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The results showed that the appropriate threshold is slightly below the local background intensity.
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For the rural background sites, the locally measured concentration was used as the local background concentration.
After ROI delineation the spill-out region is identified and for each voxel inside the spill-out region the local background is computed (see below).
Any corrected signals falling below the cut-off threshold (typically, fluorescence values less than twice the local background) were set to zero, giving final corrected signals for each fluorochrome for each spot.
The background region is defined in the same way as for the local background method.
Background refers to the local background intensity, while the standard deviation was calculated across the whole slide.
Signal intensities were corrected by subtracting the local background.
2) Subtract: this is the traditional background correction method with the local background estimate subtracted from the foreground estimate.
If the local background is indeed extremely low, treating it as background noise may not distort the actual number significantly.
Agilent-provided settings were used except for subtraction of the local background and adjustment of the global background.
Detection limits are calculated by the CodeLink software as the median intensity of the local background plus 1.5 local background standard deviations.
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