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Tumor cells are transfected with the firefly gene, which becomes part of the cells as they divide and grow, and then implanted on the mouse. The technique requires a substrate called luciferin to be added to the bloodstream. (Luciferins are a class of small-molecule substrates for their corresponding protein enzyme, luciferase. Luciferins are oxidized in the presence of luciferase to produce oxyluciferin and energy in the form of lightóbioluminescence.) It's carried to cells throughout the body and when it reaches those that have been altered to carry the firefly gene, those cells emit light. Some cancer drugs work by cutting off the blood supply to tumor cells. The researchers wanted to determine whether BLI techniques could be used to gauge the effectiveness of drugs that destroy blood vessels that feed tumors.
"Delivery of the substrate allows us to monitor the blood supply," Mason explains. "This is our contribution [to cancer research]. We had been working on vascular targeting agents. If you really want to kill a tumor, why not cut out the blood supply? It can be quite easy to destroy fragile tumors in mature vessels with no damage to healthy tissue."
Many drugs, Mason says, kill 99 percent of a tumor, but allow a few cells to survive, giving the tumor an opportunity to grow back. That's why it's critical to continue developing new chemotherapy drugs.
How effectively the luciferin reaches the tumor depends upon how the substrate is injected. "The luciferin crosses the blood-brain barrier and the placenta barrier. The kinetics may depend on how you administer it," Mason says. "Intravenously, it goes rapidly through the blood. It's not trivial. If you're two minutes too late, you may miss peak amplitude [of the light]. If you inject it peritoneally, it will go into the intestines and to the tumor. It will maintain longer. After 20 or 30 minutes the light goes away. We found that subcutaneous in the neck region diffuses into the body and goes to the tumor. Ninety-nine out of 100 times, we see what we expect. It's more robust and the light lasts as long as intra-peritoneal."
In the lab, a tumor is implanted on a mouse. When it reaches a diameter of about 10 millimeters, luciferin is administered and a BLI time course is acquired over about 30 minutes using a sensitive CCD camera. The drug to be tested is then administered and BLI repeated following administration of fresh luciferin three hours later. Measurements also are repeated regularly.