The mean fluorescence ratio for each of the cells included in these calculations is shown in Fig. 3. We note that initially, the range of fluorescence ratios is similar for the stem cells in the propagation and differentiation groups, as expected. However, as incubation time increases, we observe that the fluorescence ratio of cells in the differentiation medium shifts to values lower than that of cells in propagation medium. In fact, the mean fluorescence ratio for differentiating stem cells is statistically lower than that for the cells in propagation medium for all measurements conducted on days 8 to 21. Fluorescence ratio values are included in Fig. 3 as a function of the product of two morphological parameters that we acquire from the TPEF cell images. Specifically, we multiply the area of the cell (in thousands of pixels) with a factor indicating how circular the cells are. This factor is defined as 1 minus the eccentricity (measured as the ratio of the distance between the foci of the major and minor axies of an ellipse encompassing the cell) and is thus equal to 0 for a line and 1 for a perfect circle. These factors are used to represent quantitatively a significant increase in the area and the circularity of differentiating cells. It is interesting to note based on the results in Fig. 1 and Fig. 3 that not all cells are undergoing adipogenic differentiation at the same rate, even in simplified two-dimensional (2-D) cultures. This is important, since most molecular characterization techniques assess global changes. In addition, we note that it is possible to draw a simple line based on visual inspection of the data, to separate all but 1 out of the 117 cells in propagation medium from 10%, 23%, 70%, 77%, 96%, and 90% of the cells in differentiation medium imaged on days 2, 8, 12, 14, 18, and 21 of treatment, respectively. Adipogenic differentiation is confirmed by RT-PCR analysis [Fig. 3]. LPL, a marker of adipogenic differentiation, is expressed in hMSCs maintained in adipogenic differentiation medium while expression of AP is not detected. Only low levels of LPL and AP are expressed in stem cells maintained in propagation medium.