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Phosphorimaging

Phosphorimaging

This technique has several advantages over standard autoradiography, but the most important is that it is much more accurate in quantifying the amount of radioactivity in a substance.  This is because its response to radioactivity is far more linear than that of an x-ray film.  With standard autoradiography, a band with 50,000 radioactive disintegrations per minute (dpm) may look no darker than one with 10,000 dpm because the emulsion in the film is already saturated at 10,000 dpm.  But the phosphorimager collects radioactive emissions and analyzes them electronically, so the difference between 10,000 dpm and 50,000 dpm would be obvious.  Here is how the technique works:  We start with a radioactive sample- a blot with DNA bands that have hybridized with a labeled probe, for example.  We place this sample in contact with a phosphorimager plate, which absorbs B-rays.  These rays excite molecules on the plate, and these molecules remain in an excited state until the phosphoimager scans the plate with a laser.  At that point, the B-ray energy trapped by the plate is released and monitored by a computerized detector.  The computer converts the energy it detects to an image.  This is a false color image, where the different colors represent different of radioactivity, from the lowest (yellow) to the highest (black).

 

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