Week 5 Analyzing SEM Images

In week 4, the group began to research bacteria that could exist in water supplies and that would cause harm to the human body. Also in Week 4, the nanofibers were spun samples of PEO using different variable lengths that were stated was one of the parameters that would be altering. The spun PEO samples were at lengths 4, 6, 8, 10, 12, and 16 cm. These lengths are the distances between the syringe and the collecting plate . Altering these lengths changed the different diameters of the Electrospun fibers. The goal is to create a filter using these fibers and below is an analysis of the diameters of the fibers:

PEO - 4cm



Figure 11. PEO Spun 4cm

Figure 11 illustrates the diameters of the spun fibers at a distance of 4 cm from the needle and the collecting plate. This image is zoomed so that it would be easy to calculate the distance. Using ImageJ the group was able to estimate the distances of the threads. To take a proper estimate, the measurements were taken from 30 different threads. the average of the diameters were 67.17 nm. the standard deviation was 0.0175 μm.

PEO - 6cm

Figure 12. Spun PEO 6cm

In Figure 12 you see one nano fiber that was woven together through the electrospinning process. Using the Image J measuring tool that was used gave the results that the the fiber has diameter of 10 μm. This holds true to the research that was conducted earlier in the experiment. The diameters should increase to a certain point until they will decrease again. This comparison between 4 cm shows that the results are correct in creating new filtration technology. The measurements were taken from 30 fibers that are comming off of the long big fiber. the measurement average was. 161.49 nm. the standard deviation is 0.0801 μm.

PEO - 8cm

Figure 13. Spun PEO 8 cm

The SEM image at 8 cm also resulted in a picture of only one thread. The diameter in this thread was 7.485 μm which is a little bit finer than the thread spun at 6 cm. Although it is thinner than the thread from 6cm, the 8cm PEO thread is still thicker than the diameter of the thread spun at 4cm which was 0.5722 μm. As in the image before there was only one thread so there was no average taken and the final diameter at 8cm is 7.485 μm. 

PEO - 10cm



Figure 14. Spun PEO 10cm



 In Figure 14 the measurements were taken from 30 different fibers in the SEM image. This was just to get a better approximation of the diameters because there were more fibers spun in the 10cm sample than there was in the 4cm sample. The average of the diameters was 81.6 nm. The standard deviation 0.025 μm.

PEO - 12cm



Figure 15. Spun PEO 12 cm



The PEO results at 12 cm is the best just because of the quantity of the fibers that were spun and collected. Also the diameter is the smallest of all the fibers that were spun before. This image had lots of fibers and it was zoomed to 200nm. the average of 30 fibers is 49.52 nm. the standard deviation of the fiber diameters 14.39 nm

PEO - 16cm



Figure 16. Spun PEO 16cm 



In Figure 16 you see that the fibers don't seem to be spun as well as the fibers before. The diameters are sill very small for the fibers but the fibers do not appear to be as stable. This sample was very running and did not have good results. The fiber diameters 120.56 nm and the standard deviation is 0.036 μm.

The ideal fiber seems to be the fiber spun at the distance of 12 cm. These fibers gave the smallest diameter which is what is needed when spinning fibers for a filter. With these current results it shows that the fiber chosen to spin would be a filter using a 12 cm distance, but there will  also testing the angle of spin to get the ideal distance and angle for a fiber. When the angle results were acquired will be spinning an ideal filter out of the fibers. Currently the only realization is that one ideal variable which is 12 cm.

Week 4 Cont. - Investigation of Filtration and Preventing Disease

The application of our nanofibers will be to create a filter that will block microbes and bacteria that cause disease. The conducted research in this specific area, more specifically, seeing what are the most common bacteria in contaminated water, what are the ranges of size of these bacteria, and the effects of the specific bacterium. The four most common bacteria in untreated water were Fecal Coliform (general), E. Coli, Enterococcus Faecium, and Giardia [14]. The overarching goal is to create a filter that has a specific pattern (most likely a square-crossing pattern) in order to block and trap the bacteria that are in the water supply. PEO is miscible in water that is why this experiment will a larger control in which other solutions that are immiscible in water can be spun and used.


Classification of Bacteria in Water that Cause Disease:

Figure 7. Fecal Coliform Colony (SEM magnified) [7]

Fecal Coliform, as seen to the left, are rod-shaped, small, non-sporulating bacterium. They are not very hazardous to human health; however, specific strains of them (E. Coli) can be very dangerous and potentially life-threatening. Failing home septic systems can allow coliforms in the effluent to flow into the water table, aquifers, drainage ditches and nearby surface waters. Sewage connections that are connected to storm drain pipes can also allow human sewage into surface waters [14]. Animal waste as well as agriculture waste from fertilizers contribute to the formation of coliform. Severe diseases can arise from lethal strains of this bacterium such as: ear infections, dysentery, typhoid fever, viral and bacterial gastroenteritis, and hepatitis A [14]. Without proper treatment, deadly genetic mutations of the RNA inside the bacterium will be certain death. This specific bacterium is approximately 0.60~0.70 μm in diameter and has an approximate volume of 0.816 cubic μm. The nanofibers spun must be so the diameter of each fiber is approximately 0.50 μm and the area of the square pattern is smaller than the diameter of one of these bacteria.

Figure 8. E. Coli under SEM Imaging [10]

A specific form of Fecal Coliform is E. Coli which is known to be a deadly without treatment. Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis.[14]. E. Coli is approximately 2.0 μm in length, has a diameter of 0.50 μm, and volume of 0.70 cubic μm. Although E. Coli is relatively easy to treat if found in the early stages of the disease, it can be lethal if found too late. E. Coli is especially tricky to diagnose because the symptoms arise right before serious illness so there is a insufficient amount of time to kill the bacterium.The image on the right shows how E. Coli are rod-shaped and also are always in colonies.


The last two common bacteria found in contaminated water are Enterococcus Faecium and Giardia. E. Faecium is quite large; with a length of 1 to 2 mm [14]. They can move very fast and are known to target human arteries and hearts. They usually overtake and replace Fecal Coliform in water supply by engulfing them through phagocytosis (cell membrane forming around a substance or foreign object) [14]. Giardia cysts are 8 to 14 μm and the actual bacterium is about 5 μm in diameter. Giardia has symptoms that mimic other gastrointestinal problems and the common symptoms are persistent diarrhea, weight loss, abdominal cramps, nausea, and dehydration. In general, the symptoms begin within a week after exposure and the acute symptoms can last for up to 2 weeks, but chronic symptoms can last for up to 2 months [14]. Usually it is not life-threatening but treatment must be given to get rid of both bacteria.

Figure 9. E. Faecium infecting pulmonary tissue [10]

Figure 10. Giardia in water supply [10]

Since the smallest form of Fecal Coliform is the smallest parameter of bacteria size in water supply, a filter must be created to take the size and dimensions of the bacteria into account. The filter mechanism is actually possible to create because the size and pattern shape can be created through mechanical/technological systems.

Week 4 - SEM Imaging

The SEM produces 6 different signals which include:  secondary electrons, back-scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence), specimen current and transmitted electrons. The SEM that will be used, transmits secondary electron detector signals. There are 3 different types of microscopes that can be used which range from LM, TEM, SEM as shown in figure 5 [12].



Figure 5. Functionality of LM, TEM, SEM [12]

 

All three microscopes begin with the light source at the top illuminating to a condenser lens. During the next step the LM and TEM microscopes vary from the SEM because the specimen is between the first condenser lens and the objective lens. In the SEM microscope there is a second condenser lens for a better image. The LM microscope requires you to use your eye to look at the projection lens while the TEM uses a fluorescent screen [13]. The SEM makes a crisper image because it has the specimen after the objective lens, and instead the specimen then emits a signal to a signal detector which creates a crisp image as shown below.  



Figure 6. Difference between optical & SEM of a small marine organism [13]

The SEM imaging is the ideal choice in this case because as shown in figure 6 the image is more detailed than an average optical micrograph. It uses the electron's wavelength to send a signal to the specific specimen being observed. The wavelength bounces off and the resultant signal is transmitted, filtered, and an image is forced. This process is different from optical microscopes and results in less focus because the wavelength is much higher than an electron's [12]. In the experiment the SEM imaging is used to measure the diameter, and count the nanofibers in the electrospun strand. Some important details about the SEM microscope are: working distance, depth-of-focus, and the secondary electron. The working distance is important because it is the distance between the lower surface of the objective lens and the surface of the specimen and altering this distance will change the clarity of the picture. The depth-of-focus is plainly the focus of the SEM image. Finally, the last variable  is the secondary electron which are the electrons ejected by the specimen durring the inelastic scattering of the energetic beam electrons. The more electrons ejected the clearer the image. In week 4, Evan and Tristan are using the SEM to complete these viewing tasks.

Week 3 Cont. - Spinning Process *Detailed*

A major component that was not taken into account was the constants was the needle gauge. We chose a 21 gauge that had an inner diameter of 14.5 mm. The higher the gauge, the smaller the diameter becomes. It is important to keep the gauge constant because different sized gauges will change the diameter of respective nanofibers. This will cause innaccurate comparisons between the different spun fibers [11]. This was an important constant to add to the list because it is an important part of the apparatus. The rotational mixer used to mix the solutions was made by Elmeco Engineering Inc and the specific model was the Arma-Rotator A-1. When the spinning began some measurements were taken: the temperature was 24.3 degrees Celsius and the humidity was 26%.

Figure 3. Our Electrospinning Apparatus

Evan and Tristan began to spin the mixed solution that they created on the previous day. They started off by changing the distance variable and completed the first 6 deliverables. They spun at 4 cm, 6 cm, 8 cm, 10 cm, 12 cm, and 16 cm. These distances represented the distance of the syringe's needle tip to the collecting plate. The purpose of changing the distance was to see the effect on the fiber's diameter, abundance, and pattern that formed. In Figure 3 (to the left), the final apparatus can be seen. The syringe contains the PEO solution and the needle slowly releases the solution at a rate of 1 mL/hr. The red wire is the voltage that hits the solution and begins the fiber forming process. The silver square sheet is actually aluminum foil that is wrapped around a brown metallic sheet. This will keep the charged PEO fiber to stick onto the collecting plate and it will allow for a sample to form. The other wire (on the right) keeps the collecting plate charged for this exact reason. In the back, the holder allows the plate to be held at a certain elevation. The holder is wrapped so no current is able to pass through it.

Figure 4. 5% PEO solution used during spinning process

Evan and Tristan used the solution they made the day before. The solution was 5% PEO in water and the figure on the right shows the color of the solution as well as the solution itself. The solution was clear and is perfectly mixed because it was placed in the rotational mixer. This solution was then put into the syringe to be hit by high voltage as mentioned above.

Week 3 - Beginning to Spin

Figure 1: Ethylene oxide with aid of catalyst becomes PEO [1]

On April 16, in the lab Evan and Tristan mixed two identical solutions of PEO by dissolving 0.5 grams of 600,000 molecular weight PEO into 10 mL of water.  The outcome of this is a 5% molecular weight PEO in water solution which was placed on a rotational mixer to mix overnight. The rotational mixer helped thoroughly mix the solution and the particles of water with PEO. The process was new and required some getting used to understand exactly how PEO is made. The picture on the left demonstrates how it is created with an aid of a catalyst [4]. First ethylene oxide is the monomer that is combined with itself with a help of a catalyst. The catalyst allows for an addition polymer to be formed; in this case, polyethylene oxide (PEO). April 17 Evan and Tristan began spinning the created PEO solution.
Evan and Tristan will be spinning PEO fibers at six different angles in addition to six different distances. Each fiber will be spun for 30 min and stored for SEM imaging later in the week or durring week 4. Through extensive research increasing the will make the diameter smaller and increasing the distance should also make the diameter smaller. The ideal situation in our experiment is to get the thinest PEO fibers possible with some textile strength to make a proper filter. The textile strength only has to be strong enough to handle strong water flow and if time permits these fibers will be tested for their strength after SEM imaging with a tention meter.

Week 2 - Composing Project Proposal

      In week 2 consisted of additional research about the apparatus and materials used for electrospinning. Through the research we found the different variables of spinning were; time, solute, solvent, voltage, distance, temp, humidity, viscosity, and angle. In the project, these variables are tested to see the effect the spun fibers. After consulting with Marjorie about the limited time constraints it was decided to change the distance and the angles at which the different fibers are spun at. This initial testing is to do a general test on how well a fiber can be used for filtering a water solution. The main goal in the project is to create a filter out of fibers that would make cleaner drinking water. Since PEO dissolves in water, a different polymer can replace the PEO and be spun with the same variables for an ideal filter. The ideal variables will be found through our experimentation and we will implement those variables on another polymer to create this filter. Hopefully if time allows, we will be able to create this filter. 

      Below is a video of the Electrospinning Apparatus and it's functionality: 

Video #1 : Apparatus Design

Week 1 - Deciding Freshman Design III Topic

The class topics ranged from designing a building using sustainable materials to audio cheapskates and tweaks and finally to polymer composite fibers created from electrospinning. The topic chosen ended up being electrospinning of a specific polymer called poly(ethylene oxide) by changing the variables and testing the strength of the different fibers to see which is best for military purposes. Electrospinning involved all of the team member's majors in various forms.The majors of the group members are biomedical engineering, chemical engineering, and electrical engineering. The spinning part of the experiment includes concepts from electrical engineering due to the electrical current passing through the solution to create the fiber. The polymer, PEO, involves chemical engineering because to understand the solution chemical properties need to be analyzed. Finally it includes biomedical engineering because a large amount of the fibers can be used to create artificial tissue. With this being said the initial topic goal is to vary the distance from the needle to the collecting dish, changes the angle of the needle, varying the concentration of the solution, and finally varying the voltage of the current.