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June 17th, 2013 by Lauren

Experiments, experiments, experiments

This last week has been a flurry of experiments. Professor Meyer’s experience with live imaging is limited, so I’ve been conducting a variety of logistical experiments to figure out ideal imaging conditions.

For instance, I’ve been trying to find the best conditions for incubating the embryos in the fluorescent Hoechst dye. The better the dye is absorbed by the cells during incubation, the brighter and clearer the fluorescent images will be. Since we’ve been experiencing difficulties with the dye fading too rapidly (making multi-hour movies impossible), I’ve also been trying to find the conditions under which the dye will last the longest. Better absorption and longer fluorescent will enable us to take longer, more informative movies.

Thus far, I’ve experimented with exposure time, incubation temperature, and cell permeability.

Early last week, I conducted a simple multivariate incubation experiment. Embryos were collected and placed in six separate dishes. Three dishes were incubated in the Hoechst solution at 16C (~61F), while the other three dishes were incubated at room temperature (21C, 70F). In each of the temperature groups, one dish was incubated in the dye for 1 hour, one dish was incubated for 2 hours, and one dish was incubated for 4 hours.

At the end of each dish’s incubation period, we took time-zero images of the embryos. This was just to compare the initial brightness of the Hoechst. There was a very noticeable difference in the initial brightness of the dye between groups. All the embryos incubated at room temperature had brighter fluorescence than the embryos incubated in the cooler refrigerator, regardless of incubation time. This would seem to suggest that higher temperatures, at least within the tested range, lead to better uptake of the dye.

Below, on the left, is a time-zero fluorescent image of an embryo incubated in Hoechst for 1 hour at 16C. On the right is a time-zero image of an embryo incubated in Hoechst for 1 hour at 21C. The bright green dots are cell nuclei where the dye has been absorbed. Note the difference in the brightness between the two images.

Time-zero fluorescent image of an embryo incubated in Hoechst for 1 hour at 16C

After 1 hour at 16C

der

After 1 hour at 21C

 

Longer exposure time had mixed results… The 2-hour groups were brighter than the 1-hour groups, but the 4-hour groups were about the same, if not dimmer in some cases, compared to the 2-hour groups. These results were boggling, so we repeated a simplified version of the experiment. We compared two groups: embryos were incubated in the dye at room temperature for either 1 hour or 4 hours. This time, the 4-hour group did appear to be noticeably brighter.

I also experimented with increasing the embryos’ permeability, or the ease with which molecules can pass through the cells’ membranes. Higher permeability means that more molecules (e.g. more of the dye) can more easily be absorbed into the cell. A group of embryos were briefly exposed to a solution of sodium citrate sucrose, which softens the egg envelope and increases permeability. The sodium-citrate-sucrose-treated embryos were then incubated in the Hoechst dye and imaged. Compared to a control group (only incubated in Hoechst, with no exposure to the sodium citrate sucrose), the treated embryos showed significantly brighter fluorescence. The dye also remained brighter and more visible for the duration of the multi-hour movie. Yes! Success at last!

This treatment may be the key to creating ideal dye brightness for our movies! The downside is that the treated embryos are extremely tricky to work with; their softened egg envelopes become sticky and very vulnerable to damage. I accidentally ‘retired’ an entire dish of treated embryos when I attempted to pipette them into a fresh dish. Every single embryo got stuck to the inside of the pipette tip and wouldn’t budge. When I tried to shake them loose by pumping water in and out of the pipette, the embryos all lysed (their cells burst open and the organisms were instantly killed). …Oops.

Fortunately (or unfortunately…?), despite my remarkably quick and efficient manslaughter of innocent worm babies, this blunder wasn’t a particularly novel mishap. Worm death is not an uncommon tragedy in the annelid lab. The vast majority of the worms born in the lab are euthanized in the pursuit of scientific research*. On the bright side, our lab still boasts a far lower mortality rate than the worms’ natural habitat. It isn’t perfect, but it’s a fair sight better than the salt marshes. (I’m sure the worms would tell you so themselves, but they’re too busy bumming around in cozy, smelly mud.)

Til next time,

- Lauren

(*As an important disclaimer, all worms are treated humanely and ethically, in living and in death.)

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