Science!

Today was very productive, in that I Learned A TON!

I worked with Rachid, mainly in a dark lab putting together slides or diluting solutions. But right at the end I got to do a tiny bit of analysis too.

So, first steps upon entering the lab. Turn on the water, the camera, and the switch for the laser. But keep the laser shuttered. Put the microscope on eye mode. Then prepare your DNA or Proteins. Generally when you make them, you make enough to last several days so often this step can be skipped. Today we made a DNA sample first by diluting DNA with water into 8 different containers. Afterwards we made the protein samples. We diluted the protein with a buffer that we also prepared. The buffer came from a mix of HEMES, NaCl, and water. One of each sample went into our ice bucket and the rest went into the freezer. Its important to keep each dark because the florescent molecules that are attached to the dna and the proteins have a two second lifetime under light.

Then we took some clean circular slides and locked them in between two metal circular objects. its important to center them first on one and to never touch the top side. We use a delicate pair of tweezers. Later in the day I tried to pick up one of these slides with the tweezers and squeezed to hard so it broke right there. I was more afraid of breaking them when screwing them in between the circular objects since it has to be tight so the substrate wont leak onto the microscope and change youre refraction angle and thus obscure your results.

Now in a different solution entirely there are quantum dots. So first put a small amount of these onto one of the slides you prepared (we could do up to five at a time) or onto a regular rectangular slide. Place it delicately on the microscope and lock it into place. Now switch the microscope to camera mode and make sure you notice it start on the computer. Now unshutter the laser. Initially it is probably going straight up and through the substrate, which means you are only performing epi-fluorescent microscopy. Move the platform that the final mirror that reflects the laser into the microscope is on. As you move it the light going through the substance changes angle until it is at 90 degrees and the critical angle is obtained. Now you are achieving Total Internal Relflection Fluorescent Microscopy!

The quantum dots are unaffected by the amount of light they have recieved so you should be able to fine tune your microscope until you see a thin layer of them blinking. Once you have acheived that you know that the microscope and camera are both working normally. Now test the Dna by using a pipette to drop a small amount onto the same coverslip but in a different area. move the slip so this drop is focused over the lens and make sure you can focus on the DNA and that there is an appropriate concentration.

Now you drip a little bit of DNA onto one of the circle slide, I think it was 50 nL? Let it sit for two inutes in the dark so that the DNA attach to the surface of the glass. Then wash it out three to four times with this special vacuum they have. Then place it into the groove over the lens. Before putting anything on top of the lens always! always! always! put a little bit of this special oil on to it first so that the refraction angle will be the same and to protect it. If you can find a good spot of dna on the camera focus in on it. Then turn the laser off, move the slide a bit to a new spot without dna that have already lost their fluorescence from the laser. Now start recording a video, turn on the laser, focus the microscope in on the DNA points, and wait for them to appear and fade. When all of them have faded you can move in to the next part. If there are too many of them you can flash the cover slip so that is fully dark again. Now drop an appropriate amount of the protein solution on to the slide. and watch as they glow. After most of them are gone stop the video, turn off the laser, and remove the slide. re-cover the microscope. Save the video to a .tif format. and voila!

Tomorrow: All the things that can go wrong!

Premier Jour!

So today we made it to the labs! I met Dr. Cedric Bouzigues and a graduate student, Rachid, with whom I'll be working all summer. My project is basically the current stage of his Thesis. Dr. Bouzigues took me on a quick tour of the facility, focusing on the room my desk is in and the lab the TIRF microscope is in. I worked with Rachid in the lab and already got to work with the microscope . I was trying to focus it on a line drawn on a cover slip first with 20X which was easy and then 100X which was trickier. Rachid says it will take some practice to get a feel for when to move closer or further away from the slide with the scope. Then as we discussed what was actually going to be done, I discovered that my project was quite a bit different from what I originally imagined which was probably part of the point of our whole presentations. However all the background research that I did was definitely helpful! I was able to answer a lot of questions correctly such as why we needed a liquid on the slides (so the refractive index is the same as the cover slip, the refractive index of the air is too different), knew that the slide was lighted from below by a laser, understood the overall objective of the project (to study how proteins repair broken dna), what gain is, and a couple other things I do not remember right now.

I will be spending my time this summer split between two general activities, taking data and analyzing it, for each stage. For taking data I will be preparing slides and then using the camera to take pictures and videos of the interactions between dna and proteins. I can not actually see the dna or the proteins but rather if either one is interacting they light up the entire pixel they are in. One of the sources of error in this project is the fact that both of these molecules are significantly smaller than the pixel itself so its not very well localized. Then when I analyze, I will be using matlab programs that Rachid has written to watch the pixels that seem to be dna and determine how many possible interactions each has per trial and how long these interactions last. When you first select the dna spot to study you determine its precise location with a matlab program. This is also within a margin of error because of the fact that the dna is smaller than a pixel but you obviously cant see smaller than a pixel and another error arises for this calculation because the slide is always moving a little bit from natural oscillations. One of the possible solutions to this problem would be to use quantum dots as markers for the slide and then measuring the relative positions of the dna and proteins to those quantum dots. Quantum dots are like the other molecules that have been dyed fluorescent but unlike those, their fluorescence doesn't ever die (the fluorescence of the dna and proteins disappears after an average of two seconds because of interactions with air molecules) because they are composed of inorganic materials.

To begin this summer my project is too optimize the surfaces to produce the most dna and protein interactions. To achieve this we are going to try covering the surface with a thin layer of proteins. The other proteins will not be able to interact with these proteins and so will hopefully be attracted more often to the broken dna "flaps". I am not yet sure how we cover a slide with proteins but I did get to watch Rachid prepare a slide today and place it on the microscope.

After we achieve this, I will be working with Rachid on studying the length of time interactions take to occur. The resulting average could help distinguish the strength of the current contradictory theories for the proteins behavior.

A whirlwind tour of Paris

One of the famous living statues of Paris

So yesterday after my favorite international flight ever, we arrived in Paris! After reaching the hotel our new ENSTA friends, Charles, Antoine, Neda, et Rania showed us all over Paris. We saw Le Tour Eiffel, L'Arc de Triomphe, Le Louvre, Notre Dame, et Le Champs-Elysses. But we didn't officially go into anything so there will still be plenty to explore in the coming weeks. Along the way we saw a lot of street performers including one of the Living Statues which was really cool also. I have never spent significant time in such a large city and am really enjoying the atmosphere. I am sure we will have a lot of adventures exploring the city further.

Tomorrow we are all meeting with our advisors and begin our main adventure. I am so ready to begin work on my project. I am sure at the beginning will be an adjustment period where I am asking a ton of questions and reading more papers in order to understand what exactly is going on. I am looking forward to perhaps a week from now when things have settled into more of a routine. Right now I am envisaging it being something like running or swimming in the morning, traveling to L'Ecole Polytechnique, working hard all day, traveling back, blogging and taking a small break, having dinner somewhere new with the other students or cooking something in the dorms, then studying for the Physics GRE for 1-2 hours, and then all hanging out or just relaxing, until we repeat it again. Weekends will probably be split between visits to other parts of France (a vineyard is high on my list!) or Europe, and exploring Paris in depth. I am sure the schedule will vary as we become absorbed in our projects and find we want to stay later some days to run a few more tests or something and other unprecedented events but I hope to study for the Physics GRE at least 4 times every week and work out in some fashion at least 3 times every week.

à tout à l'heure!

My Project Expecations Part Deux

Dna in an average human cell breaks 10 times per day. Natural proteins repair this damage and in general keep your genome intact. When mistakes are madem cell mutations can occur leading to serious problems such as cancer.

The various proteins that work to maintain your DNA in all of its proper coding are known but are not very well understood. Using TIRF microscopy this project is going to investigate the behavior of some of these proteins.

Figure 3: Mre11-2 and Mre11-1 repair broken DNA.

In the image above you can see the protein Mre11-1 acting to repair a strand of broken DNA. Mre11-1 is part of the MRN complex.

I will be working with the reparation protein NucS and observing its interactions with a free single strand of DNA. This is analogous to the types of DNA breaks that are seen in nature. In my case the protein will be fluorescently labeled so that we can see it clearly with TIRF. The goal will be to identify and study the mechanisms that NucS uses to repair the DNA.If this goes well we might move on to observing the interactions and cooperation among various proteins in the complex.

My Goals for the Summer:

-Learn how to prepare substrates

-Gain comfort and proficiency performing TIRF microscopy

-Increase independent thought in developing analysis
-Acquire a sense for graduate work abroad