nm of Ti and 15 nm of Au onto the slide using an E-beam evaporator17.</li>
			<li>Soak the slide overnight in a glass beaker containing photoresist remover solvent to remove the gold resting on top of the NFR resist.</li>
			<li>Retrieve the slide, flush it with acetone followed by IPA, and dry it with N2 flow.</li>
			<li>Dry the slide on a hot plate at 115 &#176;C before cleaning it in the oxygen plasma asher at 100 W for 90 s.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Molecular-assembly patterning by lift-off (MAPL) 
	Note: The area of each fibronectin-coated island is set to be within 700-900 &#181;m2 to facilitate adequate HeLa cell spreading in a square region while minimizing the chances of multiple cells aggregating on the island. A schematic diagram for the preparation of cell-patterning islands is shown in Figure 3b.
	<ol>
		<li>Spin coating
		<ol>
			<li>Repeat step 1.1.2, but use S1813-positive photoresist and a temperature of 115 &#176;C.</li>
		</ol>
		</li>
		<li>Aligned photolithography
		<ol>
			<li>Mount the chrome mask onto the mask aligner and make sure that the side with the pattern is facing down towards the slide with the gold dots.</li>
			<li>Set the photolithography recipe to hard exposure mode with 9 s of UV exposure.</li>
			<li>Align the top mask with the bottom slide using alignment marks, located around the edge of the mask, as a spatial reference.</li>
			<li>Check the central portion of the mask and verify that the pattern features are aligned correctly.</li>
			<li>Complete the UV exposure without a post-bake.</li>
		</ol>
		</li>
		<li>Development
		<ol>
			<li>Repeat step 1.1.3 but with 45 s of development before drying on a hotplate and preserving in N2 storage.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Chemical treatment
	<ol>
		<li>Prepare the passivating solution: 0.5 mg/mL PLL-g-PEG in 10 mM HEPES buffer.</li>
		<li>Retrieve the slide from N2 storage and clean it using RIE with the parameter setting: 500-Torr pressure, 100-W power, and 90-s duration.</li>
		<li>Pipette one drop of passivating solution onto a piece of paraffin film.</li>
		<li>Sandwich the solution with the film and the slide, making sure that the side with the pattern features is facing down towards the film; avoid the formation of bubbles.</li>
		<li>Wait for 45 min before removing the slide from the film.</li>
		<li>Soak the slide consecutively in photoresist remover, photoresist remover and DI water (1:1), and DI water, and agitate it in an ultrasound bath for 90 s for each soak.</li>
		<li>Dry the slide on a hotplate to remove the moisture before sealing it in a desiccator and storing it in a refrigerator.</li>
	</ol>
	</li>
	<li>Chip assembly
	<ol>
		<li>Repeat steps 1.1.1-1.1.4, but with SU8-2025-negative photoresist and the parameter setting referred called the Microchem protocol18, to prepare a silicon mold with a photomask.</li>
		<li>Fabricate a PDMS microchannel (40 mm x 25 mm x 5 mm, L x W x H) and a small PDMS slab (800 &#181;m-wide groove structure) using soft lithography.</li>
		<li>Punch the microchannel for fluid access ports and clean it with tape and then with IPA.</li>
		<li>Shield the patterned area of the glass substrate with the PDMS slab, and apply RIE (100 W, 500 mTorr, 60 s) to remove PLL-g-PEG from the peripheral area.</li>
		<li>Treat the microchannel with a reduced dose of RIE (25 W, 500 mTorr, 25 s), and then align it to the patterned glass substrate (with the small PDMS slab removed); bring the microchannel and the patterned glass substrate in conformal contact under a stereoscope.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55106/55106fig3.jpg" /> 
Figure 3: Schematic diagrams for microfabrication and chip assembly. a) Pattern of gold dots on the glass substrate. b) Preparation of the glass substrate for cell patterning via MAPL. c) Assembly of the microfluidic channel with plasma bonding. See the Materials List for the definitions of the abbreviations. <a href="http://cloudfront.jove.com/files/ftp_upload/55106/55106fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Cell attachment 
	NOTE: HeLa cells are routinely maintained in DMEM supplemented with 10% FBS and 1% antibiotic/antimitotic solution in a cell culture incubator. A schematic diagram for the chip assembly and cell attachment is shown in Figure 3c.
	<ol>
		<li>Prime the chip with PBS for 30 min at 1 &#181;L/min, and then infuse fibronectin solution (50 &#181;g/mL in PBS, 1 &#181;L/min) for 45 min.</li>
		<li>While waiting, trypsinize cell culture with 0.25% trypsin-EDTA, wash them in culture medium, centrifuge individual cells, and reconstitute the cell suspension in pre-warmed (37 &#176;C) culture medium at 5x106 cells/mL.</li>
		<li>Replace the fibronectin solution with PBS and flush the chip at 10 &#181;L/min for 5 min.</li>
		<li>Inject the prepared cell suspension into the chip, stop the flow, clamp the outlet, and maintain the chip in a cell culture incubator for 30 min.</li>
		<li>Release the outlet and flush the chip with cell culture medium at 10 &#181;L/min for 5 min. Reduce the perfusion flow rate of the culture medium to 0.75 &#181;L/min and maintain the cell culture for 2 h in the incubator.</li>
	</ol>
	</li>
</ol>
2. Bubble Generation and Flow Visualization
NOTE: A schematic of the experimental setup is shown in Figure 4a for tandem bubble generation and the resultant flow interaction with a single cell grown nearby in a microfluidic channel.
<ol>
	<li>Generation of tandem bubbles with two lasers and timing control
	<ol>
		<li>Place the microfluidic chip on the stage of an inverted microscope and align the foci of two pulsed Nd:YAG lasers (&#955; = 532 nm, 5-ns pulse duration) on a pair of patterned gold dots separated by 40 &#181;m.</li>
		<li>Use a digital delay generator to trigger the two lasers with a time delay about 2.5 &#181;s to produce two bubbles initiated at the gold dots.</li>
		<li>Adjust the laser output energy (~10 &#181;J) to produce a maximum diameter of the bubbles within 50 &#177; 2 &#181;m.</li>
		<li>Synchronize a high-speed camera (200-ns exposure and 2 million frames per second (fps)) to the lasers and capture the dynamics of bubble expansion, collapse, bubble-bubble interaction, and jet formation.</li>
	</ol>
	</li>
	<li>Quantification of jet velocity
	<ol>
		<li>Record the position of the jet tip (J1) at the proximal end of the first bubble (B1) and the distal end of B1, starting at the generation of the second bubble (B2) and ending with the touchdown of J1 towards the distal end of B1; see the schematic in Figure 5a.</li>
		<li>Linearly fit the time course of position for both the proximal (jet tip) and the distal ends. Calculate the slope of the tip position versus time curve for the proximal end to determine the jet speed. The intersection of the two lines indicates the jet touchdown time. More details can be found in Reference 19.</li>
	</ol>
	</li>
	<li>Visualization of the tandem bubble-induced flow field
	<ol>
		<li>Prepare 1 &#181;m of polystyrene (PS) bead suspension in DI water (2.6% w/v) and inject the beads into the microfluidic chip.</li>
		<li>Generate tandem bubbles as described in step 2.1.1-2.1.3.</li>
		<li>Record the tandem bubble dynamics using a high-speed video camera at a framing rate of 5 M fps with a 100-ns exposure time.</li>
		<li>Upload the acquired image sequence into a commercial PIV software.</li>
		<li>Divide each image into smaller interrogation windows of 16 x 16 pixels (px) with a 75% overlap.</li>
		<li>Apply multi-pass iteration and regional filters to reduce the errors in velocity field computation, and output the results in a velocity vector map.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/55106/55106fig4.jpg" /> 
Figure 4: Schematic of the experimental setup and image recording. a) Experimental setup for tandem bubble generation. b) Time sequence used for different types of imaging acquisitions. FL: fluorescence microscopy, BF: bright field, IFT: interframe time. <a href="http://cloudfront.jove.com/files/ftp_upload/55106/55106fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Single-cell Analysis and Bioeffects
NOTE: The tandem bubble is produced next to the target cells, and the resultant bioeffects are studied in a standoff distance-dependent manner. The time sequence for different types of image acquisitions is depicted in Figure 4b.
<ol>
	<li>Membrane poration and macromolecular uptake 
	NOTE: The uptake of extracellular macromolecules into the target cell is characterized by the progressive diffusion of membrane impermeant propidium iodide (PI) through the poration site on the cell membrane.
	<ol>
		<li>Following step 1.5, replace the regular culture medium (i.e., DMEM) with PI solution (100 &#181;g/mL in DMEM) running at a perfusion flow rate of 0.5 &#181;L/min throughout the experiment.</li>
		<li>Program the microscope control software to automatically select the PI fluorescent cube on the rotating turret and synchronize the timing of the CCD camera with the PI fluorescence excitation to capture fluorescence images with an exposure time of 200 ms.</li>
		<li>After the two laser foci are aligned to a pair of gold dots next to a target cell, record both bright field (BF) and fluorescence (FL) images before the tandem bubble treatment.</li>
		<li>Use microscope control software to immediately start a time-lapse fluorescence image recording of the target cell shortly after step 2.1 to capture the history of PI uptake.</li>
	</ol>
	</li>
	<li>Membrane deformation
	<ol>
		<li>Prepare 1-&#181;m beads (1% w/v, activated with water-soluble carbodiimide) and functionalize them with Peptide-2000 (100 &#181;g/mL in PBS).</li>
		<li>Following step 1.5, incubate the attached cells with the functionalized beads at a density of 1x109 beads/mL at 37 &#176;C for 30 min.</li>
		<li>Repeat step 2.1 to produce tandem bubbles next to the target cell and record the cell deformation with an ultra-high-speed camera running at a framing rate of 5 million fps.</li>
		<li>Identify a triad of 3 beads that remain on the imaging plane throughout the experiment and compile their positions as (x1, y1) (x2, y2), and (x3, y3) in the experiment coordinates.</li>
		<li>Calculate the area of the triad before and after the tandem bubble treatment using the formula: 
		<img alt="Equation 1" src="/files/ftp_upload/55106/55106eq1.jpg" /></li>
		<li>Calculate the area strain as: 
		<img alt="Equation 2" src="/files/ftp_upload/55106/55106eq2.jpg" /></li>
		<li>Based on the coordinates of the three vertices before and after the tandem bubble treatment, calculate the local principal strain and area strain following established protocols20,21.</li>
	</ol>
	</li>
	<li>Viability and apoptosis 
	NOTE: FITC Annexin V labels cells, including apoptotic ones, through the externalization of phosphatidylserine (green fluorescence). PI labels the nuclei of necrotic cells (red fluorescence). Bright field imaging is used to document cell morphology and to assist with the identification of cell viability and apoptosis.
	<ol>
		<li>Record the position of each treated cell in the microfluidic channel (facilitated by the address labels in the channel, see Figure 2b) while repeating step 3.1.</li>
		<li>Incubate the treated cells in the cell culture medium for another 2 h before perfusing the chip with FITC Annexin V solution for 15 min. Positive cells display green fluorescence.</li>
		<li>Repeat step 3.3.2 24 h after the tandem bubble treatment to study the long-term bioeffects in the targeted cells.</li>
	</ol>
	</li>
	<li>Calcium response
	<ol>
		<li>Mix 3 &#181;L of fura-2 AM stock solution (1 mg/mL in DMSO) with 3 &#181;L of 10% w/v Pluronic F-127 in 500 &#181;L of reduced serum medium to prepare the final labeling solution (6 &#181;M).</li>
		<li>Following step 1.5, replace the cell culture medium with the labelling solution and incubate under room temperature in the dark for 40 min (1 &#181;L/min perfusion rate).</li>
		<li>Refill the channel with the reduced serum medium (0.75 &#181;L/min perfusion rate) before starting the cell experiment.</li>
		<li>Start ratiometric imaging (wavelength: 340/380 nm, 50-ms exposure) of the target cell using the commercial PTI system.</li>
		<li>After 10 s of ratiometric imaging of the cell at resting level, produce tandem bubbles as in step 2.1; first record with an interframe time (IFT) of 0.1 s for 1 min, then increase the IFT to 1 s for another min, and further increase to 5 s after 2 min.</li>
		<li>Use the PTI software to calculate the ratio R = F340/F380 in the region of interest (ROI), which is proportional to the intracellular calcium concentration.</li>
	</ol>
	</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Properties+of+the+Red+Cell+Membrane:+I.+Membrane+Stiffness+and+Intracellular+Pressure.">Mechanical Properties of the Red Cell Membrane: I. Membrane Stiffness and Intracellular Pressure.</a> Biophys J. 4 (2), 115-135 (1964).</li><li>Lim, C. T., Dao, M., Suresh, S., Sow, C. H., Chew, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+deformation+of+living+cells+using+laser+traps.">Large deformation of living cells using laser traps.</a> Acta Mater. 52 (7), 1837-1845 (2004).</li><li>Puig-de-Morales-Marinkovic, M., Turner, K. T., Butler, J. P., Fredberg, J. J., Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelasticity+of+the+human+red+blood+cell.">Viscoelasticity of the human red blood cell.</a> Am J Physiol Cell Physiol. 293 (2), C597-C605 (2007).</li><li>Kudo, N., Okada, K., Yamamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonoporation+by+single-shot+pulsed+ultrasound+with+microbubbles+adjacent+to+cells.">Sonoporation by single-shot pulsed ultrasound with microbubbles adjacent to cells.</a> Biophys J. 96 (12), 4866-4876 (2009).</li><li>van Wamel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrating+microbubbles+poking+individual+cells:+Drug+transfer+into+cells+via+sonoporation.">Vibrating microbubbles poking individual cells: Drug transfer into cells via sonoporation.</a> J Control Release. 112 (2), 149-155 (2006).</li><li>Hu, Y., Wan, J. M., Yu, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+perforation+and+recovery+dynamics+in+microbubble-mediated+sonoporation.">Membrane perforation and recovery dynamics in microbubble-mediated sonoporation.</a> Ultrasound Med Biol. 39 (12), 2393-2405 (2013).</li><li>Dijkink, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cavitation-cell+interaction:+trans-membrane+transport+and+viability+studies.">Controlled cavitation-cell interaction: trans-membrane transport and viability studies.</a> Phys Med Biol. 53 (2), 375-390 (2008).</li><li>Rau, K. R., Quinto-Su, P. A., Hellman, A. N., Venugopalan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+laser+microbeam-induced+cell+lysis:+Time-resolved+imaging+and+analysis+of+hydrodynamic+effects.">Pulsed laser microbeam-induced cell lysis: Time-resolved imaging and analysis of hydrodynamic effects.</a> Biophys J. 91 (1), 317-329 (2006).</li></ol>

The authors have nothing to disclose.

Single-cell analysis, in combination with live-cell imaging, has greatly enhanced our understanding of the dynamic and often variable processes in individual cells, such as phenotype development and immune response23. In contrast to the conventional cell culture in dishes or flasks, microfluidic systems enable precise control of the microenvironment, down to the single-cell level, in real time. Consequently, advances in microfluidic technology and techniques have largely improved the throughput and reproducibi...

There is a growing recognition that cellular heterogeneity, arising from the stochastic expression of genes, proteins, and metabolites, exists within a large cell population and serves as a fundamental principle in biology to allow for cell adaptation and evolution1. Therefore, it is often inaccurate and unreliable to use population-based bulk measurements to understand the function of individual cells and their interactions. Developing new technologies for single-cell analysis is therefore of high interest in biological and pharmacological research, and can be used, for example, to better understand the key signaling pathways and processes in stem cell biology and cancer therapy2-4. In recent years, the emergence of microfluidic platforms has greatly facilitated single-cell analysis, where the positioning, treatment, and observation of the response from individual cells have been performed with novel analytical strategies5.
Cavitation plays an important role in a diverse range of biomedical applications, including the treatment of cancers by high-intensity focused ultrasound (HIFU)6, the non-invasive fragmentation of kidney stones by shock wave lithotripsy (SWL)7, drug or gene delivery by sonoporation8, and the recently reported destruction of cells or tissues by hydrodynamic bubble cavitation9,10. Despite this, the dynamic processes of cavitation bubble(s) interactions with biological tissue and cells have not been well understood. This is due to the randomness in cavitation initiation and bubble dynamics produced by ultrasound, shock waves, and local hydraulic pressure; furthermore, there is a lack of enabling techniques to resolve the inherently complex and fast responses of biological cells, especially at the single-cell level.
Because of these challenges, it is not surprising that very few studies have been reported to investigate bubble-cell interactions under well-controlled experimental conditions. For example, membrane poration of individual cells trapped in suspension11 and the impulsive large deformation of human red blood cells12 have been demonstrated using laser-generated single bubbles in microfluidic channels. The latter technique, however, can only produce very small deformation in eukaryotic cells because of the presence of the nucleus13. Moreover, it is difficult to monitor downstream bioeffects when treating cells in suspension. In other studies, ultrasound excitation of a cell-bound microbubble (or ultrasound contrast agent) for producing membrane poration and/or intracellular calcium responses in single adherent cells has been reported8. Membrane poration of single adherent cells can also be produced by using laser-generated tandem bubbles in a thin liquid layer containing light-absorbing Trypan blue solution14, or by an oscillating gas bubble generated by microsecond laser pulses irradiating through an optically absorbing substrate in microchambers15. When compared, the optically absorbing substrate has an advantage over the laser-absorbing Trypan blue solution because the latter is toxic to cells. More importantly, laser-generated bubbles are more controllable in terms of bubble size and location than acoustically excited bubbles. Nevertheless, in all these previous studies, the cell shape, orientation, and adhesion conditions were not controlled, which may substantially influence the cell response and bioeffects produced by mechanical stresses16.
To overcome these drawbacks in previous studies, we have recently developed an experimental system for bubble generation, cell patterning, bubble-bubble-cell interactions, and real-time bioassays of cell response in a microfluidic chip constructed by using a unique combination of microfabrication techniques. Three main features that distinguish our experimental system from others in the field are: 1) the patterning of micron-sized gold dots on the glass substrate to enable localized laser absorption for bubble generation17; 2) the patterning of micron-sized islands of extracellular matrix (ECM) for cell adhesion on the same substrate to control both the location and geometry of individual cells; and 3) the compression of the dimension of the bubble-bubble-cell interaction domain from 3D to a quasi-2D space to facilitate in-plane visualization of bubble-bubble interactions, jetting flow fields, cell deformation, and bioeffects, all captured in one streamlined imaging sequence (Figure 1d).
<img alt="Figure 1" src="/files/ftp_upload/55106/55106fig1.jpg" /> 
Figure 1: The microfluidic chip and schematics of different assays. a) An assembled microfluidic chip with channels filled with blue ink for visualization. b) A region inside the microfluidic chip with patterned cells and gold dots (the distance between the two gold dots in proximity is 40 &#181;m). Many pairs of working units can be arranged in a channel. c) Close-up image of a single working unit consisting of a pair of gold dots and a HeLa cell adhered to the cell-patterning region. d) Schematic of the device operation. A single cell adheres to and spreads on the &#34;H&#34;-shaped island coated with fibronectin. A pair of cavitation bubbles (tandem bubble) with anti-phase oscillation are produced by illuminating pulsed laser beams on the gold dots (see Figure 4a), leading to the generation of a fast and localized jet moving towards the target cell nearby. The cell may be deformed, porated for macromolecular uptake, and/or stimulated with a calcium response, depending upon the standoff distance (Sd) of the cell to the tandem bubble. <a href="http://cloudflare.jove.com/files/ftp_upload/55106/55106fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This platform can be further combined with fluorescence assays and functionalized beads attached to the cell surface for cavitation-induced bioeffects. In particular, this platform opens the way for reliable and quantifiable assays at the single-cell level. Up to now, we have used the device for the analysis of tandem bubble-induced cell membrane deformation, cell poration and intracellular uptake, viability, apoptosis, and intracellular calcium response. In the following protocol, we describe the process of chip fabrication and the procedure for analyzing the various bioeffects mentioned above. Moreover, the operations of the chip are also described.

<table><tbody><tr><td>&lt;strong&gt;Reagent/Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>75 mm x 38 mm Plain Microscope Slides</td><td>Corning</td><td>2947-75X38</td><td></td></tr><tr><td>Acetone</td><td>Sigma Aldrich, Co.</td><td>320110</td><td>ACS reagent, &amp;ge;99.5%</td></tr><tr><td>Isopropyl alcohol</td><td>Sigma Aldrich, Co.</td><td>W292907</td><td>&amp;ge;99.7%, FCC, FG</td></tr><tr><td>Sulfuric acid</td><td>Sigma Aldrich, Co.</td><td>320501</td><td>ACS reagent, 95.0-98.0%</td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich, Co.</td><td>216763</td><td>30 wt.% in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Primer P-20</td><td>Microchem</td><td>MCC Primer 80/20</td><td></td></tr><tr><td>NFR photoresist</td><td>JSR</td><td>NFR016D2</td><td></td></tr><tr><td>Photomask</td><td>Photoplotstore</td><td>N/A</td><td>4x4 Direct write mask</td></tr><tr><td>MF-319 Developer</td><td>Shipley (Rohm and Haas)</td><td>Microposit MF-319</td><td></td></tr><tr><td>1165 Photoresist Remover</td><td>Dow Chemical, Co.</td><td>DEM-10018073</td><td>1-methyl-2-pyrrolidinone based</td></tr><tr><td>S1813 photoresist</td><td>Shipley (Rohm and Haas)</td><td>S1813</td><td></td></tr><tr><td>PLL-g-PEG</td><td>SuSoS</td><td>PLL(20)-g[3.5]- PEG(2)</td><td></td></tr><tr><td>HEPES</td><td>ThermoFisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Paraffin film</td><td>HACH</td><td>251764</td><td></td></tr><tr><td>SU-2025 photoresist</td><td>Microchem</td><td>SU-2025</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>Microbore Tubing</td><td>Saint-Gobain PPL Corp.</td><td>S-54-HL</td><td></td></tr><tr><td>Metal pins</td><td>New England Small Tube</td><td>NE-1300-01</td><td>Cut Tube (straight), 0.025&amp;rdquo; OD x 0.017&amp;rdquo; ID x 0.50&amp;rdquo; Long</td></tr><tr><td>HeLa cells</td><td>Duke Cell Culture Facility</td><td>(307-CCL-2) HeLa, p.148</td><td></td></tr><tr><td>DPBS (1x) buffer</td><td>ThermoFisher Scientific</td><td>14190144</td><td></td></tr><tr><td>DMEM culture medium</td><td>ThermoFisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Fibronectin Bovine Protein, Plasma</td><td>ThermoFisher Scientific</td><td>33010018</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Propidium Iodide</td><td>ThermoFisher Scientific</td><td>P21493</td><td></td></tr><tr><td>Carboxylate Microspheres 1.00&amp;nbsp;&amp;mu;m</td><td>Polysciences, Inc</td><td>08226-15</td><td></td></tr><tr><td>Carboxylate Microspheres 2.00&amp;nbsp;&amp;mu;m</td><td>Polysciences, Inc</td><td>18327-10</td><td></td></tr><tr><td>EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)</td><td>ThermoFisher Scientific</td><td>22980</td><td></td></tr><tr><td>Sulfo-NHS</td><td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td><td>24510</td><td></td></tr><tr><td>Peptite-2000</td><td>Advanced BioMatrix</td><td>5020-5MG</td><td></td></tr><tr><td>FITC Annexin V</td><td>ThermoFisher Scientific</td><td>A13199</td><td></td></tr><tr><td>Fura-2, AM</td><td>ThermoFisher Scientific</td><td>F1221</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich, Co.</td><td>D2650</td><td></td></tr><tr><td>F-127</td><td>invitrogen</td><td>P6866</td><td>0.2 &amp;micro;m filtered (10% Solution in Water)</td></tr><tr><td>Reduced serum media&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11058021</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasma asher</td><td>Emitech</td><td>K-1050X</td><td>O&lt;sub&gt;2&lt;/sub&gt; / Ar plasma ashing of photoresist and other organic materials</td></tr><tr><td>Mask aligner</td><td>SUSS MicroTec&amp;nbsp;</td><td>Karl Suss MA6/BA6</td><td></td></tr><tr><td>E-beam evaporator</td><td>CHA Industries</td><td>CHA Industries Solution E-Beam</td><td></td></tr><tr><td>RIE</td><td>Trion Technology&amp;nbsp;</td><td>Trion Technology Phantom II</td><td>(oxide/ nitride/ polymer) etching</td></tr><tr><td>Stereoscope</td><td>AmScope</td><td>American Scope SM-4TZ-FRL</td><td>Stereo Microscope</td></tr><tr><td>Syringe pump</td><td>Chemyx Inc</td><td>NanoJet</td><td></td></tr><tr><td>Cell culture incubator</td><td>NuAire</td><td>AutoFlow NU-8500 Water Jacket CO&lt;sub&gt;2 &lt;/sub&gt;Incubator</td><td></td></tr><tr><td>Biological Safety Cabinets</td><td>NuAire</td><td>NU-425-400</td><td></td></tr><tr><td>Water bath</td><td>VWR</td><td>1122s</td><td></td></tr><tr><td>Centrifuge</td><td>IEC</td><td>Centra CL2</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio Observer Z1</td><td></td></tr><tr><td>Nd:YAG laser&amp;nbsp; (laser 1)</td><td>New Wave Research</td><td>Tempest</td><td></td></tr><tr><td>Nd:YAG laser&amp;nbsp; (laser 2)</td><td>New Wave Research</td><td>Orion</td><td></td></tr><tr><td>Delay generator</td><td>Berkeley Nucleonics&amp;nbsp;</td><td>BNC 565-8c</td><td></td></tr><tr><td>Flash lamp</td><td>Dyna-Lite</td><td>ML1000 fiber-coupled flashtube</td><td></td></tr><tr><td>high speed camera</td><td>DRS Hadland</td><td>&amp;nbsp;Imacon 200</td><td></td></tr><tr><td>high speed&amp;nbsp; camera</td><td>Shimadzu</td><td>HPV-X</td><td></td></tr><tr><td>high speed camera</td><td>Vision Research</td><td>Phantom V7.3</td><td></td></tr><tr><td>PIV software</td><td>LaVision</td><td>DaVis 7.2</td><td></td></tr><tr><td>camera</td><td>Zeiss</td><td>AxioCam MRc 5</td><td></td></tr><tr><td>software</td><td>Zeiss</td><td>AxioVision</td><td></td></tr><tr><td>PTI system</td><td>Horiba</td><td>S/N: 1705 RAM-X</td><td></td></tr><tr><td>EasyRatio software</td><td>Horiba</td><td>Easy Ratio Pro 2</td><td>version 2.3.125.86</td></tr><tr><td>63&amp;times; objective</td><td>Zeiss</td><td>LD Plan Neofluar</td><td></td></tr></tbody></table>

a microfluidic system with surface patterning for investigating cavitation bubble(s)&#8211;cell interaction and the resultant bioeffects at the single-cell level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

The microfluidic platform described in this work can be used to investigate bubble-bubble interactions and to analyze a variety of cavitation-induced bioeffects at the single-cell level. Here, we present several examples to demonstrate a variety of experimental studies and bioassays that can be performed in our experimental system. We will first illustrate the transient interactions of tandem bubbles with the jet formation, the visualization of the resultant flow field, and the calculatio...

Watch this Scientific Journal Video about A Microfluidic System with Surface Patterning for Investigating Cavitation Bubblesâ€“Cell Interaction and the Resultant Bioeffects at the Single-cell Lev

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

a-microfluidic-system-with-surface-patterning-for-investigating

Research

JoVE Journal

Bioengineering

11.7K Views.  Duke University. A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

Video: A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)–Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Biology

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

Environment

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

Engineering

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant adsorption rates, evaluating equilibrium surface tensions as a function of bulk surfactant concentration, and relating how surface tension changes with changes in the interfacial area following equilibration. Simultaneous visualization of the interface using fluorescence imaging with a high-speed confocal microscope allows the direct evaluation of structure-function relationships. In the capillary pressure microtensiometer (CPM), a hemispherical air bubble is pinned at the end of the capillary in a 1 mL volume liquid reservoir. The capillary pressure across the bubble interface is controlled via a commercial microfluidic flow controller that allows for model-based pressure, bubble curvature, or bubble area control based on the Laplace equation. Compared to previous techniques such as the Langmuir trough and pendant drop, the measurement and control precision and response time are greatly enhanced; capillary pressure variations can be applied and controlled in milliseconds. The dynamic response of the bubble interface is visualized via a second optical lens as the bubble expands and contracts. The bubble contour is fit to a circular profile to determine the bubble curvature radius, R, as well as any deviations from circularity that would invalidate the results. The Laplace equation is used to determine the dynamic surface tension of the interface. Following equilibration, small pressure oscillations can be imposed by the computer-controlled microfluidic pump to oscillate the bubble radius (frequencies of 0.001-100 cycles/min) to determine the dilatational modulus The overall dimensions of the system are sufficiently small that the microtensiometer fits under the lens of a high-speed confocal microscope allowing fluorescently tagged chemical species to be quantitatively tracked with submicron lateral resolution.

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant ...

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)–Cell Interaction and the Resultant Bioeffects at the Single-cell Level

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