The authors acknowledge support from SNSF PRIMA grant 179834 (to E.S.), an ETH Research Grant ETH-15 17-1 (R. S.), and a Gordon and Betty Moore Foundation Investigator Award on Aquatic Microbial Symbiosis (grant GBMF9197) (R. S.). The authors thank Dr. Miguel Angel Fernandez-Rodriguez (University of Granada, Spain) for the SEM imaging of bacteria and for the insightful discussions. The authors thank Dr. Jen Nguyen (University of British Columbia, Canada), Dr. Laura Alvarez (ETH Z&#252;rich, Switzerland), Cameron Boggon (ETH Z&#252;rich, Switzerland) and Dr. Fabio Grillo for the insightful discussions.

1. Silicon master preparation
NOTE: The PDMS templates bearing the microfabricated traps that form the template for colloidal and microbial patterning were fabricated according to the method introduced by Geissler et al.17. The silicon master was prepared by conventional lithography in a cleanroom. See the following steps for the procedure and the Table of Materials for the equipment.
<ol>
	<li>Design the features using computer-aided design (CAD) software.</li>
	<li>Prepare the chrome-glass mask with a layer of positive photoresist by exposing the designed features with a UV direct laser writer.
	<ol>
		<li>Develop the chrome-glass mask with a spin developer using a developer at 1:4 photoresist to water ratio for 15 s.</li>
		<li>Immerse the mask in chromium etchant for 50 s.</li>
		<li>Plasma-treat a 10 cm silicon wafer for 3 min at 600 W.</li>
		<li>Vapor-deposit a layer of hexamethyldisilizane and bake at 110 &#176;C to improve the adhesion toward the photoresist.</li>
		<li>Deposit a layer of photoresist at 4,500 &#215; g for 2 min.</li>
		<li>Expose the feature to UV through the chrome-glass mask with a mask aligner for 2 s and develop with a developer as for the mask.</li>
	</ol>
	</li>
	<li>To complete the fabrication of the silicon master, etch the wafer via deep reactive ion exchange, adjusting the etching time (&#60;2 min) to achieve the desired depth (measured with a profilometer).</li>
</ol>
2. Microchannel mold preparation
<ol>
	<li>Design the channel with CAD software and slice it with a slicer software to convert the designed model into instructions for the 3D printer, setting the slicing distance at 0.05 mm.</li>
	<li>Print the channel with a 3D printer for ~1 h.</li>
	<li>Wash and postcure the mold in a dedicated curing and washing machine.
	<ol>
		<li>Put the mold in a container filled with pure isopropyl alcohol (IPA) and vortex the liquid (wash for 20 min). Take the container filled with IPA out of the machine.</li>
		<li>Once the washed mold is removed from the IPA container, put it back into the washing and curing machine and postcure it for 15 min at 35 &#176;C. 
		NOTE: The Table of Materials reports the 3D printer and the curing and washing machine used in the mold preparation protocol. The 3D model was sliced with the 3D printer&#39;s proprietary slicer software.</li>
	</ol>
	</li>
	<li>Place the print in a UV oven for 12 h and place it in an oven at 80 &#176;C for 12 h. 
	NOTE: This step ensures that all the polymer is cured and all uncured polymer is removed from the print as it would prevent PDMS from curing.</li>
	<li>Silanize the mold through vapor deposition of Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane.
	<ol>
		<li>Place the 3D mold in a vacuum desiccator along with 20 &#181;L of 100% concentrated Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane pipetted on an aluminum foil placed close to the mold.</li>
		<li>Create a vacuum in the desiccator to generate vapor and leave for 40 min.</li>
		<li>Remove the mold from the desiccator and rinse it with pure ethanol before pouring PDMS on it.</li>
	</ol>
	</li>
</ol>
3. Fabrication of the microfluidic chip
<ol>
	<li>Prepare a PDMS mixture by mixing the elastomer with its crosslinking agent (Table of Materials). Stir the mixture vigorously to blend the two components uniformly until air bubbles are formed, and the PDMS mixture looks opaque. 
	NOTE: The amount of cross-linker should be 10% by weight of the amount of elastomer.</li>
	<li>Degas the mixture of elastomer and crosslinking agent in a vacuum desiccator to remove the trapped air bubbles. Transfer the mixture in the desiccator to the container used for mixing (step 3.1). Continue the degassing process until all the bubbles have been removed and the mixture looks transparent again. 
	NOTE: The time typically required for desiccation is 30 min.</li>
	<li>Pour 3 g of the mixture on the silicon master to produce the template, which will serve as the &#34;floor&#34; of the microfluidic chip. 
	NOTE: The thickness of the PDMS layer can be tuned by changing the amount of mixture poured on the silicon master.
	<ol>
		<li>To obtain a 400 &#181;m-thick template, pour 3 g of PDMS on the silicon master, place the silicon master on a spin coater, and spin coat at 21 &#215; g for 5 s and 54 &#215; g for 10 s, and then degas again as described in step 3.2 to remove trapped air bubbles.</li>
	</ol>
	</li>
	<li>Pour 20 g of the PDMS mixture on the 3D-printed mold to make the microchannel, which will serve as the &#34;roof&#34; of the microfluidic chip, and degas it as described in step 3.2 for 30 min.</li>
	<li>Bake both the silicon wafer and the 3D printed mold at 70 &#176;C for at least 2 h.</li>
	<li>Peel the PDMS layer off the 3D printed mold, cut the PDMS with a blade around the microchannels, and punch the holes that will serve as inlet and outlet of the microfluidic channel.</li>
	<li>Peel the PDMS off the silicon master and cut the PDMS layer into smaller pieces with the same dimensions of the microfluidic channels that will be bonded on top of the templates.</li>
	<li>Gently rub the templates and microchannels using a 1% detergent solution (see the Table of Materials) for 5 min and then rinse with deionized water. Next, rinse the templates and microchannels with isopropanol and rinse them with deionized water. Dry the templates and microchannels at room temperature for 1 min with compressed air at 1 bar.</li>
	<li>Place the templates and the microchannels in a plasma cleaner with the surfaces to be bonded facing up. Turn on the plasma cleaner, and plasma treat the templates and the microchannels for 40 s. Take them out from the plasma cleaner and immediately bond the microchannels on top of the templates by putting them in contact with one another.</li>
	<li>Store the microfluidic chips in an oven at 70 &#176;C for five days to ensure PDMS hydrophobic recovery and have a receding contact angle within the optimal range, between 30 and 60&#176;10,11.</li>
</ol>
4. Bacterial patterning
<ol>
	<li>One day prior to the experiment, grow a population of Escherichia coli (strain MG1655 prpsM-GFP). Inoculate the culture directly from the frozen stock and grow overnight for 20 h in lysogeny broth (LB) medium in a shaker incubator at 37 &#176;C. Add 50 &#181;g/mL of kanamycin for the cells to retain the prpsM-GFP plasmid.</li>
	<li>On the day of the experiment, set the box incubator (Figure 2A) at 37 &#176;C several hours before the experiment to have a uniform and stable temperature before starting the experiment. Set up the syringe pumpand the heated glass plate on the microscope stage (Figure 2B,C), setting the temperature at the same temperature as the box incubator. 
	NOTE: The box incubator in which the entire system, including the microscope (Figure 2E), is enclosed ensures that a uniform and constant temperature is maintained throughout the entire experiment when the channel is flushed with medium.</li>
	<li>Ninety minutes before the experiment, put the microfluidic chip in a vessel filled with 100% ethanol (EtOH) and flush the channel with 100% EtOH for at least 10 min. Place the microfluidic chip in a vacuum desiccator and degas for at least 30 min. Exchange the EtOH with distilled water and vacuum-treat the chip for at least 30 min. Put the microfluidic chip in the oven at 70 &#176;C for 10 min to remove any traces of liquid left in the channel. 
	NOTE:&#160;This step is necessary to prevent bubble formation in the channel when flushed with culture medium. This bubble prevention protocol was adapted from Wang et al.18.</li>
	<li>Pipette 1 mL of 3-(N-morpholino)propanesulfonic acid (MOPS) medium (1x) into a centrifuge vial and add 10 &#181;L of 0.132 M potassium phosphate (K2HPO4). Take the overnight culture out of the 37 &#176;C incubator and aliquot 100 &#181;L into the centrifuge vial and centrifuge the culture at 2,300 &#215; g for 2 min. Gently pipette the supernatant out of the centrifuge vials, and resuspend the pellet in 1 mL of fresh MOPS medium with 0.015% v/v of Tween 20 and 0.01% v/v of potassium phosphate. 
	NOTE: The typical concentration of the bacterial suspension is in the range of 0.015-0.15 vol%, which ensures the formation of an extended and mobile accumulation zone and prevents the accumulation of particles on the substrate. Replacing the overnight medium with fresh medium minimizes the risks of releasing films of bacteria on the template. The lack of carbon source in the fresh medium prevents cells from growing in the suspension during template patterning. A concentration of 0.015% v/v of Tween 20 in the suspension is necessary for the contact angle of the receding liquid on the PDMS template to fall within the optimal range, between 30 and 60&#176;.</li>
	<li>Load the bacterial suspension in a 1 mL syringe and connect the syringe to the chip through microfluidic tubing.
	<ol>
		<li>To secure the connection between the syringe and the tubing, directly insert a needle with an outer diameter of 0.6 mm into the tubing. To avoid scattering any suspension remaining in the inlet vicinity across the channel, flush fresh medium through the hole used as an outlet during the patterning process (Figure 3A-III), rather than the hole used as the inlet.</li>
		<li>Mount the syringe on the syringe pump and inject the suspension into the microfluidic chip through the inlet located at the upstream part of the channel until the suspension covers the template region with traps. 
		NOTE: During the liquid injection process, air can escape through an outlet located at the downstream end of the channel.</li>
		<li>Set the syringe pump to withdraw the bacterial suspension (Figure 3A-I) at a flow rate of 0.07-0.2 &#181;L/min, which in the described geometry corresponds to a meniscus receding speed of 80-100 &#181;m/min.</li>
		<li>Monitor the patterning process via microscope software. 
		NOTE: Here, a 10x magnification was used to monitor the receding meniscus on the template, and a 20x magnification was used to monitor the deposition of individual bacteria into the microfabricated traps.</li>
	</ol>
	</li>
	<li>Once the template has been patterned with cells (Figure 3A-II), increase the withdrawal flow rate to quickly empty the microfluidic channel and flush it with fresh LB that was previously degassed for at least 30 min and prewarmed at 30 &#176;C.</li>
	<li>Set the syringe pump at a flow rate of 2 &#181;L/min to gently flush the channel. Once the channel has been filled, increase the flow rate (15 &#181;L/min) according to the specific experimental needs.</li>
	<li>Acquire images of growing bacteria at the desired magnification and time interval.</li>
</ol>
5. Colloidal patterning
<ol>
	<li>Pipette 900 &#181;L of a 0.015% v/v Tween 20 aqueous solution into a centrifuge vial and pipette 100 &#181;L of the original colloidal suspension into it. Centrifuge the suspension at 13,500 &#215; g for 1 min. Gently remove the supernatant and replace it with the aqueous Tween 20 (0.015% v/v) solution. Repeat this process three times to ensure complete replacement of the supplier&#39;s solvent from the stock suspension.</li>
	<li>Load the colloidal suspension in a 1 mL syringe and connect the syringe to the chip through microfluidic tubing. Inject the suspension into the microfluidic chip through the inlet located within the central section, at the upstream part of the channel, and gradually push the suspension until the template is covered.</li>
	<li>Withdraw the colloidal suspension at a flow rate of 0.07-0.2 &#181;L/min, which corresponds to a meniscus receding speed of 1-2 &#181;m/min, and image the patterning process via microscope software. Use 10x magnification to monitor the receding meniscus on the template and 20x magnification to observe the deposition of individual particles into the microfabricated traps. Increase the flow rate once the meniscus reaches the end of the template to empty the channel quickly. 
	NOTE: Colloidal arrays composed of several particles can be produced by running the patterning process from steps 5.1 to 5.3 in series. The channel is loaded with a new colloidal suspension at each iteration according to the desired colloidal arrays&#39; composition. Each patterning step adds one particle to the colloidal array and can be run sequentially until the traps have been filled with the desired particles&#39; sequence. The composition of the resulting colloidal arrays is solely given by the sequence of colloidal suspensions used to fill the traps (Figure 3B).</li>
</ol>

<ol>
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The authors have no conflicts of interest to disclose.

The microfluidic platform described here allows the patterning of micro-sized objects, such as colloids and bacteria, into prescribed spatial arrangements on a PDMS substrate. The full control over environmental conditions offered by microfluidics and the ability to pattern cells with micrometric precision granted by sCAPA technology makes it a very promising platform for future physiology and ecology studies.
In the experiments presented in this work, the silicon master was realized using the...

Spatial patterning of single microorganisms, particularly within experimental arenas that enable full control over environmental conditions, such as microfluidic devices, is highly desirable in a broad range of contexts.&#160;For example, arranging microorganisms into regular arrays would permit the accurate imaging of large numbers of individual cells and the study of their growth, physiology, gene expression in response to environmental stimuli, and drug susceptibility. It would also allow studying cell-cell interactions of particular interest in research into cellular communication (e.g., quorum sensing), cross-feeding (e.g., algal-bacterial symbiosis), or antagonism (e.g., allelopathy), with full control over the spatial localization of cells relative to each other. Cell physiology and evolution studies1, cell-cell interaction studies2, phenotypic differentiation screening3, environmental monitoring4, and drug screening5 are among the fields that can greatly benefit from a technology able to achieve such quantitative single-cell analysis.
Several strategies to isolate and handle single cells have been proposed in recent years, from holographic optical traps6 and heterogeneous surface functionalization methods7,8,9,10 to single-cell chemostats11 and droplet microfluidics12. These methods are either technically very demanding or affect cell physiology and fail to provide a high-throughput platform to pattern microbes that can be studied over long periods, ensuring single-cell resolution, full optical access, and control over environmental conditions. The goal of this paper is to describe a platform to pattern bacteria with micrometric precision into prescribed spatial arrangements on a PDMS surface through capillarity-assisted assembly. This platform allows precise and flexible spatial patterning of microbes and enables full optical access and control over environmental conditions, thanks to its microfluidic nature.
The technology behind this platform is an assembly technology developed in recent years, named sCAPA13,14,15 (sequential capillarity-assisted particle assembly) that was integrated into a microfluidic platform16. The meniscus of an evaporating liquid droplet, while receding over a patterned polydimethylsiloxane (PDMS) substrate inside a microfluidic channel, exerts capillary forces that trap the individual colloidal particles suspended in the liquid into micrometric wells microfabricated on the substrate (Figure 1A). Suspended particles are first transported to the air-liquid interface by convective currents and then placed into the traps by capillarity. Capillary forces exerted by the moving meniscus act on a larger scale compared to forces involved in particle interactions.
Thus, the assembly mechanism is not influenced by the material, dimensions, and surface properties of the particles. Parameters such as particle concentration, the speed of the meniscus, temperature, and surface tension of the suspension are the only parameters that influence the yield of the patterning process. The reader can find a detailed description of the influence of the aforementioned parameters on the patterning process in13,14,15. In the original sCAPA technology13,14,15, the colloidal patterning process was carried out in an open system and required a high-precision piezoelectric stage to drive the suspension across the template. This platform exploits a different strategy and allows the patterning to be carried out with standard equipment generally used in microfluidics in a controlled environment, thus minimizing the risks of contaminating the samples.
This microfluidic platform was first optimized on colloidal particles to create regular arrays of inert particles and then successfully applied to bacteria. Both microfluidic platforms are described in this paper (Figure 1B,C). Most of the preparatory steps and the experimental equipment described in the protocol are common for the two applications (Figure 2). We report colloidal patterning to demonstrate that the technique can be used to perform multiple sequential depositions on the same surface to create complex, multimaterial patterns. In particular, one single particle was deposited per trap for each step to form colloidal arrays with a specific geometry and composition, solely dictated by the traps&#39; geometry and filling sequence. As for bacterial patterning, single depositions are described, resulting in one bacterium being deposited per trap. Once cells are patterned on the surface, the microfluidic channel is flushed with medium to promote bacterial growth, the preliminary step of any single-cell study.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcatel AMS 200SE I-Speeder</td>
			<td>Alcatel Micro Machining System</td>
			<td></td>
			<td>deep reactive ion exchange system</td>
		</tr>
		<tr>
			<td>Alconox</td>
			<td></td>
			<td></td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>AZ400K developer</td>
			<td>MicroChemicals</td>
			<td>AZ400K</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 10 mL Syringe (Luer-Lock)</td>
			<td>BD</td>
			<td>300912</td>
			<td>used to flush fresh Lysogeny broth into the microfluidic channel</td>
		</tr>
		<tr>
			<td>Box Incubator</td>
			<td>Life Imaging Services</td>
			<td></td>
			<td>used to ensure a uniform and constant temperature in the channel</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td>used to replace the overnight media with fresh minimal media</td>
		</tr>
		<tr>
			<td>Centrifuge vial</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>CETONI Base 120</td>
			<td>CETONI GmbH</td>
			<td></td>
			<td>syringe pump</td>
		</tr>
		<tr>
			<td>Fluorescent PS particles of diameter 0.98 &#181;m (red)</td>
			<td>microParticles GmbH</td>
			<td>PS-FluoRed-Fi267</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent PS particles of diameter 1.08 &#181;m (green)</td>
			<td>microParticles GmbH</td>
			<td>PS-FluoGreen-Fi182</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent PS particles of diameter 2.07 &#181;m (green)</td>
			<td>microParticles GmbH</td>
			<td>PS-FluoGreen-Fi183</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent PS particles of diameter 2.08 &#181;m (red)</td>
			<td>microParticles GmbH</td>
			<td>PS-FluoRed-Fi180</td>
			<td></td>
		</tr>
		<tr>
			<td>Gigabatch 310 M</td>
			<td>PVA TePla</td>
			<td></td>
			<td>used to plasma treat a 10 cm silicon wafer</td>
		</tr>
		<tr>
			<td>H401-T-CONTROLLER</td>
			<td>Okolab</td>
			<td></td>
			<td>controller of the heated glass plate</td>
		</tr>
		<tr>
			<td>H601-NIKON-TS2R-GLASS</td>
			<td>Okolab</td>
			<td></td>
			<td>heated glass plate</td>
		</tr>
		<tr>
			<td>Heidelberg DWL 2000</td>
			<td>Heidelberg Instruments</td>
			<td></td>
			<td>UV direct laser writer</td>
		</tr>
		<tr>
			<td>Insulin syringes, U 100, with luer</td>
			<td>Codan Medical ApS</td>
			<td>CODA621640</td>
			<td>1 mL syringe used to withdraw the liquid suspension during the patterning process</td>
		</tr>
		<tr>
			<td>Klayout</td>
			<td>Opensource</td>
			<td></td>
			<td>used to design the features on the silicon master</td>
		</tr>
		<tr>
			<td>LB Broth, Miller (Luria-Bertani)</td>
			<td>Fisher Scientific</td>
			<td>244610</td>
			<td>Lysogeny broth flushed into the microfluidic channel</td>
		</tr>
		<tr>
			<td>Masterflex transfer tubing</td>
			<td>Masterflex</td>
			<td>HV-06419-05</td>
			<td>0.020&#39;&#39; ID, 0.06&#39;&#39; OD</td>
		</tr>
		<tr>
			<td>MOPS (10x)</td>
			<td>Teknova</td>
			<td>M2101</td>
			<td>diluted tenfold with milliQ water and used to replace the overnight medium</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti2</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td>microscope</td>
		</tr>
		<tr>
			<td>openSCAD</td>
			<td>Opensource</td>
			<td></td>
			<td>used to design the mold</td>
		</tr>
		<tr>
			<td>OPTIspin SB20</td>
			<td>ATM group</td>
			<td>51-0002-01-00</td>
			<td>spin developer</td>
		</tr>
		<tr>
			<td>Plasma chamber Zepto</td>
			<td>Diener Electronic</td>
			<td>ZEPTO-1</td>
			<td>used to plasma treat the template and microchannel to bond them</td>
		</tr>
		<tr>
			<td>Positive photoresist AZ1505</td>
			<td>MicroChemicals</td>
			<td>AZ1505</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>P3786</td>
			<td>added to MOPS 1x</td>
		</tr>
		<tr>
			<td>Prusa curing and Washing machine CW1S</td>
			<td>Prusa</td>
			<td></td>
			<td>used to ensure all polymer is cured and uncured polymer is removed from the mold</td>
		</tr>
		<tr>
			<td>Prusa Resin - Tough</td>
			<td>Prusa Research a.s.</td>
			<td></td>
			<td>UV photosensitive 405nm liquid resin for 3D printing</td>
		</tr>
		<tr>
			<td>Prusa SL1 3d printer</td>
			<td>Prusa</td>
			<td></td>
			<td>used to print the mold</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>VWR-CH</td>
			<td>611-2605</td>
			<td>used to weight PDMS mixture</td>
		</tr>
		<tr>
			<td>Silicon wafer (10 cm)</td>
			<td>Silicon Materials Inc.</td>
			<td></td>
			<td>N/Phos &#60;100&#62; 1-10 &#937; cm</td>
		</tr>
		<tr>
			<td>S&#252;ss MA6 Mask aligner</td>
			<td>SUSS MicroTec Group</td>
			<td></td>
			<td>used to align the chrome-glass mask and the substrate, and expose the substrate</td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dow Corning</td>
			<td></td>
			<td>silicone elastomer kit; curing agent</td>
		</tr>
		<tr>
			<td>Techni Etch Cr01</td>
			<td>Technic</td>
			<td></td>
			<td>chromium etchant</td>
		</tr>
		<tr>
			<td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma Aldrich</td>
			<td>448931</td>
			<td>used to silianize the 3D printed mold</td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>used to ensure an optimal receding contact angle during the patterning process</td>
		</tr>
		<tr>
			<td>Veeco Dektak 6 M</td>
			<td>Veeco</td>
			<td></td>
			<td>profilometer</td>
		</tr>
		<tr>
			<td>VTC-100 Vacuum Spin Coater</td>
			<td>MTI corporation</td>
			<td></td>
			<td>vacuum spin coater</td>
		</tr>
	</tbody>
</table>

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.

A microfluidic platform that exploits capillarity-assisted assembly to pattern colloidal particles and bacteria into traps microfabricated on a PDMS template was developed. Two different channel geometries have been designed to optimize the patterning of colloids and bacteria through the capillarity-assisted assembly. The first channel geometry (Figure 1B) consists of three 23 mm long parallel sections with no physical barrier between them. The two sections on the sides are 5 mm wide and 1 m...

Watch this Scientific Journal Video about Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly at JoVE.com

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

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, ...

patterning-microorganisms-microparticles-through-sequential

Research

JoVE Journal

Engineering

3.1K Views.  ETH Zurich. 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.

Video: Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Patterning via Optical Saturable Transitions - Fabrication and Characterization

This protocol describes the fabrication and characterization of nanostructures using a novel nanolithographic technique called Patterning via Optical Saturable Transitions (POST). In this technique the chemical properties of organic photochromic molecules that undergo single-photon reactions are exploited, enabling rapid top-down nanopatterning over large areas at low light intensities, thereby, allowing for the circumvention of the far-field diffraction barrier.4 Simple, cost-effective, high throughput and resolution alternatives to nanopatterning are being explored, such as, two-photon polymerization5,6, beam pen lithography (BPL)7, scanning electron beam lithography (SEBL), and focused ion beam (FIB) patterning. However, multi-photon approaches require high light intensities, which limit their potential for high throughput and offer low image contrast. Although, electron and ion beam lithographic processes offer increased resolution, the serial nature of the process is limited to slow writing speeds, which also prevents patterning of features over large areas. Beam-pen lithography is an approach towards parallel near-field optical lithography. However, the gap between the source of the beam and the surface of the photoresist needs to be controlled extremely precisely for good pattern uniformity and this is very challenging to accomplish for large arrays of beams. Patterning via Optical Saturable Transitions (POST) is an alternative optical nanopatterning technique for patterning sub-wavelength features1-3. Since this technique uses single photons instead of electrons, it is extremely fast and does not require high light intensities1-3, opening the door to massive parallelization.

We report that the diffraction limit of conventional optical lithography can be overcome by exploiting the transitions of organic photochromic derivatives induced by their photoisomerization at low light intensities.1-3 This paper outlines our fabrication technique and two locking mechanisms, namely: dissolution of one photoisomer and electrochemical oxidation.

This protocol describes the fabrication and characterization of nanostructures using a novel nanolithographic technique called Patterning via Optical ...

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data acquisition times with high spatial resolution. In the electronics industry there is serious interest in performing failure analysis on 3D microelectronic packages, many which contain multiple levels of high-density interconnections. Often in tomography there is a trade-off between image resolution and the volume of a sample that can be imaged. This inverse relationship limits the usefulness of conventional computed tomography (CT) systems since a microelectronic package is often large in cross sectional area 100-3,600 mm2, but has important features on the micron scale. The micro-tomography beamline at the Advanced Light Source (ALS), in Berkeley, CA USA, has a setup which is adaptable and can be tailored to a sample&#39;s properties, i.e., density, thickness, etc., with a maximum allowable cross-section of 36 x 36 mm. This setup also has the option of being either monochromatic in the energy range ~7-43 keV or operating with maximum flux in white light mode using a polychromatic beam. Presented here are details of the experimental steps taken to image an entire 16 x 16 mm system within a package, in order to obtain 3D images of the system with a spatial resolution of 8.7 &#181;m all within a scan time of less than 3 min. Also shown are results from packages scanned in different orientations and a sectioned package for higher resolution imaging. In contrast a conventional CT system would take hours to record data with potentially poorer resolution. Indeed, the ratio of field-of-view to throughput time is much higher when using the synchrotron radiation tomography setup. The description below of the experimental setup can be implemented and adapted for use with many other multi-materials.

For this study synchrotron radiation micro-tomography, a non-destructive three-dimensional imaging technique, is employed to investigate an entire microelectronic package with a cross-sectional area of 16 x 16 mm. Due to the synchrotron&#39;s high flux and brightness the sample was imaged in just 3 min&#160;with an 8.7 &#181;m spatial resolution.

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data ...

Optical Trap Loading of Dielectric Microparticles In Air

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

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

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

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. Customized channel layout designs are necessary for specific functions and integrated performance of microfluidic devices in numerous biomedical and chemical applications (e.g., cell culture, biosensing, chemical synthesis, and liquid handling). Owing to the nature of molding approaches using silicon wafers with photoresist layers patterned by photolithography as master molds, the microfluidic channels commonly have regular cross sections of rectangular shapes with identical heights. Typically, channels with multiple heights or different geometric sections are designed to possess particular functions and to perform in various microfluidic applications (e.g., hydrophoresis is used for sorting particles and in continuous flows for separating blood cells6,7,8,9). Therefore, a great deal of effort has been made in constructing channels with various sections through multiple-step approaches like photolithography using several photoresist layers and assembly of different PDMS thin sheets. Nevertheless, such multiple-step approaches usually involve tedious procedures and extensive instrumentation. Furthermore, the fabricated devices may not perform consistently and the resulted experimental data may be unpredictable. Here, a one-step approach is developed for the straightforward fabrication of microfluidic channels with different geometric cross sections through PDMS sequential wet etching processes, that introduces etchant into channels of planned single-layer layouts embedded in PDMS materials. Compared to the existing methods for manufacturing PDMS microfluidic channels with different geometries, the developed one-step approach can significantly simplify the process to fabricate channels with non-rectangular sections or various heights. Consequently, the technique is a way of constructing complex microfluidic channels, which provides a fabrication solution for the advancement of innovative microfluidic systems.

Several methods are available for the fabrication of channels of non-rectangular sections embedded in polydimethylsiloxane microfluidic devices. Most of them involve multistep manufacturing and extensive alignment. In this paper, a one-step approach is reported for fabricating microfluidic channels of different geometric cross sections by polydimethylsiloxane sequential wet etching.

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. ...

Drawing and Hydrophobicity-patterning Long Polydimethylsiloxane Silicone Filaments

Polydimethylsiloxane (PDMS) silicone is a versatile polymer that cannot readily be formed into long filaments. Traditional spinning methods fail because PDMS does not exhibit long-range fluidity at melting. We introduce an improved method to produce filaments of PDMS by a stepped temperature profile of the polymer as it cross-links from a fluid to an elastomer. By monitoring its warm-temperature viscosity, we estimate a window of time when its material properties are amendable to drawing into long filaments. The filaments pass through a high-temperature tube oven, curing them sufficiently to be harvested. These filaments are on the order of hundreds of micrometers in diameter and tens of centimeters in length, and even longer and thinner filaments are possible. These filaments retain many of the material properties of bulk PDMS, including switchable hydrophobicity. We demonstrate this capability with an automated corona-discharge patterning method. These patternable PDMS silicone filaments have applications in silicone weavings, gas-permeable sensor components, and model microscale foldamers.

Here, we present a protocol to produce long filaments of polydimethylsiloxane (PDMS) silicone by gravity-drawing through a furnace. Filaments are on the order of hundreds of micrometers in diameter and tens of centimeters in length and are hydrophobically patternable via an Arduino-controlled corona discharge system.

Polydimethylsiloxane (PDMS) silicone is a versatile polymer that cannot readily be formed into long filaments. Traditional spinning methods fail because ...

Study of a Dot-patterning Process on Flexible Materials using Impact Print-Type Hot Embossing Technology

Here we present our study on an impact print-type hot embossing process which can create dot patterns with various designs, widths, and depths in real time on polymer film. In addition, we implemented a control system for the on-off motion and position of the impact header to engrave different dot patterns. We performed dot patterning on various polymer films, such as polyester (PET) film, polymethyl methacrylate (PMMA) film, and polyvinyl chloride (PVC) film. The dot patterns were measured using a confocal microscope, and we confirmed that the impact print-type hot embossing process produces fewer errors during the dot patterning process. As a result, the impact print-type hot embossing process is found to be suitable for engraving dot patterns on different types of polymer films. In addition, unlike the conventional hot embossing process, this process does not use an embossing stamp. Therefore, the process is simple and can create dot patterns in real time, presenting unique advantages for mass production and small-quantity batch production.

Impact print-type hot embossing technology uses an impact header to engrave dot patterns on flexible materials in real time. This technology has a control system for controlling the on-off motion and position of the impact header to create dot patterns with various widths and depths on different polymer films.

Here we present our study on an impact print-type hot embossing process which can create dot patterns with various designs, widths, and depths in real ...

Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid samples, such as chemical reagents and cell cultures, is predominantly accomplished through steady flows that are typically driven by syringe pumps, gravity, or capillary forces. The use of complementary oscillatory flows is seldom considered in applications despite its numerous advantages as recently demonstrated in the literature. The significant technical barrier to the implementation of oscillatory flows in microchannels is likely responsible for the lack of its widespread adoption. Advanced commercial syringe pumps that can produce oscillatory flow, are often more expensive and only work for frequencies less than 1 Hz. Here, the assembly and operation of a low-cost, plug-and-play type speaker-based apparatus that generates oscillatory flow in microchannels is demonstrated. High-fidelity harmonic oscillatory flows with frequencies ranging from 10-1000 Hz can be achieved along with independent amplitude control. Amplitudes ranging from 10-600 &#181;m can be achieved throughout the entire range of operation, including amplitudes &#62; 1 mm at the resonant frequency, in a typical microchannel. Although the oscillation frequency is determined by the speaker, we illustrate that the oscillation amplitude is sensitive to fluid properties and channel geometry. Specifically, the oscillation amplitude decreases with increasing channel circuit length and liquid viscosity, and in contrast, the amplitude increases with increasing speaker tube thickness and length. Additionally, the apparatus requires no prior features to be designed on the microchannel and is easily detachable. It can be used simultaneously with a steady flow created by a syringe pump to generate pulsatile flows.

The protocol demonstrates a convenient method to produce harmonic oscillatory flow from 10-1000 Hz in microchannels. This is performed by interfacing a computer-controlled speaker diaphragm to the microchannel in a modular manner.

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid ...

Modeling and Experimental Analysis of the Single-Shaft Coaxial Motor-Pump Assembly in Electrohydrostatic Actuators

An electrohydrostatic actuator (EHA) can be the most promising alternative compared with the traditional hydraulic servo actuators for its high power density, ease of maintenance, and reliability. As the core power unit that determines the performance and service life of the EHA, the motor-pump assembly should simultaneously possess a wide speed/pressure range and a high dynamic response.
This paper presents a method to test the performance of the motor-pump assembly through simulation and experimentation. The flow output characteristics were defined through simulation and analysis of the assembly at the beginning of the experiment, leading to the conclusion of whether the pump could meet the requirements of the EHA. A series of performance tests were conducted on the motor-pump assembly via a pump test bench in the speed range of 1,450-9,000 rpm and the pressure range of 1-30 MPa.
We tested the overall efficiency of the motor-pump assembly under various working conditions after confirming the consistency between the test results of the flow output characteristics with the simulation results. The results showed that the assembly has higher overall efficiency when working at 4,500-7,000 rpm under the pressure of 10-25 MPa and at 2,000-2,500 rpm under 5-15 MPa. Overall, this method can be utilized for determining in advance whether the motor-pump assembly meets the requirements of EHA. Moreover, this paper proposes a rapid test method of the motor-pump assembly in various working conditions, which could assist in predicting EHA performance.

We built a simulation model to evaluate pump flow characteristics and performance of the single-shaft coaxial motor-pump assembly in electrohydrostatic actuators and investigate the overall efficiency in a wide set of working conditions of the motor-pump assembly experimentally.

An electrohydrostatic actuator (EHA) can be the most promising alternative compared with the traditional hydraulic servo actuators for its high power ...

Frugal Imaging Technique of Capillary Flow Through Three-Dimensional Polymeric Printing Powders

The development of novel imaging techniques of molecular and colloidal transport, including nanoparticles, is an area of active investigation in microfluidic and millifluidic studies. With the advent of three-dimensional (3D) printing, a new domain of materials has emerged, thereby increasing the demand for novel polymers. Specifically, polymeric powders, with average particle sizes on the order of a micron, are experiencing a growing interest from academic and industrial communities. Controlling material tunability at the mesoscopic to microscopic length scales creates opportunities to develop innovative materials, such as gradient materials. Recently, a need for micron-sized polymeric powders has been growing, as clear applications for the material are developing. Three-dimensional printing provides a high-throughput process with a direct link to new applications, driving investigations into the physio-chemical and transport interactions on a mesoscale. The protocol that is discussed in this article provides a non-invasive technique to image fluid flow in packed powder beds, providing high temporal and spatial resolution while leveraging mobile technology that is readily available from mobile devices, such as smartphones. By utilizing a common mobile device, the imaging costs that would normally be associated with an optical microscope are eliminated, resulting in a frugal-science approach. The proposed protocol has successfully characterized a variety of combinations of fluids and powders, creating a diagnostic platform for quickly imaging and identifying an optimal combination of fluid and powder.

The proposed technique will provide a novel, efficient, frugal, and non-invasive approach for imaging fluidic flow through a packed powder bed, yielding high spatial and temporal resolution.