> Howard Robinson writes: > > Problem:The signals in the Cy5 and PE-Cy5 channels [on a Cytomation MoFlo, > equipped with a Melles Griot 35mW Helium Neon laser (Laser #2)] are very low. > On unstained cells the signal only occupies 20% to 30% of the first decade on > the display. > Essentially all the events in these channels are cramped down near the zero > channel. This is a real problem with dual parameter histograms such as FITC vs > Cy-5. When the Cy-5 negative population is so low, the FITC (pos)/ Cy-5 > (neg) population is difficult to discern. > In discussing this problem with Cytomation, increasing PMT voltages to push out > the negative population causes thermeonic emissions in theses channels. > So, we are faced with either negative populations that are too low to view data > or we are faced with creating an erroneous population. > Question: > How can we move out the negative population without thermeonic emissions > creating a ghost population? Howard, Are you sure this isn't a calibration problem? Assuming we're talking log amps, there isn't a zero -- only logs that we choose to include. If you shift the offset to the "low" end of the log range, your signals should come of the baseline . . . something to ask Cytomation about. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry <http://www.cancer.med.umich.edu/flow_cytometry> phone: 734-647-3216 fax: 734-936-7376 kukuru@umich.edu
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