Looking back to the past weeks of working in a laboratory, I cannot believe how much my supervisor managed to train me and guide me through my project. I have learned so many different and most advanced techniques used in cell biology research directly from him and this made Laidlaw summer internship more precious than I have ever expected. One of the most challenging technique of all I have learned was to perform Cryo-Electron Microscopic imaging.
The elements that are involved in Cryo EM starts from culturing the cells properly. Once you defrost the cells that were kept in the liquid nitrogen tank, you must take care of them by regularly changing the media (liquid with nutrients and growth factors that help the cells grow and multiply in numbers), checking that they don’t have fungal infection or dividing them into flasks for more space to grow. For my experiment, I had to treat appropriate chemicals to my cultured cells to differentiate them into a more mature form of neuronal cells. This added up extra bits of care and procedures to be done before actually running any experiments on them.
Once you fix the cells after the experiment, you would scrape them off the dish they were growing on, and spin them down in a centrifuge to make a palette of cells which will be as small as a grain of rice. And this is where things get more challenging. The next step is to load the palette on a metal stub. This sounds quite simple, but you are expected to deal with a very small, slippery and fragile palette of cells and load them in a shape of a mountain on a stub that is barely 2mm wide. This has to be done within approximately 30 seconds, otherwise, the palette will crumble and become like a jam (which cannot be sectioned and thus will be disposed). Once you shaped the palette on the stub correctly, this is to be frozen instantly by going into liquid nitrogen.
If the techniques mentioned above do not sound challenging to you, here comes nanometer level of samples and thousand pounds worth of diamond knives to deal with. The tiny frozen cell palettes are now ready to be sectioned into 70nm thickness. The machine that you will be handling (a cryostat) has two very small diamond knives (one for trimming to make blocks, and the other for actual sectioning). They are so delicate, needless to say, you should never touch the tip of the knives, but you use a stick with an eyelash attached at the tip to handle the sectioned samples. I would have to say I still feel nervous after handling them for weeks. Once you finally start sectioning the blocks, you will have to fight the static, delicacy of the 70nm thickness, the condition of the knives, temperature setting, weather of the day and all sorts of factors that can change the quality of the section. I usually take about 1 to 2 hours to find a perfect combination for the day that would actually lead to proper sectioning. After all this, you need to pick up the section with a small metal loop with less than 10 microliters of liquid blob. By very gently touching the sectioned sample, it will attach to the blob which will then be moved to 2mm diameter copper grid.
The copper grids with sectioned cells on it are then to be washed, labeled with antibody and gold particles, stained and dried. This takes about three hours to complete as long as you do not lose the grids in between each step. Prepared grids are to be imaged in an electron microscope, which the machine itself takes up a whole room and is also, very delicate to control.
Starting from growing cells to finally imaging the results take nearly two weeks for each cycle, and I personally found it slightly stressful that not a single step during the two weeks are any less delicate and challenging than the other. Nonetheless, it is unbelievably satisfying when you image the grids and finally get to see the result of your experiment with your own eyes.