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If you look at a slide of cells stained with a fluorescent dye under a fluorescence microscope, you are likely to notice that, each time you move to a new field of view, the fluorescence from the cells in the new field is more intense than the fluorescence from the field that you had been looking at immediately before, which has undergone some photobleaching. This effect makes it difficult to get precise quantitative measurements of fluorescence intensity from cells in a static or scanning cytometer if you have to find the cells by visual observation before making the measurement, because the extent of photobleaching prior to the measurement will differ from cell to cell. In a flow cytometer, each cell is exposed to excitation light only for the brief period during which it passes through the illuminating beam, usually a few microseconds, and the flow velocity is typically nearly constant for all the cells examined. These uniform conditions of measurement make it relatively easy to attain high precision, meaning that one can expect nearly equal measurement values for cells containing equal amounts of fluorescent material; this is especially desirable for such applications as DNA content analysis of tumors, in which the abnormal cells’ DNA content may differ by only a few percent from that of normal stromal cells.

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