Concentration VS Intensity in TE Experiments
Hello Everyone,
Please note that Figures are all shown in the powerpoint file.
The goal of these experiments was to plot a standard curve for concentration and intensity in my Track Etched Membranes. In order to that, we decided to dry droplets on the membranes with different dilution, then check the fluorescent intensity of them and also image them with SEM to count the number of beads, therefore we can have a standard curve for intensity and concentration. Using standard curve, we can have a rough estimate for number of particles captured, released or washed away in my system.
The problem for drying the beads droplets was coffee ring effect leading to non-uniform deposition of beads on the surface of the membrane. After a meeting with Dr. Schertzer, he suggested trying to run these experiments on high temperature using hot plate. According to a paper published in 2016 in Nature (ScientificReport) by Li et al which can be found here: https://www.nature.com/articles/srep24628 , Raising the temperature is useful to prevent the coffee ring effect. ”When the interface descends more quickly than particle diffusion, particles will be captured by the interface, forming a quasi-steady-state semi-solid layer, which prevents the particles from transporting to the drop edge. Just like fishing, if the net closes so fast that there is little time for the fishes to escape, they will be captured by the net.”
So I tried to run these experiments using the hot plate, and i tested different concentration of beads as you can see in the table from the power point file. In all of my sample, i still saw an outside ring which was expected, but a very uniform deposition of dried beads inside the ring. Obviously, there is a loss of beads happening in the ring, rings are always more concentrated (Multilayer deposition) compared to the inside region. So i imaged different regions of each droplet, and from each image, the intensity of different regions of interest was measured and averaged in the table. So, as you can see, the plot looks pretty linear but not perfect, it seems that my 2 first data points are off from the other fours. The next step was to look at these regions below SEM. SEM showed exactly what we expected, a ring which is more concentrated and packed compared to inner regions, Besides the ring, it is worth it to mention that my first 2 data points (25X and 50X were formed multilayered deposition structure which can be the reason for being off from the expected linear fit. So i extruded these 2 data points from my analysis, and you can see the plot shows a perfect linear fit for my remained data points (very Promising). This plot is named reported concentration plot.
Then, using ImageJ, i tried to count the number of beads on the membrane by making the images binary and deciding the whole surface area of beads by the one single bead surface area, then i estimated the concentration of beads in the droplets using the magnification and the area of the droplets. It is important to mention that all the SEM images shown are taken in 10KX for a fair comparison. Finally, the whole concentration was calculated and the standard curve was plotted as you can see and is named counted concentration plot. This plot is not as perfect as the reported concentration plot. The calculated concentration is in the same order of magnitude as the reported concentration which is good, and the difference can be caused by loss of beads in tubing and device and also the formed ring in all samples which is more concentrate.