The microfluidic device is made up of channels that are designed to capture images and videos. The viewing area occurs in the central channel region and uses high resolution to capture the images. Inlet and outlet placement makes room for the buffer solution to be fed through the device. Also, through the central channel, single cell bacteria is inserted so the environment can be manipulated by the solution. The feeder channels are parallel to the central feeder channel viewing area and are joined by nanopores. Nanopores gives the side channels access to the viewing area to observe the bacteria once the solution diffuses the area. The rate in which the solution was diffused has to do with the amount of dyes injected into the solution. Five μm of Fluorescein and 5 μm of Texas Red made up the dye formula. Once the dyes were injected in the feeder channels, the colors which turned out to be green and red, were able to be seen through the optical filters one dye at a time. Tracking the dyes separately allows the microscope view to determine the diffusion rate into the central swimming channel.
The solution consists of 14 grams of polydimethylsiloxane (PDMS) and 1.4 grams of curing agent. Once the mixture was prepared, it was poured over the top of the silicon chip. The solution was dehydrated and had to be solidified by baking at a temperature of 60 °C. The moldings were removed from the microfluid device and then the inlet and outlet holes were created. The device was cleaned with plasma to assist with removing surface debris from the PDMS. Cleaning the surface alters the use of the device and glass so the two can bond together. Once the bond is determined, the device and the glass slide are paired then placed in the oven for half an hour at 60 °C to solidify the bond. When the time approached, liquids were poured into the chemotaxis device using 1 ml syringes and 0.3 mm syringe tips and tubing. A syringe pump was used to control the flow of the liquid and kept at a constant rate of 30 μl per hour. Once the device was assembled, it was filled with 0.1% bovine serum albumin and rinsed with distilled water after 10 minutes of inactivity. Bacterial solution was poured in the central channel while the inlets and outlets holes were stuffed with bits of PDMS. This was accomplished by filling the syringes with its respective solution and connected to the inlet holes through tubing. The outlet holes served as waste output using tubing made of Teflon.
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The process started with filling the central viewing area with cultured cells. The video in the central viewing area was able to capture data on the cultured cells by viewing the single cell motility. Acetic acid was placed into the environment in the central channel so the viewing area could record the bacteria’s reaction to the change. The quantitative date was able to be extracted based on the reaction of the new synthesis. Once the videos were recorded, they were divided into short 10 second instances and then each session was processed individually. The videos created images useable for flat field correction. This was achieved by removing the background and cells without motility. A threshold algorithm was applied to convert the video to binary to track the cells frame by frame using the Manual Tracking Plugin. The data was processed and extracted to the Chemotaxis and Migration Tool.
In order to determine the traits that make up cellular movement, the Forward Migration Index (FMI) was utilized. The FMI calculates cell movement and their relation to the X and Y axes. The formulas FMI X and FMI Y gather speed instances from both ends to communicate cell speed and data. The FMI further explains the space surrounding each individual cell, the motion of entire cell groups, total distance or length travelled by the cell and cell endpoints. Furthermore, the Euclidean distance is considered in conjunction with the FMI formula. Euclidean distance calculates the length of the line segment that connects the starting and ending points of the total cell route. It also accounts for the velocity and directness through measuring these cell routes. This is determined by comparing Euclidean and accumulated distances.
