Effect of imaging depth on lens performance. Three-dimensional stacks of images of small fluorescent beads immersed in water were acquired, using an oil-immersion objective lens, a water-immersion objective, and an objective designed for use with a silicone oil as the immersion medium (refractive index ∼1.4). The graph shows the intensity of the bead image in each focal plane of the 3D stacks from three beads located at different distances from the coverslip surface (at depth = 0). For objects located immediately adjacent to the coverslip, the oil-immersion lens gives a brighter signal and sharper axial response than the other lenses. However, its performance is rapidly degraded by spherical aberration with increasing depth, whereas the other two objectives are less sensitive, so that at depths corresponding to typical eukaryotic cells the performance ranking is reversed.