The overall goal of this experiment is to evaluate the effect of the drying air liquid crystalline or air-LC interface on orientation of megamolecular biopolymers such as polysaccharides, microtubules, and DNA as visualized dynamic behaviors in millimeter scale. This message can help answer key questions in the dispersive structure fields such as self-organization of biopolymers under a drying environment where living organisms have crawled onto ground approximately 500 million years ago. The main advantage of this technique is that it allows side views of the sample to be taken during the drying process without drying on the horizontal plane.
To begin this procedure add 0.5 grams of sacran to 100 milliliters of water. Cover the container with plastic wrap and stir the solution at approximately 80 degrees celsius for more than 12 hours. After allowing the sacran and xantham gum solutions to cool, centrifuge the sacran solution to remove impurities.
Now, prepare a 0.5 weight percent tubulin solution in Britton-Robinson buffer on ice. Mix 50 microliters of the tubulin solution with 5 microliters of GpCpp. Then incubate the solution at 37 degrees celsius for three hours to obtain a stable microtubule or MT nucleus.
Following this, mix 950 microliters of the tubulin solution and 50 microliters of the GpCpp containing tubulin solution. Then, allow the solution to sit for one day at approximately 25 degrees celsius to obtain a stable 0.5 weight percent MT solution. Next, prepare a 0.5 weight percent DNA solution and Tris-EDTA buffer solution.
Keep the 0.5 weight percent biopolymer sample solutions at 25 degrees celsius for the drying experiment. Cut a silicon sheet into an appropriate shape with a thickness of 1 millimeter. Assemble a one side open cell composed of the silicon spacer and two non-modified glass slides.
Fix both sides of the cell with double clips to keep the sample solutions from leaking out. Using a one millimeter pore size pipette tip slowly add 100 to 300 microliters of each 0.5 weight percent biopolymer solution to each cell at approximately 25 degrees celsius. Remove air bubbles from the cell using a syringe needle.
Next, place the cells in an oven with an air circulator for 18 hours at 60 degrees celsius under atmospheric pressure for evaporation. An operation direction is opposite to that of gravity. This placement is also applied to the monitoring for the latter reviews.
Use a 100 watt halogen lamp with a flat surface light source over a wide area to provide straight visible light. Adjust the polarizers to 45 degrees and 135 degrees using the holders. Fix the positions of the light source, polarizers, sample stage, and camera using an optical rail and rod stance.
Place the sample stage between the two polarizers. Then, place the camera approximately 20 centimeters from the sample stage to allow focusing. At the given times indicated in the text protocol, place the biopolymer samples between the polarizers on the stage parallel to the XZ plane and cover the device with a black curtain.
Finally, photograph the samples through linear crossed polarizers using a digital single lens reflex camera with a standard zoom lens. Self integration from microdomain to macrodomain on a drying air-LC interface was evaluated. Before drying, bright regions with transmitted light in a scattered state were observed.
After drying, the region beneath the air-LC interface and the sacran solution became high and the xanthine solution intensity decreased with small macrodomains. Spatiotemporal analysis showed that the peak intensity around the air liquid interface and the thickness increased, indicating that the microdomains start to orient from the air-LC interface and grow into a milli-scale domain parallel to the interface. Polarization microscopic images of the sacran fluid phase showed one blue region, meaning that a single macrodomain formed.
The xanthan gum fluid phase showed blue, yellow, and pink regions, meaning that the multiple macrodomains formed with arbitrary orientations. Before drying, the sacran and MT solutions showed similar LC states with scattered domains. During drying, the MT solution underwent self-integration and the scattered domains were integrated into a single macrodomain.
The DNA solution showed no specific orientation in the liquid phase. The sacran drying record exhibited significant birefringence and wavy bundles were observed for the MT drying record. The DNA drying record showed grain shaped macrodomains in arbitrary directions.
After its development, this technique paved a way for researchers in the field of biomedical engineering using biopolymers to explore a visualization of air stream dynamics on the both drying and wetting processes in geometric approaches.
