Labyrinth

The whole embryos at various ages were immersed in 4% paraformaldehyde overnight at 4°C, after which either the whole embryos (prior to E12.5) were analyzed together or the inner ear was carefully dissected out first (after E13.5). The inner ear was then immersed in 4% paraformaldehyde for 3 hours at 4°C. For trans-section analysis, the whole embryo or inner ear was immersed in 30% sucrose overnight at 4°C and then embedded in Optimum Cutting Temperature compound, frozen in dry ice, and cut into sections 12 μm thick. For whole-mount analysis, the whole cochlear duct and corresponding medial spiral ganglion tissues were divided into three parts, the basal, middle, and apical turns, with the exception that the E13.5 cochlear duct was maintained as a whole.
Both whole mounts and trans-sections were permeabilized and blocked at room temperature for 1 hour in solutions containing 1% bovine serum albumin and 1% Triton X-100 in 10 mM phosphate-buffered saline (PBS, pH 7.4). Tissues were then incubated with primary antibodies in blocking solution (1% bovine serum albumin and 0.1% Triton X-100 in 10 mM PBS) overnight at 4°C, followed by 3 washes for 10 minutes each in 10 mM PBS. Then, tissues were incubated with secondary antibodies in the same blocking solution overnight at 4°C, followed by 3 washes for 10 minutes each in 10 mM PBS. Tissues were then incubated for 30 minutes at room temperature in Hoechst 33342 in 10 mM PBS (Invitrogen, H3570, 1:1,000), followed by 3 washes for 10 minutes each in 10 mM PBS, and finally were mounted in ProLong Gold antifade reagent (Invitrogen, P36934). Samples were dried at room temperature for at least 24 hours. All whole-mount samples were analyzed with a Zeiss META 510 confocal microscope. Trans-section samples at E17.5 were analyzed with the Zeiss META 510 confocal microscope, and those at other embryonic ages were analyzed with our regular fluorescence microscope.
The following primary antibodies were used: anti-myosin-VIIa (rabbit, 1:200, Proteus Bioscience, 25-6790), anti-GFP (chicken, 1:1000, Abcam, ab13970), anti-Sox10 (goat, 1:250, Santa Cruz Biotechnology, sc-17342), anti-NeuroD1 (goat, 1:100, Santa Cruz Biotechnology, sc-1084), anti-Sox2 (goat, 1:1000, Santa Cruz Biotechnology, sc-17320), anti-beta-galactosidase (rabbit, 1:500, ICN, 55976), and anti-TUJ1 (mouse, 1:1000, MMS-435P, Covance). The following secondary antibodies from Invitrogen Company were used: donkey anti-rabbit Alexa Fluor 647 (1:1000, A-31573), donkey anti-goat Alexa Fluor 568 (1:1000, A11057), goat anti-chicken Alexa Fluor 488 (1:1000, A11039), goat anti-mouse Alexa Fluor 568 (1:1000, A11031), and goat anti-rabbit Alexa Fluor 568 (1:1000, A11036).

The process used to automatically identify the labyrinth, ossicles, and external auditory canal relies on atlas-based registration, a common technique in the field of medical imaging. The principle of atlas-based registration is that an image of a known subject can be transformed automatically such that the anatomical structures of the known subject are made to overlap with the corresponding structures in the image of an unknown subject. Given a perfect registration, transformed labels from the known atlas exactly identify the location of the structures in the unknown image. Figure 1 shows an example of atlas-based registration as is typically used in neurosurgical applications. The underlying assumption of this method is that the images of different subjects are topologically similar such that a one-to-one mapping between all corresponding anatomical structures can be established via a smooth transformation. For patients with normal anatomy, this assumption is valid in the anatomical regions surrounding the labyrinth, ossicles, and external auditory canal. Using atlas-based registration methods described previously [6 ,7 ,8 ] and an atlas constructed with a CT of a “normal” subject, we created a registration approach to allow labeling of these structures on temporal bone CT’s.
For the anatomical region surrounding the facial nerve and chorda tympani, topological similarity between images cannot be assumed due to the highly variable pneumatized bone. Therefore, the facial nerve and chorda tympani are identified using another approach, the navigated optimal medial axis and deformable-model algorithm (NOMAD) [8 ]. NOMAD is a general framework for localizing tubular structures. Statistical a-priori intensity and shape information about the structure is stored in a model. Atlas-based registration is used to roughly align this model information to an unknown CT. Using the model information, the optimal axis of the structure is identified. The full structure is then identified by expanding this centerline using deformable-model (ballooning) techniques.
To validate our process, we quantified automated identification error as follows: (1) The temporal bone structures were manually identified in all CT scans by a student rater then verified and corrected by an experienced surgeon. (2) Binary volumes were generated from the manual delineations, with a value of 1 indicating an internal voxel and 0 being an external voxel. (3) Surface voxels were identified in both the automatic and manually generated volumes. (4) For each voxel on the automatic surface, the distance to the closest manual surface voxel was computed. We call this the false positive error distance (FP). Similarly, for each voxel on the manual surface, the distance to the closest automatic surface voxel was computed, which we call the false negative error distance (FN) (See Figure 2). We compute both FP and FN errors because, as shown in Figure 2, the FP and FN errors are not necessarily the same for a given point. In fact, to properly characterize identification errors, computing both distances is necessary.