Nick Terry writes: >IMHO adequate DNA measurements can only be made if the >cross section of your interogating beam is substantially narrower than the >diameter of the objects that you are measuring. > >(Waiting for flames.) > >Under these conditions 2 G1 cells stuck together will have a very similar >integral (yes - mostly the same as area) signal as a true single G2/M cell. >The peak signal from a G2/M cell will however be significantly greater than >that from a G1 cell and from the sequential peak signals from a G1G1 >doublet. But, the latter is only visible if the exciting beam is narrow. >In comparisons between area/width and integral (area)/peak discriminating >ability on an instrument with 5 micron excitation optics the integral peak >analysis gave the best discrimination. It is even possible to recognize >doublets that are not progressing linearly through the beam due to >turbulence or other factors. > >How anyone manages to discriminate doublets from true G2 cells with a 40 >micron beam, or nuclei with a 15-20 micron beam is beyond me. (tactfully). > >(Pondering the likelihood that there is a greater proportion of tetraploid >tumors in the literature than in reality.) Yes, one does need a narrow beam for doublet discrimination, but the most precise DNA measurements are made with arc source systems, notably the Partec, but also Lindmo and Steen's (Skatron, Bruker, Bio-Rad, etc.) design, where the beam is substantially larger than the cells, and the nondirectional illumination is important in getting the precision... Only a warmish breeze; not a flame, I hope. -Howard
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