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Keys to Successful Densitometry

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Integrated density is defined as the sum of pixel values minus a background value for each pixel within a bounded area. For a given area, increased brightness or size of a band or spot correlates to higher integrated density measurements. Integrated density measurements are useful for comparing band or spot intensities on images where no samples of known concentration (standards) are included. The Integrated Density function of Eagle Sight software version 3.0 allows rapid comparison between bands or spots within a single image from the Eagle Eye system.

figure 1

Determining integrated density with Eagle Sight software begins with choosing the Integrated Density function from the toolbar or Analyze menu (figure 1 (30k), RED). Integrated density information can then be viewed either by clicking directly on the band or spot or by clicking within a user-defined region of interest. This information can then be written on the image overlay, saved with the image and printed to preserve a hard copy of the final data.

Pixel Saturation: When acquiring an image for integrated density determinations, the user must keep the Saturation Warning function of the View menu enabled to avoid saturating pixels within bands or spots of interest. Enabling the Saturation Warning function results in image pixels with values of 255 to be displayed in red. Because 255 is the largest possible pixel value, increasing the exposure time of an image that already contains saturated pixels will not increase these pixel values further. However, pixel values below 255 will continue to increase with longer exposures until they too become saturated. Integrated density calculations on bands or spots having saturated pixels will produce measurements that are lower than expect
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