Computer-based image analysis of cartilage differentiation in embryonic limb bud micromass cultures

A computer imaging system was used to analyse the effects of benzamide (BAM), sodium butyrate and dimethyl sulphoxide (DMSO) on chondrogenesis in culture. Embryonic chick limb bud cells were used as a source of undifferentiated mesenchyme, and cultures were prepared using a micromass culture technique. The degree of chondrogenesis is directly related to the amount of Alcian blue staining. Standard assays include either manual tally of individual cartilage nodules, which is both tedious and time-consuming, or extracting bound dye for spectrophotometric analysis, which obscures individual variations because it requires pooled samples. In the present study, low-magnification images of individual micromass colonies were converted into derived images based on their percentage transmittance relative to the background. These derived images were analysed for relative degree of chondrogenesis, using area and integrated optical density (IOD) as descriptors. While both types of data were useful for ranking the degree of chondrogenesis in culture, IOD was the preferred descriptor because of its accuracy over a wide range of threshold values. The area and IOD data both demonstrate that BAM produces marked enhancement of chondrogenesis in culture, sodium butyrate causes marked inhibition and DMSO produces mixed results (enhancement at the high dose, inhibition at the low dose). While the present study demonstrates the usefulness of computer-aided microscopy for analysis of low-magnification images, the same descriptors (area and IOD) should be useful in quantifying data from a variety of objects (cells, nuclei, etc.) which can be stained in a selected, quantitative fashion.