This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferationmodifying agents.Key terms: Proliferation rate, correlative microscopy, tetrazolium dye, growth factors, antiproliferative drugs, drug response, computer simulation, heterogeneityThe presence of variant cells within a population derived from a common ancestor is known as cellular heterogeneity (8,27). This situation represents a significant problem in cancer therapeutics, for example, in that diversely responsive subpopulations within a target tissue may allow for selection of resistant cells during therapy (5). To study cellular heterogeneity directly, it is necessary to have methods that are capable of defining cellular characteristics and behavior a t the level ofthe individual cell. Advances in flow cytometry and image analysis are making such studies increasingly possible.Proliferation is an important cell behavior in cancer research, inasmuch as cancer is primarily a disease of growth control, and anticancer therapies are usually aimed at killing, or otherwise arresting the growth of, malignant cells. Unusual cell proliferation also plays a role in arteriosclerosis (7), and thus is a cell behavior with relevance to more than one significant health problem. Although cytometric systems, such as flow approaches, permit multiple parameter measurements on individual cells virtually instantaneously, such measurements do not permit quantification of cell proliferation.A method commonly used for evaluation of proliferative potential of individual cells is the colony formation assay, wherein immobilized cells are allowed to proliferate, and growth is quantified by enumeration of progeny. Computer-assisted evaluation of colony formation assays has been employed for colony counting (10,141, for total colony volume analysis (29,311, and for histogram analysis of colony sizes (12). These approaches permit assessment of growth kinetics of populations and estimation of growth rate of individual colonies by inference. A manual approach involving colony isolation and cell enumeration has been used to determine numbers of cells in colonies in soft agar (29); a similar approach has been used to describe heterogeneity in growth rate among individual colonies growing on plastic (15). Time-lapse cinematographic approaches (1 1,211 and time-lapse video approaches (6,7) have been used to determine the growth rate of individual cells throu...
