Update on the Status of my Research
For the first few weeks, I had major problems seeing my fluorescence in the confocal microscope. The microscope isn’t supposed to work miracles; you have to be able to see the image through the eyepieces before you take a picture of the image. My problem was that the GFP (green) and tomato (red) fluorescence was dull so I couldn’t really see my cells. The only thing I could see was the Hoechst nuclear stain (blue). I learned that it is quite difficult to image and it takes much longer than it is supposed to when the fluorescence is dull. It feels like you’re looking for something that isn’t there; thus, it was difficult to even find the dorsal lateral, dorsal medial, and ventral parts of the striatum quickly. Another thing I had to check for was bleedthrough in the channels. I had to make sure that the blue channel was not bleeding through to the red and the green and the same for the other channels. I did not want to get false positive signals and analyze them incorrectly.
This is when I truly learned that science research goes quite slowly because of all the unexpected issues and troubleshooting that needs to be done. My mentor and I brainstormed about what sorts of issues could have caused this decreased fluorescence (which used to be quite bright). The main issue was whether it was a problem in the fixation/processing of the tissue or if it was a problem with the biology in the animal. We headed to the epifluorescence microscope to take a look at our samples. One of the guys in the lab upstairs looked at our slides and mentioned that it seemed as if our slices were uneven- thinner at the top and thicker at the bottom. The postdoc I’m working with said that even if the fluorescent signal is dull, the cells should still look healthy; instead, our cells looked unhealthy, prompting him to think that it was a slicing issue. It occurred to us that our vibratome was having issues and was significantly affecting people’s results. As soon as we got it fixed, our fluorescence has been much brighter!
I have gone back to the confocal to image the new slices. However, the Zeiss 410 confocal that I have been using is becoming obsolete because there is a new confocal, the Zeiss 510. The Zeiss 410 confocal scans a little off- the top of my blue channel for my nuclear stain is bright but it dims as it continues scanning to the bottom, so I have a hard time adjusting the brightness and contrast to an appropriate level. Thus, I will be training to use the Zeiss 510 on Monday (the earliest training date available because they are backed up with all the new users). Then, I will need to count my cells and look at colocalization.
I definitely have my plate full for the next two weeks. I know there will be many hours spent in front of the confocal. In addition, I have been learning to clip the toes of my mice and genotype them for my project.
The Zeiss 510 confocal that I will be using shortly.