Figure 1. Extended imaging of the midgut in live Drosophila adults.

(A) Adult female midgut in situ, sagittal view. The white highlighted area indicates region R4a-b, also known as P1-2, (Buchon et al., 2013a; Marianes and Spradling, 2013)) of the midgut that will be exposed for imaging. (B–C) The midgut is accessed through a small cuticular window cut in the back of a live animal. (B) (Top) Schematic of the imaging apparatus. The animal is affixed to a modified petri dish ‘mount’. The chamber of the mount contains media. The underside of the mount supports a feeder tube. See and Fig. 1-fig. supplement 2. (Bottom) Dorsal (left) and ventral (right) views of an animal in the mount. In the left panel, the exposed midgut is outlined by the magenta dotted line. Scale bars: 0.25 mm (left), 0.5 mm (right). See Video 4. (C), Steps in preparing the midgut for imaging. See Video 1 tutorial. (D–F) Registration macros are applied post-acquisition to correct the blurring caused by tissue movements. (D), Before registration, blurring and duplications (arrowheads) are evident. This panel is a raw z-series projection of one movie time point. (E), During registration, two ImageJ plugins are applied in series. (1) 'StackReg' corrects for tissue movement during z-stack acquisition at a single time point. (2) 'Correct 3D Drift' corrects for global volume movements over multiple time points. (F), After registration, blurring and duplications are negligible. Cyan, all nuclei (ubi-his2av::mRFP); yellow, stem cells and enteroblasts (esg >LifeactGFP). Scale bars, 20 μm. See Video 6.

Figure 1—figure supplement 1. Mounts for upright, inverted and light-sheet microscopes.

(A–B) Mount for upright microscopes. (A) Schematic of an animal in the mount on a microscope stage. (A′) Isometric illustrations of mount components: (1) modified petri dish, (2) metal shim with cutout for Drosophila abdomen (Fig. 1-fig. supplement 2Figure 1—figure supplement 2), (3) feeder tube, and (4) bottom chamber with wet Kimwipes (light blue). (Bottom chamber is not shown in (A).) (B) Schematic of the humidity box that encloses the mount. Unassembled (B) and assembled (B′) views are shown. (C) Mount for inverted microscopes. The midgut is imaged through a glass-bottomed petri dish. To elevate the animal, two spacers are glued to the bottom of the dish, and the metal shim is affixed to the spacers. Media is added to the level of the spacers. (D) Mount for light-sheet microscopes. The barrel of a 1 ml syringe is modified to fit the metal shim. The animal and feeder tube are inside the barrel, and the dorsal surface and exposed midgut are outside the barrel. The barrel is submerged in media with one end remaining open to the air. 3D, side and end-on views are shown.

Figure 1—figure supplement 2. Specifications for abdomen cutouts.

The metal shim of the imaging mount includes a cutout through which the dorsal abdomen is inserted. ‘Fat’ (left) and ‘skinny’ (right) cutouts accommodate differently sized female abdomens. This diagram can be used as a CAD file for automated laser cutters.

Figure 1—figure supplement 3. Cell viability during extended imaging.

Cell viability during extended imaging was evaluated using the cell-death stain Sytox Green. (A) Positive control. To induce cell death, midguts were dissected out of the animals and cultured ex vivo. Sytox⁺ cells (green) are rare at the start of culture (0 hr) but became abundant after 2.5 hr. (B) Appearance of a Sytox⁺ cell during extended live imaging. In (A–B), arrowheads point to the same cells before and after becoming Sytox⁺. Nuclei are colored magenta (ubi-his2av::mRFP). Scale bars, 20 µm. See Video 5. (C) Timeline for the appearance of Sytox⁺ cells during extended live imaging of three midguts. Each vertical line marks the time at which one Sytox⁺ midgut cell became visible. A dot marks the end of each imaging session. Yellow box in Midgut 1 marks the timepoints that are shown in Video 5 (10.6–12 h).