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? Unstained cells shouldn't be very fluorescent in the emission range associated with Cy5 (and PE-Cy5), so one would not expect them to occupy more than a fraction of the first decade on the display. Essentially, there isn't a signal to amplify, so the only way you can move the negative population where you like it is to increase the gain, and what you see will be noise due to thermionic emission and other electronic sources. But what do you mean by a "ghost population"? If you mean that some of the negative cells come off the base line and some don't, that effect is typically an artifact of fluorescence compensation. The only way to make it go away on the display is to add random noise to the signal or random numbers to the digitized data. Just as is the case when you turn the PMT gain way up, you are making the data look better by adding noise. On a Cy5 channel, one can increase the apparent signal from unstained cells by using an emission filter which lets some scatter through - one would generally prefer to avoid doing this, because, again, you're adding noise, and this will decrease the separation between the Cy5-positive and Cy5-negative cells (as will all the other methods of adding noise). As long as your gate on a 2-D histogram can include the axes, there shouldn't be a problem with analyzing the data as you get them; if that's a problem, gate out the fluorescein-positive population on a 2-D histogram of green fluorescence vs. scatter and do a 1-D histogram of Cy5 to find positives and negatives. -Howard Shapiro
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:55:38 EST