A method for the blind correction of the effects of attenuation and shading in light micrographs based upon moderated...

A means of correcting for the effects of attenuation and shading in multi-dimensional, digital, light micrographs, blindly, i.e. without the need for additional control sets of image data that record these effects, is described. The method, termed trans-elemental moderated histogram equalization (TEMHE), works with all three types of image that are collected in light microscopy: bright objects viewed against a dark background, bright and dark objects set against a grey background and darker objects set against a light background. In its most simple form TEMHE requires that the features of interest are distributed widely and evenly throughout the image data. If, however, the pattern of attenuation or shading is extracted, smoothed and the result used to correct the original set of image data, then the only restriction is that when different classes of feature are present the boundaries between them are not approximately parallel to the axes of one, or more, of the dimensions to be corrected. Moreover, when it is possible to formulate a simple model of the pattern of attenuation or shading this is no longer a constraint. The method does need to analyse a large number of elements of image data (pixels, voxels, etc.) to function correctly but it will correct shading in single frames of image data providing that they are quite large and the overall signal-to-noise ratio is relatively high.