Three-dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy

Abstract

Three-dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step-by-step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings.

Fig. 1

Tissue preparation of both embryonic and adult ears. Inner ears must first be removed from the head and as much tissue as possible should be removed, although the otic capsule itself should not be compromised. Additionally, slight excess of tissue increases the ease of ear movement throughout the remainder of steps (A, B, D). For immunochemistry, remove portions of otic capsule but do not compromise the three-dimensional structure (C). After dissection of the ear from the head, it may be necessary to decalcify the inner ear. This may not be necessary at earlier stages such as E15.5 or P0 (A' and B'), but at later stages such as P21, decalcification is essential (D', 4 consecutive days of decalcification). As the tissue becomes decalcified, the opaque bony structures become translucent (B' and B”). After decalcification and dehydration, the ears are very lightly stained in an Ethanol/Rhodamine solution (A”, B”, D”). It is critical with confocal microscopy to not overstain the specimens. The last step of inner ear preparation is to clear the ear with MSBB/Spalteholz solution (Santi et al., 2009). Ears should be transparent except for a very light pink hue (A'”, B'”, D'”). Scale bar = 10 μm.

Fig. 2

Imaging and 3D reconstruction of the inner ear. Prepared ears are imaged through confocal microscopy and ventral images of the reconstructed inner ear are shown at E15.5 (A), P0 (B) and P21 (D). A properly stained ear should be easily visualized at both the top and bottom of the stack without a change in gain or in clarity. After imaging, regions of interest must be segmented (scala media segmentation shown in A', B', D'). Segmentation is the process of manually tracing regions of interest in programs such as Amira. The segmented images can then be easily reconstructed and viewed in a variety of ways, including, reconstruction of the entire endolymphatic space (A”), reconstruction of both the endolymphatic space and the otic capsule (B”), or reconstruction of any specific region of interest, such as the scala media (D'”). After segmentation and reconstruction, a number of additional analyses are possible, such as endolymphatic space measurements of WT at different ages or WT comparisons to a mutant (MT), in this case, Pax2-Cre N-Myc f/f L-Myc f/f (A'”) or length of the scala media at different ages (D'”). Additionally, volume rendering of Myosin7a immunochemistry allows visualization of hair cells in the inner ear (C) with original IC in insert. Scale bar = 10 μm. Scale bar (C) volume render = 100 μm.

Fig. 3

Flexibility of 3D reconstruction of confocal images. In addition to 3D reconstruction of the inner ear endolymphatic space of mice at any age, confocal imaging can be used to reconstruct a variety of tissues in not only mouse but of other species. Shown here are mouse auditory neurons as they enter the hindbrain and bifurcate in the auditory nucleus (red fibres innervate the basal turn of the cochlea; green fibres innervate the apical portion of the cochlea (A), ear of a Xenopus laevis embryo (B) and ear of a hagfish (C). Scale bar for A and B = 100 μm. Scale bar for C = 1 mm.