Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 123)

Section 7 Calibrating the Instrument
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3. Click Take an Exposure. The Image screen displays the fluorescence detected by the CCD
camera for each of the 96 wells on the plate. Pixels that are saturated will be shown in
pink
4. Click Show Mask. The Mask is displayed as an array of green boxes (Figure 7.2).
Fig. 7.2. Camera Image with Overlying Mask Array.
5. If any pink (saturated) pixels are present in the mask, reduce the exposure time and
retake an exposure. Keep retaking the exposure until no pink pixels are present.
6. Click Optimize Mask. Each individual box should be centered over the well so that all the
fluorescent signal from the well falls within the box.
7. Click Save Mask to save mask-data.xml file in the Mask folder of the iQ5 Folder.
The Mask can be displayed or hidden using the Show Mask button. The Mask array can be moved
as a group by clicking on the Up, Down, Left or Right buttons. An individual box within the array
can also be moved by first clicking on that box to select it, then using the Up, Down, Left or Right
buttons to move it.
The Mask can be restored to the last saved Mask by clicking Reload Mask. The default, factory
Mask can be restored by clicking Restore Factory Mask.
This completes Mask Alignment calibration. Next, perform background calibration.
7.4.2 Performing Background Calibration
Background calibration is performed to account for fluorescence in the experimental system that
is due to the reaction vessel and sealing mechanism.
What You Will Need
You need the following to calibrate the camera:
• 96-well PCR plate or preferred reaction vessel (referred to as “plate”)
• Optical-quality sealing tape or preferred sealing method
Before you perform background calibration, complete the following steps:
1. Prepare a background calibration plate (see section 7.3)
2. Ensure that the mask has been aligned.