Quantification of the degree of cell spreading of human fibroblasts by semi‐automated analysis of the cell perimeter

Abstract

Cell flattening and spreading on a substratum is of major importance in cellular and developmental biology. To study the mechanisms of cell spreading, quantitative and reproducible measures of the degree of cell spreading must be available. Normal human fibroblasts, spreading on a substratum, were fixed with glutaraldehyde, stained with acridine orange and photographed (×40) under a fluorescence microscope. The photonegatives (containing 10–30 cells) were scanned with a drum scanner and a complete picture containing 128 gray levels was constructed. Each cell contour was calculated with the use of a local threshold. The image and the superimposed cell contours were displayed on a television screen (16 gray levels) and errors were corrected interactively. With this system the spreading of normal human skin fibroblasts as a function of time could be quantified reproducibly. Compared to surface area or shape, the cell perimeter proved to be a very sensitive parameter of the degree of spreading. By using cell perimeter measurements, differences in the degree of spreading on various substrata could be quantified.