Difference picture algorithms for the analysis of extracellular components of histological images

A computer-assisted method for objectively identifying and displaying the distribution of molecules that can only be positively identified by a combination of staining characteristics and susceptibility to specific enzymatic digestion or chemical degradation is presented. The visual image of an enzymatically digested tissue section is subtracted from that of an adjacent buffer-incubated control section and the distribution of the extracellular molecules removed from the tissue section displayed. Photomicrographs are taken using white light and narrow bandwidth filters of wavelengths at or near the maximum absorbance for the dye products used to visualize the extracellular matrix and cells. Each negative is standardized using reference gray levels. The cell and matrix images of both digested and undigested sections are then registered. The locations of cells in both control and digested sections are identified and set to an undefined gray level value in the matrix images. The cell-removed images of the control and digested sections are then registered and the difference in gray levels between the two images calculated and displayed. The validity of results obtained is primarily dependent on the soundness of the histological visualization and digestion techniques used, but is independent of investigator interpretation.