Adjusting The Mask - BIO RAD iCycler iQ Packing List And Installation Manual

6.3 Positioning the Mask
Every time data are collected, two separate readings are taken for each of the 96 wells: the
inner reading and the outer reading. The inner reading is the average of all the pixels in the area
defined by the mask as the inner well region. The outer reading is the average of all the
pixels in the region immediately surrounding the inner well region and represents background.
The net reading for a well is the difference between the inner and outer reading for that well.
The area of each well inner and outer is defined by the mask file. It is from the net readings
in each well that the PCR Amp Cycle plots are created and thresholds cycles are determined.
1. From the Image menu bar, select Tools, then select Mask Editor. An Open dialog box
will appear showing files in the Ini folder. A file called mask96.ini should listed be in
the dialog box. If it is not, locate it in:
c:\Program Files\Bio-Rad\iCycler\Ini\mask96.ini
2. Double click on the mask96.ini file. The Mask Editor dialog box will open and two
concentric squares will appear around the lower left well (see Figure 6.3). The Row and
Column text boxes on the right side of the Mask Editor dialog box indicate the well
position. Note that the image is upside down and in Figure 6.3 well A1 appears in the
lower left corner. The column on the left side of the Mask Editor dialog box shows the size
of the mask over well A1. The inner square is 8 x 8 pixels and the outer square is
16 x 16 pixels for each of the four corners. For all other wells, the inner squares are 9 x 9
and the outer squares are 17 x 17.
When the mask is correctly adjusted, the entire image of the well should be within the
inner boundary. The area between the inner square and the outer square is used for
determining the background for calculations made during data analysis. Do not adjust the
sizes of these inner and outer boundaries.
Fig. 6.3. iQ configuration window with the calibration plate image showing a mask over well A1 and
with the mask editor dialog box.
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