Name: patterning of microorganisms and microparticles through sequential capillarity-assisted assembly
Uploaded: 2021-11-04T13:00:00
Description: Watch this Scientific Journal Video about Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly at JoVE.com

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.
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	<li>Design the features us...

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C., Vieu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+assembly+of+bacteria+on+chemical+patterns+using+soft+lithography.">Controlled assembly of bacteria on chemical patterns using soft lithography.</a> Colloids and Surfaces: B Biointerfaces. 65 (2), 285-291 (2008).</li><li>Rozhok, S., 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=Attachment+of+motile+bacterial+cells+to+prealigned+holed+microarrays.">Attachment of motile bacterial cells to prealigned holed microarrays.</a> Langmuir. 22 (26), 11251-11254 (2006).</li><li>Xu, L., 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=Microcontact+printing+of+living+bacteria+arrays+with+cellular+resolution.">Microcontact printing of living bacteria arrays with cellular resolution.</a> Nano Letters. 7 (7), 2068-2072 (2007).</li><li>Ingham, C. 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B., Cluzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-cell+chemostat:+an+agarose-based,+microfluidic+device+for+high-throughput,+single-cell+studies+of+bacteria+and+bacterial+communities.">The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities.</a> Lab on a Chip. 12 (8), 1487-1494 (2012).</li><li>Ni, S., Isa, L., Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+assembly+as+a+tool+for+the+heterogeneous+integration+of+micro-+and+nanoscale+objects.">Capillary assembly as a tool for the heterogeneous integration of micro- and nanoscale objects.</a> Soft Matter. 14 (16), 2978-2995 (2018).</li><li>Ni, S., Leemann, J., Buttinoni, I., Isa, L., Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+colloidal+molecules+from+sequential+capillarity-assisted+particle+assembly.">Programmable colloidal molecules from sequential capillarity-assisted particle assembly.</a> Science Advances. 2 (4), 1501779 (2016).</li><li>Ni, S., Leemann, J., Wolf, H., Isa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+Insights+into+mechanisms+of+capillary+assembly.">Nanoparticle Insights into mechanisms of capillary assembly.</a> Faraday Discussions. 181, 225-242 (2014).</li><li>Pioli, 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=Sequential+capillarity-assisted+particle+assembly+in+a+microfluidic+channel.">Sequential capillarity-assisted particle assembly in a microfluidic channel.</a> Lab on a Chip. 21 (5), 888-895 (2021).</li><li>Geissler, M., 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=Fabrication+of+metal+nanowires+using+microcontact+printing.">Fabrication of metal nanowires using microcontact printing.</a> Langmuir. 19 (15), 6301-6311 (2003).</li><li>Wang, Y., 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=Systematic+prevention+of+bubble+formation+and+accumulation+for+long-term+culture+of+pancreatic+islet+cells+in+microfluidic+device.">Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device.</a> Biomedical Microdevices. 14 (2), 419-426 (2012).</li><li>Crowley, L. C., 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=Measuring+cell+death+by+propidium+iodide+uptake+and+flow+cytometry.">Measuring cell death by propidium iodide uptake and flow cytometry.</a> Cold Spring Harbor Protocols. 7, 647-651 (2016).</li></ol>

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, ...

This method allows producing user-defined patterns of microorganisms within a microfluidic channel. Once patterned, the microorganisms can be monitored to evaluate their long-term physiology and interaction with high throughput. The use of capillary forces enables a nonspecific route for patterning, which is applicable across different classes of materials.For example, colloidal particles and bacteria. Moreover, it allows to produce large arrays with exquisite control of the spatial arrangement of the material of interest. To date, we tested and applied the method on colloidal particles and microbial cells.As a result, it can find applications in material engineering and fields requiring quantitative single-cell analysis like drug screening. To begin, prepare a PDMS mixture by mixing the elastomer with its cross-linking agent. Then stir the mixture vigorously to blend the two components uniformly until air bubbles are formed and the PDMS mixture looks opaque.Now, degas the mixture in a vacuum desiccator until all air bubbles are removed and the mixture looks transparent again. To obtain a 400 micrometer thick template floor of the microfluidic chip, pour three grams of the mixture on the silicon master and place the silicon master on a spin coater to spin coat at 21 times G for five seconds and 54 times G for 10 seconds. Degas again for removing the trapped air bubbles as described earlier.Pour 20 grams of the PDMS mixture into the 3D printed mold to make the microchannel which will serve as the roof of the microfluidic chip and degas it for 30 minutes as described earlier. Bake the silicon wafer and the 3D printed mold at 70 degrees Celsius for at least two hours, then cut the PDMS along the outline of the 3D printed mold and peel it off. Cut the PDMS with a blade around the microchannels and punch the holes that will serve as inlet and outlet of the microfluidic channel.Now cut the PDMS and peel it 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. Gently rub the templates and microchannels using a 1%detergent solution for five minutes and then rinse with deionized water. Next, rinse the templates and microchannels with isopropanol, before rinsing them with deionized water.Now dry the templates and microchannels at room temperature for one minute with compressed air at one bar. Place the templates and the microchannels in a plasma cleaner with the bonding surfaces facing up. After turning on the plasma cleaner, plasma treat the templates and microchannels for 40 seconds, then take them out from the plasma cleaner and immediately bond the microchannels on top of the templates.Store the microfluidic chips in an oven at 70 degrees Celsius for five days to ensure PDMS hydrophobic recovery. On the day of the experiment, set the box incubator at 37 degrees Celsius several hours before the experiment, then set up the syringe pump and the heated glass plate on the microscope stage, setting the same temperature as that of the box incubator. 90 minutes before the experiment, put the microfluidic chip in a vessel filled with 100%ethanol and flush the channel with 100%ethanol for at least 10 minutes, then place the microfluidic chip in a vacuum desiccator and degas for at least 30 minutes.Now exchange the ethanol with distilled water and vacuum treat the chip for at least 30 minutes. Put the microfluidic chip in the oven at 70 degrees Celsius for 10 minutes to remove any traces of liquid left in the channel. Pipette one milliliter of MOPS medium into a centrifuge vial and add 10 microliters of 0.132 molar potassium phosphate.Aliquot 100 microliters of the overnight culture into the centrifuge vial and centrifuge the culture at 2, 300 times G for two minutes. Gently discard the supernatant to resuspend the pellet in one milliliter of fresh MOPS medium with 0.015%of Tween 20 and 0.01%of potassium phosphate and load the bacterial suspension in a one milliliter syringe. To secure the syringe and the tubing connection, directly insert a needle into the tubing.Now 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. Set the syringe pump at a flow rate of 0.07 to 0.2 microliters per minute to withdraw the bacterial suspension and monitor the patterning process via microscope software. Once the template has been patterned with cells, 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 minutes and prewarmed at 30 degrees Celsius.Now set the syringe pump at a flow rate of two microliters per minute to gently flush the channel. Once the channel has been filled, again increase the flow rate. Acquire images of growing bacteria at the desired magnification and time interval.Pipette 900 microliters of a 0.015%Tween 20 aqueous solution into a centrifuge vial and then pipette 100 microliters of the original colloidal suspension into it. Centrifuge the suspension at 13, 500 times G for one minute and gently replace the supernatant with the aqueous Tween 20 solution. Load the colloidal suspension in a one milliliter 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. Withdraw the colloidal suspension at a flow rate of 0.07 to 0.2 microliters per minute and image the patterning process via microscope software. Increase the flow rate once the meniscus reaches the end of the template quickly.The straight channel geometry was exploited to pattern stationary phased cells of a fluorescent Escherichia coli strain depositing 83%of the 5, 000 analyzed traps. Patterned bacteria resumed growth at different times within 1.5 hours from when the channel was filled with fresh LB.Once growth was resumed, single bacterial cells started forming individual colonies which expanded until a surface layer was formed and the single cell resolution was lost. Analysis conducted over 55, 000 traps showed that green-red dimers made by two and one micrometer diameter particles were formed in 93%and 89%of analyzed traps respectively.And green-red-green trimmers were formed in 52%of the traps. The distance between patterned particles can be precisely controlled by performing two depositions in opposite directions, thus trapping such particles at the opposite ends of each trap. It is important to ensure uniform temperature across the template to prevent condensation during the patterning process.To this end, we place a heated glass plate underneath the template and set it at the same temperature as the box incubator. This method can be used in a variety of biological studies involving quantitative single-cell analysis. A unique advantage is that its high throughput nature which allows patterning of thousands of cells providing large statistics within the same experimental arena.

patterning-microorganisms-microparticles-through-sequential

Research

JoVE Journal

Engineering

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 ...

Visualizing Hyporheic Flow Through Bedforms Using Dye Experiments and Simulation

Advective exchange between the pore space of sediments and the overlying water column, called hyporheic exchange in fluvial environments, drives solute transport in rivers and many important biogeochemical processes. To improve understanding of these processes through visual demonstration, we created a hyporheic flow simulation in the multi-agent computer modeling platform NetLogo. The simulation shows virtual tracer flowing through a streambed covered with two-dimensional bedforms. Sediment, flow, and bedform characteristics are used as input variables for the model. We illustrate how these simulations match experimental observations from laboratory flume experiments based on measured input parameters. Dye is injected into the flume sediments to visualize the porewater flow. For comparison virtual tracer particles are placed at the same locations in the simulation. This coupled simulation and lab experiment has been used successfully in undergraduate and graduate laboratories to directly visualize river-porewater interactions and show how physically-based flow simulations can reproduce environmental phenomena. Students took photographs of the bed through the transparent flume walls and compared them to shapes of the dye at the same times in the simulation. This resulted in very similar trends, which allowed the students to better understand both the flow patterns and the mathematical model. The simulations also allow the user to quickly visualize the impact of each input parameter by running multiple simulations. This process can also be used in research applications to illustrate basic processes, relate interfacial fluxes and porewater transport, and support quantitative process-based modeling.

This manuscript describes how to create regular bedforms in a flume, visualize flow through the bedforms, and use computer simulations to simulate the hyporheic flow. The computer simulations compare well with the experimental observations. This coupled simulation and experiment is well-suited for both research and educational purposes.

Advective exchange between the pore space of sediments and the overlying water column, called hyporheic exchange in fluvial environments, drives solute ...

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 ...