Obtaining the most accurate densitometry measurements

Below are some tips for making the most accurate spot densitometry measurements:

1. Scan the image at the highest depth and resolution possible. Ten bits/pixel and 300 dpi should be considered minimum values.
2. If using ``Auto background'', select an area approximately twice the size of the spot. Ideally the number of background pixels and spot pixels should be the same. The ``Area Partitioning Statistics'' window gives this information.
3. Make several repeated measurements and take the average.
4. Although it is more tedious, the most accurate technique is to use strip densitometry instead of spot densitometry. This gives a density profile from which the optimal background value can be subtracted manually. (See figure below in ``Strip Densitometry'', Sec.8.12).

Notes:

• If all density values come out as negative numbers, either the ``background value'' or the ``maximum signal'' is set incorrectly.
• If all density values come out as identical numbers, the most likely cause is low contrast in the original image. This will not necessarily be visually apparent, because an optimal grayscale map is calculated automatically before displaying the image. Try clicking on ``Contrast..Maximize Value''.

Area measurements

Procedure for making area measurements:

1. Before starting, obtain a histogram on the image to find the desired background value for use with automatic spot detection (select ``Color...Histogram''). Alternatively Use ``Draw...Sketch'' before starting, and completely encircle the objects to be measured with a white line. Outlining the objects manually will obviate the need to select an appropriate background value.
2. Select ``Image...Spot densitometry''. Leave all the settings at their defaults except for the Background Value.
3. To identify the spot automatically, set the background value to the pixel value that corresponds to the brightest pixel in the background. Alternatively, the regions of interest can be demarcated by an outline beforehand, and the background value set to any large number (for example, if the areas were outlined with white (=255), set the background to 254).
4. Click on OK, then click again to dismiss the message box.
5. Click anywhere inside the object to measure. The area being measured will temporarily be painted in black, then a list box will appear displaying the area.
6. Click on the background (or the upper left corner of the list box) to hide the list box. Then click on the next object to measure.
7. Press Esc when done.