Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging

Abstract

The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a specific plane, which may be inaccessible due to tissue size or shape. Recreating high-resolution 3D images is not possible because the point-spread function of light reduces the resolution in the Z-axis about 3-fold, compared to XY, and light scattering obscures signal deep in the tissue. However, the XY plane of interest can be chosen if embedded samples are precisely oriented and sectioned prior to imaging (Figure 1). Here we describe the preparation of frozen tissue sections of the Drosophila wing imaginal disc, which allows us to obtain high-resolution images throughout the depth of this folded epithelium.

Keywords: Confocal microscopy; Cryosection; Drosophila; Frozen sections; Wing imaginal disc.

Figure 1.. The epithelial structure and undistorted folding pattern are revealed in its entire depth in this frozen section of developing Drosophila wing.

A-D. Transverse dorsoventral sections through the wing pouch. A. Cryosection reveals nuclei (A, green) and subcellular distribution of α-catenin (A’, A”, magenta) with signal throughout the depth of the epithelium. The basal surface is clearly detectable (arrows). A” is digitally enhanced image of A’. B. A Z-stack of images collected in a top-down view displayed as XZ orthogonal view reveals nuclei (B) but little discernable detail for α-catenin (B’, B”) and even the digitally enhanced image (B”) fails to reveal the basal epithelial surface (arrow). C. Transverse dorsoventral section displaying the Distal-less (Dll, green) gradient in the wing pouch and subcellular localization of DE-Cadherin (magenta) throughout the epithelium. D. View of the wing pouch. Dorsal is to the left; apical is up. Scale bars are 1 µm in A, B, 11 µm in C, 5 µm in D.

Figure 2.. Schematic representation of the gelatin embedding process.

A. Transfer a wing disc to the gelatin by pipette. B. Remove excess sucrose solution from around the disc. C. Add an addition 300 µl of gelatin on top of the disc.

Figure 3.. Orientation of wing discs in cut gelatin cubes.

Wing disc stained with DAB to visualize expression of Wingless & Patched, for illustration, in trimmed gelatin blocks. A. Top-down view of a wing disc in gelatin cube. The red line indicates the face of the cube which should be placed face-down into the embedding capsule to acquire a dorsoventrally aligned section. B. ‘edge-on’ view of the wing disc from the direction indicated by the arrow in A. C. An alternatively angled cube which would allow for sections aligned 45° to the dorsoventral boundary. The red line indicates face of the cube which should be placed downward.

Figure 4.. Preparation of plastic embedding capsule.

A. Plastic embedding capsule (Step B8 above). B. Cut the conical tip off from the capsule using a razor blade (the arrow indicates movement of the blade). Discard the conical tip. C. Place the sample with the desired cutting face downward onto the lid of the capsule (red cube, indicated by arrow). D. Close the capsule and fill from the top with tissue freezing medium (arrow indicates where to add medium).

Figure 5.. Preparing samples for cryosectioning.

A. An empty microtome sample holder; B. Sample affixed to holder using TFM; C. Holder with sample in the cryostat chamber.

Video 1.. Operation of the cryostat.

The anti-roll plate, which is lowered at the beginning of the video, ensures the section remains flat. Note tissue sections will adhere to the microscope slide after gently coming in contact.

Figure 6.. Tissue Section Examples.

A. A near-perfect section through the wing pouch along the anteroposterior compartment boundary. The wing imaginal disc is a relatively flat sac, where the disc proper consists of a pseudostratified columnar epithelium; a simple cuboidal epithelium provides the transition to the simple squamous epithelium that makes up most of the (covering) peripodial membrane (PM). The wing pouch is dome-shaped and surrounded by folds of the future wing hinge (the ventral hinge region has a characteristic folding pattern in sections). The position of the dorsoventral compartment boundary is indicated (arrow, ‘D/V’); dorsal is to the left and ventral to the right. B. An oblique section through the wing pouch is evident from its uneven shape and nuclear layer, the epithelium is too high and of uneven height. C. Although largely in the dorsoventral direction, the section cuts slightly ‘diagonal’ across the disc. No clear parallel cell outlines are evident. The epithelium appears too high and of uneven height. Uneven depth and increased number of nuclei (> 4 nuclear profiles) identify an oblique cut. The sectioned wing pouch appears at an angle to the remainder of the disc, revealing a cut deviating from the dorsoventral axis. Scale bars are 15 µm in A, 9 µm in B, 12 µm in C.