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June 10th, 2013 by Lauren

What cheese slices and fluorescent microscopes have in common

For the past week I’ve been working on creating live imaging movies in the lab with C. teleta embryos. The first step is soaking the embryos in a fluorescent dye (in our case, Hoechst dye). Hoechst is a nuclear dye, meaning it’s soaked up by the DNA in the cells’ nucleuses. Later on, when we look at the embryos under a fluorescent microscope, the cells’ nucleuses will appear as bright dots inside the organism.

After the embryos have soaked in the dye for a few hours, we plate them on an airtight glass slide to keep them from drying out while we film our movie. The tools of the trade include vacuum grease (to form the seal), modeling clay (to make “feet” for the coverslip), and eyelash brushes. Yes, eyelash brushes are exactly what they sound like: a tiny brush made with a single human eyelash. They’re used to rotate and position the microscopic embryos once they’re on the slide. Professor Meyer described hairloop brushes, another common tool of cellular biologists. Researchers will select a piece of their own hair (hopefully clean!), form a small loop, and secure it to the end of a narrow tool. The taut loop is then used to cut microscopic cells and organisms. I had never realized how weird and creative lab tools can be!

In any case, back to the embryos.

The embryos (now properly plated on a slide) are placed in the fluorescent microscope. The software can be programmed to take a photo of the slide at specific time intervals (in our case, every 15 minutes). Additionally, the microscope can take “Z stacks”, or multiple stacked photos in the Z plane. The Z axis, in the 3D world, is up and down. Therefore Z planes are basically slices, or cross-sections, that stack vertically (like a packet of cheese slice singles). When you take a Z stack, you end up with is a series of images that, together, create a 3D model of an organism. So instead of a single 2D image of the organism at a particular depth (cheese slice #12, from the middle of the package), you get numerous slices covering a range of depths (cheese slices #1-25).

In our case, many of our initial movies had the microscope taking a Z stack of ~20 simultaneous photos every 15 minutes. In tiny C. teleta embryos, our slices were only a few micrometers thick (very very very thin cheese slices).

We imaged the live embryos for about 2 hours. Since the organisms were still alive and developing, the resulting movies allowed us to watch real-time embryonic development, in 3D, with visible nucleuses showing the movement and division of individual cells. That’s some seriously awesome stuff for the first week.

If this sounds too good to be true, that’s because it is. The main problem with our movies is that the Hoechst dye is fading too quickly. Below are fluorescent images from the lab demonstrating this fading. Both images are of the same Z slice, in the embryo, over time. The first image, on the left, is from time zero (the beginning of the imaging movie). The image on the right is from 1.5 hours later. Note the significant decrease in dye brightness.

Embryo at time zero

Embryo at time zero

Embryo after 1.5 hours

Embryo after 1.5 hours

Often the dye is completely bleached (no longer visible) within 30 minutes to an hour. That means we can no longer observe nucleuses, ergo cell movement and division. A maximum of one hour is an extremely small time window and doesn’t allow us to film actual development. This upcoming week, I’ll be conducting a few small scale experiments with the Hoechst dye. My goal is to find ideal conditions for incubating the embryos in the dye. I’ll be trying to find a way to make the dye soak in better or last longer, allowing us to create longer, more useful live imaging movies.

I’ll be mostly experimenting with incubation temperature, exposure time, and embryo stage. Wish me luck!

Til next week!

- Lauren

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