Active matter has been used in various applications like molecular shadows. To bring the application to the next level, we need to develop the ability to control the active matter locally. We provide an easy to use method that doesn't require modification of the active fluid.
Nor the need to modify the optical path of microscope to achieve the local control of the active fluid. Our method can be applied to a wide range of systems where the main reactions obey the erroneous law such as the microtubule gliding assay or enzyme based systems. The setup involves a water circulation system.
If leaks occur, water can damage the microscope. Therefore, before adopting our protocol, it is important to ensure the system is leak free. Our protocol requires mounting the glass sample on a temperature stage.
While the manuscript describes the mounting procedure, it lacks mechanical subtleties such as securing the sample to the stage. Demonstrating the procedure, will be Teagan Bate, Edward Jarvis, and Megan Varney. Teagan and Edward are graduate students, and Megan is an undergraduate student from a laboratory.
To prepare active fluids in an Eppendorf tube, mix 16 point seven microliters of eight milligrams per milliliter microtubules with six point seven microliters of one point eight micromolar kinesin motor clusters, and one point one microliters of 500 millimolar DTT in high-salt M2B. Bundle microtubules by adding 11 point four microliters of seven percent weight by weight polyethylene glycol. Then activate kinesin motors by adding two point eight microliters of 50 millimolar ATP.
Maintain the ATP concentrations by adding two point eight microliters of stock pyruvate kinase/lactate dehydrogenase, and 13 point three microliters of 200 millimolar phosphenol pyruvate. Reduce the photo bleaching effect by adding 10 microliters of 20 millimolar Trolox, one point one microliters of three point five milligrams per milliliter catalase, one point one microliters of 20 milligrams per milliliter glucose oxidase, and one point one microliters of 300 milligrams per milliliter glucose. Track the motion of the fluid by adding one point six microliters of zero point zero two five percent volume by volume tracer particles.
Add high-salt M2B to achieve a total volume of 100 microliters. Next, rinse a polyacrylamide coated glass slide and cover slip with DI water. Dry the glasses with pressurized air.
Place the glasses on a clean, flat surface. Cut a three millimeter wide channel in the wax film. The total width is the same width as the glass cover slip at 20 millimeters.
Insert the wax between the slide and cover slip to form a flow cell channel for fluid. Adhere the glass to the wax film by placing the glass wax complex on an 80 degree Celsius hot plate to melt the wax. During the melting, press the cover slip gently with a pipette tip to uniformly adhere the wax film to the glass surfaces.
After adhesion, cool the glass complex to room temperature. Promptly load the prepared active fluids to the flow channel with the pipette tip at an angle away from the channel opening and in contact with the glass slide surface. Seal the channel with UV glue.
After building a temperature control setup, place the active fluid sample on the sapphire surface with the slide side contacting the surface. Secure the glass slide with paper tape. Using copper tape, attach the ThermoSensor to the cover slip surface.
To mount the setup on a microscope stage, with the cover slip slide facing toward the objectives, secure the setup with microscope stage needle clamps. Turn on the temperature controller and fish tank pump. Follow the manufacturers guide to set the target temperature.
Enable temperature control. And record ThermoSensor temperature data. Image the sample with a constant time interval on a fluorescent microscopy equipped with a GFP filter cube to monitor the Alexa 488 labeled tracer particles in the active mix.
Adjust the time interval to allow the tracer displacement between frames to be within nine pixels. For imaging tracers moving at 10 micrometers per second, using a four times objective, the time interval is recommended to be one to five seconds. Save the images as TIF files, name the files based on frame number, and store them in a separate folder.
For this kinesin driven microtubule based active fluids, the temperature was controlled at 10, 20, 30, and 40 degrees Celsius. The fluctuation in temperature was within zero point one to zero point three degrees Celsius for four hours demonstrating the stability and reliability of this temperature control setup. The tracers were imaged every two seconds, which the sequential images allowed for tracking tracer trajectories.
The mean speeds measured at 20 to 36 degrees Celsius, appeared to be nearly time independent at zero to two hours. Where as at 10 and 40 degrees Celsius, the mean speeds decayed quickly caused by microtubule depolymerization below 16 degrees Celsius and the kinesin clusters malfunctioning above 36 degrees Celsius respectively. When the system temperatures were alternated between 20 and 30 degrees Celsius every 30 minutes, the mean speeds of the active fluids did not only accelerate and decelerate accordingly, but they also responded to the temperature change within 10 seconds.
Active fluids begin to malfunction, when cooled below 16 or heated above 36 degrees. So when manipulating the temperature controller, ensure the temperature is between 16 and 36 degrees. The ability of tuning active fluid locally, allows for directing the fluid power such as delivering cargos from point A to point B or developing a microfluidity device that doesn't require a physical valve.
The acrylamide used to coat glassware is a neurotoxin which can absorb through the skin. Wear proper protective equipment such as gloves, lab coats and safety goggles to minimize risk.
