iCys User Guide

May 6, 2005 Generating Analytical Data
Using Contouring
Following are some examples of how contouring can be used.
Comparing the Size of the Nucleus to the Amount of Cytoplasm
You can set up contours to compare the size of the nucleus to the amount of
cytoplasm. The key here is that by generating the contours, you can determine the
integral areas for both the nucleus and the cytoplasm, as follows:
❑ Threshold contour isolates individual cells around the nuclear stained area.
❑ Integral contour begins a specified distance from the threshold and integrates
fluorescence in the nucleus.
❑ Peripheral contours begin farther out from the threshold contour, and need not
extend to the end of the cytoplasm. Pixel values within the peripheral contours are
integrated, providing a measure of cytoplasmic fluorescence.
❑ Background contours begin just outside the peripheral contours for background
sampling.
Peripheral Contouring and Background Correction
When using peripheral contours, you usually do not want to subtract background from
the detector that measures the cytoplasmic stain of interest. For example, consider a
specimen where the primary contour is set to segment events based on the Blue
detector and the cytoplasmic staining is measured in the Green detector. To prevent
the background value, which is determined for each detector by the pixels in the
event's background contours, from subtracting too much from your peripheral
integral, deselect the Green detector in the Corrected Channels list box on the
Background tab of the Event module.
Revision B iCys Research Imaging Cytometer User Guide 4-13