Figure 4. Minimization of motion artifacts. (A) Custom-made holders were built to accommodate mice and rat salivary glands (left panels), rat kidney (center right panel) or mouse tongue (right panel). (B–E) Anesthetized rats were either injected with Hoechst (B, C and E) or the salivary glands were exposed and bathed with Texas Red-dextran to label the stroma (D). Time-lapse imaging was performed by two-photon microscopy (excitation 800 nm for B, C and E; 930 nm for D). Salivary glands were exposed and imaged directly with a 60x water immersion objective (NA 1.2, Olympus) (B), or exposed and placed in a custom made holder without (C) or with a coverglass to minimize the motion (D and E). In (B), motion artifacts create distortions within the frames (arrows), shifts in the xy plane and changes in the focal plane. In (C), although several structures are still in register (arrowheads) distortions within the frames are observed (arrows). In (D), only shifts in the xy plane are observed and can be corrected by using software such as ImageJ (Stack-ref plug ins) (E). Bars, 20 µm.