Part 2: Microscopy
When observing your cells under fluorescence excitation, you should work with the room lights off for best results. A member of the teaching faculty will be with you to help you make the most of your 20-25 minutes.
- Prior to the first group using the microscope, the teaching faculty will turn on the microscope and allow it to warm up for 15-20 min. First, on the mercury lamp that is next to the microscope, the ‘POWER’ switch will be flipped. Next, the ‘Ignition’ button will be held down for about a second, then released.
- When you arrive, the lamp ready and power indicators should both be lit – talk to the teaching faculty if this is not the case.
- Place your first sample slide on the microscope, coverslip-side up, by pulling away the left side of the metal sample holder for a moment.
- Begin your observations with the 10X objective.
- Turn on the illumination using the button at the bottom left of the microscope body (on the right-hand side is a light intensity slider).
- Next, turn the excitation light slider at the top of the microscope to ‘DIA-ILL’ (position 4).
- Try to focus your sample. However, be aware that the contrast is not great for your cells, and you might not be able to focus unless you find a piece of debris. Whether or not you find focus, after a minute or two, switch over to fluorescence. Your cells will be easier to find this way.
- First, turn the white light illumination off.
- Next, move the excitation slider to ‘FITC’ (position 3). You should see a blue light coming from the bottom part of the microscope.
- This light can excite both the green and the red dye in the viability kit, and the associated filter allows you to view both colors at once.
- Finally, you must slide the light shield (labeled ‘SHUTTER’) to the right to unblock it. Now you can look in the microscope, and use the coarse focus to find your cells (which should primarily be bright green), then the fine focus to get a clearer view.
- You can also switch the excitation slider over to ‘EthD-1’ (position 2) to see only the red-stained cells. Some of your cells may appear to be dimly red, but the dead ones are usually obviously/brightly stained.
- Be aware that the dyes do fade upon prolonged exposure to the excitation light, so don’t stay in one place too long, and when you are not actively looking in the microscope, slide the light shield back into place.
- You can try looking at your cells with the 40X objective as well if you have time. As you move between objectives and samples, choose a few representative fields to take pictures of. As a minimal data set, try to get 3 fields at 10X of both of your samples.
- To take a picture, remove one eyepiece from the microscope, and replace it with the camera adaptor. Be sure to keep the light shield in place until you are ready to take the picture (to avoid photobleaching)!
- Note that 10X images will reveal a broader field, but 40X images may have better contrast.
- Check with the teaching faculty if you are having difficulty getting clear pictures.
- Later in the module, you will compare the average cell numbers in each sample using the statistical methods we discussed during Module 2.
- Post two well-captioned pictures to the wiki before leaving (one of each sample), so we can discuss the class data in our next lecture. Be sure to note whether the image is at the surface or core of the bead.
- If you are one of the last two groups to use the microscope, you may post your data within 24 hours instead.