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Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy
Hearing Research 171 (2002) 119^128 www.elsevier.com/locate/heares
Imaging the intact guinea pig tympanic bulla by orthogonal-plane £uorescence optical sectioning microscopy Arne H. Voie
Spencer Technologies, 701 16th Ave., Seattle, WA 98122, USA Received 11 September 2001; accepted 18 April 2002
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 Wm. A description of the imaging method and specimen preparation procedure is presented, as are images of features from the cochlea, ossicles, and vestibular system. 2 2002 Elsevier Science B.V. All rights reserved. Key words: Cochlea; Vestibular system; Ossicles ; Guinea pig; Imaging ; Microwave
1. 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 ¢delity 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).
* Web address: www.spencertechnologies.com; Tel.: +1 (206) 329 7220; Fax: +1 (206) 329 7230. E-mail address: [email protected] (A.H. Voie). Abbreviations: BM, basilar membrane; CCD, charge-coupled device; CT, computed tomography; EDTA, ethylenediaminetetraacetic acid; MR, magnetic resonance; MRI, magnetic resonance imaging; OPFOS, orthogonal-plane £uorescence optical sectioning; OW, oval window; RITC, rhodamine isothiocyanate; RW, round window; SSC, semicircular canal; V, wavelength
To analyze the ¢ner 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 Wm. 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 e¡orts 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 sec-
0378-5955 / 02 / $ ^ see front matter 2 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 2 ) 0 0 4 9 3 - 8
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Fig. 1. 3D reconstruction of guinea pig scala tympani from OPFOS images. Also shown are the cochlear aqueduct, BM and round window membrane. Increments on coordinate axes are millimeters.
tion 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 di⁄culties 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 di⁄cult to track (and subsequently reconstruct) in this manner. Whole-specimen MR microscopy overcomes these di⁄culties and has been used to great advantage for volumetric measurements and 3D visualization of cochlear features (Henson et al., 1994). In general, the trade-o¡ 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 Wm voxel) is about 86 Wm, 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 £uid 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 £uorescence 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-in¢nite 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 Wm. 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 (V) = 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 ¢lled 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 £uorescent dye in the tissue is excited (absorption V = 550 nm) by the laser light but only in the very thin region de¢ned by the geometry of the beam. The £uorescent light (V = 585 nm) passes through the surrounding transparent tissue, relatively unimpeded and undistorted. When a camera lens is focused on this thin £uorescing 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 sacri¢ced 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 £uid 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.
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Fig. 2. Overall schematic of the OPFOS imaging system. The coordinate axes represent the three directions of translation and two directions of rotation in which the specimen can be moved. The green He^Ne laser is focused into a thin sheet of light using a cylindrical lens. This light traverses the cleared tissue which has been treated with RITC. The resulting £uorescent light forms an image of the optical section on the CCD sensor of the video camera, and is displayed on the monitor of the image-processing computer.
2. Materials and methods 2.1. Specimen preparation The tympanic bulla was harvested from guinea pigs sacri¢ced under deep anesthesia, ¢xed in 10% neutral bu¡ered formalin, and placed in a jar containing 100 ml 10% ethylenediaminetetraacetic acid (EDTA) solution (Fisher Scienti¢c, S657-3) for decalci¢cation. Tissue decalci¢cation 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 holes to admit a temperature sensor, air-agitation tube and air circulation. The microwave oven was set to maintain an EDTA solution temperature of 45‡C, and to run for the maximum programmable time of 6.6 h, after which the EDTA solution was changed. After the fourth such change, decalci¢cation was su⁄cient to visualize trapped air within the bulla. Two small holes were made in the bulla wall away from internal structures to allow these bubbles to escape. Decalci¢cation was complete after eight microwave periods, after which the bulla was dehydrated using a succession of ethanol-distilled water solutions,
24 h each. The ethanol fraction in the dehydration succession was 25%, 50%, 75%, 100%, 100%. Spalteholz £uid, a 5:3 solution of methyl salicylate (Sigma, M-6752) and benzyl benzoate (Sigma, B-6630) was used to render the tissue transparent using the classic Spalteholz clearing technique (Spalteholz, 1914). The bulla was cleared using a succession of Spalteholz-ethanol solutions, 24 h each. The Spalteholz £uid fraction in the clearing succession was 25%, 50%, 75%, 100%, 100%. After clearing, the bulla was treated with the £uorescent dye rhodamine-b isothiocyanate (Sigma, R-1755). The dye bath was prepared by dissolving 0.5 mg rhodamine isothiocyanate (RITC) into 0.5 ml ethanol, followed by dilution in 100 ml of Spalteholz £uid. The cleared bulla remained in the dye bath for 4 days, after which it was removed and placed in 100% Spalteholz £uid. Finally, the bulla was cemented to a nylon ¢xture designed to ¢t into the positioning chuck of the microscope sample chamber. The ¢xture was made from a 3/16Q nylon machine screw. The head of the screw was cut o¡, leaving the shaft. A high-speed drill and dental burr were used to shape this cut end so that a portion of the cleared temporal bone sample would ¢t in. The bone was then cemented to the nylon ¢xture with a
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Fig. 3. OPFOS image of a right guinea pig bulla in cross-section. Constructed of a montage of 15 images taken while moving the specimen in a raster pattern in the x^y plane. A combination of 1U macro lens and 200 mm focal length cylindrical lens was used. The actual dimensions spanned by the image are 12.8 mm (horizontal) by 16.3 mm (vertical). Spatial resolution is 27 Wm. One of the holes drilled to allow air to escape is seen at upper left. Horizontal streaks in the image are the result of scattering and absorption of the laser light as it traverses the specimen. The direction of the laser light is from left to right in the OPFOS images.
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Fig. 4. OPFOS image through the vestibule. Single optical section image showing portions of the tympanic membrane, middle ear ossicles, OW and RW, saccule and utricle. Frame width of image is 5.3 mm, and spatial resolution is 27 Wm. A combination of 1U macro lens and 200 mm focal length cylindrical lens was used.
non-acrylic dental cement (Durelon). The cementing site was chosen so as not to obscure key temporal bone features. It was also considered desirable to orient the modiolar axis of the cochlea parallel and if possible coaxial with the axis of the mounting ¢xture. The threads of the ¢xture provided a convenient site to grasp with long forceps and so guide the bone into the positioning chuck. 2.2. Instrument 2.2.1. Specimen chamber The specimen chamber was machined from a solid block of Delrin. A glass window at the top of the chamber admits the laser light (see Fig. 2). Windows on the side allow £uorescent light to pass out of the chamber and into the imaging optics. The chamber is ¢lled with clearing solution. The top window is removed to attach the specimen to the end of the rotation shaft within the chamber. After the specimen is secured, the top window is returned to its position, the clearing solution is topped o¡ (removing air bubbles), and the chamber is sealed.
The rotation shaft extends through the wall of the specimen chamber and is ¢xed to a manual rotation drive (Newport URM80AMS, Newport Corp., Irvine, CA, USA), which allows rotation about the z-axis. An optical encoder (Newport CV1000) provides a digital readout of rotation angle (a) to 0.001‡. The chamber itself is mounted on a rotation stage that allows rotation (K) about the x-axis (Newport 481-A). This allows the specimen to be imaged through either of the side windows. The chamber and rotation stage are mounted on two horizontal translation stages to provide movement along the y- and z-axes (Oriel 16123). The video camera is mounted on a translation stage that provides movement along the x-axis. 2.2.2. Illumination system The illumination system consists of a 1.5-mW green helium-neon laser (Newport U-1676), beam forming optics, positioning and alignment components. The beam forming components consist of a 10U beam expander (Newport 781-10U) and cylindrical lens. Three cylindrical lenses, with focal lengths of 200 mm (Newport CKX 537-C), 150 mm (Newport CKX 534-C), and
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Fig. 5. OPFOS image of utricular macula and ampullar cristae. Frame width is 2.7 mm, and spatial resolution is 28 Wm. Vertical lines are a result of image averaging. A 2U macro lens was used in combination with a 150 mm focal length cylindrical lens.
100 mm (Newport CKX 531-C) were used interchangeably in this system, to provide beam thickness of 20, 15, and 10 Wm respectively. 2.2.3. Imaging optics The imaging optics consists of a macro lens, long pass ¢lter, and charge-coupled device (CCD) video camera. Macro lenses were chosen to provide su⁄cient magni¢cation, depth of ¢eld, and working distance (Rodenstock 1U, 2U, 4U, Linos Photonics, Inc., Rockford, IL, USA). A 560 nm long pass ¢lter (Thin Film Imaging Technologies, Inc., Green¢eld, MA, USA) was placed into a 5 mm extension tube and ¢tted onto the C-mount of the lens. The ¢lter blocked scattered laser light (543.5 nm) from entering the camera while admitting £uorescent light (585 nm). Lens and ¢lter were ¢tted to a monochrome CCD video camera (Cohu 4915, Cohu, Inc., Calle Fortunada, CA, USA). 2.2.4. Image resolution The resolution and magni¢cation of the OPFOS images are determined by the combination of cylindrical lens used to form the laser beam, and the properties of the imaging lens used with the video camera. The imaging lens determines the lateral resolution of the OPFOS
system. This is a ¢xed characteristic of the lens ^ a function of its focal length and f-number (Inoue and Spring, 1997). This was 18, 24, and 12 Wm, for the 1U, 2U and 4U lenses, respectively (provided by manufacturer). The axial resolution is determined by the beam thickness, a function of the cylindrical lens focal length (Siegman, 1986). As stated, the three cylindrical lenses produce beam thicknesses of 20, 15 and 10 Wm. The usual combinations of lenses used were 200, 150, 100 mm cylindrical lenses with 1U, 2U, and 4U imaging lenses, respectively. The overall spatial resolution of the images was the geometric mean of the lateral and axial resolutions. The above combinations corresponded to spatial resolutions of 27, 28, and 16 Wm, respectively. The care and use of the animals used in this study were approved by the Institutional Animal Care and Use Committee of RpR Rabbitry, Stanwood, WA, USA.
3. Results Fig. 3 is a montage of optical sections through the right bulla of a guinea pig. The bulla was translated in a
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three-by-¢ve raster pattern while keeping the laser plane ¢xed. 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 Wm, su⁄cient to detect Reissner’s membrane and the membranous vestibular structures including a portion of the ductus reuniens. The horizontal lines or stripes seen in Figs. 3^6 are caused by non-uniform absorption and scattering of the laser beam as it traverses the tissue. Gray-scale levels in the images help to di¡erentiate tissue types. The dye binds in a non-speci¢c manner (Haugland, 1981), so higher concentration results where the tissue is more dense. Though decalci¢ed, the formerly bony tissue is still a dense collagen matrix, and appears brighter in the images than soft tissues such as membranes, nerves and ligaments and cartilage. Fluid-¢lled spaces appear dark. It should be noted that image gray-scale levels are not solely a function of tissue density. In the dye bath the RITC di¡uses from the outer tissue surfaces inward, and the brightness gradient in the image re£ects this. Fig. 4 is an optical section that shows portions of the
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incus and stapes, RW and OW, and saccule and utricle within the vestibule. The union of the lenticular process of the incus with the head of the stapes is clearly seen. The stapes appears narrow because the sectioning plane is passing through just the anterior crus, with the inferior crural arch on the left and the superior crural arch on the right. To the left of the head of the stapes is the anterior portion of the RW niche, and part of the tympanic membrane is seen in the upper portion of the image. The vestibule of the cochlea is the dark region to the left of the stapes footplate and OW. Reissner’s membrane may be seen in the upper right of the vestibule, just to the left of the RW niche. The saccule is above and to the left of the utricle, at the lower portion of the vestibule. The saccule lies against the lateral wall of the vestibule, and the slightly darker portion of the wall to the left of the saccule is the macula. Nerves leading to the saccular macula may be seen just above and to the left of the macula. The optical section of Fig. 5 transects portions of the utricular macula and the ampullar cristae. The footplate of the stapes is at the upper left in this image. The dark space immediately below the stapes is the lumen of the vestibule, and the dark elongated shape below this is the utricle in cross-section. The utricular
Fig. 6. OPFOS image of ampullar cristae. Frame width is 810 Wm, and spatial resolution is 16 Wm. A 4U macro lens with 60 mm extender tube was used in combination with a 100 mm focal length cylindrical lens.
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macula appears as a bright region in the boundary tissue between the utricle and vestibule. The otolithic membrane is just discernable over the utricular macula. To the right of the utricle are the ampullar cristae of the lateral (above) and superior (below) semicircular canals (SSCs). The connective tissue supporting the membranous walls of the utricle and superior SSC appears along the bottom of the image, with capillaries showing up as a somewhat brighter, lacy network within this tissue. Fig. 6 is a high-magni¢cation section through the ampullar cristae of the superior and lateral SSCs. The sensory epithelial layers containing the hair cells are visible, as are the ¢bers of the vestibular nerve leading to them. This image magni¢cation was achieved using a 60 mm extension tube with the 4U lens.
4. 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 di¡erent ratios (Culling, 1957; Emmel and Cowdry, 1964; Tompsett, 1970) and indeed most recent workers report using a modi¢ed Spalteholz technique that uses only methyl salicylate for tissue clari¢cation (Bissonette and Fekete, 1996; Bever and Fekete, 2000). The author has used the composition described in this paper since 1986 with satisfactory results and has stayed with it for that reason. Tissue preparations have been stored for over 10 years with no discernable degradation or loss of £uorescence. Tissue photo-bleaching may result after prolonged exposure to laser light, and so should be avoided.
The OPFOS images shown above are at in increasing levels of magni¢cation and spatial resolution. The features of the vestibular system were chosen to demonstrate high-magni¢cation capability because they present challenges for imaging in general. The endolymphatic membrane-bounded spaces, and the sensory organs contained within, are di⁄cult to visualize. These features are beyond the resolving capabilities of CT and MRI, while histology sections cannot easily be oriented to pass through all vestibular system sensory organs. The technique of clearing the tissue and ¢lling the membranous labyrinth with paint (Bissonette and Fekete, 1996) allows the vestibular system to be seen in outline but the sensory tissues are obscured in the process. This work demonstrates that the OPFOS technique may be used to appreciate the £uid spaces, membranous boundaries and sensory tissues of the vestibular system. Since the specimen can be translated and rotated, it is possible to acquire data for 3D reconstruction and quantitative measurements of the middle ear and vestibular system as well as for the cochlea. As mentioned above, the horizontal lines that appear in Figs. 3^6 are due to non-uniform absorption of the laser light as it traverses the tissue. When the laser light traverses tissue with a higher regional dye concentration, more of the light is absorbed and less is available for more distal areas. This gives rise to narrow shadow lines in the direction of the laser illumination. Bringing additional laser light from the opposite direction to more uniformly illuminate the focal zone theoretically could reduce this e¡ect. Alignment of multiple beam focal zones to overlap precisely would be challenging, however. Vertical lines are evident in Figs. 5 and 6, which are the higher-magni¢cation OPFOS images. Because less
Fig. 7. Comparison of three cochlear imaging modalities. Shown are mid-modiolar sections of approximately equal scale. (A) Histological serial section. (B) MR microscopy section. (C) OPFOS section. The images in frames (A) and (B) are reprinted with permission from the Cochlear Fluids Research Laboratory website (http://oto.wustl.edu/cochlea).
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£uorescent light enters the macro lens as the imaging region becomes smaller, the brightness of an OPFOS image is inversely proportional to its magni¢cation. By increasing the gain of the video camera, small and faint structures may be visualized. However, this introduces noise, in the form of random speckling, to the image. Averaging reduces the noise, but this in turn produces vertical lines in the image, which are associated with the sensing elements of the CCD camera. It may be possible to reduce this e¡ect with a more sensitive imaging device, but this would probably be an expensive upgrade. Another approach to improving the sensitivity of this inherently low-light process is to use an imaging lens of higher numerical aperture. The challenge to this is the requirement of a long working distance for the lens, which must be located outside of the sealed Spalteholz-¢lled sample chamber. Typically, lens numerical aperture is inversely proportional to its working distance. Comparing the imaging technique of OPFOS to MR microscopy and histological serial sections, the following statements can be made. OPFOS provides better resolution than MR, but not as good as histology. This is evident in Fig. 7, which shows images (of di¡erent specimens of guinea pig cochleae) from these modalities side-by-side for comparison. The resolution of the histological section (A) and the OPFOS image (C) is clearly higher than that of the MR image (B). Comparing frames (A) and (C), the superior spatial resolution of the histological section is evident when inspecting the organ of Corti. In (A) the tunnel of Corti and supporting cells are clearly seen. If the goals of a study require resolution to the cellular level and beyond, or if 3D macro-anatomy need not be preserved, then histological techniques have compelling advantages. On the other hand MR and OPFOS can lead to an understanding of 3D macroanatomy that is much more intuitive than from histological sections. A comparison of the time involved with each technique for 3D reconstruction requires the process to be broken into stages of image acquisition, feature extraction and data analysis. Image acquisition by MR probably requires the least time of these methods. There is little tissue preparation necessary beyond ¢xation and trimming of excess bone to ¢t the sample into the chamber of the MR device (Thorne et al., 1999). The time from tissue harvest to completion of imaging may be on the order of days. The time to complete the imaging process for OPFOS and histological sectioning may be several weeks, taking the tissue processing time into account. Feature extraction and data analysis is probably equivalent for the three methods, requiring a fair amount of trained human interaction such as tracing of boundaries. Comparing the whole specimen techniques, OPFOS
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provides greater resolution than MR, however the tissue preparation and image acquisition time is faster with MR. An advantage of the OPFOS technique is the simplicity of the apparatus, most of which can be built from relatively inexpensive components (the system used in this work, including host computer, frame grabber, software, laser, video camera, optical table, optical components and positioning equipment, was assembled by the author for under US$25 000). This makes control of the imaging process available to the researcher, who would otherwise need to take the tissue to one of a limited number of centers that have the expensive MR microscopy apparatus. Control of the imaging process is one of the major advantages of the OPFOS system, in that the device produces images in real time, giving it a unique interactive quality that facilitates exploration and education.
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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Imaging the intact guinea pig tympanic bulla by orthogonal-plane £uorescence optical sectioning microscopy Arne H. Voie
Spencer Technologies, 701 16th Ave., Seattle, WA 98122, USA Received 11 September 2001; accepted 18 April 2002
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 Wm. A description of the imaging method and specimen preparation procedure is presented, as are images of features from the cochlea, ossicles, and vestibular system. 2 2002 Elsevier Science B.V. All rights reserved. Key words: Cochlea; Vestibular system; Ossicles ; Guinea pig; Imaging ; Microwave
1. 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 ¢delity 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).
* Web address: www.spencertechnologies.com; Tel.: +1 (206) 329 7220; Fax: +1 (206) 329 7230. E-mail address: [email protected] (A.H. Voie). Abbreviations: BM, basilar membrane; CCD, charge-coupled device; CT, computed tomography; EDTA, ethylenediaminetetraacetic acid; MR, magnetic resonance; MRI, magnetic resonance imaging; OPFOS, orthogonal-plane £uorescence optical sectioning; OW, oval window; RITC, rhodamine isothiocyanate; RW, round window; SSC, semicircular canal; V, wavelength
To analyze the ¢ner 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 Wm. 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 e¡orts 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 sec-
0378-5955 / 02 / $ ^ see front matter 2 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 2 ) 0 0 4 9 3 - 8
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Fig. 1. 3D reconstruction of guinea pig scala tympani from OPFOS images. Also shown are the cochlear aqueduct, BM and round window membrane. Increments on coordinate axes are millimeters.
tion 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 di⁄culties 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 di⁄cult to track (and subsequently reconstruct) in this manner. Whole-specimen MR microscopy overcomes these di⁄culties and has been used to great advantage for volumetric measurements and 3D visualization of cochlear features (Henson et al., 1994). In general, the trade-o¡ 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 Wm voxel) is about 86 Wm, 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 £uid 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 £uorescence 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-in¢nite 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 Wm. 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 (V) = 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 ¢lled 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 £uorescent dye in the tissue is excited (absorption V = 550 nm) by the laser light but only in the very thin region de¢ned by the geometry of the beam. The £uorescent light (V = 585 nm) passes through the surrounding transparent tissue, relatively unimpeded and undistorted. When a camera lens is focused on this thin £uorescing 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 sacri¢ced 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 £uid 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.
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Fig. 2. Overall schematic of the OPFOS imaging system. The coordinate axes represent the three directions of translation and two directions of rotation in which the specimen can be moved. The green He^Ne laser is focused into a thin sheet of light using a cylindrical lens. This light traverses the cleared tissue which has been treated with RITC. The resulting £uorescent light forms an image of the optical section on the CCD sensor of the video camera, and is displayed on the monitor of the image-processing computer.
2. Materials and methods 2.1. Specimen preparation The tympanic bulla was harvested from guinea pigs sacri¢ced under deep anesthesia, ¢xed in 10% neutral bu¡ered formalin, and placed in a jar containing 100 ml 10% ethylenediaminetetraacetic acid (EDTA) solution (Fisher Scienti¢c, S657-3) for decalci¢cation. Tissue decalci¢cation 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 holes to admit a temperature sensor, air-agitation tube and air circulation. The microwave oven was set to maintain an EDTA solution temperature of 45‡C, and to run for the maximum programmable time of 6.6 h, after which the EDTA solution was changed. After the fourth such change, decalci¢cation was su⁄cient to visualize trapped air within the bulla. Two small holes were made in the bulla wall away from internal structures to allow these bubbles to escape. Decalci¢cation was complete after eight microwave periods, after which the bulla was dehydrated using a succession of ethanol-distilled water solutions,
24 h each. The ethanol fraction in the dehydration succession was 25%, 50%, 75%, 100%, 100%. Spalteholz £uid, a 5:3 solution of methyl salicylate (Sigma, M-6752) and benzyl benzoate (Sigma, B-6630) was used to render the tissue transparent using the classic Spalteholz clearing technique (Spalteholz, 1914). The bulla was cleared using a succession of Spalteholz-ethanol solutions, 24 h each. The Spalteholz £uid fraction in the clearing succession was 25%, 50%, 75%, 100%, 100%. After clearing, the bulla was treated with the £uorescent dye rhodamine-b isothiocyanate (Sigma, R-1755). The dye bath was prepared by dissolving 0.5 mg rhodamine isothiocyanate (RITC) into 0.5 ml ethanol, followed by dilution in 100 ml of Spalteholz £uid. The cleared bulla remained in the dye bath for 4 days, after which it was removed and placed in 100% Spalteholz £uid. Finally, the bulla was cemented to a nylon ¢xture designed to ¢t into the positioning chuck of the microscope sample chamber. The ¢xture was made from a 3/16Q nylon machine screw. The head of the screw was cut o¡, leaving the shaft. A high-speed drill and dental burr were used to shape this cut end so that a portion of the cleared temporal bone sample would ¢t in. The bone was then cemented to the nylon ¢xture with a
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Fig. 3. OPFOS image of a right guinea pig bulla in cross-section. Constructed of a montage of 15 images taken while moving the specimen in a raster pattern in the x^y plane. A combination of 1U macro lens and 200 mm focal length cylindrical lens was used. The actual dimensions spanned by the image are 12.8 mm (horizontal) by 16.3 mm (vertical). Spatial resolution is 27 Wm. One of the holes drilled to allow air to escape is seen at upper left. Horizontal streaks in the image are the result of scattering and absorption of the laser light as it traverses the specimen. The direction of the laser light is from left to right in the OPFOS images.
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Fig. 4. OPFOS image through the vestibule. Single optical section image showing portions of the tympanic membrane, middle ear ossicles, OW and RW, saccule and utricle. Frame width of image is 5.3 mm, and spatial resolution is 27 Wm. A combination of 1U macro lens and 200 mm focal length cylindrical lens was used.
non-acrylic dental cement (Durelon). The cementing site was chosen so as not to obscure key temporal bone features. It was also considered desirable to orient the modiolar axis of the cochlea parallel and if possible coaxial with the axis of the mounting ¢xture. The threads of the ¢xture provided a convenient site to grasp with long forceps and so guide the bone into the positioning chuck. 2.2. Instrument 2.2.1. Specimen chamber The specimen chamber was machined from a solid block of Delrin. A glass window at the top of the chamber admits the laser light (see Fig. 2). Windows on the side allow £uorescent light to pass out of the chamber and into the imaging optics. The chamber is ¢lled with clearing solution. The top window is removed to attach the specimen to the end of the rotation shaft within the chamber. After the specimen is secured, the top window is returned to its position, the clearing solution is topped o¡ (removing air bubbles), and the chamber is sealed.
The rotation shaft extends through the wall of the specimen chamber and is ¢xed to a manual rotation drive (Newport URM80AMS, Newport Corp., Irvine, CA, USA), which allows rotation about the z-axis. An optical encoder (Newport CV1000) provides a digital readout of rotation angle (a) to 0.001‡. The chamber itself is mounted on a rotation stage that allows rotation (K) about the x-axis (Newport 481-A). This allows the specimen to be imaged through either of the side windows. The chamber and rotation stage are mounted on two horizontal translation stages to provide movement along the y- and z-axes (Oriel 16123). The video camera is mounted on a translation stage that provides movement along the x-axis. 2.2.2. Illumination system The illumination system consists of a 1.5-mW green helium-neon laser (Newport U-1676), beam forming optics, positioning and alignment components. The beam forming components consist of a 10U beam expander (Newport 781-10U) and cylindrical lens. Three cylindrical lenses, with focal lengths of 200 mm (Newport CKX 537-C), 150 mm (Newport CKX 534-C), and
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Fig. 5. OPFOS image of utricular macula and ampullar cristae. Frame width is 2.7 mm, and spatial resolution is 28 Wm. Vertical lines are a result of image averaging. A 2U macro lens was used in combination with a 150 mm focal length cylindrical lens.
100 mm (Newport CKX 531-C) were used interchangeably in this system, to provide beam thickness of 20, 15, and 10 Wm respectively. 2.2.3. Imaging optics The imaging optics consists of a macro lens, long pass ¢lter, and charge-coupled device (CCD) video camera. Macro lenses were chosen to provide su⁄cient magni¢cation, depth of ¢eld, and working distance (Rodenstock 1U, 2U, 4U, Linos Photonics, Inc., Rockford, IL, USA). A 560 nm long pass ¢lter (Thin Film Imaging Technologies, Inc., Green¢eld, MA, USA) was placed into a 5 mm extension tube and ¢tted onto the C-mount of the lens. The ¢lter blocked scattered laser light (543.5 nm) from entering the camera while admitting £uorescent light (585 nm). Lens and ¢lter were ¢tted to a monochrome CCD video camera (Cohu 4915, Cohu, Inc., Calle Fortunada, CA, USA). 2.2.4. Image resolution The resolution and magni¢cation of the OPFOS images are determined by the combination of cylindrical lens used to form the laser beam, and the properties of the imaging lens used with the video camera. The imaging lens determines the lateral resolution of the OPFOS
system. This is a ¢xed characteristic of the lens ^ a function of its focal length and f-number (Inoue and Spring, 1997). This was 18, 24, and 12 Wm, for the 1U, 2U and 4U lenses, respectively (provided by manufacturer). The axial resolution is determined by the beam thickness, a function of the cylindrical lens focal length (Siegman, 1986). As stated, the three cylindrical lenses produce beam thicknesses of 20, 15 and 10 Wm. The usual combinations of lenses used were 200, 150, 100 mm cylindrical lenses with 1U, 2U, and 4U imaging lenses, respectively. The overall spatial resolution of the images was the geometric mean of the lateral and axial resolutions. The above combinations corresponded to spatial resolutions of 27, 28, and 16 Wm, respectively. The care and use of the animals used in this study were approved by the Institutional Animal Care and Use Committee of RpR Rabbitry, Stanwood, WA, USA.
3. Results Fig. 3 is a montage of optical sections through the right bulla of a guinea pig. The bulla was translated in a
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three-by-¢ve raster pattern while keeping the laser plane ¢xed. 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 Wm, su⁄cient to detect Reissner’s membrane and the membranous vestibular structures including a portion of the ductus reuniens. The horizontal lines or stripes seen in Figs. 3^6 are caused by non-uniform absorption and scattering of the laser beam as it traverses the tissue. Gray-scale levels in the images help to di¡erentiate tissue types. The dye binds in a non-speci¢c manner (Haugland, 1981), so higher concentration results where the tissue is more dense. Though decalci¢ed, the formerly bony tissue is still a dense collagen matrix, and appears brighter in the images than soft tissues such as membranes, nerves and ligaments and cartilage. Fluid-¢lled spaces appear dark. It should be noted that image gray-scale levels are not solely a function of tissue density. In the dye bath the RITC di¡uses from the outer tissue surfaces inward, and the brightness gradient in the image re£ects this. Fig. 4 is an optical section that shows portions of the
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incus and stapes, RW and OW, and saccule and utricle within the vestibule. The union of the lenticular process of the incus with the head of the stapes is clearly seen. The stapes appears narrow because the sectioning plane is passing through just the anterior crus, with the inferior crural arch on the left and the superior crural arch on the right. To the left of the head of the stapes is the anterior portion of the RW niche, and part of the tympanic membrane is seen in the upper portion of the image. The vestibule of the cochlea is the dark region to the left of the stapes footplate and OW. Reissner’s membrane may be seen in the upper right of the vestibule, just to the left of the RW niche. The saccule is above and to the left of the utricle, at the lower portion of the vestibule. The saccule lies against the lateral wall of the vestibule, and the slightly darker portion of the wall to the left of the saccule is the macula. Nerves leading to the saccular macula may be seen just above and to the left of the macula. The optical section of Fig. 5 transects portions of the utricular macula and the ampullar cristae. The footplate of the stapes is at the upper left in this image. The dark space immediately below the stapes is the lumen of the vestibule, and the dark elongated shape below this is the utricle in cross-section. The utricular
Fig. 6. OPFOS image of ampullar cristae. Frame width is 810 Wm, and spatial resolution is 16 Wm. A 4U macro lens with 60 mm extender tube was used in combination with a 100 mm focal length cylindrical lens.
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macula appears as a bright region in the boundary tissue between the utricle and vestibule. The otolithic membrane is just discernable over the utricular macula. To the right of the utricle are the ampullar cristae of the lateral (above) and superior (below) semicircular canals (SSCs). The connective tissue supporting the membranous walls of the utricle and superior SSC appears along the bottom of the image, with capillaries showing up as a somewhat brighter, lacy network within this tissue. Fig. 6 is a high-magni¢cation section through the ampullar cristae of the superior and lateral SSCs. The sensory epithelial layers containing the hair cells are visible, as are the ¢bers of the vestibular nerve leading to them. This image magni¢cation was achieved using a 60 mm extension tube with the 4U lens.
4. 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 di¡erent ratios (Culling, 1957; Emmel and Cowdry, 1964; Tompsett, 1970) and indeed most recent workers report using a modi¢ed Spalteholz technique that uses only methyl salicylate for tissue clari¢cation (Bissonette and Fekete, 1996; Bever and Fekete, 2000). The author has used the composition described in this paper since 1986 with satisfactory results and has stayed with it for that reason. Tissue preparations have been stored for over 10 years with no discernable degradation or loss of £uorescence. Tissue photo-bleaching may result after prolonged exposure to laser light, and so should be avoided.
The OPFOS images shown above are at in increasing levels of magni¢cation and spatial resolution. The features of the vestibular system were chosen to demonstrate high-magni¢cation capability because they present challenges for imaging in general. The endolymphatic membrane-bounded spaces, and the sensory organs contained within, are di⁄cult to visualize. These features are beyond the resolving capabilities of CT and MRI, while histology sections cannot easily be oriented to pass through all vestibular system sensory organs. The technique of clearing the tissue and ¢lling the membranous labyrinth with paint (Bissonette and Fekete, 1996) allows the vestibular system to be seen in outline but the sensory tissues are obscured in the process. This work demonstrates that the OPFOS technique may be used to appreciate the £uid spaces, membranous boundaries and sensory tissues of the vestibular system. Since the specimen can be translated and rotated, it is possible to acquire data for 3D reconstruction and quantitative measurements of the middle ear and vestibular system as well as for the cochlea. As mentioned above, the horizontal lines that appear in Figs. 3^6 are due to non-uniform absorption of the laser light as it traverses the tissue. When the laser light traverses tissue with a higher regional dye concentration, more of the light is absorbed and less is available for more distal areas. This gives rise to narrow shadow lines in the direction of the laser illumination. Bringing additional laser light from the opposite direction to more uniformly illuminate the focal zone theoretically could reduce this e¡ect. Alignment of multiple beam focal zones to overlap precisely would be challenging, however. Vertical lines are evident in Figs. 5 and 6, which are the higher-magni¢cation OPFOS images. Because less
Fig. 7. Comparison of three cochlear imaging modalities. Shown are mid-modiolar sections of approximately equal scale. (A) Histological serial section. (B) MR microscopy section. (C) OPFOS section. The images in frames (A) and (B) are reprinted with permission from the Cochlear Fluids Research Laboratory website (http://oto.wustl.edu/cochlea).
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£uorescent light enters the macro lens as the imaging region becomes smaller, the brightness of an OPFOS image is inversely proportional to its magni¢cation. By increasing the gain of the video camera, small and faint structures may be visualized. However, this introduces noise, in the form of random speckling, to the image. Averaging reduces the noise, but this in turn produces vertical lines in the image, which are associated with the sensing elements of the CCD camera. It may be possible to reduce this e¡ect with a more sensitive imaging device, but this would probably be an expensive upgrade. Another approach to improving the sensitivity of this inherently low-light process is to use an imaging lens of higher numerical aperture. The challenge to this is the requirement of a long working distance for the lens, which must be located outside of the sealed Spalteholz-¢lled sample chamber. Typically, lens numerical aperture is inversely proportional to its working distance. Comparing the imaging technique of OPFOS to MR microscopy and histological serial sections, the following statements can be made. OPFOS provides better resolution than MR, but not as good as histology. This is evident in Fig. 7, which shows images (of di¡erent specimens of guinea pig cochleae) from these modalities side-by-side for comparison. The resolution of the histological section (A) and the OPFOS image (C) is clearly higher than that of the MR image (B). Comparing frames (A) and (C), the superior spatial resolution of the histological section is evident when inspecting the organ of Corti. In (A) the tunnel of Corti and supporting cells are clearly seen. If the goals of a study require resolution to the cellular level and beyond, or if 3D macro-anatomy need not be preserved, then histological techniques have compelling advantages. On the other hand MR and OPFOS can lead to an understanding of 3D macroanatomy that is much more intuitive than from histological sections. A comparison of the time involved with each technique for 3D reconstruction requires the process to be broken into stages of image acquisition, feature extraction and data analysis. Image acquisition by MR probably requires the least time of these methods. There is little tissue preparation necessary beyond ¢xation and trimming of excess bone to ¢t the sample into the chamber of the MR device (Thorne et al., 1999). The time from tissue harvest to completion of imaging may be on the order of days. The time to complete the imaging process for OPFOS and histological sectioning may be several weeks, taking the tissue processing time into account. Feature extraction and data analysis is probably equivalent for the three methods, requiring a fair amount of trained human interaction such as tracing of boundaries. Comparing the whole specimen techniques, OPFOS
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provides greater resolution than MR, however the tissue preparation and image acquisition time is faster with MR. An advantage of the OPFOS technique is the simplicity of the apparatus, most of which can be built from relatively inexpensive components (the system used in this work, including host computer, frame grabber, software, laser, video camera, optical table, optical components and positioning equipment, was assembled by the author for under US$25 000). This makes control of the imaging process available to the researcher, who would otherwise need to take the tissue to one of a limited number of centers that have the expensive MR microscopy apparatus. Control of the imaging process is one of the major advantages of the OPFOS system, in that the device produces images in real time, giving it a unique interactive quality that facilitates exploration and education.
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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