>Given that the delay between a cell passing through laser A and then laser >B is in the milliseconds range won't the fluorochromes excited in the first >beam still be fluorescing to a certain extent when they pass through the >second ? The delay is microseconds to tens of microseconds, not milliseconds. The fluorescence lifetimes of fluorochromes commonly used in flow cytometry are nanoseconds to tens of nanoseconds, so the fluorochromes will not be fluorescing when they pass through the second beam. >I may be talking rubbish, but when doing fluorescence microscopy and you >turn the source off, the "glow" doesn't disappear immediately. >Maybe someone more into the physics of flow could answer this. > Given the facts stated above about fluorescence lifetimes, some of the "glow" could be due to phosphorescence, which has a much longer lifetime than fluorescence. The illumination times in fluorescence microscopy are very long (seconds or more) compared to the few microseconds a cell spends in the beam of a flow cytometer, and many of the fluorescent molecules in a sample may be bleached while the cell is under the microscope. Bleaching, as Tomas Hirschfeld noted years ago, preferentially removes the molecules with higher quantum efficiencies. Therefore, although phosphorescence is generally a lower probability event than fluorescence, as you leave the illumination on for longer and longer times, the proportion of phosphorescent molecules will increase. The red "fluorescence" of acridine orange probably includes a substantial amount of phosphorescence, and some fairly strange luminescence effects have been observed in phycobiliproteins by people in the Herzenberg lab at Stanford, although I don't think they've published on the subject. Then, of course, there's also some persistence of vision on neurologic rather than quantum mechanical grounds, which could also explain some of the lingering glow. -Howard
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