Sparkly nail polish
This past week has primarily been spent conducting further calibration experiments, this time with the equipment itself rather than the experiment parameters. We’ve noticed that the microscope we use for live imaging movies seems to have an issue with focal plane slippage. When we film movies of animals over long periods of time (often a minimum of 1.5 hours), the focal planes seem to slowly slide out of focus. By the end of the movie, almost nothing is in focus, and it’s extremely difficult to observe development over time.
Below are two images of the same animal from a single movie. The first image is one Z-slice at time zero (the beginning of the movie). The second image is the same Z-slice after about an hour and a half (the end of the movie). Notice how the second image is out of focus and the animal seems to be further away from the lens.
Since we were filming live animals suspended in water, we initially assumed this slippage was due to the animals themselves sinking in the water over time, slowly moving away from the microscope lens above them. However, this same issue occurred even when the animals were positioned flush against the glass slide, thereby making it impossible for them to sink any further.
It was therefore concluded that the issue lay with the microscope itself. Perhaps the stage (the platform on which the slides rest) was sinking over time, or the grease & clay on the slides were heating up and melting, or the lens (which shifts up and down to film the Z-stack) wasn’t realigning correctly… or, crazily enough, the air conditioning vent on the ceiling, its breeze barely noticeable to human senses, was actually pushing the stage down on a microscopic level. The possibilities were nearly endless. We needed to solve this mystery before we proceeded with further live imaging movies. Otherwise, all our experiments would continue to be mucked up.
So, obviously, we conducted some experiments with sparkly nail polish.
Sparkly nail polish has the benefit of being inanimate, so we know it won’t move on its own. It also has conveniently suspended particles in clear enamel. Perfect.
We filmed several movies with the nail polish, experimenting with the size of the Z-stack, its depth and location, the thickness of the Z-slices, the length of the movie, whether or not the air vent was covered with a box… In all of these movies, we still observed a significant shift in the focal planes over time. Observe the images below. The nail polish itself it obviously not moving, yet the sparkles get very out of focus over time.
However, on the bright side, the degree of slippage has been dramatically reduced since we’ve updated the microscope software. Previously, we’d see shifts of ~17 micrometers*, a huge shift on the microscopic scale. Now, the overall shift is less than 2 micrometers. It’s now fairly manageable rather than completely detrimental.
The images below are from another nail polish movie, after the software update. In the previous nail polish movie, there was an overall shift of 8-10 micrometers. These two images show a shift of less than 2 micrometers. While still apparent, it’s not nearly as severe.
Unfortunately, the ultimate cause of this problem remains unknown. We weren’t able to draw any solid conclusions about the cause of the focal plane slippage from the nail polish movies. None of the tested factors seemed to have any consistent, noticeable effect. Professor Meyer believes it may simply be due to background vibration caused by people walking around in the lab, an unavoidable complication.
Our microscope mystery now somewhat solved, I’ll be returning to regular experiments. This week I’ll be continuing work on Hoechst concentrations, incubation times, and permeability.
Til next week,
(* 1 micrometer is one-thousandth of a millimeter, or about 0.000039 inches. Needless to say, it’s a very small unit of length. When you’re working with embryos, however, 17 micrometers is huge.)