new toy   Leave a comment

The cells have been mostly ok- I lost one dish to bacteria, and another contracted a case of mold, which I’m treating with fungizone.

In the meantime, while the cells are doing their thing, I’ve updated my fluorescence setup.

Fluorescence is used as an imaging technique in my lab- basically, I can ‘paint’ specific molecules a certain color, and then use the microscope optics to only image those molecules. As an example, DAPI (“dap-ee”) binds to the DNA in a cell and thus, by looking only at DAPI, I can clearly see the nucleus of cells. As other examples, I can paint acetylated alpha-tubulin (the protein that makes up the cilia ‘skeleton’), actin (the cytoskeleton), ZO-1 (the junctions between cells), SSTR3 (a molecule thought to hang out on cilia), etc. etc. I should post some images…. The point is, fluorescence imaging lets us image a specific molecule in the cell.

The technique is very standard, and so is widely used in biomedical research. One of the easy pieces of information that is discovered by this technique is where proteins are located in a cell- many proteins can move from the membrane to the nucleus, and this is evidence that the cell is “doing something”. It’s possible to watch proteins being made-and once made, get moved around to their final destination- Jennifer Lippincott-Schwartz has taken some amazing videos.

But, there are some limitations. For one, there’s a limited ‘color palate’. That is, if one color is too close to another color (say green-blue and blue-green), then there’s no way to distinguish the two colors in the images, meaning I can’t tell which molecule is which. It’s fairly easy to use three colors- red, green, blue- and not much more effort is needed to squeeze in a fourth- either deep blue or deep red- meaning that usually we can only track 3 or 4 molecules at a time. It would be nice to watch 10 or 20!

The companies that make the color filters (there aren’t that many) have been making the filters better and better over time, and I just got a new filter that allows me to image 5 different colors. That means I need only 1 filter to look at 5 distinct colors: deep blue, blue, green, red, and deep red, and I can look at them all *simultaneously*.

There’s another advantage to this filter (a quad Sedat filter), in that I removed 3 filters that were occupying space in the microscope. So, not only can I image more colors than I could before, but I have additional space (which I filled already- yellow, Fura-2, and a laser tweezer cube) that lets me perform additional functions.

I’ll have to post some images… I’ll sacrifice some cells tomorrow or Friday and stain them.

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Posted September 29, 2010 by resnicklab in Physics, Physiology, Science

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