Non-uniform illumination results in nonuniform fluorescence. All images were collected using a microscope (model TE2000E; Nikon), a Plan-Apochromat 20x 0.75 NA objective lens, a camera (ORCA-AG; Hamamatsu Photonics), and MetaMorph software. (A) An image of a field of fluorescent beads, using wide-field illumination. Individual beads contain a similar concentration of fluorophore (clumps of beads appear brighter, as is seen near the center of the image). A pseudo-color displaying the range of intensity values (see inset) was applied. Note that beads in the top left have different intensity values than the beads in the bottom right. (B) An image of a uniform field of fluorophore taken with the same microscope optics and conditions as A, showing uneven illumination across the field of view. This nonuniform illumination explains the nonuniform fluorescence from the beads of similar fluorophore concentration shown in A. (C) After flat-field correction (Zwier et al., 2004; Wolf et al., 2007), the image intensity values more accurately reflect the real fluorescence in the specimen. This image was obtained using the image arithmetic function in image processing software (in this case, MetaMorph) to divide the image in A by the image in B. Bar = 50 µm.