BIO RAD iCycler iQ Packing List And Installation Manual page 21

a. In the Cam Filter box, click Home and then select the radio button for
position 2. If the filter returns to blank when you select position 2, check the
interlock connector at the Reaction Module to be sure that it is properly attached.
b. In the Lamp Filter box: click Home and then select the radio button for position 2.
4. From the menu bar at the top of the Image dialog box, select Expose. This displays the
Expose: Image dialog box (see Figure 6.2).
5. Select an exposure time of 640 ms and click Expose.
6. You must now set the DC offset.
The DC offset controls the camera bias and affects the intensity of each pixel. When the
DC offset is set properly, you can place the cursor on points in the dark area, surrounding
the image of the 96-well plate, and the pixel values will be in the range of 40–70 counts.
You can check the pixel intensity by looking at the bar at the top of the image: the X and
Y coordinates of the cursor are displayed along with the intensity (I) of the pixel underneath
the cursor.
You can also assess the setting of the DC offset (bias) from the Gain box. The Gain box
presents the distribution of the pixel intensity in a histogram with intensity ranging from
0 to 1023, bottom to top. The White line at the top shows the white level (1023 counts)
and the Black line at the bottom shows the black (0 counts) level. The width of the
histogram correlates with the number of pixels at each level. There is always a large peak
very near the black line at the bottom of the Gain box. The goal of adjusting the DC
offset is to set it so that the black line cuts across the histogram about 1/3 of the way down
the large peak. See the figures below:
When the DC offset is set properly:
• The Black and White lines are as far apart as possible.
• The Black line cuts the large peak in the histogram about 1/3 of the way down from
the peak.
• The majority of pixels outside the image of the 96-well plate are in the range of
40–70 counts.
To set the DC offset:
1. Double click in the DC offset field in the Expose dialog box. Type in a new DC offset.
2. Click Expose and look at the histogram in the Gain box.
3. Continue adjusting the DC offset until it is optimized.
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