Elsevier

Hearing Research

Volume 171, Issues 1–2, September 2002, Pages 119-128
Hearing Research

Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy

https://doi.org/10.1016/S0378-5955(02)00493-8Get rights and content

Abstract

Orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy was developed for the purpose of making quantitative measurements of the intact mammalian cochlea and to facilitate 3D reconstructions of complex features. A new version of this imaging apparatus was built with a specimen chamber designed to accommodate samples as large as the intact guinea pig bulla. This method left the cochlear connections with the vestibular system and with the ossicles of the middle ear undisturbed, providing views within the cochlea with no breaches of its structural integrity. Since the features within the bulla were not physically touched during the preparation process, the risk of damage was minimized, and were imaged in relatively pristine condition with spatial resolution to 16 μm. A description of the imaging method and specimen preparation procedure is presented, as are images of features from the cochlea, ossicles, and vestibular system.

Introduction

The study of cochlear anatomy in 3D poses several challenges: microscopic dimensions; fragile, membranous features; complex ultrastructure; spiral geometry; all encased in temporal bone or an otic capsule. The accuracy with which these features may be studied is primarily a function of the imaging system resolving power and the fidelity of the tissue preparation process. The in vivo 3D imaging modalities of computed tomography (CT) and magnetic resonance (MR), with spatial resolution on the order of 1 mm, can reproduce only the large features of the cochlea such as the outlines of the scalae (Tomandl et al., 2000, Nanawa et al., 1999). To analyze the finer structures, cochleae typically are excised post-mortem where other imaging techniques may be brought to bear. These ex vivo techniques can be categorized broadly as deriving from histological serial sections, or from whole-specimen imaging.

Before the development of whole-specimen imaging, the primary source of 2D images for 3D cochlear reconstructions was from histological sections. Standard histological sections and light microscopy can resolve to about 1 μm. This approach was used to reconstruct the basilar membrane (BM) of the gerbil and the guinea pig (Plassman et al., 1987, Wada et al., 1998). Other efforts have been directed at the 3D reconstruction of the human cochlea. The external boundaries of the cochlea have been reconstructed as part of the larger reconstruction of the temporal bone (Harada et al., 1988, Lutz et al., 1989, Takahashi et al., 1989a, Green et al., 1990). Internal cochlear features have also been reconstructed. These include the organ of Corti and spiral ganglion (Takagi and Sando, 1989, Ariyasu et al., 1989), membranous labyrinth (Harada et al., 1990), round window membrane (Takahashi et al., 1989b), scala vestibuli (Takahashi et al., 1990), scala tympani and BM. Transmission electron microscopy serial section techniques, with spatial resolution on the order of hundreds of nanometers, have been used to produce 3D reconstructions of outer hair cells in the Japanese macaque (Sato et al., 1999).

There are, however, serious shortcomings inherent with extracting data from histological slides for 3D reconstructions. These shortcomings include the difficulties of restoring spatial register, and the tissue distortions induced by the passage of the microtome blade through the specimen. From the standpoint of 3D reconstruction, it is problematic that the sectioning process forces a single parallel-plane data sequence. Cochlear features that follow spiral or tortuous courses are difficult to track (and subsequently reconstruct) in this manner.

Whole-specimen MR microscopy overcomes these difficulties and has been used to great advantage for volumetric measurements and 3D visualization of cochlear features (Henson et al., 1994). In general, the trade-off between the serial-section and the whole-specimen techniques is one of high 2D resolution vs. volumetric integrity. The spatial resolution of the MR image (computed as twice the 3D diameter of the 25 μm voxel) is about 86 μm, an improvement over in vivo MRI (imaging) by an order of magnitude, but less than that of serial sectioning by some two orders of magnitude. MR microscopy has been used to reconstruct and measure the scalae and fluid spaces of several species (Thorne et al., 1999), plus the RW and cochlear aqueduct in the guinea pig (Ghiz et al., 2001).

Another whole-specimen imaging technique, orthogonal-plane fluorescence optical sectioning (OPFOS) was designed to view thin regions of the intact, excised cochlea (Voie et al., 1993). The optical section planes may be chosen using three axes of translation and two axes of rotation, allowing a near-infinite number of repetitive sectioning orientations and sequences. It has been used to gather data about cochlear dimensions and to facilitate 3D computer reconstructions of selected features (see Fig. 1) (Voie and Spelman, 1995, Voie, 2002). A new OPFOS instrument has been built that incorporates several technical advances, including a sample chamber that accommodates a larger tissue sample, and optics that enable spatial resolution to 16 μm.

Fig. 2 shows an overall schematic of the OPFOS imaging system and illustrates key principles of its operation. OPFOS works by passing a thin sheet of laser light (laser wavelength (λ)=542.5 nm) through a tissue sample that has been rendered transparent by chemical means. The tissue to be imaged is secured within the specimen chamber, which is filled with clearing solution and sealed. Components of the illumination system form laser light into an ultra-thin sheet that passes through the relatively transparent tissue. A slight amount of fluorescent dye in the tissue is excited (absorption λ=550 nm) by the laser light but only in the very thin region defined by the geometry of the beam. The fluorescent light (λ=585 nm) passes through the surrounding transparent tissue, relatively unimpeded and undistorted. When a camera lens is focused on this thin fluorescing region within the tissue, from an axis perpendicular to the sheet of laser light, a sharply focused image results. This is due to the fact that only light from the focal zone contributes to the image, so there is no out-of-focus component.

This paper describes the use of the OPFOS technique to examine not only the cochlea of the guinea pig, but also to include the structures of the middle ear and bulla. The tympanic bulla is removed intact from the sacrificed guinea pig, and with the exception of removing extraneous soft tissue from its outer surface and drilling two small holes to allow air to escape from its interior, no further trimming is done. The tympanic membrane is undisturbed, as are all middle ear ossicles and other structures. The cochlear apertures of oval (OW) and RW are not breached so air bubbles do not enter to hinder the imaging process. The elimination of cochlear trimming leaves the vestibular system intact so these membranous structures may be viewed in their natural positions, continuous with the fluid spaces of the cochlea. The improved resolution of the OPFOS system facilitates visualizing various tissue structures, such as the neuroepithelium of the crista ampullaris and the ductus reuniens joining the saccule and the scala media.

Section snippets

Specimen preparation

The tympanic bulla was harvested from guinea pigs sacrificed under deep anesthesia, fixed in 10% neutral buffered formalin, and placed in a jar containing 100 ml 10% ethylenediaminetetraacetic acid (EDTA) solution (Fisher Scientific, S657-3) for decalcification. Tissue decalcification was enhanced by microwave energy (Madden and Henson, 1997) (Pelco 3451 Laboratory Microwave System with load cooler and power controller, Ted Pella, Inc., Redding, CA, USA). The lid of the jar had three small

Results

Fig. 3 is a montage of optical sections through the right bulla of a guinea pig. The bulla was translated in a three-by-five raster pattern while keeping the laser plane fixed. The cochlea is seen in mid-modiolar section, with the tympanic membrane and external meatus to the right and portions of the vestibular system below. The ossicles do not appear in this image plane. One of the two holes drilled in the bulla to allow air to escape is at the upper left. The resolution of this image is 27

Discussion

As noted in the Specimen Preparation section, the clearing solution was a 5:3 mixture of methyl salicylate and benzyl benzoate. It should be mentioned, however, that other sources suggest different ratios (Culling, 1957, Emmel and Cowdry, 1964, Tompsett, 1970) and indeed most recent workers report using a modified Spalteholz technique that uses only methyl salicylate for tissue clarification (Bissonette and Fekete, 1996, Bever and Fekete, 2000). The author has used the composition described in

Acknowledgements

Special thanks to Mr. Marc McDaniel for his help in the design, machining, and fabrication of the OPFOS specimen chamber. This work has been supported by NIH grants 1R43DC03623-01 and 2R44DC03623-02.

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