ArticlePDF Available

Micro-optical coherence tomography of the mammalian cochlea

Authors:

Abstract and Figures

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 offer 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, fixed 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 fibers, hair cells, and supporting cells. Visualization of these structures, via volumetrically-reconstructed image stacks and endoscopic perspective videos, represents an improvement over previous efforts using conventional OCT. These are the first μOCT images of mammalian cochlear anatomy, and they demonstrate μOCT’s potential utility as an imaging tool in otology research.
Content may be subject to copyright.
1
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
www.nature.com/scientificreports
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 oer
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 eorts
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 patients
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 sucient 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 cochleas
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 Hensens 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% aerent innervation, and three
rows of outer hair cells (OHC), which receive 90% eerent innervation. e inner hair cells transduce sound via
mechanical shearing forces imparted by the basilar membranes vibration and the cells’ protruding actin stere-
ocilia. Stereocilia deection 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
www.nature.com/scientificreports/
2
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
neurites, generating electrical signals in response to frequency-specic 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 diusion barrier separating the contents of two of the cochleas 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 1010 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-
tication 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-specic 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 zebrash 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
Figure1a–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. Figure1a 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 Reissners 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. Figure1b 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.
Figure2a 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. Figure2b shows a single plane image of the region where
outer hair cells reside – individual outer hair cells are identiable. For reference and orientation, Fig.2c,d show
immunohistochemically-labeled cells and neuronal processes in the guinea pig organ of Corti.
Aer 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 eerent nerve
bers, based on its radial trajectory and location within the tunnel28. A dierent 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 specic location of this bundle are characteristic of the tunnel spiral
www.nature.com/scientificreports/
3
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 eerent bers are consistent with previously reported arbori-
zation patterns of eerent 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 identiable 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 OCTs 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 decit 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 signicant 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 aords 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. Magnication = 2×; Scale = 1 mm. (b) A single half-turn
of the guinea pig cochlea, representing the region boxed in blue in (a). Magnication = 10×; Scale = 200 μ m.
(c) Zoomed-in view of the organ of Corti (boxed in red in (b)). Colored arrows point to specic 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. Magnication = 10× ; Scale = 200 μ m.
www.nature.com/scientificreports/
4
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
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 signicant 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 conguration to the constraints of the small size and embedded
location of the human cochlea remains a signicant technical challenge.
Clinical realization of μ OCT cochlear imaging faces signicant 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 neurolament-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.
www.nature.com/scientificreports/
5
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
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 neurolament-H
(green) marks neuronal bers. Scale = 50 μ m.
www.nature.com/scientificreports/
6
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
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-specic 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
eerent 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)) specic 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.
www.nature.com/scientificreports/
7
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
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, aords the resolution and speed necessary to be considered a promis-
ing candidate for otologic imaging development; however, signicant progress remains to be made. Future eorts
should aim to improve μ OCTs resolution and penetration depth, determine whether similar imaging results can
be reproduced in living animal subjects, and dene 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 Inrmary (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 MEEIs 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 soware-controlled galvanometer scanning motors (orlabs, Newton, NJ); the same soware 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), dened 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 Buered 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.
www.nature.com/scientificreports/
8
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
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 soware 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-
cied 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× magnications for Fig.1a,b, respectively. Figure1b 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 reected 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 reected by the RM. e return light is collimated and directed by the beam-
splitter (BS) towards a diraction 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.
www.nature.com/scientificreports/
9
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
Immunohistochemistry and confocal imaging. Guinea pig cochleae were extracted, xed, and decalci-
ed as described above. Under a stereomicroscope, the decalcied 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.
Aer 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 neurolament-H (pol-
yclonal chicken; AB5539, Lot #2701573; EMD Millipore, Temecula, CA) overnight. Aer 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 Scientic, Inc., Waltham, MA) diluted in 1% NHS with 0.4% Triton X-100 for 90 minutes
in combination with 1:200 rhodamine phalloidin (ermoFisher Scientic, 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 briey
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 soware. e nal images were
cropped and scale bars were retraced in Photoshop CS5.1.
References
1. Types of Hearing Loss. Center for Diease Control and Prevention, National C enter on Birth Defects and Developmental Disabilities,
http://www.cdc.gov/ncbddd/hearingloss/types.html. Accessed February 29, 2016 (2015).
2. Ali, Douraghy & ArionF., Chatziioannou. Basic Sciences of Nuclear Medicine, (Springer-Verlag Heidelberg Dordrecht, 2011).
3. Neolla, S. G. & Saraste, A. In Cardiac CT, PET, and M (ed. Dilsizian, V. & Pohost, G. M.) 301–333 (Wiley-Blacwell, West Sussex,
2010).
4. van der Jagt, M. A. et al. Visualization of human inner ear anatomy with high-resolution M imaging at 7T: initial clinical
assessment. AJN. American journal of neuroradiology 36, 378–383 (2015).
5. aphael, Y. & Altschuler, . A. Structure and innervation of the cochlea. Brain research bulletin 60, 397–422 (2003).
6. Picles, J. O. An Introduction to the Physiology of Hearing, (Academic Press Inc., London, 1982).
7. Fujimoto, J. G., Pitris, C., Boppart, S. A. & Brezinsi, M. E. Optical coherence tomography: an emerging technology for biomedical
imaging and optical biopsy. Neoplasia 2, 9–25 (2000).
8. Huang, D. et al. Optical coherence tomography. Science 254, 1178–1181 (1991).
9. Spaide, . F., oizumi, H. & Pozzoni, M. C. Enhanced depth imaging spectral-domain optical coherence tomography. American
journal of ophthalmology 146, 496–500 (2008).
10. Shah, S. U. et al. Enhanced depth imaging optical coherence tomography of choroidal nevus in 104 cases. Ophthalmology 119,
1066–1072 (2012).
11. Mogensen, M., rane, L., Jorgensen, T. M., Andersen, P. E. & Jemec, G. B. OCT imaging of sin cancer and other dermatological
diseases. Journal of biophotonics 2, 442–451 (2009).
12. Pierce, M. C., Strasswimmer, J., Par, B. H., Cense, B. & de Boer, J. F. Advances in optical coherence tomography imaging for
dermatology. e Journal of investigative dermatology 123, 458–463 (2004).
13. Boppart, S. A. et al. In vivo cellular optical coherence tomography imaging. Nature medicine 4, 861–865 (1998).
14. Povazay, B. et al. Submicrometer axial resolution optical coherence tomography. Optics letters 27, 1800–1802 (2002).
15. Leitgeb, . A., Villiger, M., Bachmann, A. H., Steinmann, L. & Lasser, T. Extended focus depth for Fourier domain optical coherence
microscopy. Optics letters 31, 2450–2452 (2006).
16. Wong, B. J., de Boer, J. F., Par, B. H., Chen, Z. & Nelson, J. S. Optical coherence tomography of the rat cochlea. Journal of biomedical
optics 5, 367–370 (2000).
17. Wong, B. J. et al. Imaging the internal structure of the rat cochlea using optical coherence tomography at 0.827 microm and 1.3
microm. Otolar yngology-head and nec surgery: ocial journal of American Academy of Otolaryngology-Head and Nec Surgery 130,
334–338 (2004).
18. Lin, J., Staecer, H. & Jafri, M. S. Optical coherence tomography imaging of the inner ear: a feasibility study with implications for
cochlear implantation. e Annals of otology, rhinology, and laryngology 117, 341–346 (2008).
19. Gao, S. S. et al. Quantitative imaging of cochlear so tissues in wild-type and hearing-impaired transgenic mice by spectral domain
optical coherence tomography. Optics express 19, 15415–15428 (2011).
20. Subhash, H. M. et al. Volumetric in vivo imaging of intracochlear microstructures in mice by high-speed spectral domain optical
coherence tomography. Journal of biomedical optics 15, 036024 (2010).
21. Wang, . . & Nuttall, A. L. Phase-sensitive optical coherence tomography imaging of the tissue motion within the organ of Corti
at a subnanometer scale: a preliminary study. Journal of biomedical optics 15, 056005 (2010).
22. Hong, S. S. & Freeman, D. M. Doppler optical coherence microscopy for studies of cochlear mechanics. Journal of biomedical optics
11, 054014 (2006).
23. Liu, L. et al. Imaging the subcellular structure of human coronary atherosclerosis using micro-optical coherence tomography. Nature
medicine 17, 1010–1014 (2011).
24. ashiwagi, M. et al. Feasibility of the assessment of cholesterol crystals in human macrophages using micro optical coherence
tomography. PloS one 9, e102669 (2014).
25. Liu, L. et al. Met hod for quantitative study of airway functional microanatomy using micro-optical coherence tomography. PloS one
8, e54473 (2013).
26. Liu, L. et al. An autoregulatory mechanism governing mucociliary transport is sensitive to mucus load. American journal of
respiratory cell and molecular biolog y 51, 485–493 (2014).
27. Cui, D. et al. Dual spec trometer system with spectral compounding for 1-mum optical coherence tomography in vivo. Optics letters
39, 6727–6730 (2014).
28. Brown, M. C. Morphology of labeled eerent bers in the guinea pig cochlea. e Journal of comparative neurology 260, 605–618
(1987).
29. Dunn, . A. A comparison of Golgi-impregnated innervation patterns and ne structural synaptic morphology in the cochlea of the
cat. PhD thesis, Harvard University (1975).
30. Liberman, M. C. Eerent synapses in the inner hair cell area of the cat cochlea: an electron microscopic study of serial sections. Hear
es 3, 189–204 (1980).
www.nature.com/scientificreports/
10
Scientific RepoRts | 6:33288 | DOI: 10.1038/srep33288
31. Yuan, W. et al. Optimal operational conditions for supercontinuum-based ultrahigh-resolution endoscopic OCT imaging. Optics
letters 41, 250–253 (2016).
32. Oishi, N. & Schacht, J. Emerging treatments for noise-induced hearing loss. Expert Opin Emerg Drugs 16, 235–245 (2011).
33. Yang, H., Zhao, B., Qiurong, Y., Liu, Y. & Hu, H. Ghosting phenomena in single photon counting imagers with Vernier anode. ev
Sci Instrum 82, 023110 (2011).
34. Landegger, L. D., Psaltis, D. & Stanovic, . M. Human audiometric thresholds do not predict specic cellular damage in the inner
ea r. Hear es 335, 83–93 (2016).
35. Schunecht, H. F. Further Observations on the Pathology of Presbycusis. Archives of otolaryngology 80, 369–382 (1964).
36. Schunecht, H. F. & Gace, M. . Cochlear pathology in presbycusis. e Annals of otology, rhinology, and laryngology 102, 1–16
(1993).
37. Tonndorf, J. Acute cochlear disorders: the combination of hearing loss, recruitment, poor speech discrimination, and tinnitus. e
Annals of otology, rhinology, and laryngology 89, 353–358 (1980).
38. Winter, H. et al. Deafness in Tbeta mutants is caused by malformation of t he tectorial membrane. e Journal of neuroscience : the
ocial journal of the Society for Neuroscience 29, 2581–2587 (2009).
39. ujawa, S. G. & Liberman, M. C. Synaptopathy in the noise-exposed and aging cochlea: Primary neural degeneration in acquired
sensorineural hearing loss. Hear e s 330, 191–199 (2015).
40. Schunecht, H. F. et al. Atrophy of the stria vascularis, a common cause for hearing loss. e Laryngoscope 84, 1777–1821 (1974).
41. Chen, F. et al. In viv o imaging and low-coherence interferometry of organ of Corti vibration. Journal of biomedical optics 12, 021006
(2007).
42. Chen, F. et al. A dierentially amplied motion in the ear for near-threshold sound detection. Nat Neurosci 14, 770–774 (2011).
43. Choudhury, N. et al. Low coherence interferometry of the cochlear partition. Hear e s 220, 1–9 (2006).
44. Biret, S. E. et al. A functional anatomic defect of the cystic brosis airway. Am J espir Crit Care Med 190, 421–432 (2014).
45. Pococ, G. M. et al. Hig h-resolution in v ivo imaging of regimes of laser damage to the primate retina. Journal of ophthalmology 2014,
516854 (2014).
46. Yin, B. et al. μ OCT imaging using depth of focus extension by self-imaging wavefront division in a common-path ber optic probe.
Optics express 24, 5555–5564 (2016).
47. Chu, . . et al. In vivo imaging of airway cilia and mucus clearance with micro-optical coherence tomography. Biomed. Opt. Express
7, 2494–2505 (2016).
48. Fujita, T. et al. Surgical Anatomy of the Human ound Window egion: Implication for C ochlear Endoscopy rough the External
Auditory Canal. Otology & neurotology: ocial publication of the American Otological Society, American Neurotology Society and
European Academy of Otology and Neurotology 37(8), 1189–94 (2016).
49. Haghpanahi, M., Gladstone, M. B., Zhu, X., Frisina, . D. & B orholder, D. A. Noninvasive technique for monitoring drug transport
through the murine cochlea using micro-computed tomography. Annals of biomedical engineering 41, 2130–2142 (2013).
50. Gao, S. S. et al. In vivo vibrometry inside the apex of the mouse cochlea using spectral domain optical coherence tomography.
Biomed. Opt. Express 4, 230–240 (2013).
51. Izumiawa, M. et al. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nature
medicine 11, 271–276 (2005).
52. Furness, D. N. & Hacney, C. M. Cross-lins between stereocilia in the guinea pig cochlea. Hear e s 18, 177–188 (1985).
53. ompson, A. C. et al. Infrared neural stimulation fails to evoe neural activity in the deaf guinea pig cochlea. Hear  es 324, 46–53
(2015).
54. Yamano, T., Higuchi, H., Ueno, T., Naagawa, T. & Morizono, T. Trial of Micro CT Scanner SYSCAN1176 for the Imaging of the
Guinea Pig Cochlea in vivo. Nihon Jibiinoa Gaai aiho 119, 129–133 (2016).
55. Wojtowsi, M. et al. Ultrahigh-resolution, high-speed, Fourier domain optical coherence tomography and methods for dispersion
compensation. Optics express 12, 2404–2422 (2004).
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).
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
© e Author(s) 2016
... In the field of cardiology, OCT is also investigated to obtain coronary intravascular images [10,11] . In the field of otology, OCT has been used to observe the intracochlear morphology and mechanics in animal models [12][13][14][15][16][17] has enabled the identification of larger structures, including the Reissner's [3,[12][13][14][15][16] , basilar [3,[12][13][14][15][16] , and tectorial membranes [3,[14][15][16] , the spiral ligament [14,16] , the 3 scalae of the cochlea [12,[14][15][16] , region of sensory epithelium [3,14,15] , and the tunnel of Corti [3,16] . However, owing to the resolution threshold of OCT, these studies are limited similarly to conventional imaging methodology and are unable to demonstrate visualization of smaller anatomical features, including the inner hair cells (IHC), outer hair cells (OHC), and auditory nerve fibers. ...
... In the field of cardiology, OCT is also investigated to obtain coronary intravascular images [10,11] . In the field of otology, OCT has been used to observe the intracochlear morphology and mechanics in animal models [12][13][14][15][16][17] has enabled the identification of larger structures, including the Reissner's [3,[12][13][14][15][16] , basilar [3,[12][13][14][15][16] , and tectorial membranes [3,[14][15][16] , the spiral ligament [14,16] , the 3 scalae of the cochlea [12,[14][15][16] , region of sensory epithelium [3,14,15] , and the tunnel of Corti [3,16] . However, owing to the resolution threshold of OCT, these studies are limited similarly to conventional imaging methodology and are unable to demonstrate visualization of smaller anatomical features, including the inner hair cells (IHC), outer hair cells (OHC), and auditory nerve fibers. ...
... In the field of cardiology, OCT is also investigated to obtain coronary intravascular images [10,11] . In the field of otology, OCT has been used to observe the intracochlear morphology and mechanics in animal models [12][13][14][15][16][17] has enabled the identification of larger structures, including the Reissner's [3,[12][13][14][15][16] , basilar [3,[12][13][14][15][16] , and tectorial membranes [3,[14][15][16] , the spiral ligament [14,16] , the 3 scalae of the cochlea [12,[14][15][16] , region of sensory epithelium [3,14,15] , and the tunnel of Corti [3,16] . However, owing to the resolution threshold of OCT, these studies are limited similarly to conventional imaging methodology and are unable to demonstrate visualization of smaller anatomical features, including the inner hair cells (IHC), outer hair cells (OHC), and auditory nerve fibers. ...
Article
Full-text available
Objectives: This study aimed to investigate the feasibility of using optical coherence tomography (OCT) to provide information about cochlear microanatomy at a cellular level, specifically of cochlear hair cells in mammals. Materials and methods: A total of 10 Sprague-Dawley rats were divided into 2 experimental groups for comparing the arrangement of normal and damaged hair cells. Postnatal day 3 Sprague-Dawley rats were used to test the swept-source OCT system, and the images recorded were compared with fluorescence microscope images. Results: Intracochlear structures (the inner hair cells, outer hair cells, and auditory nerve fibers) were clearly visualized at the individual cellular level. Conclusion: These images reflect the ability of OCT to provide images of the inner hair cells, outer hair cells, and auditory nerve fibers (ex vivo). OCT is a promising technology, and these findings could be used to encourage research in the area of cochlear microstructure imaging in the future.
... A region of approximately 300 µm width in the sensory epithelium (red line) was subjected to a B-scan, and the unaveraged image is displayed in Fig. 4c. In this image, we identified the outer tunnel, the tunnel of Corti, and OHCs based on the schematic in Fig. 4a and literature [12,15,17,20,22,38]. The OHC diameter and length at the basal turn of the guinea pig cochlea are ~ 7 μm and ~ 40 μm, respectively [16], whereas the ideal lateral resolution of our system was 1.95 μm. ...
... Because of the features and adjustments above, the system that we constructed successfully captured high-resolution images and sub-nanoscale vibrations in the apical and basal regions of OHCs in vivo. An ultra-high-resolution OCT with an SC source was previously applied to record the image for guinea pig cochlea ex vivo [38]. However, its sampling rate was 40 Hz, which was insufficient for highfrequency vibrometry. ...
Article
Full-text available
Sound evokes sub-nanoscale vibration within the sensory epithelium. The epithelium contains not only immotile cells but also contractile outer hair cells (OHCs) that actively shrink and elongate synchronously with the sound. However, the in vivo motion of OHCs has remained undetermined. The aim of this work is to perform high-resolution and -accuracy vibrometry in live guinea pigs with an SC-introduced spectral-domain optical coherence tomography system (SD-OCT). In this study, to reveal the effective contribution of SC source in the recording of the low reflective materials with the short total acquisition time, we compare the performances of the SC-introduced SD-OCT (SCSD-OCT) to that of the conventional SD-OCT. As inanimate comparison objects, we record a mirror, a piezo actuator, and glass windows. For the measurements in biological materials, we use in/ex vivo guinea pig cochleae. Our study achieved the optimization of a SD-OCT system for high-resolution in vivo vibrometry in the cochlear sensory epithelium, termed the organ of Corti, in mammalian cochlea. By introducing a supercontinuum (SC) light source and reducing the total acquisition time, we improve the axial resolution and overcome the difficulty in recording the low reflective material in the presence of biological noise. The high power of the SC source enables the system to achieve a spatial resolution of 1.72 ± 0.00 μm on a mirror and reducing the total acquisition time contributes to the high spatial accuracy of sub-nanoscale vibrometry. Our findings reveal the vibrations at the apical/basal region of OHCs and the extracellular matrix, basilar membrane.
... Considering the bone of the cochlear capsule, the external auditory canal, and other structures have to be removed during the imaging process, which inevitably causes a certain degree of damage. Therefore, this imaging method is only used to observe the isolated human cochlea [66] and has not yet been applied in living humans. At the same time, it may be used to avoid residual hearing loss during cochlea implant (CI) insertion [67] and may provide real-time 3D images in the future. ...
Article
Full-text available
Optical coherence tomography (OCT) has become a novel approach to noninvasive imaging in the past three decades, bringing a significant potential to biological research and medical biopsy in situ, particularly in three-dimensional (3D) in vivo conditions. Specifically, OCT systems using broad bandwidth sources, mainly centered at near-infrared-II, allow significantly higher imaging depth, as well as maintain a high-resolution and better signal-to-noise ratio than the traditional microscope, which avoids the scattering blur and thus obtains more details from delicate biological structures not just limited to the surface. Furthermore, OCT systems combined the spectrometer with novel light sources, such as multiplexed superluminescent diodes or ultra-broadband supercontinuum laser sources, to obtain sub-micron resolution imaging with high-speed achieve widespread clinical applications. Besides improving OCT performance, the functional extensions of OCT with other designs and instrumentations, taking polarization state or birefringence into account, have further improved OCT properties and functions. We summarized the conventional principle of OCT systems, including time-domain OCT, Fourier-domain OCT, and several typical OCT extensions, compared their different components and properties, and analyzed factors that affect OCT performance. We also reviewed current applications of OCT in the biomedical field, especially in hearing science, discussed existing limitations and challenges, and looked forward to future development, which may provide a guideline for those with 3D in vivo imaging desires.
... allowing visualization of the cochlear microanatomy at a cellular level (Iyer et al., 2016). While 43 the resolutions of µOCT images are the highest yet achieved for OCT imaging, these 44 measurements were from ex vivo cochleae that were chemically fixed. ...
Preprint
Full-text available
Because it is difficult to directly observe the morphology of the living cochlea, our ability to infer the mechanical functioning of the living ear has been limited. Nearly all of our knowledge about cochlear morphology comes from postmortem tissue that was fixed and processed using procedures that possibly distort the structures and fluid spaces of the organ of Corti. In this study, optical coherence tomography was employed to obtain in vivo and postmortem micron-scale volumetric images of the high-frequency hook region of the gerbil cochlea through the round-window membrane. The anatomical structures and fluid spaces of the organ of Corti were segmented and quantified in vivo and over a 90-minute postmortem period. The results show that some aspects of the organ of Corti are significantly altered over the course of death, such as the volumes of the fluid spaces, whereas the dimensions of other features change very little. We postulate that the fluid space of the outer tunnel and its surrounding tectal cells form a resonant structure that can affect the motion of the reticular lamina and thereby have a profound effect on outer-hair-cell transduction and thus cochlear amplification. In addition, the in vivo fluid pressure of the inner spiral sulcus is postulated to effectively inflate the connected sub-tectorial gap between the tectorial membrane and the reticular lamina. This gap height decreases after death, which is hypothesized to reduce and disrupt hair-cell transduction
... In 2011, a new mode of OCT termed micro-OCT (µOCT) was demonstrated with a resolution of 1-2 µm (28). The initial µOCT technology was implemented using a bench-top microscope system and has shown broad utility for a variety of in vitro and ex vivo studies and applications (28)(29)(30)(31)(32). Recently, to implement µOCT clinically, a single fiber optic µOCT probe and intracoronary catheter have been created (33, 34)-the technology is now poised to be used in coronaries in vivo (35). ...
Article
Full-text available
Intravascular optical coherence tomography (IVOCT) that produces images with 10 μm resolution has emerged as a significant technology for evaluating coronary architectural morphology. Yet, many features that are relevant to coronary plaque pathogenesis can only be seen at the cellular level. This issue has motivated the development of a next-generation form of OCT imaging that offers higher resolution. One such technology that we review here is termed micro-OCT (μOCT) that enables the assessment of the cellular and subcellular morphology of human coronary atherosclerotic plaques. This chapter reviews recent advances and ongoing works regarding μOCT in the field of cardiology. This new technology has the potential to provide researchers and clinicians with a tool to better understand the natural history of coronary atherosclerosis, increase plaque progression prediction capabilities, and better assess the vessel healing process after revascularization therapy.
... Although the spatial resolution of both techniques is high, the imaging depth is limited to 300 µm. Several recent studies have also explored the feasibility of using OCT for otorhinolaryngology applications [12]; in these studies, the anatomic features of the guinea pia cochlea, such as the scala vestibuli (SV), scala media (SM), scala tympani (ST), modiolus, and spiral-like conduit inside the cochlea [13][14][15] can be clearly identified. Moreover, studies have investigated the potential of using OCT as the image guidance tool in the cochlear implant surgery [16,17]. ...
Article
Currently, the cochlear implantation procedure mainly relies on using a hand lens or surgical microscope, where the success rate and surgery time strongly depend on the surgeon's experience. Therefore, a real-time image guidance tool may facilitate the implantation procedure. In this study, we performed a systematic and quantitative analysis on the optical characterization of ex vivo mouse cochlear samples using two swept-source optical coherence tomography (OCT) systems operating at the 1.06-µm and 1.3-µm wavelengths. The analysis results demonstrated that the 1.06-µm OCT imaging system performed better than the 1.3-µm OCT imaging system in terms of the image contrast between the cochlear conduits and the neighboring cochlear bony wall structure. However, the 1.3-µm OCT imaging system allowed for greater imaging depth of the cochlear samples because of decreased tissue scattering. In addition, we have investigated the feasibility of identifying the electrode of the cochlear implant within the ex vivo cochlear sample with the 1.06-µm OCT imaging. The study results demonstrated the potential of developing an image guidance tool for the cochlea implantation procedure as well as other otorhinolaryngology applications.
Article
Since it has been difficult to directly observe the morphology of the living cochlea, our ability to infer the mechanical functioning of the living ear has been limited. Nearly all our knowledge about cochlear morphology comes from postmortem tissue that was fixed and processed using procedures that possibly distort the structures and fluid spaces of the organ of Corti. In this study, optical coherence tomography was employed to obtain volumetric images of the high-frequency hook region of the gerbil cochlea, as viewed through the round window, with far better resolution capability than had been possible before. The anatomical structures and fluid spaces of the organ of Corti were segmented and quantified in vivo and over a 90-min postmortem period. We find that the arcuate-zone and pectinate-zone widths change very little postmortem. The volume of the scala tympani between the round-window membrane and basilar membrane and the volume of the inner spiral sulcus decrease in the first 60-min postmortem. While textbook drawings of the mammalian organ of Corti and cortilymph prominently depict the tunnel of Corti, the outer tunnel is typically missing. This is likely because textbook drawings are typically made from images obtained by histological methods. Here, we show that the outer tunnel is nearly twice as big as the tunnel of Corti or the space of Nuel. This larger outer tunnel fluid space could have a substantial, little-appreciated effect on cochlear micromechanics. We speculate that the outer tunnel forms a resonant structure that may affect reticular-lamina motion.
Chapter
inner ear immunology in vestibular migraine and meniere disease, difrential proinflammatory profile
Article
Full-text available
Sensorineural hearing loss (SNHL) is one of the most profound public health concerns of the modern era, affecting 466 million people today, and projected to affect 900 million by the year 2050. Advances in both diagnostics and therapeutics for SNHL have been impeded by the human cochlea’s inaccessibility for in vivo imaging, resulting from its extremely small size, convoluted coiled configuration, fragility, and deep encasement in dense bone. Here, we develop and demonstrate the ability of a sub-millimeter-diameter, flexible endoscopic probe interfaced with a micro-optical coherence tomography (μOCT) imaging system to enable micron-scale imaging of the inner ear’s sensory epithelium in cadaveric human inner ears.
Article
Optical coherence tomography (OCT) has been increasingly utilised to guide percutaneous coronary intervention (PCI). Despite the diagnostic utility of OCT, facilitated by its high resolution, the impact of intracoronary OCT on clinical practice has thus far been limited. Difficulty in transitioning from intravascular ultrasound (IVUS), complex image interpretation, lack of a standardised algorithm for PCI guidance, and paucity of data from prospective clinical trials have contributed to the modest adoption. Herein, we provide a comprehensive up-do-date overview on the utility of OCT in coronary artery disease, including technical details, device set-up, simplified OCT image interpretation, recognition of the imaging artefacts, and an algorithmic approach for using OCT in PCI guidance. We discuss the utility of OCT in acute coronary syndromes, provide a summary of the clinical trial data, list the work in progress, and discuss the future directions.
Article
Full-text available
We investigated the optimal operational conditions for utilizing a broadband supercontinuum (SC) source in a portable 800 nm spectral-domain (SD) endoscopic OCT system to enable high resolution, high-sensitivity, and high-speed imaging in vivo. A SC source with a 3-dB bandwidth of ∼246 nm was employed to obtain an axial resolution of ∼2.7 μm (in air) and an optimal detection sensitivity of ∼-107 dB with an imaging speed up to 35 frames/s (at 70 k A-scans/s). The performance of the SC-based SD-OCT endoscopy system was demonstrated by imaging guinea pig esophagus in vivo, achieving image quality comparable to that acquired with a broadband home-built Ti:sapphire laser.
Article
A survey of the temporal bone collection at the Massachusetts Eye and Ear Infirmary reveals 21 cases that meet the criterion for the clinical diagnosis of presbycusis. It is evident that the previously advanced concept of four predominant pathologic types of presbycusis is valid, these being sensory, neural, strial, and cochlear conductive. An abrupt high-tone loss signals sensory presbycusis, a flat threshold pattern is indicative of strial presbycusis, and loss of word discrimination is characteristic of neural presbycusis. When the increments of threshold loss present a gradually decreasing linear distribution pattern on the audiometric scale and have no pathologic correlate, it is speculated that the hearing loss is caused by alterations in the physical characteristics of the cochlear duct, and the loss is identified as cochlear conductive presbycusis. It is clear that many individual cases do not separate into a specific type but have mixtures of these pathologic types and are termed mixed presbycusis. About 25% of all cases of presbycusis show none of the above characteristics and are classified as indeterminate presbycusis.
Article
We have designed and fabricated a 4 mm diameter rigid endoscopic probe to obtain high resolution micro-optical coherence tomography (µOCT) images from the tracheal epithelium of living swine. Our common-path fiber-optic probe used gradient-index focusing optics, a selectively coated prism reflector to implement a circular-obscuration apodization for depth-of-focus enhancement, and a common-path reference arm and an ultra-broadbrand supercontinuum laser to achieve high axial resolution. Benchtop characterization demonstrated lateral and axial resolutions of 3.4 μm and 1.7 μm, respectively (in tissue). Mechanical standoff rails flanking the imaging window allowed the epithelial surface to be maintained in focus without disrupting mucus flow. During in vivo imaging, relative motion was mitigated by inflating an airway balloon to hold the standoff rails on the epithelium. Software implemented image stabilization was also implemented during post-processing. The resulting image sequences yielded co-registered quantitative outputs of airway surface liquid and periciliary liquid layer thicknesses, ciliary beat frequency, and mucociliary transport rate, metrics that directly indicate airway epithelial function that have dominated in vitro research in diseases such as cystic fibrosis, but have not been available in vivo.
Article
Objective: To enable development of an endoscope for cellular-level optical imaging of the inner ear. Study design: A prospective study of 50 cadaveric human temporal bones to define detailed surgical anatomy of the round window (RW) region and the range of angles necessary to reach the RW membrane perpendicularly via the external ear canal. Main outcome measure: The transcanal angle to the RW membrane was surgically measured in 3D intact specimens, and correlated with the angle calculated from temporal bone computed tomography (CT) scans of the same specimens obtained before and after measurements in situ. Results: Surgically measured transcanal angles to the RW membrane correlated well with the radiographically measured angles. The angles ranged from 110 to 127 degrees, with the median of 115 degrees and the middle 50% ranging from 109 to 119 degrees. Four temporal bones were excluded because of pathology. The opening of the RW niche was located posteriorly in six bones (13%), inferiorly in 18 bones (39%), and postero-inferiorly in 22 bones (48%). The angles were not statistically different among the three orientations of the RW niche. Conclusions: By correlating measurement from cadaveric human temporal bones and their CT scans, we defined key parameters necessary for designing an endoscope for intracochlear imaging using a minimally invasive approach through the external auditory canal. The excellent correlation between the measurement on the CT scan and the actual shape of the probe that was able to reach the RW through the ear canal enables selection of the probe using the CT data.
Article
The usefulness of the micro CT scanner system SKYSCAN1176 was evaluated for the study of the guinea pig cochlea. Each slice of the section was 9 μm and we were able to identify each ossicles, modiolus, upper, middle, and basal turn of the cochlea. This scanner enables us to observe inner ear structure repeatedly in vivo.
Article
Optical coherence tomography (OCT) is an attractive medical modality due to its ability to acquire high-resolution, cross-sectional images inside the body using flexible, small-diameter, scanning fiber optic probes. Conventional, cross-sectional OCT imaging technologies have approximately 10-μm axial resolution and 30-μm lateral resolution, specifications that enable the visualization of microscopic architectural morphology. While this resolution is useful for many clinical applications, it is insufficient for resolving individual cells that characterize many diseases. To address this gap, a supercontinuum-laser-based, μm-resolution OCT (μOCT) system and a 500 μm-diameter, extended depth of focus single fiber optic probe for endoscopic and intravascular imaging were designed and fabricated. At the distal tip of the fiber optic probe, a cylindrical waveguide was used to divide the wavefront to provide multiple circular propagation modes. Once transmitted through a relatively high NA lens (NA >0.1), these modes were projected as multiple coaxial foci (∼3 μm full width at half maximum (FWHM)) over a greatly extended focal depth range. The distal tip of the probe also contained a common-path reference reflectance to minimize polarization and dispersion imbalances between sample and reference arm light. Measurements showed that the probe provides a 20-fold depth of focus extension, maintaining a 3-5 μm lateral resolution (FWHM of PSF) and a 2 μm axial resolution over a depth range of approximately 1 mm. These results suggest that this new optical configuration will be useful for achieving high-resolution, cross-sectional OCT imaging in catheter/endoscope-based medical imaging devices.
Article
At present there is some debate as to the processes by which infrared neural stimulation (INS) activates neurons in the cochlea, as the lasers used for INS can potentially generate a range of secondary stimuli e.g. an acoustic stimulus is produced when the light is absorbed by water. To clarify whether INS in the cochlea requires functioning hair cells and to explore the potential relevance to cochlear implants, experiments using INS were performed in the cochleae of both normal hearing and profoundly deaf guinea pigs. A response to laser stimulation was readily evoked in normal hearing cochlea. However, no response was evoked in any profoundly deaf cochleae, for either acute or chronic deafening, contrary to previous work where a response was observed after acute deafening with ototoxic drugs. A neural response to electrical stimulation was readily evoked in all cochleae after deafening. The absence of a response from optical stimuli in profoundly deaf cochleae suggests that the response from INS in the cochlea is hair cell mediated. Copyright © 2015. Published by Elsevier B.V.
Article
The classic view of sensorineural hearing loss (SNHL) is that the "primary" targets are hair cells, and that cochlear-nerve loss is "secondary" to hair cell degeneration. Our recent work in mouse and guinea pig has challenged that view. In noise-induced hearing loss, exposures causing only reversible threshold shifts (and no hair cell loss) nevertheless cause permanent loss of >50% of cochlear-nerve / hair-cell synapses. Similarly, in age-related hearing loss, degeneration of cochlear synapses precedes both hair cell loss and threshold elevation. This primary neural degeneration has remained hidden for three reasons: 1) the spiral ganglion cells, the cochlear neural elements commonly assessed in studies of SNHL, survive for years despite loss of synaptic connection with hair cells, 2) the synaptic terminals of cochlear nerve fibers are unmyelinated and difficult to see in the light microscope, and 3) the degeneration is selective for cochlear-nerve fibers with high thresholds. Although not required for threshold detection in quiet (e.g. threshold audiometry or auditory brainstem response threshold), these high-threshold fibers are critical for hearing in noisy environments. Our research suggests that 1) primary neural degeneration is an important contributor to the perceptual handicap in SNHL, and 2) in cases where the hair cells survive, neurotrophin therapies can elicit neurite outgrowth from spiral ganglion neurons and re-establishment of their peripheral synapses. Copyright © 2015 Elsevier B.V. All rights reserved.