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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). Ova tehnika ima nekoliko prednosti nad standardnom autoradiography, ali najvažnije je da je mnogo točniji quantifying u iznosu od radioaktivnosti u tvar. To je zbog svojih odgovor na radioaktivnost je daleko više linearno, nego da je x-ray film. Uz standardne autoradiography, band sa 50000 radioaktivni disintegrations u minuti (dpm) svibanj izgleda ne tamniji od jednoga sa 10000 dpm jer emulzija u film je već zasićena i 10.000 dpm. Ali radioaktivno emisija phosphorimager prikuplja i analizira ih elektroničkim putem, pa je dpm razlika između 10000 i 50000 dpm bi biti očito. Ovdje je kako se radi tehnika: Mi početi s radioaktivnim sample-a izbrisat s DNA bendova koji su hybridized s označenom kao sonda, na primjer. ovom smo mjestu uzorak u kontakt s phosphorimager plate, koji apsorbira B-zrake. Ove zrake nadražiti molekula na tanjuru, i ove molekule ostaju u pobuđeno drzave do phosphoimager skenira plate sa laserom. U tom trenutku, B-ray energy zarobljeni na plate je izdana i pratio je kompjuterizirana detektor. računalo pretvara u energiju se otkrije da jedna slika. Ovo je lažna slika u boji, gdje je drugačije boje predstavljaju različite od radioaktivnosti, od najnižih (žuta) do najviše (crna).