Fig 1.
Application of fluorescent dyes to the pupal wing tissue and the larval wing imaginal disk.
(a) A pupa with a dye-containing solution (a red arrow) sandwiched between wings. The panels a, c, and d are images also shown in a previous study for an introductory purpose. (b) A treated pupa in a simple humidified chamber, an upside-down glass cup. (c) An operated pupa with its forewing and hindwing on a piece of slide glass. (d) Pupal wings seen through a glass slide. (e) An operated larva with its wing imaginal disk exposed (a red arrow). (f) An operated larva in contact with a dye-containing solution.
Fig 2.
Triple staining of a larval wing imaginal disk.
The tissue was stained with Hoechst 33342 for nuclei, BODIPY FL Thapsigargin for ER, and MitoTracker Orange CMTMRos for mitochondria. Yellow arrows indicate peri-nuclear signals. White arrows indicate endosome-like or autophagosome-like unstained structures. Blue arrowheads indicate cells with small double nuclei. (a) Low magnification view. (b) High magnification view.
Fig 3.
Triple staining of a pupal wing tissue.
The tissue was stained with Hoechst 33342 for nuclei, BODIPY FL Thapsigargin for ER, and MitoTracker Orange for mitochondria. White arrows indicate endosome-like or autophagosome-like unstained structures. (a) Low magnification view. (b) High magnification view. Original scanning images were acquired with 1.0 μm intervals, and those 23 images were stacked to produce the final image of (b) and (d). (c) Hoechst 33342 staining. A part of the image shown in (a) was enlarged and its contrast was enhanced. Right and left images are identical except that nuclei with possible circular arrangements are highlighted in white. (d) 3D image of an epithelial tissue.
Fig 4.
Double staining of a larval wing imaginal disk and pupal wing tissue.
The tissue was stained with SYBR Green I for nuclei and MitoTacker Orange for mitochondria. Scale bars indicate 10 μm. (a) Larval wing imaginal disk. A portion is enlarged in the right panel. An optical vertical cross-section image shown at the bottom was made along a blue broken line. Thickness of a cell was approximately 2.9 μm. The image was constructed by stacking 28 images down to 5.5 μm in depth with 0.2 μm intervals. (b) Pupal wing tissue. The pupal head is positioned to the left. A portion is enlarged in the right panel. An optical vertical cross-section image shown at the bottom was made along a blue broken line. Thickness of a cell was more than 11.4 μm (the deepest portion not detected). The image was constructed by stacking 58 images with 0.2 μm intervals.
Fig 5.
Deep serial cross-sections of pupal epithelial cells triple-stained with Hoechst 33342, BODIPY FL Thapsigargin, and MitoTracker Orange.
Numbers at the right upper corner indicate the depth from the apical cellular surface. White arrows indicate a large cell, likely a prospective scale cell. Asterisks indicate a large endosome-like or autophagosome-like unstained structure (see Fig 6). Red dots within a nucleus are not explainable. Also see S1 Movie.
Fig 6.
Deep serial cross-sections of pupal epithelial cells triple-stained with Hoechst 33342, BODIPY FL Thapsigargin, and LysoTracker Red.
A 3D image was reconstructed first, from which 2D cross-section images were presented in (a) and (b). (a) Serial images. Numbers at the right upper corner indicate the depth from the apical cellular surface. Small lysosomal bodies are strongly stained in red. Large endosome-like or autophagosome-like structures are weakly stained in red inside but their membranous structures are seen in orange. Hemocytes are strongly stained in red (arrows). (b) Enlarged image of (a), 5 μm in depth. Small lysosomal bodies are indicated by red arrows, whereas endosome-like or autophagosome-like structures are indicated by black arrows. (c) 3D image of an epithelial sheet. Stained hemocytes are indicated by arrows.
Fig 7.
Serial cross-sections of double-stained pupal epithelial cells.
Cells were double-stained with BODIPY FL Thapsigargin for ER and MitoTracker Orange for mitochondria. Extensive overlapping of green and red signals is largely because of highly intermingled mitochondria and ER. Cell A and cell B exhibited extensive connections via epidermal feet. A white arrow indicates mitochondria inside an epidermal foot.
Fig 8.
Deep serial cross-sections of pupal epithelial cells and a reconstruction of a stacking 3D image.
Cells were stained with CFSE for multiphoton microscopy. (a) Sections are presented from the top to the bottom. Also see S2 Movie. (b) Reconstruction of a stacking image from the bottom to the top. Also see S3 Movie.
Fig 9.
A 3D image of pupal epithelial cells of J. orithya.
An image is shown in a cube with the following dimensions: 143.36 μm (width), 143.36 μm (height), and 130.04 μm (depth). Calibration: 0.28 μm (x axis), 0.28 μm (y axis), and 2.36 μm (z axis). Ripples in deep positions are artifacts caused by sample movement.
Fig 10.
Reconstruction of a stacking 3D image of a pupal wing tissue using a different species, Z. maha.
Reconstruction of a stacking image from the bottom to the top. Cells were stained with CFSE. Also see S6 Movie.
Fig 11.
Wide-area images of deep sections of a pupal wing tissue.
Cells were stained with CFSE. (a) A basal wing area. Possible cellular clusters are indicated by white arrows. (b) A prospective eyespot area (white arrows). A bubble is trapped (an arrowhead). The rightmost panel is a synthetic image, showing a cone-like structure.
Fig 12.
Staining pattern of a prospective eyespot area and edge spots.
(a) A larval wing imaginal disk. Stained with SYBR Green I for nuclei and MitoTracker Orange for mitochondria. A white arrow indicates a prospective eyespot area, as judged by venation pattern. (b) Pupal wing tissue stained with SYBR Green I for nuclei and MitoTracker Orange for mitochondria. A white arrow indicates a prospective eyespot area. (c) High magnification of a prospective eyespot area shown in (b). (d) Peripheral area of a pupal wing tissue stained with CFSE. Arrowheads indicate pupal edge spots.