Plasmid K401-BCCP-H6 was a gift from Dr. Zvonimir Dogic. This research was supported by Dr. Kun-Ta Wu's start-up fund in Worcester Polytechnic Institute. We thank Dr. Zvonimir Dogic for the protocols to purify and label tubulin and to synthesize active fluids. We are grateful to Dr. Marc Ridilla for his expertise in protein expression and purification. We thank Dr. William Benjamin Roger for assisting us with building the temperature-controlled stage. We acknowledge Brandeis MRSEC (NSF-MRSEC-1420382) for use of the Biological Materials Facility (BMF). We acknowledge the Royal Society of Chemistry for adapting the figures from Bate et al. on Soft Matter33.

The authors have nothing to disclose.

Controlling active matter in situ opens the door to directed self-organization of active matter4,5,24,28,54. In this article, we present a protocol for using temperature to control kinesin-driven, MT-based active fluids in situ, based on the Arrhenius characteristic of the system29,30,<sup c...

Active matter is differentiated from conventional passive matter due to its capability to convert chemical energy into mechanical work. A material that possesses such capability can consist of living or non-living entities such as bacteria, insects, colloids, grains, and cytoskeletal filaments1,2,3,4,5,6,7,8,9,10. These material entities interact with their neighbors. At a larger scale, they self-organize into either turbulent-like vortices (active turbulence) or material flows11,12,13,14,15,16,17,18,19,20. An understanding of self-organization of active matter has led to various applications in molecular shuttles, optical devices, and parallel computation21,22,23. To bring applications to the next level requires control beyond self-organization. For example, Palacci et al. developed a hematite-encapsulated colloid that self-propelled only when exposed to manually controlled blue light, which led to the emergence of living crystals24. Morin et al. established the control of rolling Quincke colloids by using a tunable external electric field, resulting in colloidal flocking in a racetrack-like channel25. These previous works demonstrate the role of local control in applications and advance the knowledge base of active matter.
In this article, we focus on the controllability of kinesin-driven, microtubule (MT)-based 3D active fluids. The fluids consist of three main components: MTs, kinesin molecular motors, and depletants. The depletants induce a depletion force to bundle the MTs, which are later bridged by motor clusters. These motors walk along the MTstoward the plus end. When a pair of bridged MTsis antiparallel, the corresponding motors walk in opposite directions. However, the motors are bound in a cluster and are unable to walk apart, so they cooperatively slide apart pairs of MTs (interfilament sliding, Figure 1A). These sliding dynamics accumulate, causing bundles of MTsto extend until reaching their buckling instability point and break (extensile bundles, Figure 1B)26. The broken bundles are annealed by the depletion force, which subsequently extends again, and the dynamics repeat. During the process of the repeating dynamics, the bundle movements stir the nearby liquid, inducing flows that can be visualized by doping with micron-scale tracers (Figure 1C). Sanchez et al. and Henkin et al. have characterized the mean speeds of tracers, finding that the speeds were tunable by varying the concentrations of adenosine triphosphate (ATP), depletants, motor clusters, and MTs19,27. However, such tunability existed only prior to active fluid synthesis. After synthesis, the tunability was lost, and the fluids self-organized in their own way. To control active fluid activity after synthesis, Ross.et al. reported a method using the light-activated dimerization of motor proteins, allowing fluid activity to be tuned on and off using light28. While light control is convenient in terms of locally activating the fluids, the method requires redesigning the structures of motor proteins, along with modifying the optical paths in a microscope. Here, we provide an easy-to-use method for locally controlling fluid flows without microscope modification while keeping the motor structure intact.
Our method of locally tuning active fluid flow is based on the Arrhenius law because the kinesin-MT reaction has been reported to increase with temperature29,30,31,32. Our previous studies showed that the temperature dependence of the mean speed of an active fluid flow followed the Arrhenius equation: v = A exp(-Ea/RT), where A is a pre-exponential factor, R is the gas constant, Ea is the activation energy, and T is the system temperature33. Therefore, fluid activity is sensitive to the temperature environment, and the system temperature needs to be consistent to stabilize the motor performance, and consequently, the fluid flow speed34. In this article, we demonstrate the use of the motor&#39;s temperature dependence to continuously tune the flow speeds of active fluids by adjusting the system temperature. We also demonstrate the preparation of an active fluid sample, followed by mounting the sample on a microscope stage whose temperature is controlled via computer software. Increasing the temperature from 16 &#176;C to 36 &#176;C speeds up the mean flow speeds from 4 to 8 &#181;m/s. Additionally, the tunability is reversible: repeatedly increasing and decreasing the temperature sequentially accelerates and decelerates the flow. The demonstrated method is applicable to a wide range of systems where the main reactions obey the Arrhenius law, such as the MT gliding assay29,30,31,32.

<table><tbody><tr><td>(&amp;plusmn;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td><td>Sigma-Aldrich</td><td>238813</td><td>Trolox</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate, 98%, ACROS Organics</td><td>Fisher Scientific</td><td>AC216550050</td><td></td></tr><tr><td>3.2mm I.D. Tygon Tubing R-3603</td><td>HACH</td><td>2074038</td><td>Water tubes</td></tr><tr><td>31.75 mm diameter uncoated, sapphire window</td><td>Edmund Optics</td><td>43-637</td><td>Sapphire disc</td></tr><tr><td>3M 1181 Copper Tape - 1/2 IN Width X 18 YD Length - 2.6 MIL Total Thickness - 27551</td><td>R.S. HUGHES</td><td>054007-27551</td><td>Copper tape</td></tr><tr><td>Acetic Acid</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>Acrylamide Solution (40%/Electrophoresis), Fisher BioReagents</td><td>Fisher Scientific</td><td>BP1402-1</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate dipotassium salt hydrate</td><td>Sigma-Aldrich</td><td>A8937</td><td>ATP</td></tr><tr><td>Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td><td>Thermo Fisher Scientific</td><td>A20006</td><td>Far-red fluorescent dye. Alexa 647 can be pre suspended in dimethylsulfoxide (DMSO) before mixing with microtubules (1.3.3.2.)</td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Unit</td><td>Sigma-Aldrich</td><td>UFC801024</td><td>Centrifugal filter tube. Cutoff molecular weight: 10 kDa</td></tr><tr><td>Ammonium Persulfate, 100g, MP Biomedicals</td><td>Fisher Scientific</td><td>ICN802829</td><td>APS</td></tr><tr><td>Ampicillin Sodium Salt (Crystalline Powder), Fisher BioReagents</td><td>Fisher Scientific</td><td>BP1760</td><td>Ampicillin</td></tr><tr><td>Antivibration Table</td><td>Nikon</td><td>63-7590S</td><td></td></tr><tr><td>Avanti J-E Centrifuge</td><td>Beckman Coulter</td><td>369001</td><td></td></tr><tr><td>Bacto Agar Soldifying Agent, BD Diagnostics</td><td>VWR</td><td>90000-760</td><td>Agar</td></tr><tr><td>Biotin</td><td>Alfa Aesar</td><td>A14207</td><td></td></tr><tr><td>Bucket-plastic white - 2 gallon</td><td>Bon</td><td>84-715</td><td>Water bucket</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>746495</td><td>CaCl2</td></tr><tr><td>Catalase from bovine liver</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>CFI Plan Apo Lambda 4x Obj</td><td>Nikon</td><td>MRD00045</td><td>4x air objective</td></tr><tr><td>C-FLLL-FOV GFP HC HC HISN ero Shift</td><td>Nikon</td><td>96372</td><td>GFP filter cube</td></tr><tr><td>CH-109-1.4-1.5</td><td>TE Technology</td><td>CH-109-1.4-1.5</td><td>Thermoelectric Cooler (TEC)</td></tr><tr><td>Chloramphenicol, 98%, ACROS Organics</td><td>Fisher Scientific</td><td>C0378</td><td></td></tr><tr><td>Cooling block</td><td>N/A</td><td>N/A</td><td>Custom milled aluminum</td></tr><tr><td>Coomassie Brilliant Blue R-250 #1610400</td><td>Bio-Rad</td><td>1610400</td><td>Triphenylmethane dye</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Dimethyl Sulfoxide (Certified ACS), Fisher Chemical</td><td>Fisher Scientific</td><td>D128</td><td>DMSO</td></tr><tr><td>DL-1,4-Dithiothreitol, 99%, for biochemistry, ACROS Organics</td><td>Fisher Scientific</td><td>AC165680050</td><td>DTT</td></tr><tr><td>DOWSIL 340 Heat Sink Compound</td><td>Dow</td><td>1446622</td><td>Thermal paste</td></tr><tr><td>ETHYL ALCOHOL, 200 PROOF ACS/USP/NF GRADE 5 GALLON POLY CUBE</td><td>Pharmco by Greenfield Global</td><td>111000200CB05</td><td>Ethanol</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#039;,N&#039;-tetraacetic acid</td><td>Sigma-Aldrich</td><td>E3889</td><td>EGTA</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>798681</td><td>EDTA</td></tr><tr><td>Fisher BioReagents Microbiology Media Additives: Tryptone</td><td>Fisher Scientific</td><td>BP1421</td><td>Tryptone</td></tr><tr><td>Fisher BioReagents Microbiology Media Additives: Yeast Extract</td><td>Fisher Scientific</td><td>BP1422</td><td>Yeast extract</td></tr><tr><td>Fluoresbrite YG Microspheres, Calibration Grade 3.00 &amp;micro;m</td><td>Polysciences</td><td>18861</td><td>Tracer particles</td></tr><tr><td>Glucose Oxidase from Aspergillus niger</td><td>Sigma-Aldrich</td><td>G2133</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>GpCpp</td><td>Jena Bioscience</td><td>NU-405L</td><td>Guanosine-5&amp;rsquo;[(&amp;alpha;,&amp;beta;)-methyleno]triphosphate (GMPCPP)</td></tr><tr><td>GS Power&#039;s 18 Gauge (True American Wire Ga), 100 feet, 99.9% Stranded Oxygen Free Copper OFC, Red/Black 2 Conductor Bonded Zip Cord Power/Speaker Electrical Cable for Car, Audio, Home Theater</td><td>Amazon</td><td>B07428NBCW</td><td>Copper wire</td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate</td><td>Sigma-Aldrich</td><td>G8877</td><td>GTP</td></tr><tr><td>Hellmanex III</td><td>Sigma-Aldrich</td><td>Z805939</td><td>Detergent</td></tr><tr><td>HEPES Sodium Salt (White Powder), Fisher BioReagents</td><td>Fisher Scientific</td><td>BP410</td><td>NaHEPES</td></tr><tr><td>High performance blender machine</td><td>AIMORES</td><td>AS-UP1250</td><td>Blender</td></tr><tr><td>His GraviTrap</td><td>GE Healthcare</td><td>11003399</td><td>Gravity Column</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I6758</td><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside</td></tr><tr><td>Isopropyl Alcohol 99%</td><td>Pharmco by Greenfield Global</td><td>231000099</td><td>Isopropanol</td></tr><tr><td>JA-10 rotor</td><td>Beckman Coulter</td><td>369687</td><td></td></tr><tr><td>L-Glutamic acid potassium salt monohydrate</td><td>Sigma-Aldrich</td><td>G1501</td><td>K-Glutamate</td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>M2670</td><td>MgCl2&amp;bull;6H2O</td></tr><tr><td>MES sodium salt</td><td>Sigma-Aldrich</td><td>M5057</td><td>2-(N-Morpholino)ethanesulfonic acid sodium salt</td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td>3-(N-Morpholino)propanesulfonic acid</td></tr><tr><td>MP-3022</td><td>TE Technology</td><td>MP-3022</td><td>Thermocouple</td></tr><tr><td>N,N,N&#039;,N&#039;-Tetramethylethylenediamine 99%, ACROS Organics</td><td>Fisher Scientific</td><td>AC138450500</td><td>TEMED</td></tr><tr><td>Nanodrop 2000c UV-VIS Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>E112352</td><td>Spectrometer</td></tr><tr><td>Nikon Ti2-E Nikon Inverted Microscope</td><td>Nikon</td><td>MEA54000</td><td></td></tr><tr><td>Norland Optical Adhesive 81</td><td>Norland Products</td><td>NOA81</td><td>UV glue</td></tr><tr><td>Novex Sharp Pre-stained Protein Standard</td><td>Thermo Fisher Scientific</td><td>LC5800</td><td>Protein standard ladder</td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels, 1.5 mm, 10-well</td><td>Thermo Fisher Scientific</td><td>NP0335BOX</td><td>SDS gel</td></tr><tr><td>Optima L-90K Ultracentrifuge</td><td>Beckman Coulter</td><td>365672</td><td></td></tr><tr><td>Parafilm PM996 Wrap, 4&quot; Wide; 125 Ft/Roll</td><td>Cole-Parmer</td><td>EW-06720-40</td><td>Wax film</td></tr><tr><td>Pe 300 ultra Illumination System Single Band, 3mm Light Guide control Pod power supply</td><td>Nikon</td><td>PE-300-UT-L-SB-40</td><td>Cool LED Illuminator</td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma-Aldrich</td><td>78830</td><td>PMSF</td></tr><tr><td>Phosphoenolpyruvic acid monopotassium salt, 99%</td><td>BeanTown Chemical</td><td>129745</td><td>PEP</td></tr><tr><td>Pierce Coomassie (Bradford) Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23200</td><td></td></tr><tr><td>Pierce Protease Inhibitor Mini Tablets</td><td>Thermo Fisher Scientific</td><td>A32953</td><td></td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P6757</td><td>1,4-Piperazinediethanesulfonic acid</td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>Poly(ethylene glycol)</td><td>Sigma-Aldrich</td><td>81300</td><td>PEG. Average molecular weight 20,000 Da</td></tr><tr><td>Potassium Hydroxide (Pellets/Certified ACS), Fisher Chemical</td><td>Fisher Scientific</td><td>P250-500</td><td>KOH</td></tr><tr><td>PowerEase 300W Power Supply (115 VAC)</td><td>ThermoFisher Scientific</td><td>PS0300</td><td>DC power supply of the gel box</td></tr><tr><td>PS-12-8.4A</td><td>TE Technology</td><td>PS-12-8.4A</td><td>DC power supply of the temperature controller</td></tr><tr><td>Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td><td>Sigma-Aldrich</td><td>P-0294</td><td>PK/LDH</td></tr><tr><td>Quiet One Lifegard Fountain Pump, 296-Gallon Per Hour</td><td>Amazon</td><td>B005JWA612</td><td>Fish tank pump</td></tr><tr><td>Rosetta 2(DE3)pLysS Competent Cells - Novagen</td><td>Millipore Sigma</td><td>71403</td><td>Competent cells</td></tr><tr><td>Sharp Microwave ZSMC0912BS Sharp 900W Countertop Microwave Oven, 0.9 Cubic Foot, Stainless Steel</td><td>Amazon</td><td>B01MT6JZMR</td><td>Microwave for boiling the water</td></tr><tr><td>Sodium Chloride (Crystalline/Certified ACS), Fisher Chemical</td><td>Fisher Scientific</td><td>S271-500</td><td>NaCl</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td>SDS</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>S8282</td><td>NaH2PO4</td></tr><tr><td>Streptavidin Protein</td><td>Thermo Fisher Scientific</td><td>21122</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>TC-720</td><td>TE Technology</td><td>TC-720</td><td>Temperature controller</td></tr><tr><td>Tris Base, Molecular Biology Grade - CAS 77-86-1 - Calbiochem</td><td>Sigma-Aldrich</td><td>648310</td><td>Tris-HCL</td></tr><tr><td>Type 45 Ti rotor</td><td>Beckman Coulter</td><td>339160</td><td></td></tr><tr><td>Type 70 Ti rotor</td><td>Beckman Coulter</td><td>337922</td><td></td></tr><tr><td>Type 70.1 Ti rotor</td><td>Beckman Coulter</td><td>342184</td><td></td></tr><tr><td>VWR General-Purpose Laboratory Labeling Tape</td><td>VWR</td><td>89097-916</td><td>Paper tapes</td></tr><tr><td>VWR Micro Cover Glasses, Square, No. 1 1/2</td><td>VWR</td><td>48366-227</td><td>Glass coverslips</td></tr><tr><td>VWR Plain and Frosted Micro Slides, Premium</td><td>VWR</td><td>75799-268</td><td>Glass slides</td></tr><tr><td>XCell SureLock Mini-Cell</td><td>ThermoFisher Scientific</td><td>EI0001</td><td>Gel box</td></tr><tr><td>ZYLA 5.5 USB3.0 Camera</td><td>Nikon</td><td>ZYLA5.5-USB3</td><td>Monochrome CCD camera</td></tr></tbody></table>

controlling flow speeds of microtubule-based 3d active fluids using temperature

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4&#8211;8 &#181;m/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.

Preparing the kinesin-driven, MT-based active fluids requires both kinesin and MTs. The MTs were polymerized from labeled tubulins (steps 1.3 and 1.4) that were purified from bovine brains (step 1.1, Figure 2A), followed by recycling to enhance purity (step 1.2, Figure 2B). The kinesin motor proteins were expressed in and purified from E. coli (steps 2.1 and 2.2, Figure 2B)...

Watch this Scientific Journal Video about Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature at JoVE.com

Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method ...

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7.1K Views.  Worcester Polytechnic Institute. The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

Video: Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Biology

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3, drug delivery4, improved hydrocarbon recovery5-7, and flowable electrodes for energy storage8. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius&#160;and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9.&#160;Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37.&#160;In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.

Confocal microscopy is used to image quiescent and flowing colloid-polymer mixtures, which are studied as model systems for attractive suspensions. Image analysis algorithms are used to calculate structural and dynamic metrics for the colloidal particles that measure changes due to geometric confinement.

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed ...

Chemistry

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

Environment

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.

The present protocol describes the crystallization of microscopic ice crystals and clathrate hydrates in microfluidic devices, enabling liquid exchange around the formed crystals. This provides unparalleled possibilities to examine the crystallization process and binding mechanisms of the inhibitors.

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Biochemistry

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Bioengineering

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Forming, Confining, and Observing Microtubule-Based Active Nematics

The formation of biopolymer-based active phases has become an important technique for researchers interested in exploring the emerging field of active liquid crystals and their possible roles in cell biology. These novel systems consist of self-driven sub-units that consume energy locally, producing an out-of-equilibrium dynamic fluid. To form the active liquid crystal phase described in this report, purified protein components including biopolymers and molecular motors are combined, and the active nematic phase spontaneously forms in the presence of adenosine triphosphate (ATP). To observe the nematic state, the material must be confined in a suitable geometry for microscopy at a high enough density. This article describes two different methods for the formation of an active nematic phase using microtubules and kinesin motors: assembly of a two-dimensional active layer at an oil and water interface and assembly under an oil layer using an elastomeric well. Techniques to insert the active material into small wells of different shapes are also described.

Presented here are methods for preparing active nematics from microtubules and kinesin motors, including protein preparation and construction and the use of wells for active nematic confinement.

The formation of biopolymer-based active phases has become an important technique for researchers interested in exploring the emerging field of active ...