Microscopy is not for the faint of heart

One last Eccentric Postdoc story before I switch gears. Eccentric Postdoc was really a nice lady, despite her eccentricities, many of which were due to cultural and language barriers. I’m sure we seemed equally weird to her. Especially those of us who appeared to be witches. And Eccentric Postdoc also helped me learn the best way to do immunofluorescent staining of my cell cultures, so I can forgive a lot of eccentricities for that.

Immunofluorescent staining of cells is a fairly exhausting time-intensive experiment. When I first learned the procedure, I was taught a protocol for single-day experiments. This turned out to be a bad way to do it. The staining involved about five hours of washing and incubation, followed by microscopy work. The first time I did the experiment, I, like many before me and many after me, thought that the hard work was done by the time I got to the microscope, and all that was left was snapping a few pictures. Oh, the naivete. Experienced grad students know that microscopy work is actually very difficult. There’s a lot of scanning, adjusting, focusing, and switching of filters that takes much longer than a novice might expect. And our lab had the added challenge of combating the microscope gremlins who came in every night to misalign the lamp and loosen all the screws. Eventually, I learned to allot at least thirty minutes on the microscope per sample, even though I was only trying to get five images from each one. I also learned to make sure I signed up for the scope an hour or two early so that I could realign the lamp and tighten all the screws.

But when Eccentric Postdoc started working in our lab, I had not yet learned how to streamline my microscope experience. So with the single-day staining protocols, I faced hours of microscopy work at the end of a very long day of staining cells. One night, I was in the process of trying to mount my samples to a glass slide for the first time. Up to that point, I had been doing the microscopy on wet samples still in the dishes. With wet samples, after taking the micrographs, there was nothing to do but throw the samples away. Mounted samples, on the other hand, could be saved in the refrigerator for future observation, a much safer scenario. Unfortunately, I was having some difficulty with the mounting gel. I had followed the directions, but in the microscope, the cells looked smushed. I know now that I didn’t let the gel dry for long enough, but at the time I didn’t understand why I couldn’t see anything. Enter Eccentric Postdoc.

Eccentric Postdoc wanted to help. She helped me readjust the lab’s microscope. She helped me re-mount my samples. She brought me over to another lab where she sometimes worked to try their microscope. At first, I was grateful for the assistance, but after about an hour of fruitless searching for decent cells, I was ready to call it a day. Eccentric Postdoc would have none of it.

“Oh, well,” I kept saying. “I guess it didn’t work this time. I’ll try again tomorrow.”

“Nooo,” Eccentric Postdoc intoned. “We will try again.” And then she’d reset the microscope stage with another sample, and I would cry a little inside.

This was a difficult problem to deal with, because she was acting solely on my behalf. It really was in my best interest to acquire usable data from the experiment I had already invested so much time into. But I was also facing what The Doktah and I called, in calculus terms, “a globally bad, locally good” situation. Yes, in the long term, I would be happy to have data. But in the short term, I would have been thrilled to stop working, go home and sleep. I had already been at the lab for ten or so hours, and I really needed a break. But Eccentric Postdoc just would not quit. Those cells were going to be observed, dammit.

Finally, after a lot of wasted time – time in which I appeared to be trying to solve the problem, but was actually thinking, “please let me stop please let me stop please let me stop” – we determined that my samples were not salvageable. I had, as previously noted, not let the gel mount dry, so the cells got smeared across the coverslip when I tried to use the oil objective to observe them. The experiment was a wash, but I did learn a few things. First, I learned give the gel mount at least two hours to set before observing the cells. Second, I learned not to ask Eccentric Postdoc for help if I secretly wanted to quit. And third, Eccentric Postdoc showed me a better way to do the staining that spread the experiment over two days, which gave me one 4-hour followed by one 8-hour day instead of one 10-hour-without-a-break day. A vast improvement.