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Keys to Successful Densitometry

vided. Once a region is defined, the software automatically calculates the modal value for this area and designates it as background. For more control, users can manually enter a numerical value for background after either viewing the pixel values in image areas near the bands of interest or viewing statistical information from defined regions. Both the Pixel Value and Statistics display functions are located in the View menu.

The size of the region of interest (ROI) is also likely to affect integrated density determinations. In general, the size of the analysis area should be only slightly larger than the bands or spots used for analyses. Analysis areas that are much larger than bands of interest have the potential to magnify errors due to small variances in background levels.

In order to compare integrated density measurements for determining relative intensities between bands or spots on the same image, sample areas should be kept the same size. If the dimensions of the areas are different, accurate analysis will be difficult unless the background has the same pixel value throughout the image analysis area. This is generally not the case. Preserving a rectangular analysis area of equivalent size throughout the analysis can be accomplished using the Region of Interest (ROI) function from the toolbar. The dimensions of the area can be preserved while the region is moved from one area of the image to another by simply (1) reselecting the ROI key from the toolbar, (2) moving the cursor until it is positioned over the previous ROI (this will cause the cursor to change to a hand symbol) and (3) dragging the ROI to a new position while holding down the left mouse button.

Concentration Analysis

Concentration can be determined directly from integrated density when standards of known concentration are analyzed along with samples of unknown concentration. Assuming that
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