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Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
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Micro-optical coherence
tomography of the mammalian
cochlea
Janani S. Iyer1,2,3,*, Shelley A. Batts1,2,†,*, Kengyeh K. Chu4,5, Mehmet I. Sahin1,2,
Hui Min Leung4,5, Guillermo J. Tearney4,5,‡ & Konstantina M. Stankovic1,2,3,‡
The mammalian cochlea has historically resisted attempts at high-resolution, non-invasive imaging
due to its small size, complex three-dimensional structure, and embedded location within the temporal
bone. As a result, little is known about the relationship between an individual’s cochlear pathology and
hearing function, and otologists must rely on physiological testing and imaging methods that oer
limited resolution to obtain information about the inner ear prior to performing surgery. Micro-optical
coherence tomography (μOCT) is a non-invasive, low-coherence interferometric imaging technique
capable of resolving cellular-level anatomic structures. To determine whether μOCT is capable of
resolving mammalian intracochlear anatomy, xed guinea pig inner ears were imaged as whole
temporal bones with cochlea in situ. Anatomical structures such as the tunnel of Corti, space of Nuel,
modiolus, scalae, and cell groupings were visualized, in addition to individual cell types such as neuronal
bers, hair cells, and supporting cells. Visualization of these structures, via volumetrically-reconstructed
image stacks and endoscopic perspective videos, represents an improvement over previous eorts
using conventional OCT. These are the rst μOCT images of mammalian cochlear anatomy, and they
demonstrate μOCT’s potential utility as an imaging tool in otology research.
Few treatments for human hearing loss exist, largely because the relationship between an individual patient’s
cochlear pathology and their degree of hearing loss is poorly understood. A large obstacle to achieving this under-
standing is the inability to perform noninvasive imaging on patients’ inner ears at a resolution sucient to assess
potential physiological contributions to hearing impairment. Hearing loss can result from physiological damage
to the sensory hair cells and spiral ganglion, malformation of or damage to areas necessary for sound conduc-
tion through bone, or a mixture of these pathologies1. However, conventional clinical imaging methods, such as
magnetic resonance imaging (MRI) and computed tomography (CT), are limited in spatial resolution to approx-
imately 1 mm and 0.5–1 mm, respectively2,3. Consequently, these modalities can only detect gross abnormalities,
such as profound malformations in the bony anatomy of the cochlea. MRI and CT are largely insensitive to
intracochlear defects that fall beneath this detection range, such as missing and damaged cells within the cochlea’s
sensory epithelium, the organ of Corti4.
e organ of Corti is a heterogeneous matrix of sensory and non-sensory epithelial cells that contribute to
both the perception and ne-tuning of frequencies within the range of mammalian hearing5. Supporting cells,
such as pillar, Deiters, and Hensen’s cells, provide structural and molecular support to the sensory hair cells. Hair
cells are organized as a single row of inner hair cells (IHC), which receive 90% aerent innervation, and three
rows of outer hair cells (OHC), which receive 90% eerent innervation. e inner hair cells transduce sound via
mechanical shearing forces imparted by the basilar membrane’s vibration and the cells’ protruding actin stere-
ocilia. Stereocilia deection prompts neurotransmitter release into the post-synaptic space near spiral ganglion
1Eaton-Peabody Laboratories and Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 243
Charles St, Boston, MA, USA. 2Department of Otolaryngology, Harvard Medical School, 25 Shattuck St, Boston,
MA, USA. 3Program in Speech and Hearing Bioscience and Technology, Harvard University Graduate School of Arts
and Sciences, 1350 Massachusetts Ave, Cambridge, MA, USA. 4Wellman Center for Photomedicine, Massachusetts
General Hospital, 50 Blossom St, Boston, MA, USA. 5Department of Pathology, Massachusetts General Hospital, 55
Fruit St, Boston, MA, USA. †Present address: Analysis Group, Inc., Health Economics and Outcomes Research, 111
Huntington Ave, 14th oor, Boston, MA, USA. *These authors contributed equally to this work. ‡These authors jointly
supervised this work. Correspondence and requests for materials should be addressed to G.J.T. (email: tearney@
helix.mgh.harvard.edu) or K.S. (email: konstantina_stankovic@meei.harvard.edu)
Received: 19 April 2016
Accepted: 23 August 2016
Published: 16 September 2016
OPEN
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Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
neurites, generating electrical signals in response to frequency-specic stimulation that is sent to brainstem and
cortical processing regions. e cochlea contains other so tissue microstructures that are critical for hearing:
Reissner’s membrane, which serves as a diusion barrier separating the contents of two of the cochlea’s uid-lled
cavities; the tectorial membrane, which contacts hair cells’ stereocilia during sound transduction; the stria vascu-
laris in the spiral ligament, which maintains ionic gradients of endocochlear uids and provides a blood barrier;
and the neurites of the spiral ganglion, which form one branch of the auditory nerve, and extend both radially and
diagonally along the sensory epithelium6. ese structures fall beneath the detection limits of both MRI and CT.
Optical coherence tomography (OCT) is a non-contact, cross-sectional imaging technique that applies low
coherence interferometry to image opaque subsurface structures with a resolution typically from 10–15 μ m in
axial and 30–40 μ m in transverse planes7,8. During OCT imaging, infrared laser light is backscattered by micro-
structural features within a structure or organ of interest. e dimensions of these features can be determined
by applying low coherence interferometry, which enables the backscattered sample light to be resolved in depth.
OCT is characterized by high detection sensitivity, as small as 10−10 of the incident optical power8, and a penetra-
tion depth of 1–3 mm, depending on tissue type7. OCT is routinely used in clinical ophthalmology to image the
retina and cornea9,10 and in dermatology11,12, and has previously been used for cellular and submicrometer imag-
ing13–15. Intracochlear morphology and mechanics have also been observed with OCT in rodent models, with
axial and lateral resolution ranging from 10–20 μ m16–20, ex vivo16,17,19 and in vivo13,18,20. OCT has enabled the iden-
tication of larger structures including Reissner’s membrane16–20, the basilar16–20 and tectorial membranes18–20, the
spiral ligament17, the three scalae of the cochlea16,18,20, and the region of the sensory epithelium18–20, in addition
to spaces between various structures such as the demarcation between the osseous and membranous labyrinths17,
the tunnel of Corti19, and spaces between regions of inner and outer hair cells19, ex vivo16,17,19 and in vivo18,20.
Cochlear mechanics and measurements of the motion and displacement of intracochlear structures in response
to frequency-specic auditory stimulation have also been studied with OCT ex vivo21,22 and in vivo20. However,
similar to traditional imaging methodology, these studies have been limited by OCT’s resolution threshold, and
have not been capable of resolving smaller anatomical features including the major therapeutic targets in hearing
loss such as inner and outer hair cells, supporting cells, and nerve bers.
To improve the spatial resolution of conventional OCT, we introduced a successor called micro-optical coher-
ence tomography (μ OCT) and demonstrated its ability to resolve individual endothelial cells, leukocytes, lympho-
cytes, and monocytes in human cadaver coronary arteries, at a resolution of 2 μ m × 2 μ m × 1 μ m (x, y, z)23. μ OCT
has more recently been utilized to detect cholesterol crystals within macrophages in atherosclerosis24, to visualize
functional anatomy, including individual beating cilia involved in mucociliary clearance and transport in airway
epithelium25,26, and to resolve cellular details in zebrash larvae in vivo27. μ OCT technology may also be suitable
to resolve cochlear microanatomy at a cellular level. us, this study’s objective was to determine whether μ OCT
was capable of resolving major and micro-anatomical structures within the mammalian inner ear, and to generate
the rst μ OCT images of xed guinea pig intracochlear anatomy in situ.
Results
Figure1a–c depicts cross-sections of a guinea pig cochlea cut along its longitudinal axis, and stained with hema-
toxylin and eosin (H&E) to highlight cellular structures. Figure1a shows eight cochlear half-turn cross-sections,
corresponding to the four cochlear turns, spiraling around the bony, neuron-lled core, the modiolus (M). e
basilar membrane (BM) and Reissner’s membrane (RM) delineate the cochlea’s three uid-lled chambers: the
scala tympani (ST), scala media (SM) and scala vestibuli (SV). e bony otic capsule surrounding the cochlear
tissue is stained purple. Figure1b zooms in on the single half-turn (boxed in blue in Fig.1), and Fig.1c zooms
in on the region boxed in red in Fig.1b, depicting the sensory cells (inner and outer hair cells) and non-sensory
cells (including inner and outer pillar cells) of the organ of Corti, as well as other supporting cells and the tectorial
membrane.
Excised guinea pig temporal bones were imaged with μ OCT via a 0.5–1 mm diameter cochleostomy in the
otic capsule, corresponding to either a) the region of the second of the four cochlear turns, exposing the area
from the top of the third turn to the top of the second turn, laterally, or b) the apex. μ OCT permitted imaging in
1 mm × 1 mm and 500 μ m × 500 μ m elds of view. Raw images of the cochlea’s apical turn are shown in Fig.2.
Figure2a reveals the region of inner hair cells and inner pillar cells, a row of outer pillar cells (OPCs), and three
rows of OHCs. e dark space between the OPCs and OHCs is the space of Nuel; the dark space between the
OPCs and the inner pillar cells is the tunnel of Corti. Figure2b shows a single plane image of the region where
outer hair cells reside – individual outer hair cells are identiable. For reference and orientation, Fig.2c,d show
immunohistochemically-labeled cells and neuronal processes in the guinea pig organ of Corti.
Aer performing a volumetric reconstruction of the raw 2D scans, neuronal ber bundles became visible at
several levels within the tunnel of Corti and space of Nuel along the length of the imaged tissue (Fig.3). Due to
their location and radial trajectory across the tunnel of Corti and space of Nuel (the endolymph-lled epithelial
lumens situated between the inner and outer pillar cells and the outer pillar and hair cells, respectively), these
nerve ber bundles are hypothesized to be synaptically connected to outer hair cells.
Visualizations 1a and 1b allow the viewer to virtually “y through” the space of Nuel and tunnel of Corti,
respectively, in volumetric reconstructions of two μ OCT imaging stacks of the guinea pig organ of Corti. e
orientation visualized here is the same as those depicted in Fig.1a–c. In Visualization 1a, bundles of neuronal
bers are observed traversing the basal region of the space of Nuel (labeled still image shown in Fig.4a). In
Visualization 1b, a single bundle of neuronal bers is observed crossing the central region of the tunnel of Corti
(labeled still image shown in Fig.4b). We hypothesize that this is a fascicle of medial olivocochlear eerent nerve
bers, based on its radial trajectory and location within the tunnel28. A dierent nerve ber bundle is observed
traveling longitudinally along the medial wall of the tunnel of Corti for the length of the reconstructed tissue
section. e longitudinal trajectory and specic location of this bundle are characteristic of the tunnel spiral
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Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
bundle (TSB), a mass of primarily lateral olivocochlear nerve bers. e TSB is also visualized in cross-section
in Fig.5a–c, which display its precise location in three 2D orientations. Our interpretation regarding the tunnel
spiral bundle and tunnel-crossing bundle of medial eerent bers are consistent with previously reported arbori-
zation patterns of eerent bers in the cat cochlea29,30.
A volumetric reconstruction of the imaged section of the guinea pig organ of Corti viewed in cross-section
revealed discernable individual cell types in situ (Fig.6). In this image, the scalae tympani and media are clearly
visualized, separated by the basilar membrane and the organ of Corti atop it. Other identiable structures include
the bony modiolus (MOD) and the spiral limbus (SL; medial and medio-apical to the basilar membrane, respec-
tively), inner and outer pillar cells (IPC and OPC, respectively), outer hair cells (OHC), the tunnel of Corti (TC),
and space of Nuel (SN; inferior and medial to the outer hair cells). e inner hair cells are medial to the tunnel
of Corti; their embedded location did not permit visualization here. Bundles of nerve bers (NF) were observed
traveling from within the spiral lamina across the space of Nuel (SN) to the region of the outer hair cells.
Discussion
In presenting the rst, to our knowledge, μ OCT images of the mammalian cochlea, this report provides evidence
of μ OCT’s utility as a high-resolution intracochlear imaging tool. Our μ OCT imaging system resolved cellular
anatomy in the guinea pig organ of Corti, including nerve ber bundles, which have eluded conventional clinical
imaging methods thus far. Importantly, results of the present study also reveal that high resolution is not the sole
criterion for achieving informative images; indeed, our data suggest that μ OCT resolves some of the organ of
Corti’s anatomical and cellular features, such as lumens and bundles of neurites, more readily than other struc-
tures that are more deeply embedded in the sensory epithelium, such as inner hair cells. us, it is evident that
factors such as sample contrast and speckle noise, in addition to resolution, are important consideration for future
improvements on this technology31.
Hearing loss is the most common sensory decit in the world32 and the most common disability in the United
States33 . Because mammalian cochlear hair cells and neurons do not spontaneously regenerate, hearing loss is
permanent and irreversible in the vast majority of cases. A signicant barrier to developing otologic therapies is
a limited understanding of how cochlear pathology relates to the degree and type of hearing loss34. e cochlea
remains a “black box” in living subjects, closed to direct or conventional imaging due to its embedded location,
fragility, and complex structure. Our knowledge of cochlear physiology and morphology today thus comes pri-
marily from post-mortem analyses of human temporal bones and experiments using animal models, which have
revealed many physiological sources of hearing impairment: sensory hair cell loss or damage35,36; damage to
stereocilia due to noise over-exposure37; malformations of the tectorial membrane38; loss of auditory nerve bers
and spiral ganglion neurons39; and atrophy of the stria vascularis40.
Numerous studies have noted the value of traditional OCT imaging for the cochlea16–21,41–43; however, the
improvements that μ OCT aords over OCT in resolution and depth-of-focus (DOF) make it better-suited for
imaging the cochlea. e anatomical undulations in the cochlea require high DOF to simultaneously capture peaks
Figure 1. Micrographs of a sectioned guinea pig cochlea. (a) Cross-section of a guinea pig cochlea, stained
with H&E, and cut along its longitudinal axis. M: modiolus. BM: basilar membrane; RM: Reissner’s membrane,
ST: scala tympani; SM: scala media; SV: scala vestibuli. Magnication = 2×; Scale = 1 mm. (b) A single half-turn
of the guinea pig cochlea, representing the region boxed in blue in (a). Magnication = 10×; Scale = 200 μ m.
(c) Zoomed-in view of the organ of Corti (boxed in red in (b)). Colored arrows point to specic cell types: inner
(red) and outer (blue) hair cells, inner (green) and outer (orange) pillar cells, and neuronal bers (turquoise),
which travel through the tunnel of Corti (TC) and space of Nuel (SN). Pink arrows: supporting cells; purple
arrow: tectorial membrane. Magnication = 10× ; Scale = 200 μ m.
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and valleys; OCT’s shorter DOF may be responsible for limiting earlier studies to 5–10 μ m resolution. We have
previously reported concurrent gains in DOF and resolution in μ OCT and established the utility of this enhanced
performance in cardiovascular23 and airway imaging applications44. Compared to other conventional imaging,
the advantages of μ OCT include that it (1) can be conducted on the benchtop, (2) requires no contrast agent,
(3) can image whole structures within its detection eld near-instantaneously, and (4) employs primarily infra-
red and near-infrared light sources, theoretically safer than higher-energy (lower wavelength) laser exposure45.
While signicant development is required before this technology can be employed to assess cochlear pathology
in living humans, the present study demonstrates μ OCT’s potential to image this organ and motivates further
miniaturization of μ OCT technology. We have recently reported progress in the development of miniaturized
μ OCT instrumentation for in vivo applications46,47 and in human cochlear endoscopy via the external auditory
canal48. However, the adaptation of our imaging conguration to the constraints of the small size and embedded
location of the human cochlea remains a signicant technical challenge.
Clinical realization of μ OCT cochlear imaging faces signicant surgical and engineering hurdles; however,
applications for otolaryngology research in animals may be more immediately realized. In contemporary studies
Figure 2. μ OCT images and immunohistochemically-stained regions of sensory and supporting cell rows
within the guinea pig organ of Corti. (a) Single 2D image from a μ OCT imaging stack, depicting the regions
of the inner pillar cells and inner hair cells (red arrow), outer pillar cells (orange arrow), and 3 rows of outer hair
cells (blue arrows). e schematic in the top right-hand corner shows the orientation of the plane (pink) along which
the image was sectioned relative to the orientation of the cochlea. Scale = 100 μ m. (b) Single 2D image depicting
individual outer hair cells (examples indicated with blue arrows). Scale = 50 μ m. (c) Immunohistochemically-
stained guinea pig organ of Corti whole mount, corresponding to the orientation presented in (a). Cytoskeletal
actin within hair cells and supporting cells is labeled with rhodamine phallodin (red), neuronal processes are
labeled with neurolament-H (green), and cell nuclei are labeled with Hoechst stain (blue). Scale = 50 μ m.
(d) Zoomed-in view of immunohistochemically-stained guinea pig organ of Corti depicting the orientation
presented in (b). Color convention as in (c). Scale = 25 μ m.
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employing animal models of hearing impairment, the techniques most commonly used to detect and visualize
changes in cochlear morphology include confocal and two-photon microscopy in tandem with histology and
immunohistochemistry. ese techniques are applied post-mortem, and both specimen preparation and imag-
ing itself may be extremely time-consuming. An imaging technique such as μ OCT, capable of resolving cochlear
microanatomy in experimental animals as genetic models of human hearing loss, could potentially be adapted
Figure 3. μOCT image of nerve ber bundles traversing the tunnel of Corti and space of Nuel to innervate
outer hair cells (500 μm × 500 μm). (a) Volumetric reconstruction of maximum-projected μ OCT image stack,
depicting bundles of nerve bers traversing the organ of Corti towards the outer hair cell region. e schematic
in the top right-hand corner shows the orientation of the virtual sectioning plane. Scale = 150 μ m. (b) Schematic
representation of the microanatomy in the top panel, with bundles of nerve bers (NF) crossing the tunnel of
Corti (TC) and/or the space of Nuel (SN). OPC = outer pillar cells. Scale = 150 μ m. (c) For reference, a confocal
laser scanning microscopy image of the guinea pig organ of Corti. Rhodamine phalloidin (red) marks outer and
inner pillar cells (OPC and IPC, respectively), Hoechst stain (blue) marks cell nuclei, and neurolament-H
(green) marks neuronal bers. Scale = 50 μ m.
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for in vivo longitudinal analysis of cochlear anatomy in healthy and pathological states. Potential in vivo applica-
tions in animals include monitoring physiological changes resulting from genetic mutations associated with pro-
gressive hearing loss, facilitating surgical guidance when advancing therapy-delivering probes, targeting distinct
intracochlear spaces or frequency-specic locations through narrow cavities in the ear49, assessing and measuring
vibration in the organ of Corti prior to and post exposure to noise or regenerative therapies50, and acquiring an
improved understanding of the natural progression of age-related hearing loss. Future directions of this work
include conducting in vivo μ OCT experiments in guinea pigs, to determine optimal surgical approaches and the
instrumentation’s performance in a living subject.
e present work is subject to several limitations, namely that the described experiments were conducted in an
animal model and within healthy, normal cochleae; the specimens used were excised, partially dissected, and xed
for ease of manipulation; and a small number of cochleae (7) were examined. Nevertheless, these ndings represent
an important incremental advance regarding μ OCT’s ability to perform cellular-level imaging of the mammalian
cochlea, which we hope will accelerate the technology’s improvement and customization for broader applications.
Figure 4. 3D volumetric reconstruction revealing bundles of nerve bers traveling through the tunnel of
Corti and space of Nuel. (a) A labeled, colorized still from a 3D volumetric reconstruction of a μ OCT image
stack (Visualization 1a), “ying through” the space of Nuel (SN), showing bundles of nerve bers (NF, blue)
crossing the basal region of the SN. (b) A labeled, colorized still from a 3D volumetric reconstruction of a μ OCT
image stack (Visualization 1b), “ying through” the tunnel of Corti (TC), showing a single bundle of medial
eerent nerve bers crossing the central region of the TC, and the tunnel spiral bundle (TSB, yellow) running
along the tunnel’s medial wall. Please refer to the Supplemental Materials to view Visualizations 1a and b.
500 μm × 500 μ m eld of view.
Figure 5. Two-dimensional μOCT images of the tunnel spiral bundle (TSB) (500 μm × 500 μm) within
the guinea pig organ of Corti, in three perspectives. Yellow cross hairs in (a) (cross-section of the organ of
Corti and two uid lumens, separated by a pillar cell) and (c) (looking down from above the organ of Corti)
indicate the TSB’s (medial section, shown in (b)) specic position along the medial wall of the tunnel of Corti.
e schematics in the top right-hand corner of each panel show the orientation of the 2D plane depicted,
respectively. Scale = 100 μ m.
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Conclusions
Innovation of imaging systems capable of resolving mammalian middle and inner ear anatomy is essential for
understanding the link between otopathology and hearing function. μ OCT, shown here to be capable of resolving
microanatomy in the mammalian cochlea, aords the resolution and speed necessary to be considered a promis-
ing candidate for otologic imaging development; however, signicant progress remains to be made. Future eorts
should aim to improve μ OCT’s resolution and penetration depth, determine whether similar imaging results can
be reproduced in living animal subjects, and dene the limits of the instrumentation and surgical approaches to
permit optimal imaging access.
Methods
Animals. e guinea pig is a well-studied animal model that is commonly used in translational research stud-
ies on hearing and hearing loss because its frequency sensitivity and susceptibility to ototoxic medications are
similar to that in humans51–54, and its entire cochlea is surgically accessible. Albino adult male guinea pigs of
approximately 400 grams were used in this study (7 cochleae were imaged). e Institutional Animal Care and
Use Committee (IACUC) of Massachusetts Eye and Ear Inrmary (MEEI) approved all experimental protocols
for this study, and all procedures were carried out in accordance with approved institutional guidelines of the
IACUC. Guinea pigs were maintained at MEEI’s animal care facility in Boston, MA.
Instrumentation and image collection. e system was a customized, spectral-domain OCT (SD-OCT)
instrument with improvements to standard OCT that yield higher resolution in lateral and axial directions. e
instrumentation layout has been previously described23 and is illustrated in Fig.7.
In brief, a circular obscuration was created in the sample beam path to enhance axial depth-of-focus (DOF)
and eld-of-view, maintaining an extended DOF of approximately 300 μ m with a numerical aperture of 0.12. e
resulting lateral resolution was 2 μ m23. e high axial resolution of 1 μ m was derived from a high bandwidth cus-
tom OCT spectrometer, spanning 650–950 nm combined with an ultra-broadband supercontinuum laser source
(NKT Photonics, Birkerod, Denmark).
e optical power at the sample was less than 15 mW. Transverse (x, y) scanning across the sample was per-
formed using soware-controlled galvanometer scanning motors (orlabs, Newton, NJ); the same soware was
used to simultaneously acquire spectral data through the spectrometer camera. Images comprising 512 A-lines
were acquired at 40 frames per second (fps), with each frame spanning either 500 μ m or 1 mm in lateral space.
ree-dimensional (3D) volumes were acquired by scanning over a square region spanning either 500 × 500 μ m
or 1 × 1 mm. A large working distance (25 mm), dened as the distance between the objective lens and the focal
plane, was chosen to allow exible positioning of specimens on the sample stage. Cross-sectional and 3D images
are displayed using logarithmic, logarithmic inverse, or linear grayscale lookup tables, which depict liquid-lled
regions (e.g. the scalae) as dark, and highly-scattering regions (e.g. bone and tissue) as light.
Specimen processing. Guinea pig temporal bones were extracted following euthanasia (intraperito-
neal injection of Fatal-Plus Solution [0.1 mL/kg; Vortech Pharmaceuticals, Dearborn, MI]) and decapitation.
Temporal bones were dissected in cold 4% paraformaldehyde (PFA) (diluted in Phosphate Buered Saline [PBS])
Figure 6. Volumetrically reconstructed μOCT image (500 μm × 500 μm) and schematic of the guinea pig
organ of Corti in situ. (a) Volumetric reconstruction of μ OCT-visualized sensory and non-sensory cells of the
organ of Corti from the 2nd–3rd turn of the cochlea. (b) Schematic labeling structures visualized in the le panel,
such as outer hair cells (OHC), bundles of nerve bers (NF), and inner and outer pillar cells (IPC and OPC,
respectively). MOD = modiolus; SL = spiral limbus; IHC = inner hair cell; TC = tunnel of Corti; SN = space of
Nuel. Both scales = 100 μ m.
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to expose the cochlea’s interior to xative by opening the apical otic capsule and puncturing the round and oval
window membranes with forceps. Approximately 1 ml of cold 4% PFA was slowly infused into the apical hole
until it owed out of the oval and round windows. e temporal bones were immersed in cold 4% PFA in PBS for
4–8 hours on a shaker. Seven specimens were further dissected in PBS to remove some of the otic capsule, expos-
ing a section or the entire cochlea. Specimens were stored at 4 °C in PBS prior to μ OCT imaging. Two additional
specimens were placed in 0.12M EDTA for two weeks to decalcify the otic capsule for whole mount preparation
and immunostaining (see section ‘Immunohistochemistry’ below).
μOCT imaging and image analysis. During μ OCT imaging, specimens were removed from the PBS and
positioned beneath the μ OCT laser aperture in a dry plastic culture dish. e imaging apparatus was adjusted in
X, Y, and Z directions to achieve optimal focus.
Raw data were converted into tagged image le format (.tif) stacks using standard SD-OCT processing rou-
tines55 and then imported into OsiriX (Pixmeo SARL, Bernex, Switzerland), a soware program routinely used
to analyze clinical CT and MRI images. OsiriX was used to reconstruct (multiplanar reconstruction and volu-
metric rendering) the μ OCT images in 3D, generate maximum intensity projections, rotate, enlarge, and crop the
images, set image opacity, and generate scale bar measurements. Images were labeled, structures were colorized
with a partially transparent brush (for ease of visualization), and scale bars were traced in Photoshop (Adobe,
Inc., San Jose, CA). 3D volumetric μ OCT images were constructed using an endoscopy perspective in OsiriX to
create “y-through” videos (15 fps) revealing intracochlear structures.
Haematoxylin/eosin (H&E) histology. Animal euthanasia and specimen extraction and xation followed
the protocol described above. For H&E staining, guinea pig cochleae were perfused with 10% formalin and decal-
cied in 0.27M EDTA for 25 days. Specimens were then dehydrated in ethanol and embedded in celloidin (1.5%
celloidin for 1 week, 3% for 2 weeks, 6% for 3 weeks, 12% for 3 weeks). Hardened ears were mounted on a ber
block for sectioning with a sliding microtome. e sections were mounted on glass slides, stained with H&E, and
preserved in 80% alcohol. Imaging of these sectioned specimens was conducted with an Olympus BH2 micro-
scope (Olympus, Tokyo, Japan) at 2× and 10× magnications for Fig.1a,b, respectively. Figure1b was cropped
using Adobe Photoshop CS5.1 (Adobe, Inc., San Jose, CA) to highlight cellular detail (Fig.1c).
Figure 7. μOCT instrumentation. Schematic diagram of μ OCT system. Supercontinuum laser (SCL) power
is directed by collimating and focusing lenses (L) through a single mode ber (SMF). Output light from the
SMF is collimated and passed through an apodizing mirror (AM), resulting in a circular obscuration of the
transmitted light, which is steered by a galvanometer mirror (GM) through an objective lens onto the sample.
Light reected by the AM is focused onto a reference mirror (RM), and the reference lens and mirror assembly
can be translated in unison to adjust the reference path length. Back-scattered light from the sample is re-
integrated at the SMF with light reected by the RM. e return light is collimated and directed by the beam-
splitter (BS) towards a diraction grating (G). e spectrally dispersed light is then focused onto a line scan
camera (LSC), which outputs raw spectrograms through a CameraLink (CL) interface to an image acquisition
board (IMAQ) installed in a PC. e PC also controls scanning through a data acquisition card (DAQ), which
produces an analog output (AO) voltage signal that controls the GM.
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Immunohistochemistry and confocal imaging. Guinea pig cochleae were extracted, xed, and decalci-
ed as described above. Under a stereomicroscope, the decalcied otic capsule was peeled away from the cochlea,
and the 4 turns of the cochlea were sectioned into eight pieces. Each piece was further microdissected to reveal
the organ of Corti. e spiral ligament, stria vascularis, and tectorial and Reissner’s membranes were removed.
Aer being rinsed in PBS for 15 minutes, cochlear sections were blocked with 5% Normal Horse Serum (NHS;
Sigma-Aldrich, St. Louis, MO) in 1% Triton X-100 (Integra Chemical, Kent, WA), and were placed on a shaker for
30 minutes at room temperature. Sections were incubated with a primary antibody against neurolament-H (pol-
yclonal chicken; AB5539, Lot #2701573; EMD Millipore, Temecula, CA) overnight. Aer rinsing for 15 minutes
in PBS, the tissue was incubated in a secondary antibody (AlexaFluor 488 goat anti-chicken IgG, A-11039, Lot
#898239; ermoFisher Scientic, Inc., Waltham, MA) diluted in 1% NHS with 0.4% Triton X-100 for 90 minutes
in combination with 1:200 rhodamine phalloidin (ermoFisher Scientic, Waltham, MA). Finally, the tissue was
placed in Hoechst stain 33342 (Life Technologies, NY; 1 nM in PBS), and washed for 15 minutes in PBS and briey
in distilled water. Stained tissue was mounted under coverslips on glass slides with Vectashield mounting media
(Vector Laboratories, CA, #H-1000).
Cochlear whole mounts were imaged with 20X (PlanApochromat, oil immersion, NA= 0.7; #H1LG/02; Leica,
Wetzlar, Germany) and 63X (PlanApochromat, oil immersion, NA= 1.3, #506194; Leica, Wetzlar, Germany) Leica
objectives, using a Leica TCS SP5 laser-scanning confocal microscope. Images (1024 x 1024 pixels) were collected
in z-stacks and masked as maximum intensity projection images in the Leica soware. e nal images were
cropped and scale bars were retraced in Photoshop CS5.1.
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Acknowledgements
is work was supported by the Bertarelli Foundation (K.M.S.), the Wyss Center Geneva (K.M.S.), Department
of Defense grant W81XWH-15-1-0472 (K.M.S.), the Nancy Sayles Day Foundation (K.M.S.), the Lauer Tinnitus
Research Center (K.M.S.), and the MGH Research Scholars program (G.J.T.). e authors thank Dr. M. Charles
Liberman for insightful comments on the data and manuscript, Dr. Takeshi Fujita for providing the guinea pig
temporal bones, and Jennifer O’Malley at the Massachusetts Eye and Ear Otopathology Laboratory for providing
histology slides.
Author Contributions
K.M.S. and G.J.T. conceived of and supervised the work. K.M.S, G.J.T., S.A.B., K.K.C. and J.S.I. designed the
experiments. J.S.I., S.A.B., K.K.C., M.I.S. and H.M.L. performed the experiments. J.S.I., S.A.B., K.K.C., M.I.S.,
K.M.S. and G.J.T. analyzed data. S.A.B., J.S.I. and K.M.S. wrote the manuscript. All authors critically edited the
manuscript and approved the nal version.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Iyer, J. S. et al. Micro-optical coherence tomography of the mammalian cochlea. Sci.
Rep. 6, 33288; doi: 10.1038/srep33288 (2016).
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