Scanning optical coherence tomography probe for in vivo imaging and displacement measurements in the cochlea

We developed a spectral domain optical coherence tomography (SDOCT) fiber optic probe for imaging and sub-nanometer displacement measurements inside the mammalian cochlea. The probe, 140 μm in diameter, can scan laterally up to 400 μm by means of a piezoelectric bender. Two different sampling rates are used, 10 kHz for highresolution B-scan imaging, and 100 kHz for displacement measurements in order to span the auditory frequency range of gerbil (~50 kHz). Once the cochlear structures are recognized, the scanning range is gradually decreased and ultimately stopped with the probe pointing at the selected angle to measure the simultaneous displacements of multiple structures inside the organ of Corti (OC). The displacement measurement is based on spectral domain phase microscopy. The displacement noise level depends on the A-scan signal of the structure within the OC and we have attained levels as low as ~0.02 nm in in vivo measurements. The system’s broadband infrared light source allows for an imaging depth of ~2.7 mm, and axial resolution of ~3 μm. In future development, the probe can be coupled with an electrode for time-locked voltage and displacement measurements in order to explore the electromechanical feedback loop that is key to cochlear processing. Here, we describe the fabrication of the laterally-scanning optical probe, and demonstrate its functionality with in vivo experiments. © 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement


Introduction
Spectral Domain Optical Coherence Tomography (SDOCT) is a low-coherence interferometric system developed mainly for imaging, and also capable of displacement measurements, using Spectral Domain Phase Microscopy (SDPM) [1].SDOCT has a penetration depth of several millimeters, resulting from working in the infrared range, and the ability to simultaneously measure displacements at locations all along the axial-scan (A-scan).Its steep optical sectioning curve, based on its broadband light source, results in ~3 μm axial resolution [2].This resolution is adequate for displacement measurements in the sensory tissue of the cochlea, whose different structures are separated by distances on the order of 10 μm.Following pioneering work over the past decade [3][4][5], the data generated by OCT systems is having a substantial impact on the understanding of cochlear processing.Several groups, including ours, have performed SDOCT-based displacement measurements using a Thorlabs system (Telesto III), which is designed for imaging and can be tailored by the user for phase-based displacement measurements [6,7].The light source of the Telesto III is comprised of two coupled superluminescent diodes, with a central wavelength of ~1300 nm and a bandwidth of ~135 nm.The system's axial resolution, ∆z, is 3.5 μm in air and in salinerich tissue like the cochlea, the index of refraction n ~1.3, and ∆z is 2.7 μm.
To date, we have used the Telesto as a bulk-optics SDOCT system to measure displacements of the cochlea's sensory tissue through the transparent round window membrane (RWM), at the base of the cochlea [2,6].Other auditory groups have measured through the bony shell in the apical region of the cochlea where the bone is relatively thin [3].The access locations are limited because the cochlea is surrounded by bone, and damage will modify the measured displacements, in particular by reducing the active outer-hair-cell-based process termed cochlear amplification.Going from the apex to the base the frequency of sound processing increases, in humans from ~20 to ~20000 Hz and in gerbils from ~100 to 50000 Hz.The processing of sound is similar, but not identical, in the cochlear base and apex, and both regions are interesting scientifically.Our lab studies the basal cochlea, and the probe described here is being developed for measurements in that region, but could be used in the cochlear apex, as well as other applications.Here we demonstrate, through in vivo experiments, a fiber-optic-probe-based SDOCT system.In the current experiments we accessed the cochlea's sensory tissue through the RW, with intact RWM.The SMF/GRIN probe has a diameter of 140 μm, and can also be inserted via a hole (cochleostomy) drilled in the bone of ~200 x 500 μm (500 μm dimension to allow scanning).For many years our lab has used basal cochleostomies of ~200 μm diameter with fiber optic probes of ~150 μm without damaging the active cochlear process, in measurements in gerbil [8].The presence of the persistent stapedial artery makes larger holes difficult in the gerbil base, but cochleostomies of ~400 -500 μm have been made in the base and apex of chinchilla and guinea pig cochleae for imaging and motion measurements [9].
We had two motivations for developing the probe system.Firstly, it will allow us to access locations that the bulk-optics system cannot access.Secondly, we plan in the future to attach an electrode to the side of the probe, in order to make simultaneous displacement and outer hair cell (OHC)-produced voltage measurements.A similar pressure-voltage dual sensor, built around a fiber-optic probe, was developed previously by our lab [8].With our planned electrode/SD-OCT probe, the measurements of voltage and motion will be coincident in both time and space, and will allow us to explore the electro-mechanical feedback loop that results in cochlear amplification.
SDOCT displacement measurements with a fiber optic probe have been performed previously.For example, optical coherence elastography determines tissue displacement produced by compressive loading [10][11][12][13], using probe-based SDOCT.Probe-based displacement measurements have not been done in the cochlea, although several groups have imaged the cochlea with fiber optic probes that scan by rotating around the long axis [14,15], and another group has developed a fiber optic probe for middle-ear imaging [16].
In this paper, we describe the development and initial use of an SDOCT probe with controllable lateral scanning up to 400 μm.This scanning range is suitable for identifying cochlear structures.Once the structures are identified, the scanning is stopped and the probe can be precisely pointed at a specific angle for subnanometer-scale displacement measurements of the structures along that angle's A-scan.Two in vivo experiments were done to validate the probe's usage.

Probe design
The probe is composed of a micro-GRIN fiber ~140 μm in diameter, with g = 5.9 mm −1 , a length of 500 μm, fused to SMF-28 fiber.This basic component was ordered from WT&T Inc, Pierrefonds, Quebec Canada.A micrograph is shown in Fig. 1(A).A probe-measured A-scan of a water-immersed mirror is shown in Fig. 1(B).The beam profile was measured by the manufacturer with a BeamScan Optical profiler placed at the focal point (focal length = 250 μm).The prof in the x and y ray tracing m probe's focal whereas the b adhered to th bimorph, 2.5 benders are a dimensional m that is attache through the tr The probe external refer retro-reflector controlling th recognized, th piezoelectric bender).The probe pointin common-path raising the sig measurement files are shown y-axes respecti matrix analysis distance is in beam waist rem he end of a p cm x 0.6 cm available from micro-manipul ed to the optic ransparent RW 1. A. Microscope ured by the probe e in the x and y-a ion schematic for t e is used in a n rence arm cont r (Fig. 2).Th he iris diaphra he probe is ce bender) while scanning rang ng at the coch h (CP) setup (b gnal-to-noise r is then done at n in Fig. 1(C) a ively.This spo s for GRIN le ncreased by a mains unchange piezoelectric b m x 0.05 cm, Thorlabs (e.g.ator (three-axi cal table.For th M (Fig. 1(F)) f e image of the p e. The full width axes.E. Photograp the in vivo experim noncommon-p taining a polar his allows for gm and the an entered at the e scanning (w ge is gradually hlear sensory blue line).CP ratio (SNR) in t the sample ra and Fig. 1(D), ot size matches enses [17][18][19].

Scannin
To implement scanner is tap probe's scann ThorImage.T holder for sca The tapped s obtained by parameter in t μm/volt.Prob spike at the e components, ~270 Hz.A s components, outlined below A summin factor of 10 ( supply range.modified (deto a potentio controls the l variable DC v while scannin Systems, EPA scanning FOV The DC v voltage determ the sawtooth centered at th probe is ultim 2. Probe setup is n ization controller, ures are recogniz olling the piezoel ooth voltage wavef ltimately stopped t .We then switche ng t lateral scanni pped from the ned output ca The driving sig anning [20].Th signal (Fig. 3 n Fourierde of the complex A-scan gives the depth profile of the sample structures (Fig. 4(B)) [22].A series of A-scans (~10 6 for 10s of acquisition) is taken at a fixed lateral position in rapid temporal succession, called the M-scan, to acquire data for the displacement measurement.Displacement, δ(t), of the sample is found by evaluating the phase variations Θ(t) of the complex A-scan at the location of the peak in the A-scan magnitude.Unwrapping is performed to undo phase jumps > π at adjacent time points.δ(t) = Θ(t)/(2nk 0 ), where k o = 2π/λ ο , and λ ο is the center wavelength of the light source.The resulting waveform, δ(t), is then Fourier-transformed to the frequency domain in order to find the magnitude and phase of the sample.
Cochlear measurements were done in vivo on Mongolian gerbils (Meriones unguiculatus).The animal experiments were approved by the Columbia University Institutional Animal Care and Use Committee and a full description of the surgical preparation and anesthetic regimen and acoustic setup can be found in other papers from our lab [23].
The current paper is focused on the probe and the in vivo animal experiments were done to demonstrate its utility; new physiological findings are not a component of this paper.The measurements here were done in vivo in passive cochleae, following an independent set of measurements.In order to compare the results with those of the bulk optics system these data were collected with the probe imaging the OC through the round window (Fig. 4).

In vivo cochlea imaging and displacement measurements
The round window, where the probe accessed the cochlea, is indicated with a dotted line in a sketch of the cochlea in Fig. 4(A).The lateral scanning was initially ranged at 400 μm and the acquired B-scan (top image of Fig. 4(B)) can be compared to the sketch in Fig. 4(A) to identify the RWM, basilar membrane, organ of Corti, and Reissner's membrane.The probe was positioned at the desired angle with DC voltage applied to the bender and scanned with the sawtooth voltage.The lateral scanning range was gradually decreased, and ultimately stopped to point the probe at the desired location (shown in Fig. 4(B) with the reddotted line, and its corresponding A-scan).An M-scan was then acquired for displacement measurements, while stimulating the ear canal with a set of tones as described in the next section.The first structure at depth location 300 μm is the RWM, followed by the organ of Corti complex structures at depth locations between 400 and 500 μm.Reissner's membrane is at ~650 μm.The Fouri (Fig. 5) with displacement ~60 dB SPL expected in a cochlea is con displacement

In vivo c SDOCT syst
To verify that the bulk-opti obtained the (shown in the 60-frequency measurements measurement probe measur scans.We sel responses, no The displacem canal pressure amount of ga lateral positio scaled linearl linearity obse cochlea wher measurements linearity that note, in previ agrees with confidence th

Discussio
In this study, optics SDOC displacementcochlear parti in the access  and bulkging and ss to the flexibility structural identification, and then be arrested to measure displacement along a selected A-scan.In addition, in the future an electrode can be coupled to the probe in order to simultaneously measure mechanical and electrical cochlear responses, to explore cochlear electromechanical processing [8].Attaining a good SNR (a low displacement noise floor) for intracochlear measurements, especially deep inside the organ of Corti, is challenging because of the low reflectivity of the sensory tissue.For example, the outer hair cell reflectivity is only ~0.006% [30].Our cochlear measurements in Fig. 5 showed a noise floor of ~0.02 nm, similar to those obtained by our and other groups' vibrometry systems [25,26,31].With this noise floor, displacement can be measured at low SPL in the basal region of the gerbil's cochlea.The CP setup is used for displacement measurement to maximize the light to the sample, because with the nonCP setup, light is lost when traveling back and forth through a 75:25 fiber coupler.Additionally, by using the same optical path for both the sample and reference arms, systematic noise is reduced.On the other hand, the nonCP setup allows for adjustment of the reference beam level which is sometimes needed for real-time imaging.
As discussed in section 2.3, when using SDPM, displacement is determined from the phase variation of the complex A-scan peaks over time, and the minimum detectable phase difference σ ∆Φ in the complex A-scan sets the noise floor of the displacement measurements, δx.σ ∆Φ is directly related to the SNR of the A-scan's magnitude (SNR A = the ratio of a reflector (peak) intensity to the background intensity, and the A-scan magnitude can be used to find an approximation for the displacement noise floor.The expression is:

Conclusion
We demonstrated a lateral scanning SDOCT probe, 140 μm in diameter, coupled to a Thorlabs Telesto SDOCT, for real-time laterally-scanned B-scans within the cochlea and displacement measurements with a noise floor of ~0.02 nm.Planned work includes coupling an electrode to the probe to explore the electro-mechanical feedback loop that results in cochlear amplification.In future work, the probe could be used to image and measure displacement in locations that can be accessed through a sub-mm-sized hole.
Fig. 1 measu profile inserti ra when used in robe is held in 1(E)).The ben epoxy to an a NB2W).The pr pulator, World periments in ge ning of the bull -scan of a waterm is 12 μm.
Fig. 2 polari structu contro sawto and ul tissue Fig. 4 sensor sketch probe was g measu A speaker positioned in delivered, wit to reduce ove 37 kHz and s located at theThe Fouri (Fig.5) with displacement ~60 dB SPL expected in a cochlea is con displacement 4. A. Top shows a ry tissue through t h with the major , showing the prim gradually decrease urements (A-scan l r tube coupled n the gerbil's th each tone se erlap of nonlin spanned much 400 μm dotted ier-transformed h the stimulus frequency spe stimuli range passive prepar nsistent with p is also in line w sketch of the coch the transparent rou cochlear structure mary structures as ed (one reduction location shown wi d to a Fostex t ear canal, an et at a level of ẽar component the gerbil's he d position of Fi d time-domain s in Fig. 5(A ectrum has a n from ~0.05 to ration.A passi previous measu with previous m hlear cross-section und window.The b es labeled.B. Th in the magnified s n step shown) and ith the red dotted l tweeter

Fig. 5
Fig. 5 freque Fig. 6 RWM measu relativ on the coinci refere system 6.A.The B-scan M is at 540 μm, an ured at the same ve beam positions e sides of the B-sc ide with the struc enced to the stapes ms.
33].The predicted δx for the bright structure in the organ of Corti in the experiment of Fig. 5 is 12 nm based on the SNR A of the reflector's A-scan magnitude.Taking the frequency domain FFT lowers the noise floor because the noise is distributed among all the frequency bins (524288).This lowers (improves) the noise floor by a factor of 1 _ _ _ number of frequency bins , giving a theoretical δx of 0.0165 nm.This prediction is born out in the value of the experimental noise floor in Fig. 5(B).