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Realistic 3D computer model of the gerbil middle ear,

featuring accurate morphology
of bone and soft tissue structures

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Jan A.N. Buytaert1,, W.H.M. Salih1, M. Dierick2,

P. Jacobs2 and Joris J.J. Dirckx1

Laboratory of BioMedical Physics University of Antwerp,

Groenenborgerlaan 171, B-2020 Antwerp, Belgium
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Centre for X-ray Tomography - Ghent University
Proeftuinstraat 86, B-9000 Gent, Belgium

Running title: Accurate 3D gerbil ME model

Corresponding author:
email jan.buytaert@ua.ac.be;
telephone 0032 3 265 3553;
fax 0032 3 265 3318.

Accurate 3D gerbil ME model

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Buytaert et al.

ABSTRACT

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In order to improve realism in middle ear (ME) finite element modeling (FEM),

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comprehensive and precise morphological data are needed. To date, micro-scale X-ray

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computed tomography (CT) recordings have been used as geometric input data for FEM

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models of the ME ossicles. Previously, attempts were made to obtain this data on ME soft

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tissue structures as well. However, due to low X-ray absorption of soft tissue, quality of

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these images is limited. Another popular approach is using histological sections as data for

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3D models, delivering high in-plane resolution for the sections, but the technique is

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destructive in nature and registration of the sections is difficult.

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We combine data from high-resolution CT recordings with data from high-resolution

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orthogonal-plane fluorescence optical-sectioning microscopy (OPFOS), both obtained on the

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same gerbil specimen. State-of-the-art CT delivers high-resolution data on the three-

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dimensional shape of ossicles and other ME bony structures, while the OPFOS setup

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generates data of unprecedented quality both on bone and soft tissue ME structures.

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Each of these techniques is tomographic and non-destructive, and delivers sets of

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automatically aligned virtual sections. The datasets coming from different techniques need

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to be registered with respect to each other. By combining both datasets, we obtain a

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complete high-resolution morphological model of all functional components in the gerbil

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ME. The resulting three-dimensional model can be readily imported in FEM software and is

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made freely available to the research community.

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In this paper, we discuss the methods used, present the resulting merged model and discuss

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morphological properties of the soft tissue structures, such as muscles and ligaments.

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KEYWORDS:

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gerbil, middle ear, modeling, high-resolution, three-dimensional, soft tissue, surface mesh,

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micro-scale X-ray computed tomography (micro-CT), orthogonal-plane fluorescence optical-

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sectioning microscopy (OPFOS)

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INTRODUCTION

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The middle ear (ME) forms a small three-dimensional (3D) biomechanical system. It mainly

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consists of the tympanic membrane (TM), three ossicles malleus, incus and stapes and

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their supporting ligaments and muscles. The remarkable performance of ME mechanics is

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too complex to be understood intuitively. For better understanding, ME modeling was

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introduced. Finite-element (computer) modeling (FEM) has become an established

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numerical technique to simulate ME mechanics. In ME research, the technique was first

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introduced by Funnell and Laszlo, 1978. As one of its inputs, FEM requires 3D morphological

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computer models of the ME components. These mesh models consist of a finite number of

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elements, e.g. tetrahedra or hexahedra.

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Current morphological models are either incomplete, low resolution and/or contain

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rudimentary shapes to represent (some) ME components. Pioneering work in this field used

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manually drawn geometrical shapes in the computer to represent the ME malleus, incus and

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stapes (Wada et al., 1992; Ladak and Funnell, 1996; Blayney et al., 1997; Eiber et al., 2000;

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Prendergast et al., 2000; Koike et al., 2002). Some authors used low or modest resolution

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shapes measured with medical X-ray computed tomography (CT) (Rodt et al., 2002; Lee et

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al., 2006) or with tabletop micro-CT (CT) devices (Decraemer et al., 2002; 2003; Elkhouri et

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al., 2006; Puria and Steele, 2010; Lee et al., 2010). Other authors used histological sectioning

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(Funnell et al., 1992; Sun et al., 2002) or magnetic resonance microscopy (MRM, NMR, MRI)

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(Funnell et al., 2005; Elkhouri et al., 2006), but again with modest resolutions. In many

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models, the suspensory ligaments and muscle tendons are either omitted (Wada et al. 1992;

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Ladak & Funnell 1996; Blayney et al. 1997; Lord et al. 1999; Rodt et al. 2002) or manually

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incorporated as simple geometrical objects such as blocks, cylinders or cones (Prendergast

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et al. 2000; Beer et al. 2000; Koike et al. 2002; Sun et al. 2002; Lee et al. 2006). To the

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authors knowledge, only models by Wang et al., 2006; Gan et al., 2007; Cheng and Gan,

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2008 (using histological sectioning) and by Mikhael et al., 2004; Sim and Puria, 2008; Ruf et

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al., 2009 (using X-ray techniques) contain actually measured shapes of soft tissue structures,

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but in low resolution.

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To improve realism in FEM calculations, ME geometry models need to incorporate all and

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accurate shapes of the ossicles and suspensory soft tissue structures (Decraemer et al.,

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2003). As the computer calculating capacity has grown to a point where it can manage large

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amounts of data, and as the scientific measurement apparatus is now capable of high-

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resolution imaging on all kinds of tissue types, the time has come to incorporate realistic and

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complete morphological 3D ME models in FEM. We point out that it might not be necessary,

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even not numerically feasible, to perform FEM with all structures described in the highest

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detail. On the other hand, it is difficult to decide beforehand how precise the morphologic

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model needs to be. Therefore, we think it is important to first have a high-resolution

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morphologic model available, which can then be simplified to the modelers judgment.

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In the current paper we provide these high-quality models by combining data originating

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from two different tomographic techniques: State-of-the-art CT tomography allows to

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obtain precise data on bony structures, but due to the low X-ray absorption of soft tissue, CT

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generates poor quality images of soft tissue (Lemmerling et al., 1997). Therefore we

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combine these data with measurements from another and relatively new technique:

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Orthogonal-plane fluorescence optical-sectioning (OPFOS) microscopy or tomography. This

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method images both bone and soft tissue at the same time and in high-resolution.

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As gerbil is one of the standard laboratory animal models in fundamental hearing research,

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we chose this species for our first model.

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MATERIALS and METHODS

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Dissection

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All animal manipulations in this work were performed in accordance with Belgian

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legislation and the directives set by the Ethical committee on Animal Experimentation of our

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institution (University of Antwerp, Belgium). Three adult Mongolian gerbils (Meriones

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unguiculatus), aged between three and six months, were used. They were housed in cages

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with food and water ad libitum in our animal facility.

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The animals were euthanized using carbon dioxide, followed by a cardiac perfusion with

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physiological fluid to rinse out all the blood from the gerbil head blood vessels. This step is

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necessary to allow for OPFOS tomography (as we will explain below). The gerbils are then

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decapitated and the right temporal bones were isolated. The specimens were reduced in size

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until only the bulla was left containing the middle and inner ear, cf. Figure 1. During the

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harvesting of these bullas, continuous moistening with mist from an ultrasonic humidifier

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(Bionaire BT-204) was applied to avoid dehydration.

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Cross sectional imaging of bone with X-ray tomography

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The first stage of 3D tomographic recording of the ME was achieved using micro-scale

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X-ray computed tomography. The dissected bullae were enclosed in separate Eppendorf

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vials, together with a calibration object and a few droplets of physiological fluid at the

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bottom. In this way a 100% saturated humid environment was created to avoid dehydration

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artifacts. Another droplet of fluid was placed in the ear canal which could help to

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distinguish the outline border of the TM shape with the air-filled ME cavity. Water and air

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have a slightly different X-ray absorption coefficient, so a layer of water on the extremely

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thin TM can help to reveal its medial shape outline. In previous work, we measured the

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shape of the eardrum before and after putting fluid on the membrane: even with a 10 mm

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water column in the ear canal, no measurable deformation was found with moir

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profilometry of 15 m resolution (Buytaert et al., 2009). As the droplet of water is less than 3

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mm high (inducing a pressure load of 30 Pa) the TM deformation is well below the CT

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measurement resolution. The Eppendorf vials (made from polypropylene) are almost X-ray

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transparent. Especially bone absorbs X-rays well, thus creating a high contrast in

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transmission recordings. The small calibration objects were custom-made from polyvinyl

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chloride (PVC) in our mechanical workshop and possess about the same X-ray absorption

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properties as thin bone (Gea et al., 2005). They served as an independent calibration to

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verify the CT device specifications.

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The vials containing gerbil specimens were scanned at the UGCT scanning facility at Ghent

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University (www.ugct.ugent.be) using a custom-built CT scanner of medium energy (up to

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160 keV). The scanner has a directional X-ray tube with a feature recognition capability up to
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2 m (Masschaele et al., 2007). The scans were performed at a tube voltage of 120 kV

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(photon energy levels ranging from 0 to 120 keV) and a current of 58 A. A custom-made vial

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holder was mounted on a computer-controlled rotation table (MICOS, UPR160F-AIR). For

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each specimen a series of 1000 shadow projections of 1496x1880 pixels was recorded

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covering an object rotation of 360 degrees (or one recording every 0.36 degrees).

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Reconstruction of the tomographic data volume to serial sections was achieved using the

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back-projection algorithms of the Octopus software package (Dierick et al., 2004), resulting

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in 1780 reconstructed cross sections of 1496x1496 pixels. From these calculated cross

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sections with an isometric pixel size of 8.5 m, accurate 3D models of the three ossicles and

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other bony structures were generated. All three datasets cover a volume of 15.1 x 12.7 x

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12.7 mm (1780x1496x1496 times 8.5 m).

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Cross sectional imaging of soft tissue with optical tomography

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Due to the low X-ray absorption of soft tissue, another tomographic technique was

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needed: orthogonal-plane fluorescence optical-sectioning microscopy or OPFOS (Voie et al.,

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1993). OPFOS was initially developed to image the inner ear cochlea, but it has also been

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used in ME studies (Voie, 2002; Buytaert and Dirckx, 2007; 2009). In the OPFOS method,

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parallel optical sections through a macroscopic biomedical specimen are created by means

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of a thin sheet of laser light, and the fluorescence originating from within the cross section of

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the light sheet with the tissue is recorded in the direction perpendicular to the plane of the

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laser light. The light emitted by the specimen originate from auto-fluorescence or from

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staining the specimen with a fluorescent dye. OPFOS images both bone and soft tissue at the

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same time and in real-time, as no (back-projection) calculations are required. It allows

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region-of-interest (ROI) imaging and has both a high sectioning and a high in-plane

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resolution. Hence, perfectly and automatically aligned images of virtual cross sections can be

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obtained. OPFOS scanning was performed at the Laboratory of BioMedical Physics at the

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University of Antwerp (www.ua.ac.be/bimef) with a custom-built setup using bi-directional

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light-sheet illumination (Buytaert and Dirckx, 2009; Buytaert, 2010).

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For OPFOS imaging, an elaborate specimen preparation is needed (Voie, 2002; Buytaert and

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Dirckx, 2009), as the technique requires the specimens to be perfectly transparent. Before
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CT scanning, all blood was removed from the blood vessels, as coagulated blood cannot be

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made transparent afterwards. After CT recording, a 10% neutral buffered Formalin bath

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was applied. Next, all calcium was removed using 10% EDTA in water solution combined with

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microwaves. Because of this decalcification, the OPFOS method has to be performed second

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after CT X-ray scanning. Then, the specimens were dehydrated using a slowly graded

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ethanol series, up till 100%. Next, all tissue was refractive index matched using a slowly

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graded Spalteholz fluid series, again up till 100%. As a result, the specimens become entirely

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transparent when submerged in pure Splateholz fluid. Finally, to obtain stronger

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fluorescence, the specimens are stained with Rhodamine B.

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Both soft tissue and bone were made transparent and fluorescent; hence, both tissue types

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are visualized with the technique. We focused on region-of-interest (ROI) OPFOS imaging of

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ME ligaments, tendons and muscles, while images of the (often larger) bony structures are

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more easily obtained from CT. Comparison of high-resolution CT and OPFOS data allows

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us to distinguish bone from soft tissue in the OPFOS data. Merging of the two datasets

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generates the complete ME model with all of its functional components accounted for.

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The shape of the TM was obtained from the CT data. The OPFOS technique is able to

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visualize this extremely thin tissue when performing ROI imaging on a small part of the

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membrane, cf. Figure 2. However, to image the membrane full-field with OPFOS, one needs

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to zoom out and the resolution needed to adequately visualize this thin membrane is lost.

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Furthermore, the eardrum is prone to preparation artifacts: Because the gerbil specimens

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went through an extensive procedure of tissue fixation, decalcification, dehydration and

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Spalteholz treatment, the extremely thin TM can get deformed. Therefore, the data on

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eardrum shape are obtained from the CT images, recorded before any specimen processing

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was applied. X-rays are normally not suited to image soft tissue, especially if it is very thin,

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like the eardrum. We tried to counter this problem by applying a droplet of physiological

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fluid through the ear canal on top of the membrane. The medial border of the droplet and

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eardrum then become more easily distinguishable from air in the ME cavity. In this way, the

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membrane outline will be obtained without deformation and with adequate resolution.

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Apart from the specimen preparation, the OPFOS method has another disadvantage as it
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suffers from stripe artifacts. Opaque regions or areas of less transparency locally reduce the

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intensity of the laser light sectioning sheet, causing shadow lines or stripes in the rest of the

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image. This is partially countered by simultaneous dual light sheet illumination in our setup

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(Buytaert and Dirckx, 2009).

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Measuring and analyzing the OPFOS data is very time-consuming; therefore, only one gerbil

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ear has been processed. On the other hand, the CT data of all three gerbils was analyzed.

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Visual observations

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We performed visual observations of the orientation, location, shape and suspension

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of the ossicular chain inside opened ME bullae with an operating microscope (Zeiss, OPMI

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Sensera S7). When 3D computer data, models and results were obtained from CT or OPFOS

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with striking features, they were compared to qualitative observations of the real geometry

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in opened bullae with the operating microscope to verify their interpretation. These

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experiences gave us the necessary expertise to confirm the 3D model results and conclusions

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of the present paper. For instance, after a targeted dissection we could visually confirm that

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the posterior incudal ligament in gerbil indeed exists as one whole band instead of two

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separate structures, as we found in our OPFOS data and model.

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3D segmentation and reconstruction

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After obtaining several series of object cross sections one CT set originating from

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back projection calculations, and several ROI datasets from direct OPFOS recordings we

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identified and segmented the relevant structures in all images. The goal of segmentation is

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to locate objects boundaries, which in turn allows software to build 3D surface meshes by

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triangulation.

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In our case, segmentation was done manually for thousands of sections using the

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commercial image segmentation and three-dimensional surface mesh generating software

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package Amira 5.3 (Visage Imaging). Manual segmentation might seem primitive and time-

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consuming; but using our morphological expertise, manual segmentation delivers better

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results than purely automated segmentation based on thresholding of gray scale values. The
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Amira software package uses the marching cubes algorithm for triangulation. It takes eight

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neighboring voxel locations at a time (forming an imaginary cube), after which the

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polygon(s) needed to represent the part of the isosurface that passes through this cube are

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determined. The individual polygons are finally fused into the intended surface. This leads to

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subvoxel triangulation that easily manages sharp angles. When smoothing or simplification

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(reduction of the number of triangles) is used, the program takes the steepness of the

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surface into account: Flat surface parts are more reduced than curved parts.

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As final result, we end up with triangulated surface meshes for the CT and OPFOS datasets.

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These can be further developed into finite-element volume meshes using Amira or other

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packages. On the website of the Laboratory of BioMedical Physics group we suggest some

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powerful and open-source volume generating software, e.g. PreView.

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Merging of CT and OPFOS models

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All cross sections in a CT dataset, and therefore all models of ME components

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originating from it, are inherently perfectly aligned within the data stack. The OPFOS

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datasets were focused on the soft tissue by separate ROI recordings. However, parts of the

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bone are included in the OPFOS ROI recordings as well. The cross sections within each ROI

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OPFOS data stack are also perfectly aligned, but the resulting mesh models per stack are

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unrelated to the other OPFOS datasets (because of different ROI zooming and/or other

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slicing orientation) and unrelated to the CT dataset and models.

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To merge the OPFOS data with the CT data, the CT dataset was used as a reference. We

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did not merge the 2D image cross sections, but the 3D mesh models: All partial bone models

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from ROI OPFOS were three-dimensionally aligned to corresponding parts of the CT models

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using an iterative spatial transformation least-squares minimization process of the Amira

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software package. This process uses the Iterative Closest Point (ICP) algorithm to minimize

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the difference between two point clouds (e.g. all surface nodes of respectively an OPFOS and

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a CT mesh model). ICP iteratively revises the spatial transformation (6 degrees of freedom

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for translation and rotation) needed to minimize the Euclidean distance between the points

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of two datasets. This concept is referred to as the Procrustes superimposition method: The
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root-mean-square (RMS) of the distances between corresponding points of the two surfaces

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are evaluated. Corresponding point pairs are created by finding the closest point of the

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reference (CT) surface mesh for each point of the other (OPFOS) surface mesh. When the

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two surfaces are identical and perfectly superimposed, the RMS of all corresponding point

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distances will be zero. In the case of the OPFOS versus the CT stapes model for instance, we

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obtained a root-mean-square difference of 17 m (or two CT voxels). After obtaining such a

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good match between the OPFOS and CT bone model, we applied the same spatial

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transformation to the OPFOS soft tissue mesh(es) from that OPFOS dataset. In this way, all

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OPFOS datasets were combined with CT data into one model.

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RESULTS

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Computed tomography

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Three gerbil ears were recorded with CT, delivering three isometric data stacks of

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reconstructed cross sections (pixel size 8.5x8.5 m, separated 8.5 m). To illustrate the

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image quality, we present one CT cross section in Figure 3. Full movies of all cross sections

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are available on our website and the entire dataset is available upon request. Notice how

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distinguishable the ossicle boundaries, the incudomallear and incudostapedial joint cleft and

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the annular ligament cleft are in the figure. This high contrast and resolution facilitates the

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segmentation process considerably.

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Our main attention went to the ME; but separate 3D surface meshes were also created of

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the fluid-filled bony labyrinth of the inner ear (cochlea scalae and modiolus, and vestibular

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apparatus), cf. Figure 1 and Figure 4. The ME bulla air cavities of all gerbils are modeled as

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well. They give an indication of the enclosed air volume in the ME, cf. Table 1. These

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segmented volumes include the volume of the ossicles, ligaments and muscles. Finally, a

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separate rudimentary mesh of all bone using a fixed segmentation threshold was made.

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Using transparent rendering for this large model, one can virtually look inside the bulla and

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observe the ossicles and inner ear inside, cf. Figure 1. We listed volume, dimensions and

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several other properties of the ossicles, the TM and the ME bulla cavity in Table 1 to Table 3.

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These and other quantitative data is readily and accurately available from our models.
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The mass of malleus, incus and stapes are respectively 1.145 mg, 0.633 mg and 0.116 mg as

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reported by Nummela, 1995. Adopting these representative values for our specimen in

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combination with the volumes given in Table 1, we get an average ossicle bone density of

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1.37 10 kg/m for the stapes and 1.74 10 kg/m for incus and malleus.

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Note that the outline of the TM was surprisingly but successfully visualized using CT. The

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resolution was just high enough to show the shape outline of the extremely thin membrane.

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Thickness information could not be obtained. Using a fluid droplet in the ear canal to aid in

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distinguishing the medial border of the eardrum partially failed, as can be seen in Figure 3:

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Fluid is not covering the entire membrane surface in the ear canal because of an air bubble.

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Finally, we could observe channels (blood vessels) inside the ossicles, occurring especially in

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the incus and malleus bone, cf. Figure 5. The ossicular surface shapes are almost identical

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between all three animals, and the same is true for the size, volume and branching layout of

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the major channels inside them.

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OPFOS tomography

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We will now discuss all identified (soft) tissue structures of the ME of gerbil 2,

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measured with OPFOS.

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Posterior incudal ligament

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Using CT, the posterior incudal ligament cannot be found, cf. Figure 6A-B, while using

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OPFOS it is clearly visible, cf. Figure 6C-E. This comparison between the two tomographic

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imaging techniques clearly demonstrates the usefulness of combining the two methods.

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After segmentation and 3D representation, cf. Figure 6F-G, one can see that the ligament is

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built as one whole part and forms one sickle-shaped band of fibrous tissue. Its tiny volume

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amounts to 0.013mm. The sickle has its smallest thickness (orthogonally to the image plane

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of Figure 6F) of 42 m near the incus short process and broadens to 190 m towards the

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bulla edge. The contact area at the middle-ear cavity wall is also a bit larger than the contact

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area on the incus crus.

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Anterior mallear ligament

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The anterior process of the malleus has the shape of a (partially opened) hand-held Japanese

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folding fan, reaching towards the anterior bulla wall, cf. Figure 7 and Figure 8. The

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connective soft tissue of the anterior mallear ligament, which should connect the process to

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the bulla, is undistinguishable from bone, both in the OPFOS as in the CT recordings. This

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ligament is probably more ossified or cartilaginous than in some other species and no

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separate soft tissue model could be made.

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Superior mallear and incudal ligament and lateral mallear ligament

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According to real-time OPFOS observations, no superior mallear and incudal ligament are

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present in the gerbil ME, which is confirmed by visual observations with the operating

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microscope. In addition, no lateral mallear ligament could be discerned with either method.

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Tensor tympani muscle and tendon

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Figure 2A shows a high-resolution OPFOS section image through the tensor tympani muscle

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and tendon, the TM, the malleus manubrium and the bulla. This image demonstrates

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OPFOS capability to image bone and soft tissue in high-resolution.

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After the segmentation and triangulation process, the volume of the tensor tympani muscle

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and tendon can be calculated from the obtained 3D model, and was found to be 0.486 mm.

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The distance between the two most distant points on the combined structure is 3.25 mm.

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The diameter of the muscle tendon varies between 50-80 m.

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The cross-sectional area of a muscle (rather than volume or length) determines the amount

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of force it can generate. A first rough estimate of the order of magnitude of the maximum

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generated force of the muscle can be derived as follows. By dividing the muscle belly volume

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by an average muscle fiber length of 400 m (estimated from the OPFOS images), we end up

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with a cross sectional area of 1.2 10-2 cm. A common conversion factor from this area to

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the maximal isometric contraction force is given by 25 N/cm for skeletal muscle (Nigg and

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Herzog, 1999), giving a maximally generated force of this muscle of 0.3 N. An interesting

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comparison of the effect of this force on the malleus can be made by translating this number
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into a corresponding static pressure working on the TM from the ear canal side. Dividing the

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force of 0.3 N by the (projected) area of the pars tensa of the TM of gerbil 2 (13.64 mm, cf.

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Table 2), we obtain a (maximum) static pressure of 22 kPa. The magnitude of this pressure

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falls in the range of static pressures associated with scuba diving or taking an airplane.

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The final merged 3D model shows that the tensor tympani muscle belly is larger than

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expected from visual observations. Its main part is hidden as it is situated in a gap between

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the spiraled cochlear dome and the bulla wall.

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Stapedial artery

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A typical anatomical feature of the gerbil ME is the stapedial artery running through a bony

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channel on the surface of the first cochlear turn and passing through the stapes crura in the

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ME air cavity. Using OPFOS, it was possible to image this relatively large stapedial artery, cf.

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Figure 9. We could even distinguish and separately model the stapedial artery soft tissue

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wall (the actual blood vessel) and its fluid-filled lumen.

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The diameter of the blood vessel was the smallest in between the crura and amounted to

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355 m with (i.e. outer diameter) and 275 m without (i.e. inner diameter) the blood vessel

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soft tissue wall. The wall had a thickness of about 40 m.

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Stapedius muscle and tendon

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After segmentation of the stapedius muscle and tendon, we end up with the mesh shown in

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Figure 9. The tiny volume enclosed in this (tendon & muscle) mesh amounts to 0.085 mm

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and the two most distant points on the combined structure are 1.81 mm apart. The diameter

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of the tendon varies between 40-55 m. If we again divide the volume by an estimated

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average muscle fiber length of 350 m, we get a cross sectional area of 2.4 10-3 cm.

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Multiplying this value by 25 N/cm gives an estimation of 0.06 N for the maximum force the

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muscle can produce.

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The merged 3D model shows that the stapedius muscle body is attached to the lateral

430

(horizontal) semi-circular canal, cf. Figure 9. In the figure, a gap is seen between the semi-

431

circular canal and the muscle because only the fluid-filled cavity of the canal is shown. When
12

Accurate 3D gerbil ME model

Buytaert et al.

432

showing bone as well, one sees the muscle clasps firmly around the lateral semi-circular

433

canal wall.

434
435

Joint clefts

436

As can be seen in Figure 2B, the incudomallear and incudostapedial joints can be easily

437

distinguished on high-resolution OPFOS cross sections and appear to form a tight

438

connection. CT data also show both clefts, from which we made three-dimensional meshes.

439
440

The incudomallear joint connects the incus and malleus and has the shape of a twisted

441

saddle. The gap or cleft between the ossicles could contain synovial fluid as it is considered a

442

synovial joint; however, this is not confirmed from our OPFOS measurements nor CT data

443

in gerbil. No fluid or open space is detected in the joint cleft, and the joint seems quite rigid.

444

This rigidness was already reported for other species by Guinan and Peake, 1967; Gundersen

445

and Hgmoen, 1976. The thickness of the joint varies from nearly zero to 51 m. The gap or

446

joint tissue is thinner at the lateral side.

447
448

The incudostapedial joint connects the incus lenticular process with the head of the stapes.

449

Our model of this synovial joint shows an oval disk with an approximately even thickness of

450

25.5 m. Again, the joint cleft seems to possess no synovial fluid and forms a rigid

451

connection, which has also been reported in cat (Funnell et al., 2005).

452
453

OPFOS also visualized the stapedial annular ligament cleft in which the annular stapedial

454

ligament is situated, forming a syndesmosis joint. A syndesmosis is a slightly movable

455

articulation where bony surfaces are tightly united by a fibrous tissue ligament (Laurent,

456

1998). The high resolution of the OPFOS data allows to make a 3D mesh of this thin

457

structure, cf. Figure 10. The thickness of the ligament varies between 8 to 18 m, confirmed

458

by the gap seen in the CT cleft model which is about 12-18 m.

459
460

Chorda Tympani

461

The chorda tympani nerve branches from the facial nerve and runs through the ME air

462

cavity. In gerbil, the nerve jumps from a sort of support beam at the superior bulla wall to

463

the malleus where it is tightly connected to the malleus neck in the vicinity of which the
13

Accurate 3D gerbil ME model

Buytaert et al.

464

tensor tympani muscle connects as well, cf. Figure 7. It hangs in the ME air space passing the

465

incudal long process laterally and the manubrium medially. It rounds the malleus neck from

466

the posterior to the anterior side, passing the tensor tympani tendon inferiorly. At the

467

anterior side, it lies on the anterior process sheet until it disappears in a fissure of the bulla

468

wall again. It was unexpected that the chorda tympani could be visualized so well in OPFOS

469

cross sections, cf. Figure 8, because myelin nerve sheets can in principle not be made

470

transparent by the Spalteholz process. Apparently, because the nerve is thin enough, the

471

blurring effect of the less transparent chorda tympani was negligible.

472
473
474

Merging of CT and OPFOS models

475

As described before, we obtained a series of cross sectional images from CT with

476

bone only, cf. Figure 3, and from OPFOS with bony and soft tissue structures, cf. Figure 2,

477

Figure 6 and Figure 8. With OPFOS, we performed ROI recording of all soft tissue structures,

478

so only incomplete parts of the ossicles were measured. However, using these partial models

479

of ossicles and/or bulla bone that were recorded together with the soft tissue, we could

480

align these bony structures (and thus the soft tissue structures as well) to the CT bone

481

models, cf. Figure 11, using the Procrustes superimposition method.

482
483

The merging and alignment of bony structures revealed that some shrinking of the gerbil 2

484

specimen had occurred despite of our careful efforts during preparation. Using the warping

485

procedure in Amira (similar to the Procrustes superimposition method, only allowing for

486

scaling in every dimension as well), we found a shrinking factor of 8.4% in all three

487

dimensions. After applying the spatial transformation and up-scaling, the OPFOS soft tissue

488

meshes fit rather well in between the CT bone mesh models. For instance, corresponding

489

bony parts of the malleus from OPFOS using a scale-factor of 8.4% were aligned with the

490

malleus from CT. After applying the same scaling and spatial transformation to the tensor

491

tympani, its tendon attaches to the malleus, cf. Figure 11, and at the other side its muscle

492

body inserts nicely in a bony cavity of the bulla of the inverse shape, cf. Figure 12. This and

493

similar facts give us confidence in the merging of the data.

494
495
14

Accurate 3D gerbil ME model

496

Buytaert et al.

DISCUSSION

497
498

Imaging method

499

Several methods exist to measure and image the ME for the creation of FEM models.

500

CT in itself is mainly suited to image the bony structures. CT using contrast agents is a

501

valuable alternative to our combined approach (Metscher, 2009). However, it is difficult to

502

discriminate between bone and soft tissue, so it would be necessary to do CT scans before

503

and after staining, and merge the data as we now did with OPFOS. OPFOS offers a resolution

504

down to 2 micrometer, which is seldom achieved in CT. For this reason, we preferred

505

OPFOS to obtain the soft tissue data. Multiple energy CT techniques have also proven to be a

506

valuable method for discriminating between soft tissue and bone in CT images (Johnson et

507

al., 2007; Granton et al., 2008). For large macroscopic structures the technique is indeed

508

feasible, however, it becomes more difficult in the case of microscopic samples. The position

509

of the micro-focus spot changes in an X-ray tube when its energy or source is altered. As a

510

result, the datasets are slightly shifted in a complicated way, and tissue discrimination can

511

no longer be done by simple subtraction or division. Gradually, these technical issues are

512

being solved, so in the future dual-energy CT may be used to measure and discriminate soft

513

tissue and bone.

514
515

The most used alternative to our method is conventional histological sectioning, which is

516

unsurpassed in resolution and produces data on the bone and soft tissue simultaneously.

517

Both the histological method as our combined method need a similar specimen preparation

518

that can induce shrinking (Lane and Rli, 1983; Henson et al., 1994). Our method is

519

considered non-destructive (as multiple measurements can be done on the sample) while

520

histology can only measure the sample once and in one slicing orientation because of the

521

need for physical cutting of the specimen. Furthermore, these 2D slices are often deformed

522

during slicing, requiring difficult image processing and registration of all slices before

523

generating a 3D model. CT and OPFOS each deliver perfectly and automatically aligned

524

cross sections that require no post-processing. Instead of registering every 2D slice, our

525

method only needs to register complete 3D meshes of all submodels to one another. OPFOS

526

further allows real-time virtual sectioning and imaging.

527
15

Accurate 3D gerbil ME model

Buytaert et al.

528
529

Human versus gerbil

530

When using animal models, it is important to be aware of the differences with human

531

ME morphology. Figure 13 shows a schematic representation of all human ME components.

532

In addition to the data prepared in this paper, we confirmed our findings in other gerbil ears

533

during other studies using OPFOS and visual inspection with the operation microscope.

534
535

We found that in gerbil no superior incudal, no superior mallear and no lateral mallear

536

ligament are present, contrary to the case in humans. The presence and/or function of

537

superior attachments to malleus and incus as suspensory structures are of controversy,

538

though many mathematical models or drawings of the human ME include such structures,

539

cf. Table 2.1 in Mikhael, 2005; and Merchant and Nadol Jr., 2010.

540
541

It has been proposed by Rosowski et al., 1999 that the anterior mallear ligament is a bony

542

connection to the bulla, while Elkhouri et al., 2006 observed the presence of some

543

connective tissue. Our OPFOS measurements could not distinguish any soft tissue, and our

544

CT measurement showed an ossified or cartilaginous connection. The anterior process also

545

had a less pronounced shape in human than the Japanese fan-shaped structure in gerbil.

546
547

The posterior incudal ligament, which connects the incus short crus to the fossa incudes,

548

exists in many different configurations, as is illustrated in Figure 14 by Funnell, 1972 (based

549

on work by Kobayashi). From the OPFOS sections, cf. Figure 6C-E, the gerbil posterior

550

ligament appears to fall in the category of human and cat configurations. However, it is only

551

possible to appreciate the true configuration in 3D, cf. Figure 6F-G, which clearly places this

552

gerbil ligament in the category of guinea pig and rabbit. The posterior incudal ligament

553

consists of one sickle-shaped part. According to Sim and Puria, 2008, it has been observed

554

that in human the two parts shown in Figure 14 are also connected around the tip of the

555

short crus of the incus to form a single continuous ligament rather than two separate

556

ligaments. In this respect, gerbil and human ME then would be alike.

557
558

We also found the chorda tympani nerve to be present in a special arrangement in gerbil,

559

and more tightly connected to the malleus ossicle than in human: In human this nerve
16

Accurate 3D gerbil ME model

Buytaert et al.

560

traverses the open space of the ME cavity without actually attaching to the ossicles. In

561

gerbil, there exists a tight connection with the malleus neck and the nerve lies on top of the

562

Japanese fan-shaped anterior process sheet, cf. Figure 7 and Figure 8. Furthermore, the

563

topographic relation of the chorda tympani to the tensor tympani muscle differs from

564

human. In gerbil it runs hypotensoric (inferiorly to the tensor tympani) and in between the

565

muscle and manubrium, as was confirmed in a recent publication by Ruf et al., 2009; while in

566

human it passes epitensoric (superiorly to the tensor tympani), e.g. Maier, 2008.

567
568

We derived the ossicle bone density from our volume measurements and from mass data

569

from literature. We obtained an ossicle bone density of 1.37 10 kg/m for the stapes and

570

1.74 10 kg/m for incus and malleus. In comparison to human, the averaged malleus

571

density is found to be 2.31 10 kg/m and the averaged incus density is 2.14 10 kg/m

572

(Sim and Puria, 2008). Another source mentions an average stapes density of 2.2 10 kg/m

573

in human (Kirikae, 1960; Gan et al., 2004). Hence, gerbil ossicle densities appear to be

574

significantly lower than in human.

575
576

Another contrast to human is that the stapedial artery is usually present in gerbil, while

577

seldom in human.

578
579

Finally, our observations show that the gerbil manubrium of malleus is tightly fused over its

580

full length with the TM, while in human it is mainly only fixed at the tip and lateral process of

581

the manubrium (Koike et al., 2002).

582
583
584

Resolution

585

We used state-of-the-art X-ray micro-computed tomography and the relatively new

586

orthogonal-plane fluorescence optical-sectioning microscopy on the ME. In previous CT

587

based studies of the ME, the following model resolutions were reported: 5.5 m on gerbil

588

(Elkhouri et al., 2006), 6 m on human (Hagr et al., 2004), 10 m on cat (Decraemer et al.,

589

2003) and 10 m on human (Vogel, 1999). Though these numbers are comparable to our

590

isometric 8.5 m voxel size for CT on gerbil bone, our data and models are of much higher

591

quality than those shown in previous work. One reason might be that the previous authors
17

Accurate 3D gerbil ME model

Buytaert et al.

592

stated voxel size instead of resolution, while we actually achieve a true resolution of 8.5 m.

593

Other factors such as scan parameter settings could also account for differences in image

594

quality.

595
596

ME soft tissue imaged with medical CT devices gave poor resolution (Lemmerling et al.,

597

1997), and CT delivered modest resolution (Sim and Puria, 2008). The same goes for MRI

598

measurements of gerbil soft tissue structures, e.g. 45 m (Elkhouri et al., 2006). OPFOS is

599

clearly better suited to achieve high-resolution sections on ligaments and muscles with

600

pixel sizes ranging from 1 to 5 m as can be seen from our sections and 3D models, cf.

601

Figure 2 and Figure 6 til Figure 11.

602
603

The OPFOS method itself is little known but it was the first setup of the growing field now

604

known as (Laser) Light Sheet based Fluorescence Microscopy (LSFM). The many different

605

implementations and improvements of the technique have been listed in a review article by

606

Buytaert et al., 2011. The construction of an OPFOS/LSFM setup is well feasible in the sense

607

that all parts needed are readily available on the market. Researchers interested in the

608

construction of such a setup or in collaboration are welcome to contact the authors, and

609

even the first commercial devices are becoming available (Buytaert et al., 2011).

610
611
612

Artifacts

613

Segmentation of the fluid-filled inner ear channels in the CT data showed that the

614

round window in all three models is prominently bulged inwards toward the cochlea. This

615

might indicate either a small overpressure in the ME air cavity or a loss of cochlear fluid

616

because of dehydration or leakage.

617
618

Merging of OPFOS and CT data revealed shrinking of the soft and bony tissue, most likely

619

caused by the elaborate OPFOS specimen preparation (e.g. tissue fixation, decalcification,

620

dehydration and Spalteholz treatment), though previous authors reported that this

621

procedure induced negligible shrinking (Voie, 2002; Valk et al., 2005; Hofman et al., 2008).

622

Thanks to the combination of OPFOS with CT, we have undeformed reference data that we

623

can use to derive a scaling factor. Homogeneous scaling with 8% of the OPFOS (bone and
18

Accurate 3D gerbil ME model

Buytaert et al.

624

soft tissue) models has partially corrected for the shrinking artifact. After decalcification of

625

the sample, bone is reduced to a collagen matrix. The effect of decalcification cannot be

626

investigated with CT as all calcium is removed and X-ray absorption becomes negligible. It

627

is, however, a reasonable assumption that dehydration will have a similar (and

628

homogeneous) shrinking effect on both soft tissue and decalcified bone. In histology, the

629

same specimen preparation (decalcification and dehydration) is performed and the same

630

assumption (homogeneous shrinkage) is adopted.

631
632

Another artifact related to specimen preparation was noticed on the stapes. The footplate of

633

the stapes is clearly convex and bulges inwards to the cochlea in the CT models, e.g. Figure

634

10. After decalcification, dehydration and Spalteholz treatment, the footplate of the OPFOS

635

model showed some relaxation and shriveling of its convex shape. The models available for

636

download therefore consist of CT data for bone and OPFOS data for soft tissue meshes.

637
638

Stripe artifacts in OPFOS were strongly reduced but not entirely eliminated by our bi-

639

directional illumination/sectioning OPFOS setup. In combination with manual segmentation,

640

which also partially corrects for this artifact, no effect remained in the models so no image

641

post-processing of the data was required.

642
643

OPFOS was not suited to image the TM, but the full-field outline of the TM shape was

644

obtained from CT: We could not measure a volume model of the TM with the correct

645

thickness, but only a surface model. FEM modelers can, however, use the surface shape

646

directly as a shell model, cf. Gan et al., 2004; Elkhouri et al., 2006; and apply either a uniform

647

or a varying measured thickness distribution to their own choosing (as different approaches

648

are taken by different modelers). Table 2 mentions average thickness data at three TM

649

regions, measured on eleven gerbil TMs with confocal microscopy (Kuypers et al., 2005).

650
651
652

Open source availability

653

All three-dimensional data and surface mesh models presented in this paper are

654

freely available for educational and research purposes on the website of the Laboratory of

655

BioMedical Physics: http://www.ua.ac.be/bimef/models.

19

Accurate 3D gerbil ME model

Buytaert et al.

656
657

Several educational and research 3D models have also been made available in the past

658

(Eaton-Peabody Laboratory of Auditory Physiology, Ear & Auditory Research Laboratory,

659

Auditory Mechanics Laboratory, Auditory Research Laboratory, OtoBiomechanics Group).

660
661
662

CONCLUSION

663
664

Finite-element computer modeling needs accurate three-dimensional models to obtain

665

realistic simulation results for middle ear mechanics. 3D models are also useful in medical

666

training or for the interpretation and presentation of experimental results. The middle ear

667

does not only comprise the ossicles but also consists of soft tissue: tympanic membrane,

668

ligaments, muscles, tendon and blood vessels.

669
670

In this paper, we presented an accurate and complete morphological 3D middle (and inner)

671

ear model of gerbil. The model is freely available to the research community at our website.

672

The presented model quality is unprecedented. The position, orientation and size of all

673

components making up the gerbil middle ear are now accurately known and individually

674

discussed.

675
676
677

ACKNOWLEDGEMENTS

678
679

We gratefully acknowledge the financial support of the Research Foundation Flanders and

680

the Fondation Belge de la Vocation. We thank Pieter Vanderniepen for his assistance in

681

operating the CT device, Magnus Von Unge and Wim Decraemer for their feedback on

682

human and gerbil anatomy, Fred Wiese for manufacturing the vial holder, Robert Funnell for

683

the use of Figure 14, and Peter Aerts for feedback on muscle functionality.

684
685
686
687
20

Accurate 3D gerbil ME model

Buytaert et al.

688

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Rodt T, Ratiu P, Becker H, Bartling S, Kacher D, Anderson M, Jolesz F, Kikinis R (2002) 3D

visualisation of the middle ear and adjacent structures using reconstructed multi-slice CT
datasets, correlating 3D images and virtual endoscopy to the 2D cross-sectional images.
Neuroradiology 44:783790

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Rosowski JJ, Ravicz ME, Teoh SW, Flandermeyer D (1999) Measurements of middle-ear
function in the Mongolian gerbil, a specialized mammalian ear. Audiology & neuro-otology
4:129-136

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Ruf I, Frahnert S, Maier W (2009) The chorda tympani and its significance for rodent
phylogeny. Mammalian Biology - Zeitschrift fur Saugetierkunde 74:100-113

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Sim JH, Puria S (2008) Soft Tissue Morphometry of the MalleusIncus Complex from MicroCT Imaging. JARO-Journal of the Association for Research in Otolaryngology 9:521

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Sun Q, Gan RZ, Chang K-H, Dormer KJ (2002) Computer-integrated finite element modeling
of human middle ear. Biomechanics and modeling in mechanobiology 1:109-122

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Valk WL, Wit HP, Segenhout JM, Dijk F, Want JJL van der, Albers FWJ (2005) Morphology
of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops. Hearing
research 202:180-7

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Vogel U (1999) New approach for 3D imaging and geometry modeling of the human inner
ear. ORL 61:259267

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Voie AH (2002) Imaging the intact guinea pig tympanic bulla by orthogonal-plane
fluorescence optical sectioning microscopy. Hearing research 171:119-128

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Voie AH, Burns DH, Spelman FA (1993) Orthogonal-plane fluorescence optical sectioning:
three-dimensional imaging of macroscopic biological specimens. Journal of microscopy
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Wada H, Metoki T, Kobayashi T (1992) Analysis of dynamic behavior of human middle ear
using a finite-element method. The Journal of the Acoustical Society of America 92:31573168

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Wang H, Northrop C, Burgess B, Liberman MC, Merchant SN (2006) Three-dimensional

virtual model of the human temporal bone: a stand-alone, downloadable teaching tool.
Otology & neurotology: official publication of the American Otological Society, American
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REFERENCES to WEBSITES with MODELS

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Eaton-Peabody Laboratory of Auditory Physiology: 3D Virtual Models of the Human

Temporal Bone and Related Structures
http://research.meei.harvard.edu/Otopathology/3dmodels/download.html

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OtoBiomechanics Group: microCT data and 3D reconstructions

http://www.stanford.edu/~puria1/Site/Imaging.html

Auditory Research: The Vertebrate Ear and Temporal Bone

http://cbaweb2.med.unc.edu/henson_mrm/
Auditory Mechanics Laboratory: 3D Ear Human Ear
http://audilab.bmed.mcgill.ca/~daren/3Dear/
Ear & Auditory Research Laboratory: 3D Overview of Ear Anatomy
http://ear-lab.medicine.dal.ca/3D/3D-Overview.htm

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TABLE CAPTIONS

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Table 1: Volume, surface area and number of triangles for gerbil 1 (G1), gerbil 2 (G2) and

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gerbil 3 (G3) ear components, derived from the 3D surface meshes obtained from CT. The

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ME cavity volume incorporates the air, ossicles and ME soft tissue volume.

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Table 2: Geometry parameters of the TM for gerbil 1 (G1), gerbil 2 (G2) and gerbil 3 (G3) ear

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components, derived from the 3D surface meshes obtained from CT.

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(*Mean thickness data from confocal microscopy on 11 gerbils by Kuypers et al., 2005.)

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Table 3: 3D length of the manubrium (umbo tip till lateral process tip, cf. figure 5) and 3D

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height of the stapes (medial footplate till tip stapes head) for gerbil 1 (G1), gerbil 2 (G2) and

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gerbil 3 (G3), derived from the 3D surface meshes obtained from CT.

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FIGURES

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Figure 1: 3D model of separate surface meshes of bony middle and inner ear components of

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gerbil 2, obtained from CT. The bulla is rendered transparent. Voxel size 8.5x8.5x8.5 m.

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Figure 2: 2D virtual cross sections delivered by the OPFOS technique. A) Tensor tympani

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tendon reaching down towards malleus. B) Incudomallear and incudostapedial articulation.

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Pixel size 1.5x1.5 m.

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Figure 3: Reconstructed CT cross section through gerbil 1 (originally 1496x1496 pixels

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cropped to 740x950 pixels). a: middle ear air cavity, c: inner ear cochlea, i: incus, m: malleus,

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o: outer ear canal, s: stapes, t: tympanic membrane outline. Pixel size 8.5x8.5 m.

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Figure 4: 3D surface meshes. Voxel size 8.5x8.5x8.5 m.

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A) Tympanic membrane + middle ear ossicles + inner ear fluid (gerbil 1).

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B) Tympanic membrane + middle ear ossicles (gerbil 2).

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C) Tympanic membrane + middle ear ossicles + inner ear fluid (gerbil 3).

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Figure 5: Mesh of the malleus (gerbil 2) rendered transparent in combination with a mesh of

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the (major) blood vessel channels running inside it. Data obtained from CT. Voxel size

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8.5x8.5x8.5 m.

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Figure 6: A) CT cross section and B) 3D CT reconstruction from automatic thresholding do

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not show the posterior incudal ligament in the bony wall recess. Arrows indicate the position

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of the invisible ligament. Pixel (and voxel) size 8.5x8.5(x8.5) m. C-E) ROI OPFOS cross

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sections from different orientations do show the ligament in the recess. F-G) 3D OPFOS

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meshes. Voxel size 0.97x0.97x2.5 m. b: bulla, I: incus, L: ligament, m: malleus.

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Figure 7: Two views of the topography of the chorda tympani in combination with the

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malleus and the tensor tympani muscle & tendon (gerbil 2). The soft tissue data originate

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from OPFOS (voxel size 2x2x4.5 m), while the malleus data come from CT (voxel size

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8.5x8.5x8.5 m). b: bulla, c: chorda tympani, m: muscle, o: malleus ossicle, t: tendon, l:

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manubrium length.

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Figure 8: OPFOS cross sections showing the course of the chorda tympani with respect to the

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malleus. A) Chorda tympani jumps from a bony support beam to the malleus neck superior

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side. B) It rounds the malleus neck below the tensor tympani. C,D,E) It continues on the

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anterior process sheet until it enters a fissure in the bulla wall. b: bulla, c: chorda tympani, o:

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malleus ossicle, t: tendon. All subfigures are of the same scale.

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Figure 9: Stapes bone, stapedius muscle and tendon, and stapedial artery models obtained

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from OPFOS (voxel size 1.5x1.5x5 m), and the fluid-filled cavity of the horizontal semi-

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circular canal from CT (voxel size 8.5x8.5x8.5 m) are shown (gerbil 2). a: artery, m:

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muscle, o: stapes ossicle, s: semi-circular canal, t: tendon, w: artery wall.

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Figure 10: A-E) OPFOS based models of the stapes and the stapedial annular ligament (gerbil

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2). F) CT based model of the stapes (gerbil 2). The footplate modeled from CT data is

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convex, while in the OPFOS model it is not. a: annular ligament, c: cochlea, o: stapes ossicle.

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The arrow indicates the end of the OPFOS dataset.

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Figure 11: Merged OPFOS-CT ME model (gerbil 2).

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Figure 12: Cross sections at different depths through the 3D merged models of the bulla

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bone (white) from CT and the tensor tympani (blue) from OPFOS. Black represents air filled

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space such as the ME air cavity. The tensor tympani fits nicely in the bone, rather touching

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the cavity wall than overlapping with it.

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Figure 13: General schematic overview of all relevant middle ear components in human.

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Figure 14: Schematic representation of different posterior incudal ligament configurations

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per species (courtesy of Funnell, 1972). Gerbil falls in the category of guinea pig and rabbit.

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40