Jun 30th, AM

We prepared glass slides to view ATTO molecules fluoresce and to measure their fluorescent lifetime. The molecules have a tendency to clump together in water on a glass slide. To get the cover-slips to be hydrophobic, it was coated with a hydrophobic chemical, OTS(octadecytrichloro-silane). The cover-slips were plasma cleaned and then washed in an ultra-sonicator bath first with cloroform, then in ethanol and finally with water. It takes a long time for the water to evaporate or dry from the slides. Next, we try to dilute the dye in ethanol which evaporates faster than water. Then it was time to go for lunch.

Jun 29th PM

We learn how to put in values and formulas into excel sheets. Will practise doing them in my math classes. Need a manual to learn to be an EXCEL master. Something like EXCEL for dummies would help. Will be reading Nature of Science articles today.

Jun 29th AM

Today, we prepare the cantilevers to measure adhesion of cantilever tip on diamond nanocrystals. We have to clean the cantilevers with a machine called the plasma cleaner. The cantilevers are placed on a glass dish and put into the machine. A partial vacuum was created and O2 was pumped into machine. The O2 becomes plasma which is blasted onto the cantilever to remove any organic matter. The cleaned cantilever was mounted on the AFM and a control adhesion baseline was measured using a clean glass-slide. A diamond layer about 800 nm thick will be put onto a glass-slide to measure the adhesive force between cantilever tip and diamond layer. Diamond layer make a very smooth surface for studying cell adhesion. Diamond consists of C atoms and the top layer of C could be replaced with H atoms which makes the top layer hydrophobic to water. Replacing the top layer of C with O atoms will make the top layer hydrophillic.

Jun 28th PM

We learn about Google docs. We created a sample test on an excel spreadsheet and learn how to gather and manipulate the data to show the results of the test. It will need some practice to digest everything and use the Google docs to make tests for the students and get their test results. Students will need to have access to computers and a Google account to use Google docs.

Jun 28th AM

Today, we looking at the binding force between an antibody and an antigen. To do this, the cantilever tip is covalently bound to an antibody using amino groups. The antigen is fixed and covalently bound to the glass-slide. The tip with the antibody is lowered onto the surface of the slide and the binding force between the antibody and the antigen can be measured. The cantilever was put in oscilating mode. As the cantilever tip is being pulled from the surface, the antibody molecule unfolds and the interaction forces between the antibody and antigen is recorded on a graph. A computer program would calculate the interaction forces which is in pN. Very specific binding would take a larger force to pull antigen apart from antibody. Non-specific binding would require less force to separate antigen from antibody. Sometimes we see much more forces and a jagged recording on the graph. This may be due to multiple antigens binding to the antibody. Proteins denature after 2 hrs. and a new sample needs to be used after 2 hours.

June 25th PM

Project idea for curriculum.
I think I can have students make a model AFM on a macroscale to demonstrate the optics of the AFM and to use it to measure different shapes objects for a geometry class. The model cantilever can be a ruler with a nail hammered onto one end as the tip. An 1 square inch mirror can be affixed to the end with the nail. A laser pointer can be positioned to point onto the mirror. The reflected beam can be projected onto a white vertical cardboard. To calibrate the model cantilever, I would put objects of known heights under the nail and measure the vertical displacement of the reflected beam from its zero height position. Students can then plot a graph of laser deflection in mm versus object height in mm with those measurements in vertical displacements. Students can draw a line of best fit on the graph. Different shapes objects could be glued to a tile that can be moved in the x and y-axis. The students can scan the different shapes in the x-axis and plot the graph to show calculated height versus scan position. Students can scan the same object in the y-axis. Based on the graphs of the x-axis and y-axis, the students can visualized the shape of the object. The scanning will be done in the contact mode.

June 25th AM

Today, we measure the adhesion properties of fibrinogen in order to study its effects on thrombus formation. A HEK293 cell(human embryonic kidney) was placed under the cantilever end. The HEK cell was placed on the cantilever end instead of a tip. The cell contains integrin proteins on its membrane which causes the cell to adhere to surfaces. The glass-slide was incubated with a thin layer of fibrinogen molecules. The cantilever was lowered onto the surface until the cell touches the surface. Adhesion of the cell to the fibrinogen surface occurs. Next, the cantilever was slowly pulled from the surface with a very small pN force. A graph of the applied force versus time was plotted on the computer to indicate the force necessary to pull the cell away from the fibrinogen surface. In previous experiments, different surfaces were used and a small microliter drop of cells were incubated with the surface. Excess cells were rinsed off to see how many cells adhere to the different surfaces. In this experiment the actual force needed to pull apart the cell from the surface was measured.

Jun 24th PM

We went to visit Dr. Culbertson's lab. The goal was to find out what our samples of objects are made of using PIXE (proton induced X-ray emission). A beam of protons is accelerated onto each sample that is placed in a vacuum. The proton hits an electron, the electron gets excited and jump to a higher energy shell. As the electron jumps back to a lower energy shell, it can emit X-rays. The X-ray emission spectrum is different for each element and we can identify the elements in each sample based on the emission spectrum of the sample. Samples of a bracelet, ring, pearls, silver dollars and pen cap were evaluated. The bracelet was found to contain Ca and P and we concluded that it was made of bones which is mainly Ca3(PO4)2. The 1929 silver dollar has real Ag in it but the 1971 silver dollar contains Ni and no Ag. The pearls showed a lot of Ca which is the material of pearls. The pen cap was from a gold cross pen and it contains real gold atoms as seen in the emission spectrum.

revised concept map

June 24th

Today, we learn how to make DNA slides, how to focus the AFM, and how to scan the DNA samples using an oscillating mode and a contact mode for the cantilever tip. DNA samples in TAE buffer were mounted onto mica sheets put on a cover-glass. The top layer of the mica was removed using scotch-tape to ensure a smooth surface to put the DNA sample in. The sample was incubated by covering the slide with a glass cover for 5 mins. The mica slide was rinsed in ethanol and dried by blowing N2 onto the slide. The slide was mounted onto the stage of the AFM and the red laser beam was adjusted to beam onto the cantilever end that has the Si tip. The DNA sample was first scanned using the tapping mode. No good picture was seen. Next, we decide to scan the DNA sample in the contact mode. The cantilever tip has to be changed to a softer tip made of silicon nitride in order for the tip not to break when it contacts the surface. The force applied was in the picoNewton level. We got a good picture of short DNA sticks which was the desired results. They look like individual E. coli bacterium seen under the electron microscope.

Jun 23rd

Today, we prepare a monolayer of ATTO molecules to be scanned with the cantilever tip touching the surface of the sample. The goal is to find a molecule that shows quenching when the tip touches the molecule. In order to demonstrate this effect, we need to remove the SiO layer that is covering the tip. The SiO layer would block the transfer of electrons to the Si tip, thereby inhibiting quenching. To remove the SiO layer, a HF & NH4F solution was used. The cantilever was put into the HF solution for 1 minute to remove the SiO layer. Used harmful HF solution was remove by pipeting it into excess CaCl2 solution. A white precipitate of harmless CaF2 was produced. The sample was scanned with a red laser beam to visualise the fluorescent molecules. Next a molecule was picked and a smaller area around the molecule was re-scan to look for quenching effect. We picked several molecules to be re-scan. The first few molecules did not show quenching. Finally,we found a molecule that demonstrated a small quenching when the cantilever tip touches the molecule.

Jun 22nd

Today we measure the fuorescence lifetime of a fuorescent dye which is an organic molecule called ATTO 655. We use a setup consisting of 2 microscope, an atomic force microscope and a confocal fluorescence lifetime microscope. We prepare the molecules of ATTO 655 by taking 5 microliters of diluted dye and 3 microliters of pure water. We mixed the 2 drops of liquids and spreaded them on a coverslip in the laminar flowhood. We left the dye molecules to spread out into individual molecules on the coverslip for 10 minutes. Meanwhile we calibrated the 2 microscopes to send out pulses of red laser beams. The laser beam excites the ATTO molecules and the molecules fluoresce as a result of the excitation. We scan the coverslip to show the individual ATTO molecules. Next, we pick an ATTO molecule and beam a laser pulse onto the molecule. The molecule gets excited and fluoresce for a certain duration of time. The duration of time that the ATTO molecule fluoresce is called the fluorescence lifetime. We picked about 10 different ATTO molecules and measured their different fluorescence lifetimes.

Mon 21st

I visited Robert Ros lab and looked at the optical microscope that uses laser beams to scan the surfaces and measures molecules in the nanometer scale. Tomorrow, I will be looking at the surface of cancer cells and looking at how adhesive the cancer cells are. I will be learning how to calibrate the expensive laser beams optical microscope , how to handle cell culture and how to use the laminar flow hood to not contaiminate myself or the cell cultures. I will be choosing my project and I would like to work on the folding of DNA into various shapes like a triangle , a box using different hybrid strands. That will be very interesting to use in a geometry curriculum.

Mon 21st

My email is clove700@msn.com.
My cell phone is 928-380-8756.
My message phone 256-498-3063.
I am in Robert Ros lab with Adrian.