Epifluorescence microscope images of eight beads of known, decreasing size. The images of the fluorescent beads are shown in green, all adjusted to have the same maximum brightness. The actual brightness varied by ∼1000-fold. The true diameter of each bead is given above its image, and the diameter measured from the image is listed below it. The four smallest beads are shown at two different magnifications. The apparent diameters measured in the image are slightly larger than the true diameters, and the apparent diameters are the same for the three smallest beads, even though their true diameters differ by more than fivefold. These three images reveal the PSF of this microscope. The images in the upper right corner, obtained with the same microscope setup, show the appearance of three gratings with spacings of 0.29, 0.25, and 0.20 µm between the white bars. Notice that the contrast between the black and white bars decreases as the spacing gets smaller. In the actual object, the grating contrast is the same for all three scales. The bead images in the upper row are displayed at the same magnification as the gratings.