3-D fluorescence imaging was performed on an inverted wide-field microscope (Diaphot 200; Nikon) with excitation by a 100-W mercury lamp. Images were obtained through a 60× objective and digitally recorded on a cooled, back-thinned charge-coupled device camera (CH250; Photometrics) with an effective pixel size at the specimen of 80 nm in x-y and a z spacing of 250 nm. This resulted in a 3-D stack of ∼40 image planes for each cell.
