Quantitation of cell movement and lamellar extension is critical to the study of growth factors, chemotactic agents and signaling cascades. Many studies are conducted by examining the size or number of lamellae in static images of cells. However, these methods do not quantify lamellar behavior over time and may overlook important changes in lamellar function. Most presently available methods for analyzing dynamic aspects of lamellar function examine changes in a cell's 2-dimensional perimeter and are best suited to the analysis of flattened lamellae. However, some cells generate 3-dimensional lamellar ruffles whose behavior is not readily detected using these methods. In the present study we analyze temperature-dependent ruffling of human SH-SY5Y neuroblastoma cells using automated digital subtraction of time-lapse images and quantitation of the resultant 'difference' image, and compare results obtained using this and other approaches. We report that ruffling behavior of SH-SY5Y cells is measurably altered by temperature changes of as little as 1 degrees C, and that these changes are best detected using difference image analysis. Our studies indicate that temperature is a critical variable in studies of SH-SY5Y behavior and that difference image analyses may be an important complement to other methods in the study of lamellar ruffling.