There are three principal approaches to correcting shading in images. The first, and the best for those cases in which is it practical, is to capture two images, one of the specimen and another with the specimen removed or replaced by a uniform target so that the pattern of nonuniformity can be recorded directly. This “background” image can then be either subtracted from or divided into the image of the specimen to produce a leveled result. This method can be very successful with photographs on a copy stand, or for some types of specimens in a transmission light microscope. This interactive tutorial illustrates background correction performed by subtracting an image of nonuniform illumination.

The tutorial initializes with a randomly selected specimen appearing in the Specimen Image window. The Choose A Specimen pull-down menu provides a selection of specimen images, in addition to the initial randomly chosen one. The radio buttons show the Original image, the measured Background image, and the resulting image after Subtraction of the background from the original.

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