Учебники / Middle Ear Mechanics in Research and Otology Huber 2006

Fig. 1 Definitions for (a) the transcranial transmission and (b) the transmission from the forehead.

3. Results

The transcranial transmission results for the ECSP and BC hearing thresholds, both with open and occluded ear canal, are shown in Fig. 2. At low frequencies, there are some di erences in the results between open and occluded ear canal. But above 800 Hz, the results from the BC hearing threshold and ECSP measurements are similar. The standard deviations are almost frequency independent with somewhat higher values for the ECSP measurements with open ear canal.

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Fig. 2 The transcranial transmission results; (a) is the median results and (b) is the standard deviations. The ECSP measurement results are the solid and dashed lines from open and occluded ear canal, respectively. The BC hearing threshold results are the dotted and dash-dotted lines from open and occluded ear canal, respectively.

The results for the transmission from the forehead are shown in Fig. 3 for all methods. As for the transcranial transmission, open and closed ear canal produced di erent transmission at low frequencies, but at higher frequencies, the results from the four methods gave similar results. As for the

4. Discussions and Conclusions

The transcranial transmission was found frequency dependent. When measured with an occluded ear canal, the contralateral ear shows 5 dB greater excitation than the ipsilateral (positive transcranial transmission) at low frequencies. The transcranial transmission decreases with frequency and is about –15 dB at 8 kHz (attenuation = 15 dB). The transcranial transmission is similar whether obtained by hearing thresholds or by ECSP. This meansthattheECSPcanbeusedtoestimaterelativeBChearing.Whenthe resultsarecomparedwiththeBCvibrationstudybyStenfeltandGoode[1] (Fig. 5), who measured the three-dimensional vibrations of both cochleae while stimulating at several positions of the skull bone, similar results are seen when the stimulation position is similar as here. This indicates that vibrations of the cochlea can provide a good measure of the relative BC hearing, at least for the di erence in BC hearing between two stimulation positions. Nolan and Lyon [2] used BC hearing thresholds to estimate the transcranial transmission, but their results are lower and frequency independent (Fig. 5).

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Fig. 5 Comparison of the transcranial transmission with results from earlier studies, [1] and [2].

There is no clear explanation for the di erences at low frequencies between openandoccludedearcanals.OneobviousexplanationisthattheBCtransmission routes to the cochlea depend on the stimulation position causing the relative di erence. However, a similar phenomenon was discovered in loudness measurements where Keidser et al. [3] present a comparison between di erent loudness studies with open and occluded ear canals; the problem is discussed but the source for the discrepancies is not found. The lower occlusion e ect with ipsilateral stimulation is also related to the differences for open and occluded ear canals at low frequencies.

References

1.Stenfelt and Goode, Transmission properties of bone conducted sound: Measurements in cadaver heads, J. Acoust. Soc. Am. 118 (2005) pp. 2373–2391

2.Nolan and Lyon, Transcranial attenuation in bone conduction audiometry, J. Laryngol. Otol. 95 (1981) pp. 597–608

3.Keidser, Katsch, Dillon, and Grant, Relative loudness perception of low and high frequency sounds in the open and occluded ear, J. Acoust. Soc. Am. 107 (2000) pp. 3351–3357

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HIGH-RESOLUTION 3-D IMAGING OF MIDDLE EAR OSSICLES AND THEIR SOFT TISSUE STRUCTURES IN INTACT GERBIL TEMPORAL BONES, USING ORTHOGONAL FLUORESCENCE OPTICAL SECTIONING

J.A.N. Buytaert, J.J.J. Dirckx

Laboratory of Biomedical Physics, Department of Physics, University of Antwerp Groenenborgerlaan 171, B 2020 Antwerp, Belgium, Email: Jan.Buytaert@ua.ac.be

Detailed models of middle ear morphology are an important input to improve realism of computer models of middle ear mechanics. Established imaging techniques all have their specific limitations. Orthogonal-plane fluorescence optical sectioning or OPFOS, is an important additional technique which, after adequate specimen preparation, produces high quality, perfectly aligned sectional images in nearly real-time of both

282bone and soft tissue simultaneously. The technique was introduced by A. Voie with a slicing resolution of 14µm. Using improved optical design and adding motorized scanning along a direction of the image plane, we were able to reduce slicing thickness to 2µm, thus creating a high-resolution OPFOS (HROPFOS) technique. Sections from HROPFOS data show histological detail, f.i. the hollow structure of the stapes head, cavities within the body of the ossicles, etc. From the section data, we reconstructed accurate 3-D models of the middle ear ossicles and its soft tissue structures, such as the stapedial muscle and artery which are nearly invisible with X-ray CT. By imaging bone as well as soft tissue, these models are an important input for finite-element modeling. Measurements were obtained in preparations of intact temporal bones of gerbils, as the technique allows region-of-interest scanning.

1. Introduction

To generate 3-D models of biomedical structures, several well established techniques are available. On the one hand, there is optical imaging of actualphysicalsections.Ontheotherhand,therearetomographictechniques which deliver 3-D data on intact specimens.

Physical sectioning of an object, followed by imaging with light microscopy, delivers very high resolution (< 1µm) data, and can easily be combined with functional staining techniques. The preparation methods andespeciallythesectioningitselfcanintroduceimportantshapeartefacts and are extremely work intensive.

Tomography leaves the specimen intact: virtual sections are obtained which are auto-registered, and slicing can be repeated along several directions.Magnetic Resonance Imaging essentially detects di erencesinwater contentinaspecimen.Imagingofstructureswithlittlewatercontent,such asbone,isdi cult.Resolutionsdownto10µmcanbeobtainedinbiomedical specimens. Calculations are needed to obtain the image information and high-resolution measurements take long. X-ray Computer Tomography (CT) is based on contrast di erences in X-ray absorption. µCT images mainly bone (resolution ±5µm) but has di culty to visualize soft tissue. Back-projection calculations are necessary and region-of-interest (ROI) imaging can only be applied to some extent. Confocal Microscopy generates images of a specimen by focusing a laser beam to a small di raction limited point (< 1µm) within the tissue, and detecting the fluorescent light which emerges from that same point. To obtain a complete image, point- by-point scanning is needed, resulting in long measurement times. Functional staining is possible, but the imaging depth is limited to ±100µm.

In 1993, Voie introduced the OPFOS-technique: Orthogonal-Plane Fluorescence Optical Sectioning microscopy [1]. In this paper, we will demonstrateahigh-resolutionOPFOSmethod(HROPFOS)withastrong-

ly reduced slicing thickness, so that axial resolution is much more adapted 283 to in-plane resolution.

2. Materials and Method

2.1 Conventional OPFOS

In conventional OPFOS [2,3], a plane of laser light is projected through a transparentandfluorescentspecimen,andthelightemittedfromthisplane within the object is observed in orthogonal direction. The spectacular advantage of the OPFOS technique is that virtual sections through the object of bone as well as soft tissue are generated in real-time.

Inrealityitisimpossibletogenerateaplaneoflaserlight:asmallnumerical aperturecylindricallensfocusesabroadenedlaserbeamalong1dimension to a hyperbolic light pattern (cross section in Fig.1A). In the vicinity XR of the minimal focus d1 created by this lens, the hyperbolic light pattern is approximatedtoaplaneofconstantthickness d=√2d1,inwhichonerecords the virtual intersections orthogonally with a CCD-camera. Using a large numerical aperture lens, a finer focus d2 can be obtained, but the imaging zone corresponding with the approximated plane becomes very small. Hence in conventional OPFOS a trade-o exists between image width and slicethicknessd.Inpreviouswork[2],fociofd1 =10µm(atthecentreofan OPFOS-image) were obtained, causing the approximated thickness of the slicing plane at the edges of the image to drop to d = 14µm.

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Fig. 1 A) Focusing profile of a small numerical aperture cylindrical lens (OPFOS). In thezone<√2d1 (~plane) a2-Dimageistaken. B)Focusingprofileof alargenumerical aperture cylindrical lens (HROPFOS). Images are built by scanning and recording 1-D pixel lines in the thin focus. C) Schematic drawing of the HROPFOS setup: laser light passes through a beam expander (BE) and a cylindrical achromat (CL) which focuses the laser along 1 dimension in the transparent and fluorescent object (O). A two-axis motorized object translation stage (OTS) scans the specimen along X and Z. A CCD on a focusing translation stage (FTS) with microscope objective lens (OL) and colour filter records a Y-pixel column.

2.2 Improved optical setup: HROPFOS

In HROPFOS we no longer record 2-D images, but we scan the object through a line of best focus, created by a large numerical aperture cylindrical lens causing a much smaller focus d2 and corresponding tight imaging width (Fig. 1B). The CCD therefore only records 1 Y-pixel column of his imaging array in the focus d2, and an entire image is built by line-scanning the specimen through the focus by means of a motorized translation stage along the X-axis (Fig. 1C). Next, the object is moved with a second motor toimageanextsectionalongZ.HROPFOSnolongercausesthesectioning thickness to be a compromise with image width, and makes this thickness in theory only di raction limited.

2.3 Specimen preparation

To apply (HR)OPFOS, the plane of laser light needs to pass through the object without scattering or refraction, and the object needs to emit fluorescent light. The technique of clearing biomedical specimens is well established and was proposed many years ago by Spalteholz [4]. In short, the gerbil temporal bone is first decalcified in a water solution of 10% EDTA (accelerated by exposing it to low power microwave radiation). After all calcium has been removed, the ear is gradually dehydrated and made completelytransparentbyputtingitinSpalteholzfluid.Finallytheearisstained by submerging it in fluorescent dye. After this preparation procedure, the temporal bone and middle ear are completely transparent and fluorescent, which makes it compatible with OPFOS.

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Fig. 2 HROPFOS-scan of 2500×1280 pixels (1.88×0.96 mm) through a right gerbil stapes showing histological detail: 1) channel within incus, 2) incus, 3) lenticular process,4)articulation,5)calcifiedcartilageinstapeshead,6)stapedialarterythrough the cruras, 7) stapes footplate, 8) footplate annular ligament. 1×1 µm in-plane and 2µm slicing resolution.

3. Results

WehaveimprovedthesectioningoraxialresolutionofOPFOSwithafactor of 7. HROPFOS obtains a (constant) slicing resolution of 2µm over the entire image because of the improved optical design and line-scanning, while conventional OPFOS performs sectioning with a slicing thickness varying from 10µm in the center to 14µm at the edges of the image. Within the sectioningplane,theresolutionisonlylimitedbydi ractionoftheimaging lens. Because of the line-scanning principle in HROPFOS, there is no longer a limitation in image width, as the slicing thickness is maintained along a scan. Extended sections can now be made of a region-of-interest inside a bigger object. Fig.2 demonstrates an example of a scan of 2500 columns with a height of 1280 pixels, showing histological detail of a right gerbil stapes still inside its temporal bone. Fig.3 shows a right gerbil cochlea, in which we can even distinguish the Reissner’s membrane.

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Fig.3 HROPFOS-scan of 1700×1280 pixels (2.55×1.92 mm) of a right gerbil cochlea: scala tympani, 2) basilar membrane, 3) cochlear duct or scala media, 4) Reissner’s membrane, 5) scala vestibuli, 6) modiolus nerve, 7) blood vessels. Lateral & axial resolution 2μm.

From a series of auto-aligned sectional images obtained with HROPFOS, we can produce accurate 3-D models, e.g. of the incudo-stapedial joint and the exact location and morphology of the stapedial muscle, tendon and artery (Fig. 4).

Fig. 4 3-D models from HROPFOS-data showing: lenticular process of the incus & stapes (purple), stapedial artery (orange), stapedial muscle (red) of a right gerbil middle ear. These models are obtained from raw data without smoothing. Voxel: 3×3×3 µm.

4. Conclusion

Wehaveintroducedahigh-resolutionopticaltechnique,HROPFOS,which allowstogenerateimagesofvirtualsectionsthroughbiomedicalspecimens. Weachievedimageresolution<2μminslicingdirection,anddownto1μm intheslicingplane.Slicingthicknessisindependentofobjectorimagesize, and high axial resolution is maintained over the entire image width. Speci-

mens need to be cleared, refractive index matched and stained. 287 Despite conceptual simplicity, HROPFOS generates high-resolution

images of bone as well as soft tissue in nearly real-time, without the need ofcalculations,andaregionofinterestcanbeimagedwithhigh-resolution withinamuchlargerspecimenunderavarietyoforientations,allofwhich is impossible with µCT (Fig. 5). There also is no out-of-focus fluorescence, which is typical for light microscopy. Hence, our technique fills a gap not yetcoveredbyexistingimagingtechniques.FromHROPFOS-sections,full 3-D shape data are obtained, which are important input for finite-element modeling, as they not only give the shape of the ossicles, but also the exact morphology and position of soft tissue elements, such as arteries, muscles and tendon.