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Keys to Successful Densitometry

ed, relative to nonsaturated samples. Therefore, spots or bands containing saturated pixels should not be used for precise densitometry analysis.

Background: The Integrated Density function sums all pixel values, minus a background value for each pixel, within an area of defined dimensions. Small differences in pixel value, even a value of 1 or 2, summed over an entire area can have a large cumulative effect on integrated density calculations. For this reason, care should be taken to have background values roughly equivalent throughout the analysis area and to properly define the background. Accurate densitometric analysis of images whose background levels vary greatly within analysis areas is difficult. In general, before beginning the analysis, the user should define the background level from an area of the image closest to the bands or spots of interest. This will ensure that the appropriate background value is subtracted from every pixel within the analysis area. If the user does not define the background, the software will automatically select the most common pixel value (the modal value) within the entire image and use this value for all background measurements.

Defining the background value before analysis can be accomplished either manually or automatically using the Set Background function located in the Analyze menu. By choosing the automatic option, users can define an area that they consider to be representative of background levels near bands of interest. Typically, this area will be right above or below the bands to be analyzed. In order to visualize background levels more readily, the False Color option from the Process menu can be used. By replacing the gradual grayscale with sharp differences in color, the False Color option displays small differences in pixel value more readily and allows the background threshold levels to be defined such that the most accurate results are pro
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