You glow in the dark   Leave a comment

That’s mostly true. Glow-in-the-dark toys use phosphorescence, which a little different than fluorescence. But, living cells can have a lot of auto-fluorescence: naturally occurring chemicals in the cells fluoresce, which can make many microscopy techniques difficult. Some of these chemicals include tryptophan and tyrosine (two amino acids that make up proteins), vitamin B6, NADH, and many others. In our cells, there are lots of mitochondria which fluoresce due to these chemical compounds.

Here is an image of living cells auto-fluorescing a bright green. This is the first image taken of a time-lapse, again to verify that the cells can survive long periods of time under the microscope.

The main difference between this test and the brightfield tests earlier is the method of illumination. When excited, the fluorescent compounds can be broken apart, which over time leads to cell damage. It’s similar to getting a sunburn. The excitation light was turned to it’s minimum setting, and the EMCCD camera captured images with 1-second integration time and high levels of on-chip multiplication. Through the eyepiece, it was impossible to see anything- that’s why we use an EMCCD for live cell imaging.

The cells survived the 5 hour time-lapse (although they were not happy) and recovered overnight. So, by being smarter about turning on the light (for example, only when taking an image and not during the 1-minute interval), the cells can be kept more comfortable.

The next step is Fura-2 imaging. We have had problems getting the dye into the cells, but hopefully with fresh chemicals we can successfully carry out calcium imaging.


Posted September 9, 2010 by resnicklab in Physics, Physiology, Science

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