Forming, Confining, and Observing Microtubule-Based Active Nematics

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.

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Active Nematics have emerged as an exciting research topic that expands the fields of nonlinear dynamics and liquid crystals. There are complex fluid that exhibit mortal topological defects and chaotic dynamics. There are many recent theoretical studies in literature focused on confined Active Nematics.

However, it has been challenging for experimentalists to confine the material in microscale geometries. Our technique make the experimental set up more straightforward so the new groups can enter the field and try these exciting materials. The techniques could be applied to other EQUIS active matter systems, for example to form a phase compose of effecting and As more active phases are developed in the future experimenters need to figure out ways to confine the material using similar nematics.

Demonstrating the procedure will be Derek Hammar and Fereshteh Memarian, graduate students from my laboratory. To prepare hydrophilic cover slips with an acrylamide coating begin by cleaning the cover slips thoroughly with soapy water, ethanol and 0.1 molar sodium hydroxide with alternate rinses using nano pure water. Once rinsed coat the cover slips with the Silane solution composed of 100 milliliters of ethanol one milliliter of ascetic acid and 500 microliters of trimethoxysilyl propyl methacrylate a for 15 minutes.

Then rinse with nano pure water. Prepare an acrylamide solution from 95 milliliters of nano pure water and five milliliters of 40 weight percent Acrylamide. Then degas the solution for 30 minutes in a vacuum oven, add 0.07 grams of ammonium per sulfite and 35 microliters of Tetramethylethylenediamine for a 2.3 millimolar final concentration.

Pour acrylamide solution over the cover slips while facing up and incubate overnight at room temperature. To prepare hydrophobic microscope slides pipette 100 microliters of a water repellent solution onto a clean glass microscope slide. Then place another clean glass slide on top.

This ensures an even coating of the water repellent solution on the surface where it sits for two minutes. Remove the second glass slide and rinse the first slide thoroughly with nano pure water. Then dry with nitrogen gas.

Prepare a mixture of engineered oil that includes 1.8%fluoro surfactant. Assemble the glass slide and cover slip using 40 micrometers double-sided adhesive spacers. Place the spacers 1.5 millimeters apart on the hydrophobic microscope slide.

Then place the acrylamide coated cover slip on top of the spacers with a treated side face down to adhere. After the flow cell has been constructed immediately pipette the oil mixture into the flow cell filling the enclosed space using a pipette in a separate vial, gently mix six microliters of active material with 3.73 microliters of mix. One microliter of microtubule solution 0.6 microliters of ATP solution, and 0.67 microliters of M two B buffer pipette six microliters of freshly mixed active material into one open end of the flow cell.

Some oil will be displaced by the aqueous solution as it is injected into the channel. This can be wicked up at the opposite end of the flow channel using a small piece of tissue paper. After filling, seal both sides of the flow cell with an epoxy glue that hardens when exposed to UV light for 20 seconds.

To confine the active layer between the two admissible fluids in a quasi 2D layer. Place the flow cell in a swinging bucket centrifuge with the aqueous phase on top and the denser oil layer underneath centrifuge at 212 G for 10 minutes. After this step is complete the flow cell can be taken to an epi-fluorescence microscope for imaging with a 10 x or 20 x magnification objective.

First, design a master mold for the PDMS. This can be achieved by 3D printing pillars on a substrate. After 3D printing the resin master mold, clean it with isopropanol and then cure the mold under a UV lamp for 45 minutes and in the oven at 120 degrees Celsius for two hours, prepare the poly dye methyl cyan using an elastomer curing agent and an elastomer base.

Mix the two components in a 1 to 10 ratio using a metal spatula. To remove these bubbles, place the mixture under a vacuum to degas for one hour after which the uncured PDMS should appear transparent. Pour the PDMS into a suitable mold and leave overnight to cure at 60 degrees Celsius.

To prepare a hydrophilic PDMS surface clean the PDMS for 10 minutes with both ethanol and isopropanol, then rinse thoroughly with deionized water three times and dry. Use a plasma cleaner for five minutes to clean the dry cured PDMS. This makes the surface more hydrophilic.

Next, prepare a silane solution and immerse the substrate in that solution for 15 minutes to prepare the acrylamide coating, rinse the substrate thoroughly with deionized water and immerse in acrylamide solution. When ready for use, rinse the surface with deionized water and dry with nitrogen for immediate use. Attach the PDMS to a glass slide with epoxy glue, pipette one microliter of the active mixture onto the PDMs substrate, and immediately add silicone oil on top of the active network droplet.

The active network will move into the well. This process takes up to 60 minutes. Place the PDMS device in a swinging bucket centrifuge with the oil layer above the water layer and spin for 12 minutes at 212 G.After this step is complete, take the material to the microscope for imaging record progress of material.

As it equilibrates. The representative image depicts short microtubules of similar lengths. The individual microtubules may be challenging to image due to their small size.

The use of a high sensitivity camera designed for fluorescence microscopy is best for this application. A well formed active pneumatic layer is homogenous in texture with no significant void areas and mobile topological defects present. Note, however, that there may be some acceptable small voids in the defect cores.

Proper surface treatment to have a hydrophilic or hydrophobic surface. With this method, roll of geometry can be discussed easily and the active structure can be controlled by only changing the physical confinement.

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Journal (Biology)

Journal (Biology)

Forming, Confining, and Observing Microtubule-Based Active Nematics

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