iCys User Guide

May 6, 2005 Generating Analytical Data
➣ To add background contours:
1. Click the Background tab of the Protocol ⇒ Event window.
2. Check the Use Dynamic background box. Background contouring will be inactive
unless this box is checked.
3. Set the following parameters:
❍ Distance—The distance between the integration contour and the first
background contour. Set the first background contour away from the
integration contour such that it is away from the event fluorescence.
❍ Width—The distance between the first and second background contour. Set
the width such that the outer contour line includes the dimmest pixels adjacent
to an event.
❍ Use Pixels Between—When determining the background, you usually want
to use those pixels that represent the mean pixel value, such as 30-70%. If,
however, the background is very bright, you might want use a lower
threshold, such as 20-50%.
❍ Color—Select the color for the Background contour using the pulldown
menu or the color palette.
4. Once the background value has been determined, you can use Corrected
channels to subtract that value from each event pixel, thereby compensating the
selected channels for the background noise.
When using dynamic background, subsequently added channels (such as virtual
or merged channels) will be background corrected by default.
For a discussion of when you might want to do this, see “Peripheral Contouring
and Background Correction” on page 4-13.
Peripheral Contours
You can use additional contour functions to add more information to your data file.
For example, Peripheral contours give you a measure of the fluorescence in a fixed
annulus outside of the threshold contour, which is useful for sampling the cytoplasm
when contouring nuclei with nucleic acid dyes.
Peripheral contour settings are available from the Peripheral tab of the Protocol ⇒
Event ⇒ Event menu, shown below.
Revision B iCys Research Imaging Cytometer User Guide 4-9