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.