In automatic area selection, the computer decides where the boundaries of the object to quantitate are, based on the background value that you provide. The background value is a number between 0 (black) and 1 (white). This number is found by moving the mouse over the background near the feature to be measured and observing the ``d'' (density) value shown in the left information window.
For example, in panel A below, the intensity of the background (pointed to by the arrow) ranged from 0.702 to 0.765 while the intensity inside the band was around 0.482. Thus, the best background value would be the background value that is closest to the signal, i.e., 0.702. So in the Spot Densitometry dialog, you would set the following:
| Maximum signal | Black (since the band is black). |
| Area selection | Automatic |
| Pixel density calib. | None |
| Bkgd.value | 0.702 |
| Calib. factor | 1 |
| Diameter | (ignored) |
| Leave area marked | (unchecked) |
| Auto bkgd. (fuzzy k means) | (unchecked) |
Keep Auto bkgd. unchecked, since you wish to use a constant background value.
Then click OK, and click in the center of the band. The boundaries of the band are automatically calculated, and the band is temporarily highlighted (B), indicating precisely what is being measured. If this is too large, the Bkgd. value should be changed. The algorithm is designed so that the computer should find the same region, and the same area, density, and signal, regardless of the exact spot you click on within the object. If you click on the background, the program will report an area of 0. As long as you continue clicking, new areas will be selected and analyzed. At any time, clicking on Cancel or pressing Esc will stop the densitometry.
Sometimes the computer may not find the correct edge of the object. It is important that there is no unbroken trail of pixels the same color as the object that extends out of the object. If there is no clear boundary around the region of interest, it may be necessary to create one before starting densitometry by sketching a black or white border around it (for example, C).