Good evening! Are you ready for the heat? Remember to drink plenty of fluids (water, not sugary soda!!!) and take breaks if you are running around.
First task up this morning was to finish taking photos of the fluorescence images under the microscope. Since yesterday was my first time doing it, there was a learning curve to the technique and what I should be looking for. In addition, I needed to photograph the images using two different filters to be able to see the two types of stains used, one to stain the nucleus and one to stain the cytoplasm.
In order to understand how the fluorescence imaging works, you need to understand how we see colors (this should be review of 6th grade science). The musical group They Might be Giants wrote a song about the color spectrum: The Electromagnetic Spectrum, check it out.
Waves are organized by their wavelength and energy on the electromagnetic spectrum. Visible light is a very thin band of wavelengths on the electromagnetic spectrum between 400 and 700 nanometers (nm). Wavelengths with higher energy have shorter wavelengths and wavelengths with lower energy have longer wavelengths. Here’s a great website from NASA
Back to the fluorescence images… We used the same microscope we used to check the growth of our cells: But instead of using a regular bulb emitting visible light, we used a mercury bulb: and it gives of a green glow: In addition, there are four different filters which can change which wavelengths of light pass through to the sample. These filters limit which wavelengths of light can pass through to the sample. Remember that I stained the cells with two stains? These stains then absorbs the shorter wavelengths (called excitation wavelength) allowed to pass through the filter and and emits or allow the longer wavelength (called emission wavelength) to pass to the sample. These longer wavelengths are the ones that allow for the different colors in our sample. Check out this site for a more detailed description and diagram: http://cse.lmu.edu/resources/MANE_Labs/Instruments/Epi-fluorescence_Microscope__EFM_.htm
So now that I understood the basics about his this imaging worked, it was time to take some more photos of the cells on the scaffold: Using the filter that allowed red light to be transmitted through my sample I took some images of the cytoplasm of the cells on the scaffold at 10x magnification: and then I switched to the filter that allowed green light to pass through the same sample to capture the nuclei stained green: Notice the photos are in black and white? That is because the camera I used only captures the differences in intensities of the sample. This is what the image looks like on the computer screen: Once I combine the two images I can then have the computer add color to show the cytoplasm as red and the nuclei as green like this: Pretty cool huh?
But that was only part of my work today. The rest of my time was spent on seeding the next type of cells on a different plate of scaffolds. This time instead of using mouse bone marrow stem cells, I used human bone marrow stem cells! I followed the same procedures I used to plate the mouse cells (remember that we want to keep everything the same between experiments). The only things that changed was the type of cells used and the media used to grow the cells. Since human cells produce and require different proteins than a mouse, we needed to change some of the nutrients used in the media.
In both of these experiments we will be measuring the amount of cell growth at certain periods of time, day 1 and day 7. Since we allow the time to change in our experiment, it is called the independent variable. Since we do not know the amount of cells grown, this is the factor we are measuring and collecting data on, this is the dependent variable. The dependent variable (the amount of cells) DEPENDS on the independent variable, time. One of the ways we will analyze our data from these experiments will be by graphing our results just like we do in class. Which variable is the x-axis and which variable is the y-axis? Remember y depends on x… We’ll get into more of this next week.
Next up, changing the media of the mouse cells and fixing, staining, and fluorescence imaging the human cells.