Progenesis SameSpots and its statistic tools have become indispensable in our gel based proteomics workflows.
Dynamic range and intensity levels in use
The Image QC stage of Progenesis SameSpots checks all your gel images to help find any quality issues that may prevent you getting the best results out of the analysis. As feedback, it shows the results of the checks. For example...
31.25% of the available dynamic range is in use
26.17% of
the available intensity levels are in use
Dynamic range
The dynamic range of an image refers to the actual range of pixel values used, which will be less than the available range of values. For example, with a 16 bit image there are 65,535 available values. Your data will lie somewhere inside that range.
A low dynamic range means less precision in the intensities represented by each pixel. More precision in the image helps differentiate low abundance spots from background, and allows greater quantitative accuracy.
You can adjust the dynamic range in CCD camera systems by altering the exposure time, or in a laser based system by fine tuning the voltage of the PMT detector. You should consult your scanner documentation, or contact your scanner supplier, for information on how to achieve this.
If we put the pixel values used into a histogram (ignoring the top and bottom 0.1%, so we don't get skewed results because of noise), we can find the range of the image data and express this as a percentage of the available range. This is how we calculate the measure for dynamic range reported in the ImageQC stage.
Intensity histogram of a typical gel image showing a dynamic range of about 30% of the available range
Intensity levels in use
The value for available intensity levels in use is the number of discrete intensity values in the image expressed as a percentage of the available range.
The histogram below illustrates a problem that wouldn't be found by the dynamic range check but does become apparent when we look at the intensity levels in use. The dynamic range is high but only a small percentage of the individual levels are present.
A low percentage of intensity values is often a result of applying some form of Contrast Stretching or Histogram Equalisation to the scanned image. These techniques are commonly used in digital imaging applications to improve images for viewing but such manipulation of raw data in your experiment should be avoided. It can lead to inaccurate spot measurements and bias your results.
