On our MoFlo, the first pathway (488nm) has 5 PMT holders for SSC and 4 FL channels. The third pathway (633nm) has 2 PMT holders. In trying to ensure I am starting with a properly aligned machine, I use spherotech 5 peak beads. When I use a 670/40 or 670/30 filter to align the system to look at Cy5 or PE-Cy5, I see mixed results. 1.) With the 670/40 or 670/30 in the last position of either pathway, a straight path, I am having great difficulty seeing more than 2 clear peaks. The 3 lowest peaks are not resolved at all. 2.) When I insert a 710DRLP and put the 670/40 or 670/30 in position to receive reflected light from the dichroic I get beautiful resolution of all peaks through the 670/40 or 670/30. The light passed on by the dichroic is then filtered through a 740LP and sent to the last detector in either pathway, again this resolution is poor (I get the 2 higher peaks broad and the lowest 3 peaks clumped together). I have adjust the PMTs voltage and physical position in the holders, switched the PMTs, adjusted the lasers, the dichroic, and I'm at a loss for why this is happening. The light reflected by the dichroic and sent through the 670/40 or 670/30 gives a better resolved signal than the direct light passed through a 670/40 or 670/30. Is this normal? Am I doing something obviously wrong? How can I ensure the resolution for the signal passed through the 740LP is optimal? Thank for any assistance you can offer and thanks for your help to this point. Howard Robinson
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