Intraoperative peripheral nerve imaging probe to guide surgical repair

Abstract

Disclosed herein are systems and methods of use of an intraoperative imaging probe including: a housing; a first arm extending from the housing including a first proximal end and a first distal end, in which a first optical assembly is disposed within the first arm; a second arm extending from the housing including a second proximal end and a second distal end, in which a second optical assembly is disposed within the second arm; a beam scanning mechanism configured to scan imaging beams from the first and second optical assemblies; and an adjustment mechanism configured to adjust a distance between the first arm and the second arm.

Description

MGH 2025-208-02Quarles 125141.04961 INTRAOPERATIVE PERIPHERAL NERVE IMAGING PROBE TO GUIDE SURGICAL REPAIR CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 753,916 filed on February 4, 2025, which is incorporated herein by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under award numbers 5P41EB015903-13 (agreement 2021A012861) from the National Institute of Biomedical Imaging and Bioengineering and FA9550-23-1-0656 from the Air Force Office of Scientific Research (agreement 2022A015364). The government has certain rights in the invention.BACKGROUND

[0003] Peripheral nerves connect the central nervous system (CNS) to every muscle, organ, and tissue in the body. However, unlike the CNS, which is mechanically protected within the skull and spine, the peripheral nervous system is exposed. Consequently, peripheral nerve injuries (PNIs) are common complications of trauma and surgery. In the US, the incidence of traumatic PNIs (excluding chronic neuropathies) is conservatively estimated at 60,000, although this likely underestimates the true incidence by a factor of at least two and as much as ten, an admittedly wide range that highlights the need for better epidemiology. Outcomes for patients with Grade IILV PNIs are unfavorable, requiring surgical repair and often resulting in debilitating weakness, numbness, tingling, autonomic dysfunction, and pain.

[0004] There is a lack of tools for intraoperative assessment. It is difficult to differentiate healthy nerve tissue and injured or partially injured tissue. This causes challenges in determining proper treatment for the nerve. Thus, there exists a need for improved nerve assessment tools that are able to assess the nerve and select the optimal intervention.SUMMARY

[0005] Disclosed herein are methods and systems for an intraoperative imaging probe including: a housing; a first arm extending from the housing including a first proximal end and a first distal end, in which a first optical assembly is disposed within the first arm; a second arm 1QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 extending from the housing including a second proximal end and a second distal end, in which a second optical assembly is disposed within the second arm; a beam scanning mechanism configured to scan imaging beams from the first and second optical assemblies; and an adjustment mechanism configured to adjust a distance between the first arm and the second arm. In various embodiments, the methods and systems may include one or more of the following.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Some embodiments of the disclosure are described herein with reference to the accompanying figures. The description and figures make apparent to a person having ordinary skill in the art how some embodiments of the disclosure may be practiced. The figures are for the purpose of illustrative discussion and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the teachings of the disclosure. Where dimensions are given in the text or figures, these dimensions are merely exemplary and do not limit the scope or spirit of the disclosed invention.

[0007] FIGS. 1A-1E show various views of an intraoperative imaging probe in accordance with some embodiments of the systems and methods described herein.

[0008] FIG. 2 shows a handheld probe comprising two arms, each equipped with optical assemblies to perform imaging from opposing surfaces and calibration elements.

[0009] FIG. 3 shows a scanning of the beam in the z direction for each handheld probe first arm and second arm using an optical scanner.

[0010] FIG. 4 shows a configuration in which light from the first arm and second arm is encoded via depth by playing additional delay within one arm.

[0011] FIG. 5 shows a multi-channel OCT interferometer system configured to acquire imaging data from the first arm and second arm simultaneously on separate receiver channels.

[0012] FIG. 6 shows an exemplary design of the optical assembly within one of the imaging arms in accordance with some embodiments of the disclosure.

[0013] FIG. 7 shows a CAD drawing of an optical assembly using the piezoelectric actuation described in FIG. 6.2QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0014] FIG. 8 shows drawings of an intraoperative imaging probe with arms in an open and closed position.

[0015] FIG. 9 shows an example schematic of an intraoperative imaging probe in communication with a data processing system in accordance with some embodiments of the disclosure.

[0016] FIG. 10 shows an exemplary data processing system in accordance with some embodiments of the disclosure.

[0017] FIGS. 11 A-l ID show imaging fascicular architecture by OCT. Fascicles are often indistinct under structural contrast. To delineate myelinated fascicles from epineurium / perineurium and connective tissues, we use polarimetric approaches. Both myelinated fascicles and the connective epineurium / perineurium tissues are birefringent, but the optic axis of myelin is orthogonal to that of the connective tissues. By generating images based on polarimetric measurement of the optic axis, we can achieve high-contrast visualization of internal fascicles. (FIG. 11 A, FIG. 11B) In vivo OCT imaging of a surgically exposed radial nerve in a non-human primate. In (FIG. HA), structural images do not reveal internal fascicles. In (FIG. 1 IB), the derived optic axis is presented, and three fascicles are revealed. In (FIG. 11C), we used this technique to reveal the three-dimensional structure of the multi-fascicular porcine vagus nerve. In (FIG. I ID), we show fascicular (F arrow) and epineurial (E arrow) components of a human cranial nerve (fixed cadaver head used for neurosurgical training).

[0018] FIGS. 12A-12C show quantifying myelination by PS-OCT. The polarimetric signal that differentiates myelinated fascicles from connective epineurium / perineurium can also be used to quantify the degree of myelination in those fascicles. In (FIG. 12A), the technique is described. The vectorial birefringence of each pixel is mapped to a 2D plane. Myelinated (healthy fascicles) generate data at A0=9O° (red dots), while demyelinated data generate data at A0=O° and slightly closer to zero (lower retardance). The distance between these extremes defines a quantitative myelination index. (FIG. 12B) The myelination index is visualized for a rat sciatic nerve 5 days after crush injury at the injury point (FIG. 12B3, FIG. 12B4), and proximal (FIG. 12B1, FIG. 12B2) and distal (FIG. 12B5, FIG. 12B6) to the injury. Histology shows myelination changes consistent with OCT measures. (FIG. 12C) Remyelination after a crush injury (Grade II) is demonstrated at day +28. Note the complete loss of myelination distal to the3QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 injury at Day +7 (Wallerian degeneration). The ability to detect remyelination progression can inform surgical decision-making.

[0019] FIGS. 13A-13G show PS-OCT imaging of nerve fibrosis in NHPs. Scarring induces strong birefringence, which can be detected by PS-OCT. Unlike the native birefringence seen in healthy nerves, the signals from fibrotic and scarred tissues are disorganized, offering an opportunity for OCT to detect and quantify this internal fibrosis. In (FIG. 13 A), we present a 3D projection of OCT data acquired from a radial nerve of an NHP 12 months after graft repair of a Grade V transection injury. Structural and polarimetric cross-sections are displayed proximal (FIG. 13B, FIG. 13C), within (FIG. 13D, FIG. 13E), and slightly distal (FIG. 13F, FIG. 13G) to the repair site. Note the normal bi-modal polarimetric data at the proximal site, which shows distinct connective and myelinated tissue. At the distal site, there are two fascicles with limited indication of myelination (failed regeneration). At the repair site, the polarimetric image shows heterogeneous signals indicative of fibrosis and scarring.

[0020] FIGS. 14A-14F show perfusion imaging by angiographic OCT. Structural and PS signals have a limited ability to detect vessels. However, angiographic methods based on signal dynamics provide highly sensitive and high-resolution detection of perfused vessels. (FIG. 14A) Wide-field angiogram of the rat sciatic nerve 7 days after autograph repair of a transection injury showing hyperfusion within the graft (FIG. 14C) relative to a site proximal to the graft (FIG. 14B). The yellow lines in (FIGS. 14B, 14C, 14E, 14F) demarcate the fascicle, allowing discrimination of intrinsic (within the nerve) and extrinsic vessels. In (FIG. 14D), angiography of an allograft repair shows hypoperfusion in the native nerve distal to graft at day +14 relative to the native nerve proximal to the repair (FIG. 14E). The vessels in these selected cross-sections were manually segmented from the angiographic cross-sections.

[0021] FIGS. 15A-15E show a preliminary embodiment of a dual-sided OCT imaging probe. To overcome limitations in imaging depth, our teams have developed dual-sided probes that interrogate the nerve from opposing surfaces. In (FIG. 15 A), a dual-sided imaging system for ex vivo samples is shown. In (FIG. 15B, FIG. 15C), the multi -fascicular architecture of the porcine vagus nerve is shown alongside matched histology. In (FIG. 15D, FIG. 15E).Myelination contrast (FIGS. 15A-15C) is shown, demonstrating an ability to discriminate between fascicles based on myelin content.4QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0022] FIGS. 16A-16C show a prototype handheld, dual-sided probe. This 3D printed version (FIG 16A, FIG. 16B) included a linear translation of an optical fiber core developed for intravascular imaging. (FIG. 16C) Ex vivo imaging of porcine ulnar nerve (from one side only) demonstrates fascicular contrast. This prototype included optics on a single side.

[0023] FIGS. 17A-17D show beam scanning and image generation are illustrated in the proposed intraoperative probe. (FIG. 17A) The probe will provide beam scanning in the (Z) and (Y) axes to generate small 3D volumes of the nerve (~ 2mm (Y) x up to 10 mm (Z)). (FIG. 17B, FIG. 17C) Beam scanning in the Y-axis will be achieved via upward translation of the optical cores. (FIG. 17D) Left and right images will be acquired separately and merged.

[0024] FIG. 18 shows an exemplary process in accordance with some embodiments of the disclosure.

[0025] FIG. 19 shows an exemplary design of calibrating structures used to provide spatial registration of the OCT imaging data to the arm and / or used to calibrate the measured birefringence orientations to the XYZ reference frame defined by each arm.

[0026] FIG. 20 shows an exemplary optical design for the handheld probe described in FIGS. lAand IB.

[0027] FIG. 21 shows an exemplary design of an arm configured to measure force or pressure applied to the sample.DETAILED DESCRIPTION

[0028] The disclosures of any of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.

[0029] Before any embodiments of the disclosure are explained in detail, it is to be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways.5QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0030] It is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0031] In accordance with some embodiments of the disclosed subject matter, mechanisms (which can include, for example, systems and methods) for an intraoperative imaging probe (also referred to generally as a probe) are described herein.

[0032] Peripheral nerves connect the central nervous system (CNS) to every muscle, organ, and tissue in the body. However, unlike the CNS, which is mechanically protected within the skull and spine, the peripheral nervous system is exposed. Consequently, peripheral nerve injuries (PNIs) are common complications of trauma and surgery. In the US, the incidence of traumatic PNIs (excluding chronic neuropathies) is conservatively estimated at 60,000, although this likely underestimates the true incidence by a factor of at least two and as much as ten, an admittedly wide range that highlights the need for better epidemiology. In approximately half of these cases, injury is confined to the myelin sheaths or axons, but the endoneurium, perineurium, and epineurium remain intact. In such cases, the nerve can heal spontaneously, and patients achieve full or nearly full recovery.

[0033] Outcomes for the other half, e.g., patients with Grade III-V PNIs, are significantly less favorable. These injuries require surgical repair and often result in debilitating weakness numbness, tingling, autonomic dysfunction, and pain. Recovery is frustratingly slow, lasting months to sometimes years, depending on the site of injury. Worst of all, at the end of this long process, most patients achieve only partial recovery; functional return at the level of approximately 50% is typical. The median age of those with major (grade III-V) PNIs is just 38 years, and patients must live with these sequelae for several decades. Our best epidemiology suggests that at least 1 million people in the US are living with unresolved symptoms from a prior major PNI. Additionally, these injuries have significant economic costs through lost work and ongoing care, estimated at $1 billion annually in the US.

[0034] Surgeons lack tools for intraoperative assessment. Decades of productive peripheral nerve research have identified the primary factors that limit regeneration after surgical repair. Unfortunately, surgeons and researchers have stalled in translating this knowledge to6QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 improved outcomes. Tn part, this is because surgeons lack the tools needed to assess the nerve and, consequently, select the optimal intervention. For example, a nerve with Grade III-IV injury is continuous but contains a zone of the fibrotic or partially fibrotic nerve. This damaged nerve tissue must be fully resected such that coaptation is performed between two healthy nerve faces. Even a small degree of residual fibrosis at one terminal nerve face will significantly inhibit axonal regeneration. The same is true for Grade V (transection) injuries, where fibrotic regions on each nerve stump must be accurately trimmed back. However, discriminating between healthy and subtly injured nerve tissue is clinically complex, influenced by factors including the mechanism of injury, timing of repair, and surgical techniques employed. Today, the peripheral nerve surgeon relies on the nerve’s external appearance and stiffness (measured by touch) to make this assessment. Once a cut has been made, the surgeon visualizes the newly created end, looking for punctate bleeding and fascicles with a yellowish coloration to indicate health. None of these methods are thought to be dependable discriminators of healthy and partially fibrotic nerves, especially for less experienced surgeons. There is a growing consensus among leading peripheral nerve surgeons that the absence of a quantitative method for intraoperatively discriminating between healthy and fibrotic nerves hinders repair quality and, consequently, limits patient outcomes.

[0035] The inability to assess nerve health also delays surgical repair for patients with Class III-IV injuries, often by several months and sometimes up to 6 months. Patients presenting with significant symptoms several weeks after injury are scheduled for a surgical exploration. During this exploration, external scarring and adhesions are removed to eliminate pathogenic tension at the injury site. At this time, the surgeon must decide whether to perform a repair or give more time for spontaneous healing. This decision should ideally be based on knowledge of the injury severity (Sunderland Grade II vs III-IV) as well as signs of regeneration through the injury site. Instead, this decision is made today based on the nerve’s external appearance (e.g., does it look healthy or ragged), its mechanical stiffness, and electrodiagnostic testing that has known limitations. Preoperative imaging by magnetic resonance neurography (MRN) or ultrasound lacks the resolution and contrast required to stage injury or detect regeneration.Absent clear assessments and given the preference for spontaneous healing when possible, surgeons will wait to allow time for functional improvement. Because nerve regeneration progresses at approximately 1 mm / day, this can take several months or longer. Not only does this 7QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 delay recovery for those who eventually require surgical repair, but it also negatively impacts final outcomes. A nerve’s regenerative capacity is highest immediately after injury and decreases slowly from there. A 3-month delay can meaningfully impact recovery. Improved tools for assessing nerve injury and regeneration could eliminate unnecessary delay, improving and accelerating recovery, as well as eliminating a second surgery.

[0036] The examples given above are among the most frequently described root causes for poor recovery after surgical repair of a major PN1. There are many additional challenges that derive from the inadequacy of current assessment tools. For example, nerve repair today is commonly performed across all fascicles, yet sometimes there is a heterogeneous injury, with one fascicle at Grade III+ and others at Grade II. Without the ability to assess individual fascicles, it is not possible to target repair only to the fascicles that need it.

[0037] Brachial plexus injuries are a second example. In these injuries, intraoperative assessment would allow localization of injury sites within the expansive and complex neural anatomy. Finally, nerve assessment after surgical treatment of compressive neuropathies, such as carpal tunnel and cubital tunnel syndrome, would be an important prognostic tool, allowing a patient to better appreciate their recovery timeline and endpoint.

[0038] In response to the unmet and long-standing need for improved nerve assessment tools, an intraoperative imaging probe is described herein. The imaging probe may use optical coherence tomography (OCT), a label-free imaging technique that is standard of care in ophthalmology, routinely used in interventional cardiology, and under clinical evaluation in dermatology, gastroenterology, and neurology, among others. Polarization-sensitive contrast mechanisms may visualize fascicular architecture, quantify fascicular myelination, and detect scarring / fibrosis. Angiographic OCT may visualize the nerve’s external vessels as well as the intrinsic vessels of the vasa nervorum. Dual-sided imaging can improve imaging in several ways, including increasing imaging depth.

[0039] FIG. 1A shows a schematic of an intraoperative imaging probe 100. The probe includes a housing 102. The housing may be configured to be handheld (e.g., be a size and shape that may be comfortably held and operated by a user, such as a researcher or physician). The housing may have a first arm 104a that extends from the housing, and a second arm 104b that extends from the housing. Each arm has a “proximal” and “distal” end. The proximal end of the8QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 arm is closest to the housing, while the distal end of the arm is furthest from the housing. Each arm defines a XYZ coordinate system as shown wherein the nerve, tendon, or other structure to be imaged has a longitudinal direction along the Z axis. The axis that defines the proximal-to-distal end of the arms defines the Y axis. Note that each arm has a unique XYZ coordinate system that is at least shifted and inverted in the X direction, but can additionally include a slight rotation in the YX plane due to a rotation of one or both arms associated with the opening and closing of the arms.

[0040] A first optical assembly 106a may be disposed within the first arm, and a second optical assembly 106b may be disposed in the second arm. The first optical assembly and second optical assembly may be configured to perform dual-sided optical coherence tomography (OCT) imaging. The intraoperative imaging probe may further include a beam scanning mechanism 108 configured to scan imaging beams from the first and second optical assemblies.

[0041] FIG. IB shows a sideways view of an intraoperative imaging probe in accordance with some embodiments of the disclosure. FIG. 1C shows a diagonal view of the intraoperative imaging probe. FIG. ID shows additional views of the intraoperative imaging probe. FIG. IE shows views of the distal end of the first and second arms of the operative probe surrounding a structure.

[0042] Imaging may be performed by translating optical assemblies within each arm, allowing dual-sided imaging that doubles the OCT image depth (see FIG. IE). With compression, the nerve will also flatten slightly, furthering the ability of the probe to image as much of the nerve as possible. To provide noise-reducing 3D acquisition, beam scanning may be enabled in both the nerve’s longitudinal direction (z) as well as height (y). Three-dimensional imaging is important in PS-OCT to reduce noise. An acquisition that is sufficient for generating fascicular architecture (FA), fascicular myelination (FM), nerve perfusion (NP), and nerve fibrosis (NF) may take about 0.5 seconds, about 1 second, about 1.5 seconds, about 2 seconds, about 2.5 seconds, about 3 seconds, about 3.5 seconds, about 4 seconds, about 4.5 seconds, or about 5 seconds.

[0043] The imaging probe may further include calibration elements 220 (also referred to as calibrating structures). FIG. 2 denotes the handheld probe comprising two arms, each equipped with optical assemblies to perform imaging from opposing surfaces. Included within9QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 each assembly are calibrating structures 220 used to provide a “ruler” for the imaging beam scanning in the y and x directions. In some embodiments, the calibration elements provide a periodic tick marks that can act as a ruler for registering and merging images, and providing a convenient scale while viewing the collected images. The calibration elements may be multilayer elements. In some embodiments, the calibration elements are configured to aid in polarization-sensitive OCT. When the calibration elements are configured to aid in polarizationsensitive OCT, the calibration elements contain a structure that is optically birefringent wherein the optic axis of the birefringence is oriented in a known relationship to the axes of the arms, such as for example at a specific orientation in the YZ plane. A transformation can be applied to measured PS-OCT data such that the optic axis of these structures is rotated to align to their known orientation, and that transformation can be applied across the image to additionally rotate the optic axis data associated with the sample 201 to be referenced to the XYZ coordinate system defined by the imaging probe. While 200 shows a calibration structure in one arm of the two-arm probe, in further embodiments a similar calibration structure is included in both arms. In this embodiment, the PS-OCT signals from both arms can be aligned to the XYZ coordinate system and thereby to each.

[0044] In FIG. 2, the calibration structure comprises three functional layers (206b). The first and second layers (210, 211) consist of birefringent media having known retardance values and defined optic axis orientations. The third layer (212) comprises a transparent medium containing periodically embedded scatterers at controlled concentrations, forming a horizontal reference with scatterer spacings in the range of 1-5 mm.

[0045] During use (e.g., surgery on a tissue), once the structure (201) is compressed, an optical beam is emitted from a translatable optical element (204), propagates through air (205b), traverses the three-layer calibration structure, and is backscattered by the compressed tissue (201b). The backscattered light is then collected by the same optical assembly for calibration and imaging.

[0046] The resulting PS-OCT image (220) contains distinct calibration features along with the sample information (201b). The two birefringent media (210 and 211) appear as distinct polarimatric signals for calibration (223 and 224). The third structural layer in the image appears as horizontal rulers (222), which are used for registration and calibration. The calibration10QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 structure may be fabricated from a variety of optically stable materials selected to provide known polarization, scattering, spectral, or geometric reference properties. Example materials include crystalline birefringent substrates (such as quartz, magnesium fluoride, or sapphire), polymer-based birefringent films (including stretched polymers or liquid crystal polymer films), and optical-grade polymers, glass, or silicone matrices. Scattering reference features may be formed using embedded microspheres, nanoparticles, diffusive pigments, patterned voids, or surfacerelief structures with controlled size, spacing, and concentration.

[0047] Additional features may include axially separated scattering fiducials to establish depth scaling, axial resolution compensation, wavelength-appropriate birefringent elements to account for source spectral bandwidth, and thermally stable or temperature-responsive materials to monitor polarization drift under operating conditions. In certain embodiments, the calibration structure may be implemented as a fixed internal reference, a removable insert, or a replaceable component to accommodate manufacturing variability, recalibration over device lifetime, or disposable clinical use. The number, thickness, ordering, and material composition of the calibration layers may be selected to match the operating wavelength range, system architecture, and anticipated tissue interaction during tissue imaging.

[0048] In some embodiments, the OCT or PS-OCT images generated by each arm are merged to generate a single PS-OCT image describing the sample. In one embodiment, the geometric position of the PS-OCT imaging data in the XYZ axes defined by each arm is known via the use of calibrating structures or a calibrated beam-scanning profile in the Y and Z axes. Further the XYZ coordinate system defined by one arm can be different from the XYZ coordinate system of the second arm due to the different locations of the arms in the X direction, and due to the potential angular tilt of the two arms relative to each other arms associated with a hinging movement of one or both arms associated with opening or closing the arms. In one embodiment, the distance between the distal ends of the arms and the angular between the arms is known, allowing the relationship of the XYZ coordinate system of the first arm to be registered to the XYZ coordinate system of the second arm. In a further embodiment, this relationship is used to align the OCT or PS-OCT imaging data associated with each arm and to thereby generate a single merged image of the sample.11QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0049] In further embodiments, structures and features within the OCT images from each arm are used in addition to other data to perform a registration of the images from each arm and to generate a merged image. In regions of the sample that contain OCT signal from both arms, the merged image can be constructed by taking the data from one image that has higher signal intensity, by averaging the signal from both images, or by performing a weighted averaging of the signals with the weight determined by an image metric such as signal intensity. Merged images can be generated that are based on OCT structural imaging data, OCT attenuation coefficient data, OCT birefringence data, such as retardance or optic axis or depolarization, or some combination thereof. In another further embodiment, the distance between the distal arms is large enough that there is a region of the sample that is not imaged by either arm. A merged image can be generated in this configuration using knowledge of the XYZ coordinate systems of each arm as described above, and showing “empty” regions within that merged image.

[0050] In an exemplary design, the OCT or PS-OCT data along the Z axis is used to provide averaging to generate a lower-noise image describing the XY structure of the sample. In another embodiment, the OCT or PS-OCT system generates volumetric datasets describing the XYZ structure of the sample.

[0051] An exemplary design of the calibrating phantom is shown in 206b with the imaging beam 205b passing through a three-layer calibrating structure comprising two birefringent layers 210 and 211, followed by a layer with periodic scattering structures 212. The birefringent axes of layers 210 and 211 are not equal and configured to allow an absolute calibration of the measured optical axis in the reference frame of the handheld probe, e.g., layer 210 has a birefringent axis oriented in the y direction and layer 211 has a birefringent axis oriented at a 45-degree angle to the y-axis and within the y-z plane. The sample is denoted in 206b as 201b. 230 shows an exemplary OCT image generated from the optical assembly of arm 201 comprising birefringent layers 204 and 203, spatial calibration in 202, and the tissue sample at 201.

[0052] In one embodiment, the arms are configured with sensors to measure the distance between arms at a known location such that the distance between the distal ends can be calculated and used to interpret and merge OCT images generated from each arm. The distance between the arms may be measured using sensors that detect the gap between the arms at a more12QB\125141.049611100748731.1MGH 2025-208-02Quarles 125141.04961 proximal point, such as, for example, within the housing of the handheld device, and calculating the gap at the distal point based on the gap at the proximal point and the known geometry of the arms. The gap between the arms at that proximal location can be measured using capacitive / inductive sensors, linear encoders, optical sensors, optical sensors based on beam deflection, and magnetic Hall-effect sensors. Further, the angle between the arms can be measured using angle-sensing techniques to calculate the gap at the distal site.

[0053] In some embodiments, the distance between the two arms could be measured directly at the distal location using non-contact sensors such as Hall-effect sensors, or could be measured using the OCT beam by locating the arm surface depth within an OCT image collected from the other arm.

[0054] Alternatively, the distance between the arms could be measured directly from an OCT image acquired at a location in the Y-axis that is free of the sample. For example, above or below the sample 201 in FIG 2. OCT directly measures distance, and the gap between the interior surface of each arm can be measured directly from the OCT image.

[0055] FIG. 3 denotes the scanning of the beam in the z direction for each handheld probe first arm and second arm as a function of time relative to the timing of the optical switch used to control the direction and receipt of light to either the first arm or the second arm. In the embodiment of FIGS. lAand IB, an optical switch is included that directs imaging light from a single input optical fiber to one of two output optical fibers that are associated with each imaging arm. Here, the switch is configured to direct light to the first arm while the beam scans from higher to lower z positions, and is configured to then direct light to the second arm while the beam scans from lower to higher z positions.

[0056] FIG. 4 shows a configuration in which light from the first arm and second arm is encoded via depth by playing additional delay within one arm (here shown as the second arm) such that the imaging data from the first arm is located at a further depth location in the resulting image relative to the second arm - the delay loop shown on the first arm means that the data from the first arm is registered in the collected image as though it came from a different depth within the sample and thus is nonoverlapping in the image.

[0057] FIG. 5 shows a multi-channel OCT interferometer system 500 configured to acquire imaging data from the first arm and second arm simultaneously on separate receiver 13QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 channels, e g., without the need for a switch or path-length encoding. The light source 501 directs light to an optical splitter 502 that directs light to a sample arm splitter 504 and a reference arm splitter 503. The sample arm splitter 504 directs a portion of light to a first sample arm circulator 506, which then directs light to the first arm and directs reflected light from the first arm to a first polarization-diverse balanced receiver 512. The sample arm splitter 504 further directs a portion of light to a second sample arm circulator 508, which then directs light to the second arm and directs reflected light from the second arm to a second polarization-diverse balanced receiver 511. The reference arm splitter 503 directs a portion of light to a first reference arm circulator 505, which then directs light to reference mirror 509 and directs reflected light from 509 to the second polarization-diverse balanced receiver 511. The reference arm splitter 503 directs a further portion of light to a second reference arm circulator 507, which then directs light to reference mirror 510 and directs reflected light from 510 to the first polarization-diverse balanced receiver 512. The first polarization diverse receiver 512 generates optical outputs (balanced) describing a first polarization state to balanced receiver 515, and generates further optical outputs (balanced) describing a second polarization state that is approximately orthogonal to the first polarization state to balanced receiver 516. The electrical output signals 519 and 520 are generated by these receivers and directed to a signal digitizer (not shown). Polarization diverse receiver 511 and associated balanced receivers 513 and 514, and output electrical signals 517 and 518 follow a similar configuration but describe signals from the second arm.

[0058] FIG. 6 shows an exemplary design 600 of the optical assembly within one of the imaging arms 602 comprising an optical scanning system 603 directing and receiving an imaging beam to and from the sample 601 through a calibrating structure 604. The optical scanning system 603 is shown in the YX plane where an optical fiber 608 is attached to a piezoelectric actuator 606 and is configured such that the optical fiber is cantilevered off the actuator by a distance L (607). The actuator 606 is configured to translate the fiber 608 in the z direction (e.g., out of the XY plane). The imaging beam emitted by the fiber 608 is thus translated in the z direction and directed toward a lens system comprising 609 and 610 and a prism 611 such that the beam 612 is directed toward the sample 601 and scanned in the z direction. The entire optical system 603 is additionally translated in the y direction to achieve scanning in both the Z (via piezoelectric actuation) and Y (via translation of 603) directions. The translation of the end of 14QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 the optical fiber 608 may be greater than the translation of the actuator 606 via driving the actuator at a mechanical resonance of the fiber structure that overhangs the actuator. FIG. 7 shows an exemplary optical assembly described in FIG. 6. In further embodiments, the optical fiber 608 can be replaced by a waveguide structure that can include an optical fiber affixed to a mechanical platform such as a V-groove, a photonic integrated circuit, or another means of delivering guided light. The waveguide structure can be translated in a similar manner as the fiber and further can be driven at a resonance frequency that amplifies the translation of the distal tip relative to the actuator. The fiber 608 or waveguide structure may be configured with an angled end-face to reduce back-reflections, and the orientation of the fiber or waveguide adjusted to configure emitted light to be transmitted nominally along the axis defined by the proximal and distal ends of the arm.

[0059] Mechanical Design

[0060] The intraoperative imaging device is configured such that the distal ends of the first and second arm “open” (e.g., increase the distance between the arms) to allow the distal ends of the arms to be placed over a structure. The arms may be used to surround and exert pressure on (e.g., squeeze) a structure. The probe may include an adjustment mechanism configured to adjust (e.g., increase or decrease) a distance between the first arm and second arm. The distance between the first arm and second arm is measured by the distance between the first distal end and the second distal end. In some embodiments, the distance between the first and second distal ends may be about 0.5 mm, 0.75 mm, 1 mm, 1.25 mm, 1.5 mm, 1.75 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm. In some embodiments, the arms can open further before closing to accept larger structures.

[0061] In some embodiments, the probe may include a physical stop that prevents the distance between the distal arms from being placed below a defined number. This stop can be located at the distal end directly or at a more proximal location. The stop distance (gap between the distal ends) can be about 0.5 mm, 0.75mm, or 1mm.

[0062] In some embodiments, structures that may be imaged may have an approximately cylindrical shape with diameters (before compression) between 1 mm and 8 mm. Larger samples may be imaged but some interior regions may fall outside of the imaging depth of the OCT from the first arm or second arm.15QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0063] In some embodiments, the imaging probe may be configured such that both the first arm and second arm pivot away from each other to open, and towards each other to close (e.g., a scissoring motion). In other embodiments, the first arm may be fixed, and the second arm may be configured to pivot away from the first arm to open, and towards the first arm to close. In other embodiments, the imaging probe may be configured such that the arms move away from one another and towards one another while remaining parallel (e.g., the distance of the proximal ends and distal ends are the same at any given time).

[0064] It is beneficial for the first and second arms to have a great enough length such that opening and closing the arms does not affect the angle between the distal ends. The length of the arms (e.g., the distance from the proximal end to the distal end of an arm) may range from 2 cm to 15 cm. In some embodiments, the length of the arms is about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, or about 10 cm.

[0065] In the case of one or more arms undergoing a pivoting motion, it is important for the hinge point of the arms to be sufficiently far enough from the distal ends that the angle between the distal ends does not significantly change regardless of the distance between the distal ends. If the angle between the distal ends does increase such that it alters imaging, there are several ways to account for this. If the intraoperative imaging probe includes a calibration element, the calibration element can be used to reconstruct the images and account for the angle between the image from the first arm and second arm. Additionally or alternatively, if the angle between the first arm and second arm is known, the images can be reconstructed using simple image transformations.

[0066] In some embodiments, arm opening is achieved manually by pressing the actuator 110 (“push to open”) on the side of the probe 100 (FIG. 1A). A preset force, which can be dialed in by the force adjust screw on the side, will close this prong when the actuator is released. This design ensures accurate control of the force applied to the nerve, e.g., the operator only controls the opening and does not control the force at which the arms squeeze the structure. A force transducer in the probe located within 110 can report real-time measurements of the applied force. The conversion of force to pressure requires knowledge of the area of the nerve between the prongs, which is a function of the nerve diameter. As such, in some embodiments, the nerve16QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 diameter may be measured before applying the probe, and a look-up table may be used to select a force corresponding to a desired pressure (in mmHg).

[0067] In some embodiments, force sensors and / or pressure sensors may be used to control the force and pressure exerted on the structure. The force and pressure applied to the sample could be measured using sensors embedded within the distal tip. These force and pressure sensors could be based on piezoelectric, capacitive, MEMS, strain gauge, magnetostrictive and magnetic sensors, and force-sensing resistors.

[0068] Alternatively, this force could be measured at a proximal location, and the force applied to the sample calculated based on this force and the known geometry. In a further embodiment, the pressure applied to the sample is calculated based on the force applied at a proximal location and an OCT-based measurement of the length of the sample between the arms over which that force is distributed.

[0069] In some embodiments, a safe maximum amount of pressure to exert on a structure may be about 30 mmHg. In some embodiments, the imaging probe may be configured to measure a duration of an applied pressure or a metric derived from the duration of applied pressure. The imaging probe may generate signals to the user based on sound or display indicators in response to the intensity or duration of the applied pressure.

[0070] Nerve assessment

[0071] The intraoperative imaging probe can be used to assess the nerve in several ways. Forms of assessment include, but are not limited to, imaging fascicular area, fascicular myelination, nerve fibrosis, nerve perfusion, or scarring. In some embodiments, peripheral nerve assessment includes imaging at least one of imaging FA, FM, NF, NP, or scarring.

[0072] Fascicular Area (FA). Fascicular atrophy is an important indicator of nerve health. The area of each fascicle within the nerve may be quantified. The OCT system will report the area of each fascicle (FA1, FA2, etc.); note that closely packed fascicles may not be distinguishable, in which case reporting would report the total fascicular area (FAt).

[0073] Fascicular Myelination (FM). Myelination is a clear indicator of Grade III and above injuries (via Wallerian degeneration), and can be used to detect incipient and progressing regeneration. The degree of myelination within each fascicle may be quantified and reported as17QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 FM1, FM2, etc. As with FA, closely packed fascicles may require reduction to a single value for the nerve.

[0074] Nerve Fibrosis (NF). The role of fibrosis as an inhibitor to regeneration is clearly understood. There are no existing clear algorithms for quantifying fibrosis. Moreover, rats are a poor model for fibrosis / scarring, and the signals we see in this study may not be representative. A quantification of fibrosis (NF) may be evaluated.

[0075] Nerve Perfusion (NP). Perfusion is used clinically to assess nerve health but is accessible only by viewing the end of the cut nerve. Using angiography methods combined with vessel segmentation, the number of perfused vessels may be determined. Using fascicle and nerve segmentation, it is possible to discriminate this count between intrinsic (vasa nervorum) and extrinsic vessels (e g., NPint and NPext).

[0076] Image analysis

[0077] Images acquired from each prong may be registered and merged following known algorithms. Manual or automated fascicle and nerve segmentation may be used to generate fascicle-specific metrics (such as FA, FM) and will allow discrimination of intrinsic and extrinsic vessels (NP). Existing methods may be used or adapted for vessel segmentation. It is not necessary to quantify vessel diameter or flow, which would be significantly more challenging. Rather, the goal is to count the number of perfused vessels. To segment vessels, we will adapt existing methods developed for vessel segmentation in OCT imaging of the retina. Note that we do not intend in this proposal to quantify vessel diameter or flow, which would be significantly more challenging than our goal of counting the number of perfused vessels. The OCT console software will be updated to generate quasi- real time displays of the acquired structural, angiographic, and PS-OCT images. The existing system already supports real-time display of structural and angiographic datasets, and thus only real-time PS processing needs to be implemented. This real-time display will be useful in integrating the intraoperative imaging probe into a surgical workflow. Segmentation will be performed offline, as will the calculation of the injury markers (FA, FM, NP, NF).

[0078] In some embodiments, algorithms may use inherent sample fiduciary markers without using calibration. In this embodiment, there must be sufficient compression of the sample. Algorithms used in this embodiment may include but are not limited to: fascicle-based 18QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 feature registration using optic-axis orientation and retardance contrast, cluster matching of segmented fascicles as fiduciary constellations, affine 2D transformation (translation, in-plane rotation, limited shear), piecewise affine refinement in regions of nonuniform compression, gradient-domain (Poisson) blending in the overlap region, feathered alpha blending weighted by polarization confidence (e.g., DOPU), mutual-information (MI) registration using multi-channel inputs (e.g., intensity, retardance, and axis orientation), RANSAC-robust keypoint matching on PS-derived edge maps (fascicle boundaries), optical-flow refinement constrained to the overlap band (small local corrections only), graph-based matching of fascicle adjacency (relative spacing / orientation invariants), or seam selection / minimum-cut blending to avoid stitching through low- SNR or defocused regions.

[0079] In some embodiments, algorithms may use inherent sample fiduciary markers as well as information from the calibration elements. In this embodiment, there must be sufficient compression of the sample. Algorithms used in this embodiment may include but are not limited to: fiducial -based geometric registration using calibration scatterers or ruler features, polarization reference frame alignment using birefringent calibration layers, affine or rigid 2D transformation after calibration-anchored alignment, fascicle-based PS-OCT refinement for residual misalignment, polarization-aware blending preserving optic-axis continuity, Stokes domain or direct merge after polarization normalization, fiducial detection via template matching / centroiding of periodic scatterers (for example, using 1-5 mm spacing) to estimate scale and rotation, RANSAC line / spacing fit to the “ruler” layer to enforce mm-per-pixel and correct shear (e.g., may use known 1-5 mm pitch as constraint), multi-fiducial homography / affine solve from >3 scatterer correspondences across the field, axial / lateral distortion correction by fitting observed scatterer grid vs expected spacing, cross-arm polarization mapping via Jones / Mueller fit to known retardance and optic-axis layers (e.g., apply per-arm correction before comparing axis maps), or residual alignment refinement using MI / NCC restricted to tissue overlap after calibration locks geometry.

[0080] In some embodiments, the sample compression may be insufficient and lack overlap (e.g., there may be a gap present). Algorithms used in this embodiment may include but are not limited to: calibration-anchored placement of each PS-OCT image into a shared coordinate frame, independent surface registration with no forced correspondence, polarization offset correction using internal polarization phantoms, composite image construction with 19QB1125141.049611100748731.1MGH 2025-208-02Quarles 125141.04961 explicit gap representation, layered fusion maintaining separate polarization maps per surface, confidence- or mask-based visualization of unobserved regions vs real sample, use of ruler fiducials (which may be 1-5 mm) to quantify separation and report absolute spacing between surfaces (mm-scale gap estimate), conservative completion (e.g., leave gap empty / unknown; optionally show a bounded “uncertainty band” rather than interpolating signal). If the analysis is required for display only, minimal-curvature bridge surface between segmented boundaries, explicitly flagged as inferred (no polarization interpolation) may be used. For polarizationconsistent rendering: map both surfaces’ optic-axis angles into the same reference frame, but keep metrics per-surface (no averaging) may be used. For seam / overlay visualization modes: side-by-side, checkerboard, or toggled overlay in shared coordinates to support surgeon interpretation without false fusion may be used.

[0081] The algorithms may be implemented using neural networks or a trained machine learning algorithm. In some embodiments, a single deep network may be used. In some embodiments, a plurality of small neural networks may be used. Using machine learning techniques requires sufficient training data.

[0082] Computer System and OCT Consoles

[0083] The handheld cable may be connected to an existing computer system including an OCT console. FIG. 9 shows an example system in which the probe 100 is connected to a data processing system 1000. The data processing system 1000 is shown in greater detail in FIG. 10.

[0084] In FIG. 10, an example 1000 of a system (e.g., a data processing system) for operating an OCT imaging probe and image analysis in accordance with some embodiments of the disclosed subject matter is shown.

[0085] In some embodiments, computing device 1004 and / or server 1016 can be any suitable computing device or combination of devices, such as a desktop computer, a laptop computer, a smartphone, a tablet computer, a wearable computer, a server computer, a virtual machine being executed by a physical computing device, etc. As described herein, system 1000 can present information about the collected OCT images to a user (e.g., a researcher and / or a physician).20QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0086] In some embodiments, communication network 1002 can be any suitable communication network or combination of communication networks. In some embodiments, communication network 1002 can be any suitable communication network or combination of communication networks. For example, communication network 1002 can include a Wi-Fi network (which can include one or more wireless routers, one or more switches, etc.), a peer-to-peer network (e.g., a Bluetooth network), a cellular network (e.g., a 4G network, a 5G network, etc., complying with any suitable standard, such as CDMA, GSM, LTE, LTE Advanced, WiMAX, etc ), a wired network, etc. In some embodiments, communication network 1002 can be a local area network, a wide area network, a public network (e.g., the Internet), a private or semi-private network (e.g., a corporate or university intranet), any other suitable type of network, or any suitable combination of networks. Communications links shown in FIG. 10 can each be any suitable communications link or combination of communications links, such as wired links, fiber optic links, Wi-Fi links, Bluetooth links, cellular links, etc.

[0087] FIG. 10 additionally shows an example of hardware that can be used to implement computing device 1004 and server 1016 in accordance with some embodiments of the disclosed subject matter. In some embodiments, computing device 1004 can be used to execute one or more set of instructions to collect OCT images using the intraoperative imaging probe. In other embodiments, computing device 1004 can be used to perform image analysis on the collected images.

[0088] As shown in FIG. 10, computing device 1004 can include one or more hardware processor 1006, one or more displays 1008, one or more inputs 1010, one or more communications 1012, and / or memory 1014. In some embodiments, processor 1006 can be any suitable hardware processor or combination of processors, such as central processing unit, a graphics processing unit, etc. In some embodiments, display 1008 can include any suitable display devices, such as a computer monitor, a touchscreen, a television, etc. In some embodiments, inputs 1010 can include any suitable input device and / or sensors that can be used to receive user input, such as a keyboard, a mouse, a touchscreen, a microphone, etc.

[0089] In some embodiments, communication systems 1012 can include any suitable hardware, firmware, and / or software for communicating information over communication network 1002 and / or any other suitable communication networks. For example, communications21QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 systems 1012 can include one or more transceivers, one or more communication chips and / or chip sets, etc. In a more particular example, communications systems 1012 can include hardware, firmware and / or software that can be used to establish a Wi-Fi connection, a Bluetooth connection, a cellular connection, an Ethernet connection, etc.

[0090] In some embodiments, memory 1014 can include any suitable storage device or devices that can be used to store instructions, values, etc., that can be used, for example, by processor 1006 to present content using display 1008, to communicate with server 1016 via communications system(s) 1012, etc.

[0091] Memory 1014 can include any suitable volatile memory, non-volatile memory, storage, or any suitable combination thereof. For example, memory 1014 can include RAM, ROM, EEPROM, one or more flash drives, one or more hard disks, one or more solid state drives, one or more optical drives, etc. In some embodiments, memory 1014 can have encoded thereon a computer program for controlling operation of computing device 1004. In such embodiments, processor 1006 can execute at least a portion of the computer program to present content (e.g., images, user interfaces, graphics, tables, etc.), receive content from server 1016, transmit information to server 1016, etc.

[0092] In some embodiments, server 1016 can include a processor 1018, a display 1020, one or more inputs 1022, one or more communications systems 1024, and / or memory 1026. In some embodiments, processor 1018 can be any suitable hardware processor or combination of processors, such as a central processing unit, a graphics processing unit, etc. In some embodiments, display 1020 can include any suitable display devices, such as a computer monitor, a touchscreen, a television, etc. In some embodiments, inputs 1022 can include any suitable input devices and / or sensors that can be used to receive user input, such as a keyboard, a mouse, a touchscreen, a microphone, etc.

[0093] In some embodiments, communications systems 1024 can include any suitable hardware, firmware, and / or software for communicating information over communication network 1002 and / or any other suitable communication networks. For example, communications systems 1024 can include one or more transceivers, one or more communication chips and / or chip sets, etc. In a more particular example, communications systems 1024 can include22QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 hardware, firmware and / or software that can be used to establish a Wi-Fi connection, a Bluetooth connection, a cellular connection, an Ethernet connection, etc.

[0094] In some embodiments, memory 1026 can include any suitable storage device or devices that can be used to store instructions, values, etc., that can be used, for example, by processor 1018 to present content using display 1020, to communicate with one or more computing devices 1004, etc. Memory 1026 can include any suitable volatile memory, nonvolatile memory, storage, or any suitable combination thereof. For example, memory 1026 can include RAM, ROM, EEPROM, one or more flash drives, one or more hard disks, one or more solid state drives, one or more optical drives, etc. In some embodiments, memory 1026 can have encoded thereon a server program for controlling operation of server 1016. In such embodiments, processor 1018 can execute at least a portion of the server program to transmit information and / or content (e.g., OCT images, measurements based on the OCT images, etc.) to one or more computing devices 1004, receive information and / or content from one or more computing devices 1004, receive instructions from one or more devices (e.g., a personal computer, a laptop computer, a tablet computer, a smartphone, etc.), etc.

[0095] In some embodiments, any suitable computer readable media can be used for storing instructions for performing the functions and / or processes described herein. For example, in some embodiments, computer readable media can be transitory or non-transitory. For example, non-transitory computer readable media can include media such as magnetic media (such as hard disks, floppy disks, etc.), optical media (such as compact discs, digital video discs, Blu-ray discs, etc.), semiconductor media (such as RAM, Flash memory, electrically programmable read only memory (EPROM), electrically erasable programmable read only memory (EEPROM), etc.), any suitable media that is not fleeting or devoid of any semblance of permanence during transmission, and / or any suitable tangible media. As another example, transitory computer readable media can include signals on networks, in wires, conductors, optical fibers, circuits, or any suitable media that is fleeting and devoid of any semblance of permanence during transmission, and / or any suitable intangible media.

[0096] Examples

[0097] Overview23QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0098] Described herein is an intraoperative imaging probe that is suitable for preclinical imaging of anesthetized animals as a handheld clinical probe compatible with the surgical workflow. Safety of this contact imaging probe and quantify its accuracy in assessing four key internal parameters: fascicular morphology, fascicular myelination, microvascular density, and fibrosis. Finally, we will evaluate if the achieved accuracy meets rationally derived minimum viable targets for use in surgical decision- making. Successful completion of these studies will provide the data needed to translate this technology into clinical trials for human use.

[0099] Our team’s prior work has established the ability of OCT to visualize internal features of peripheral nerves in rats, swine, non-human primates, and fixed human tissues. These capabilities leverage multiple modes of contrast, including structural imaging based on tissue reflectivity (e.g., conventional OCT imaging), angiographic OCT based on tissue dynamics, and polarization-sensitive OCT based on tissue birefringence. Of these, polarization-sensitive (PS) (or equivalently polarimetric) OCT is the most important for peripheral nerve applications. It is also the most complex, both to understand and implement. Stated simply, PS-OCT measures the sub-resolution microstructure of tissue. It does this by tracking how tissue affects the polarization state of the imaging light. Aligned microstructures induce polarization rotation, and this rotation can be used to infer the strength and orientation of that microstructure. In the nerve, both myelin sheaths, axons, and the endoneurium / perineurium / epineurium all affect polarization. As a result, the PS signals are sensitive reporters of internal nerve integrity and health. Tailored algorithms are employed to generate interpretable and quantifiable images and signals from the complex, multi-dimensional PS data. FIG. 11 - FIG. 13 describe the use of PS-OCT to provide fascicular architecture, quantify myelination within fascicles, and detect internal nerve fibrosis.

[0100] FIG. 14 and FIG. 15 additionally describe angiographic nerve imaging and dualsided imaging to extend imaging depth, respectively. Together, these capabilities lay the foundation for and motivate the design of the proposed intraoperative tool.

[0101] Finally, we show in FIGS. 16A-16C a prototype of the handheld design, including optics on a single side and supporting linear pullback. Imaging of porcine ulnar nerves is shown. These data, which demonstrate fascicular contrast but exhibit higher noise than the benchtop systems, motivated the inclusion of noise-reducing 3D beam-scanning in some embodiments of the disclosure.24QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0102] A key milestone is the initiation of human studies. These studies will require data in several key areas: demonstration of a working intraoperative probe, demonstration of its safety, and evidence that information provided by the probe is accurate and likely to be actionable. The proposed research is designed to generate these data.

[0103] OCT injury markers including Fascicular area, fascicular myelination, nerve fibrosis, and nerve perfusion will be generated from the probe and validated for accuracy using best-practice histology measures across a range of relevant injury models and time points.

[0104] Threshold for clinical utility

[0105] In addition to testing for the accuracy of each parameter, we will also attempt, as best as current data allows, to evaluate whether these accuracies are sufficient to guide surgical decision- making. Because there are no tools to measure many of these parameters in vivo, there is little data in the literature that specifically reports quantitative thresholds that are clinically meaningful. For example, fibrosis is well-established to inhibit regeneration, but there are no studies that quantify this relationship. However, demonstrating evidence of clinical utility is important to motivate efforts to bring this technology to human studies and, in the context of this work, to provide a basis for powering continued studies.

[0106] We will base thresholds for clinical utility on FM, in part because it is possible to derive a rational target based on known nerve physiology / pathophysiology and in part because FM is among the more developed quantifiable outputs from OCT. Our goal is to derive a minimum viable accuracy target. We will consider a clinical scenario in which FM is measured proximal and distal to a non-transection injury of unknown severity during surgical exploration, e.g., at a timepoint after Wallerian degeneration. A surgeon, using the total, evaluates the drop in myelination at the distal site relative to the proximal site. We estimate that if the tool were able to distinguish between a 10% or worse drop from a 50% or higher drop with 90% confidence, this would reasonably be a sufficient basis to select or defer repair. Assuming FM errors are normally distributed about means of 10% and 50%, a 90% confidence requires measurement errors (standard deviation) of 12%, e.g., FM measures should be accurate to within + / - 12%.

[0107] Overview of design25QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0108] The design principles of the imaging probe are summarized in FIGS. 17A-17D. The nerve will be located between two arms that can be opened manually and that close with a controlled force / pressure. Imaging will be performed by translating optical assemblies within each arm, allowing dual-sided imaging that doubles the OCT image depth. With compression, the nerve will also flatten slightly, furthering its ability to reach as much of the nerve as possible. To provide noise-reducing 3D acquisition, beam scanning will be enabled in both the nerve’s longitudinal direction (z) as well as height (y). Three-dimensional imaging is critical in PS-OCT to reduce noise. An acquisition that is sufficient for generating FA, FM, NP, and NF will require approximately 5 seconds.

[0109] Optical design and beam-scanning mechanisms

[0110] Beam scanning in the Z direction (FIGS. 17A-17D) will be achieved via a pair of MEMS scanners located in the body (e.g., housing) (FIG. 1). The imaging field in the Z direction will be approximately 2 mm. Beam scanning in the Y direction will be achieved by translating the optical cores within each prong upward using a linear actuator (Xeryon XLA-1). This will enable Y-axis beam scanning by more than 10 mm, such as 10 mm, 12 mm, 14 mm, 16 mm, 18 mm, or 20 mm. Note that only the optical cores translate upward; the outer fixture that contacts the nerve remains stationary. An optical switch (Agiltron CrystalLatch 1x2) located in the housing will rapidly switch the OCT signal between the prongs so imaging is effectively simultaneous. Switching will occur between MEMS scans, e.g., one complex forward scan of the left prong will be acquired, and then one complex MEMS scan of the right prong will be acquired. Pullback in the Y axis will be slow, approximately 3-5 mm / sec. Image acquisition time will, therefore, be a few seconds. We note that angiographic imaging will require a separate acquisition with the MEMs scanner (3D scanning) turned off; this enables the oversampling required for the angiographic processing. For both image captures (non-angio and angio), the total acquisition time will be under 10 seconds.[OHl] Mechanical design

[0112] One prong of the imaging probe can be designed to open (pivot) to allow the probe tip to be placed over a nerve. Prong opening can be achieved manually by pressing the actuator (“push to open”) on the side of the probe (FIGS. 1 A-1B). A preset force, which can be dialed in by the force adjust screw on the side, will close this prong when the actuator is released.26QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 A force transducer can be included in the probe to provide real-time measurements of the applied force at the location of the force transducer. The pressure applied to the sample can be calculated based on this force measurement, combined with knowledge of the geometry of the probe and the surface area of the sample that is in contact with the distal end of the probe. This surface area can be estimated based on a separate measurement of the nerve diameter, or based on the Y-axis extend of the nerve as calculated from the OCT image.

[0113] Integration with OCT console

[0114] The handheld probe will connect to existing OCT consoles via a handheld cable. The cable includes a single optical fiber and electrical connections for driving the MEMS mirrors, linear translators, optical switches, and for reading the force transducer output.

[0115] Image processing and analysis algorithms

[0116] Images acquired from each prong will be registered and merged following algorithms developed for our benchtop dual-sided microscope. We will use existing nerve data to begin the development of automated fascicle and nerve segmentation tools based on machine learning. These segmentations are needed to generate fascicle-specific metrics (FA, FM) and will allow discrimination of intrinsic and extrinsic vessels (NP). To segment vessels, we will adapt existing methods developed for vessel segmentation in OCT imaging of the retina. Note that we do not intend in this proposal to quantify vessel diameter or flow, which would be significantly more challenging than our goal of counting the number of perfused vessels. The OCT console software will be updated to generate quasi- real time displays of the acquired structural, angiographic, and PS-OCT images. The existing system already supports real-time display of structural and angiographic datasets, and thus only real-time PS processing needs to be implemented. Segmentation will be performed offline, as will the calculation of the injury markers (FA, FM, NP, NF).

[0117] Validation and engineering milestones

[0118] Two categories of validation studies will be performed. The first category includes engineering validations, ensuring the system meets technical performance targets. For these, we will perform phantom imaging and measure sensitivity, polarimetry noise, and angiographic noise floor. Comparisons will be made to theoretical limits based on SNR, as well as to27QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 performance achieved by our reference benchtop systems. Ex vivo nerves (discarded rat and swine samples from MGH animal research laboratories) will be imaged, and results will be compared to those obtained using the aforementioned reference benchtop systems. Qualitative differences, if present, will be investigated.

[0119] As a second category, we will conduct clinical usability studies. We will perform in vivo imaging studies in rats and gain feedback on the design and its ability to integrate effectively with surgical workflow. Barriers to clinical adoption will be identified and, if appropriate, fixed through iteration. These in vivo studies will be conducted as needed, and not only used as a final test. In these studies, we will image both healthy and injured nerves using both compression / crush and transection models. As required, these data can be used to advance and test the segmentation tools.

[0120] Establishment of safe OCT pressure thresholds on the rat sciatic nerve

[0121] In order for OCT to be clinically viable, we must understand how the application of pressure required to obtain clinically relevant images / data impacts nerve health. The pressure applied by the imaging probe is a short-term (<30 sec) analog of chronic neurostimulator implants based on a flat geometry. These implants have been studied extensively in both preclinical and clinical models and have been shown to be safe. For this reason, we do not anticipate safety concerns, and we base our studies on those used in translating the flat stimulator to human subjects.

[0122] The rat sciatic nerve serves as the ideal model for evaluating the safety of pressures applied by the OCT system required to acquire images suitable for clinical decision making, as this model has been extensively used to evaluate axon injury caused by compression. In the rat sciatic nerve, vascular changes as a result of circumferentially applied pressure begin at 20-30 mmHg, which decreases venular blood flow within the nerve. Arterial blood flow within the nerve is decreased from 50 mmHg, with complete cessation of blood flow at 80 mmHg. The threshold for nerve demyelination in an acute setting is 50 mmHg, although pressures as low as 10 mmHg have been reported. Clinically, electrophysiology is the gold standard for evaluating nerve injury; however, it is well established that a significant degree of demyelination is needed for a noticeable decrease in nerve action potentials.

[0123] Acute evaluation of OCT pressures on nerve conductivity in situ28QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0124] A pilot study will be conducted to evaluate the impact of image acquisition using OCT utilizing validated pressures reported in the literature in an acute rat sciatic nerve crush model (equal numbers male and female, n=8). Electrophysiology will serve as the clinically relevant and experimentally validated intraoperative measure of nerve function, serving as a surrogate for axon loss and demyelination. It is well established that conduction velocity decreases while latency increases as nerve injury severity increases. Baseline electrophysiology will serve as the control for repeat measures analysis. For power calculations, see the vertebrate animal document.

[0125] Surgical approach. We will perform four graded acute compression injuries using the OCT device to deliver the compression. All surgeries will be acute (terminal) in nature, performed as described as detailed in the vertebrate animal section. For accuracy of the nerve compression, an epineural suture will be placed 33 mm proximal to the location of the greatest diameter of the gastrocnemius muscle, demarcating the site where all compressions will be applied.

[0126] OCT image acquisition and pressure application. The OCT device will be used to simultaneously image nerve architecture (axonal integrity and angiography) and apply ascending compression pressures of 20, 50, 80 and 400 mmHg. Each pressure will be applied for a period of 2 minutes (6 times longer than it takes to take a clinically relevant image with the device) and released. To allow repeated measures on the same nerve, a recovery period of 30 minutes between compressions will be allotted before the next ascending pressure is applied.

[0127] Electrophysiology: We will perform clinically relevant (e.g., conventional) electrophysiology to evaluate nerve injury using our TDT system (Tucker-Davis Technologies, Florida, USA) as described in our previous work. Electrophysiology will be performed 30 minutes prior to injury and 30 minutes after injury to account for any transient focal blocks. Peripheral neuropathies caused by compression as identified by electrophysiology will be used to validate compression values in the literature and inform compression values in further experimentation and use. The specific tests include compound nerve action potentials and nerve conduction velocity.29QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0128] Compound nerve action potentials (CNAPs). Afferent and efferent CNAPs will be performed to identify axon loss, as indicated by peak amplitude. Pre-injury amplitude will serve as the control, with a >20% decrease in peak amplitude post-injury considered axonal loss.

[0129] Nerve conduction velocity (NCV). Demyelination can be identified by a reduction in NCV and / or protraction of distal latency. A 75% decrease in conduction velocity, and / or a 130% prolongation of latency from baseline values will be considered demyelination.

[0130] Evaluation of OCT pressures on healthy nerves

[0131] To assess the impact of acute compression resulting from OCT imaging on healthy nerves, animals (equal numbers of males and females, n=32) will be randomly assigned to one of four groups with pressure ranging from 20 to 400 mmHg. Pressures to be applied by the OCT device have been derived from the validated literature and will be informed by the previous pilot study. As with the pilot study, electrophysiology will serve to validate nerve function post compression. Histology will serve as the primary assessment, as it represents the gold standard in experimental evaluation of peripheral nerve injury / regeneration. The axon morphometric parameters of interest are myelin organization and axon density. Myelin is sensitive to oxygenation stress that can occur from compression or neurovascular injury in acute timeframes and will rapidly disorganize to form whorls and separations in the wraps around the axon. This can be observed in electron microscopy (EM) of thin sections of the processed nerve. Over longer timeframes, a demyelination / remyelination event is indicated by loss of the relationship between axon diameter and myelin thickness. Wallerian degeneration and regeneration are indicated by the presence of several axonal processes within a common basil lamina. A decrease in axon density indicates chronic swelling and potentially permanent loss of axons. This can be more readily observed from immunohistochemistry (IHC), specifically myelin basic protein (MBP) and neurofilament (NF). Nerve vascularization will also be quantified with antibodies against CD31 (also known as platelet endothelial cell adhesion molecule 1, PECAM-1).

[0132] Immunohistochemistry is better suited to studying a large area of the nerve crosssection, whereas electron microscopy is best suited for studying the ultrastructure of the myelin in smaller fields of view. Thus, half the nerve samples will be processed for EM, while the other30QB1125141.049611100748731.1MGH 2025-208-02Quarles 125141.04961 half will be processed for histology and THC. For power calculations, see the vertebrate animal document.

[0133] Surgical approach. We will perform four graded acute compression injuries to the sciatic nerve using the OCT device to deliver the compression. Animals will be recovered for a period of four weeks.

[0134] Electrophysiology: We will perform clinically relevant (e.g., conventional) electrophysiology to evaluate nerve injury. We will perform repeat measures electrophysiology 30 minutes pre and 30 minutes post- injury as well as at the terminal end point. Analysis of variance (ANOVA) will be used to compare outcomes between groups. Repeated measures ANOVA will be performed to assess outcomes within groups. Pre-injury (baseline) values serve as the control.

[0135] Behavioral Analysis. We will perform functional sensory and motor assessments pre- and post- injury to assess functional outcomes, as done in our prior work. Analysis of variance (ANOVA) will be used to compare outcomes between groups. Repeated measures ANOVA will be performed to assess outcomes within groups. Pre-injury (baseline) values serve as the control. Functional assessments will take place one week prior to surgery to establish baseline values with subsequent assessments at two and four weeks post-operatively as detailed below.

[0136] Motor function. Quantitative evaluation of equilibrium, balance and coordination will be assessed by a qualified researcher using our rotor-rod (Med Associates, Fairfax, VT) and validated criteria previously published by our group.

[0137] Sensory Function. Quantitative assessment of mechanical nociception, assessing for tactile hyperalgesia or allodynia will be collected using our automated dynamic plantar esthesiometer apparatus (Ugo Basile, Varese, Italy) as previously reported and according to the manufacture’s instructions.

[0138] Histomorphology. At the experimental endpoint the injured sciatic nerve and contralateral control will then be excised for histomorphology, immunohistochemistry (IHC) and electron microscopy (EM), as done in our prior work. We will use clinically relevant (e g., conventional) methods commonly used in our lab to evaluate nerve injury. Data will be analyzed31QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 by a blinded researcher or automated software and crosschecked by a qualified researcher.Methodology and parameters for evaluating nerve crush injury are detailed below. ANOVA will be used to compare between groups.

[0139] Histomorphology. We will evaluate nerve morphology with common histological stains hematoxylin and eosin (H&E) and Gomori’s Trichrome (collagen). These stains allow easy segmentation of key structural components; epineurium, outer perineurium, and inner perineurium boundaries, using the Segment Anything Model plugin to napari. We will quantify the effective circular diameters of the nerve and fascicles, fascicle count, and perineurium thickness. Qualitative assessment of scar and / or neuroma formation will be performed.

[0140] Neuroma formation is qualitatively evaluated based on the general loss of typical structures such as fascicles and the disorganization of axons, as previously reported by our group and others.

[0141] IHC. We will label myelin with MBP, axons with NF and blood vessels with CD31, allowing formal quantification of critical peripheral nerve structures. We will utilize the same segmentation tools listed above to calculate the number, size and area / volume of myelin, axons and blood vessels in each experimental group.

[0142] EM: We will utilize scanning EM of toluidine blue stained ultrathin nerve sections to qualitatively and quantitatively assess the number of axons and degree of myelination. The presence of degenerative myelin whorls will be quantified by a blinded researcher and compared to healthy control tissue.

[0143] Validate OCT injury markers in moderate (axonotmesis), severe (neurotmesis) and extreme (repair under tension) nerve injury models

[0144] For OCT to be clinically viable, we must compare its capacity to evaluate nerve health with standardized clinical (functional and electrophysiological) and experimental (histological) outcome measures. The rat sciatic nerve serves as the ideal model, as it has been used extensively used to evaluate axon injury and recovery.

[0145] The rat sciatic nerve ranges 1 -2 mm in diameter, which is comparable to the human recurrent laryngeal nerve that averages 1.75 mm diameter. In humans, the left recurrent32QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 laryngeal nerve is a small nerve, prone to compression or damage from pathologies and surgery within the vicinity of the nerve, resulting in vocal cord palsy.

[0146] Methods. Three distinct sciatic nerve injury models will be employed in 30 randomly assigned rats (equal numbers of males and females) in order to develop and validate OCT injury markers with preoperative and contralateral nerves serving as the controls as specifically stated below.

[0147] Surgical approach. We will perform three distinct surgeries commonly practiced in our laboratory to provide validated injuries of increasing severity, demonstrated by our group and others to yield greater sensory motor deficit relative to severity. Animals will be recovered for a period of 4 weeks.

[0148] Electrophysiology. We will perform clinically relevant (e.g., conventional) electrophysiology to evaluate nerve injury.

[0149] Behavioral Analysis. We will perform functional sensory and motor assessments pre- and post-injury to assess functional outcomes.

[0150] OCT analysis. Imaging will be performed across multiple nerve locations within each animal and using safe pressure. OCT injury markers (FA, FM, NP, and NF) will calculated for each imaged location. At experimental endpoints, imaged locations will be marked by ink to aid in registration of OCT to histomorphology.

[0151] Histomorphology. At the experimental endpoint the injured sciatic nerve and contralateral control will then be excised for histomorphology, IHC and EM, as done in our prior work. We will use a mix of clinically relevant (e.g., conventional) two-dimensional methods, as well as innovative three- dimensional methods pioneered by our lab. The data will be quantified by a blinded researcher or automated software and crosschecked by a qualified researcher.

[0152] Fascicular Myelination (FM). We will take a multipronged approach to evaluating myelination, including histomorphology with hematoxylin and eosin (H&E), IHC with antibodies against MBP to label the myelin and NF to label the axons and toluidine blue staining for EM. We will segment the nerve morphology — epineurium, outer perineurium, and inner perineurium boundaries — from H&E images using the Segment Anything Model plugin to napari. We will quantify the effective circular diameters of the nerve and fascicles, fascicle count,33QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 and perineurium thickness. We will segment the myelinated fibers using CellPose to obtain their diameters. G-ratio and myelination index will be calculated. These data will be directly compared to FM values derived using OCT.

[0153] Fascicular Area (FA). Fascicular area will be quantified using H&E and compared against OCT measures.

[0154] Nerve Perfusion (NP). Nerve vasculature will be assessed via blood vessel (CD31+) counts as well as area measurements (two-dimensional sections) and volume (three-dimensional reconstructions) using the same software described in the previous section. These data will be directly compared to NP values derived using OCT.

[0155] Nerve Fibrosis (NF). Nerve fibrosis will be calculated using the segmented nerve morphology method described above, from H&E images using the Segment Anything Model plugin to napari. These data will be directly compared to NF values derived using OCT.

[0156] Correlation analysis: The correlation between histomorphology and OCT measures of FM, FA, NP, and NF will be analyzed. For each measure, a linear or low-order regression model will be calculated between OCT and histomorophology. A residual analysis will be performed to estimate the measurement error for each marker. We will investigate whether measurement error is consistent across varying injury models or if, instead, there is an injury-model dependence on OCT outputs. For FM, this error will be compared against the target for clinical utility of + / -12%.

[0157] Example Method

[0158] FIG. 18 shows an example process 1800 to image a structure. At step 1802, an intraoperative imaging probe may be used to image a structure. The imaging probe may include a housing, a first arm extending from the housing, including a first proximal end and a first distal end, and a first optical assembly disposed within the first arm. The imaging probe may further include a second arm extending from the housing, including a second proximal end and a second distal end, and a second optical assembly disposed within the second arm. The imaging probe may further include a beam scanning mechanism configured to scan imaging beams from the first and second optical assemblies. The imaging probe may further include an adjustment mechanism configured to adjust a distance between the first distal end and the second distal end.34QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0159] At step 1804, the intraoperative imaging probe may be positioned such that a distance between the first and second arm is transverse to a structure. “Transverse” may be defined with respect to a longitudinal axis of the structure.

[0160] At step 1806, images of the structure may be collected using the first optical assembly and second optical assembly.Additional Embodiments

[0161] FIG. 19 shows a further embodiment showing an alternative construction of the calibrating structures 1900 described in FIG 2. In FIG 19, a calibrating structure associated with one arm is shown, but it should be understood that this calibrating structure and its use can be applied to both arms of the probe. An axial structure to be imaged 1901, such as a nerve or tendon, is located between the probe arms, with one arm 1950 shown. The arm contains a portion 1902 that is transparent to imaging light, and through which PS-OCT imaging is performed. That window 1902 is configured to include a first calibrating structure 1903a and a second calibrating structure 1903b that are oriented along the y-axis and do not cover the full x-axis extent of the window 1902, leaving a portion 1903c that is free of calibrating structures. The beam is configured to be scanned along the X and Y axis during beam scanning such that the beam path 1904 intersects with both calibrating structures 1903a and 1903b. A reflector or prism 1905 directs the beam that is scanned along the z axis through the window 1902. The calibrating structures 1903a and 1903b can be configured to be constructed from a birefringent and optically scattering material 1910 shown in the XZ plane such that PS-OCT signals are returned throughout the XZ extent of the sample and from which a measured optic axis orientation of the structure 1910 can be calculated. Alternatively, the calibrating structures 1903a and 1903b can be configured to be constructed from a first scattering layer 1911c, a non-scattering birefringent layer such as a waveplate 1911b, and a second scattering layer 1911a. PS-OCT signals from 1911a and 1911c can be measured and used to calculate the optic axis of the waveplate 1911b. A transformation of the PS-OCT signals can be calculated that rotate the measured optic axis of 1911b or 1910 to its known orientation in the XYZ axes defined by the imaging probe. In one embodiment, the optic axis orientation of calibrating structure 1903a is different from that of 1903b, such that the transformation that is calculated is unique. In one embodiment, the optic axes of 1903a and 1903c are oriented in the YZ plane but rotated by one of 45 degrees or 9035QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 degrees relative to each other. The transformation matrix can be calculated from a set of PS-OCT data obtained from signals from structures 1903a and 1903c, and because the OCT beam repeatedly samples these regions, that transformation can be updated over time as the beam is scanned in the Y axis to account for variations in the system or movement of an optical fiber that connects the imaging probe to the OCT console

[0162] The calibrating structures 1903 a and 1903 c can further include scattering variations 1913 a- 1913g that function as tick marks to act as a rule to determine the beam location in the Y direction. The calibrating structures 1903a and 1903c also can be used to measure the beam position in the Z direction through the presence or absence of these structures within the OCT or PS-OCT signals associated with a scan across the Z axis. The PS-OCT or OCT image 1920 associated with one scan from a minimal Z-axis extent to a maximal Z-axis extent in shown in the XZ plane. The sample to be imaged 1920a is located in the image between the signals 1920c and 1920b associated with the calibrating structures 1903b and 1903a.

[0163] FIG. 20 presents the optical beam path and optical design 2000 associated with the imaging probe shown in FIG. lAand FIG. IB. Two optical fibers, each associated with one of the two arms, is connected to the optical collimators 2001a and 2001b. The collimated beams are launched to a set of beam-scanners such as MEMS mirrors 2002a and 2002b. These beam scanners are configured to scan the beam out of plane to the image shown in FIG. 20 (the Z-axis of the probe). The beams are reflected by fixed reflectors 2003a and 2003b into each arm. The beam is transmitted via an optical telescope (2004a, 2004b and 2005a, 2005b) to a final focusing lens 2006a, 2006b and then to a reflector or prism 2007a and 2007b that direct the light in the X direction toward the sample.

[0164] FIG. 21 presents a design of the distal end 2100 of the optical arm 2150 including an optical window for OCT or PS-OCT imaging 2102 with a sample structure 2101 and including an array of pressure or force transducers 2110 adjacent to the window and used to measure the pressure or force applied to the sample as a function of the Y location. These force or pressure measurements can be provided to control the force applied to the closing mechanism to maintain the pressure or force applied to the sample below a given threshold such as required for example for safety. Force or pressure sensors can be based on piezoelectric, capacitive, MEMS, strain gauge, magnetostrictive and magnetic sensors, and force-sensing resistors.36QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961

[0165] A number of references to patent and non-patent documents are made throughout the publication, each of which is herein incorporated by reference in its entirety.

[0166] Thus, while the invention has been described above in connection with particular embodiments and examples, the invention is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses are intended to be encompassed by the claims attached hereto.37QB\125141.04961\100748731.1

Claims

MGH 2025-208-02Quarles 125141.04961 CLAIMSWhat is claimed is:

1. An intraoperative imaging probe, comprising:a housing;a first arm extending from the housing comprising a first proximal end and a first distal end, wherein a first optical assembly is disposed within the first arm;a second arm extending from the housing comprising a second proximal end and a second distal end, wherein a second optical assembly is disposed within the second arm,wherein the first optical assembly and the second optical assembly are configured to perform dual-sided optical coherence tomography (OCT) imaging and collect images of a sample located between the first distal end and the second distal end;a beam scanning mechanism configured to scan imaging beams from the first and second optical assemblies; andan adjustment mechanism configured to adjust a distance between the first arm and the second arm,wherein the distance between the first arm and the second arm is determined based on a distance between the first distal end and the second distal end.

2. The intraoperative imaging probe of claim 1, wherein the intraoperative imaging probe further comprises a first calibration element disposed in the first arm and a second calibration element disposed in the second arm.

3. The intraoperative imaging probe of claim 2, wherein the first calibration element and the second calibration element comprise at least one of glass or silicon.

4. The intraoperative imaging probe of claim 2, wherein the first calibration element and the second calibration element comprise periodic markers.

5. The intraoperative imaging probe of claim 2, wherein the first calibration element and the second calibration element comprise birefringent structures.38QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 6. The intraoperative imaging probe of claim 1, wherein the beam scanning mechanism of each arm is configured to scan in at least two axes relative to the axes of the proximal end to distal end of the respective arm.

7. The intraoperative imaging probe of claim 1, wherein the beam scanning mechanism scans using a physical rotation of a mirror.

8. The intraoperative imaging probe of claim 1, wherein the beam scanning mechanism scans using translation of the tip of an optical fiber or waveguide.

9. The intraoperative imaging probe of claim 1, further comprising an optical switch configured to rapidly switch an OCT signal between the first arm and the second arm such that imaging from the first and second optical assemblies is effectively simultaneous.

10. The intraoperative imaging device of claim 1, wherein the first optical assembly and the second optical assembly collect images using delay encoding.

11. The intraoperative imaging probe of claim 1, wherein the first and second optical assemblies are configured to acquire polarization-sensitive OCT data.

12. The intraoperative imaging probe of claim 1, wherein the first and second optical assemblies are configured to acquire angiographic OCT data.

13. The intraoperative imaging probe of claim 1, wherein the beam scanning mechanism is configured to generate three-dimensional volumes of approximately 2 mm in a first axis and up to 10 mm in a second axis.

14. The intraoperative imaging probe of claim 1, wherein the first and second optical assemblies each comprise transverse resolutions less than 25 pm across all scan angles.

15. The intraoperative imaging probe of claim 1, wherein the adjustment mechanism comprises a pullback actuator, force adjustor, and force transducer.

16. The intraoperative imaging probe of claim 1, wherein the intraoperative imaging probe is handheld.

17. The intraoperative imaging probe of claim 1, wherein the intraoperative imaging probe is connected to an OCT console.39QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 18. The intraoperative imaging probe of claim 1 , wherein images obtained from the first optical assembly and the second optical assembly are registered and merged to create a single image characterizing full-thickness properties of the structure.

19. The intraoperative imaging probe of claim 1, wherein the intraoperative imaging probe is configured for peripheral nerve assessment.

20. The intraoperative imaging probe of claim 19, wherein the intraoperative imaging probe is configured for at least one of imaging fascicular architecture, myelination, perfusion, or scarring.

21. A handheld optical coherence tomography (OCT) imaging device, comprisinga housing;a first arm extending from the housing comprising a first proximal end and a first distal end, wherein a first optical assembly is disposed within the first arm,a second arm extending from the housing comprising a second proximal end and a second distal end, wherein a second optical assembly is disposed within the second arm,wherein the first arm and second arm are configured to be articulated such that the first arm and second arm can be used to apply pressure to the structure;a beam scanning mechanism configured to scan imaging beams from the first and second optical assemblies; andan adjustment mechanism configured to adjust a pressure applied to the structure, wherein adjusting the pressure comprises adjusting a distance between the first distal end and the second distal end.

22. The handheld OCT imaging probe of claim 21, wherein the imaging beams are scanned in a y-direction sufficient to image a full -length of the structure.

23. The handheld OCT imaging probe of claim 21, wherein the imaging beam is scanned in the x dimension.

24. The handheld OCT imaging probe of claim 21, wherein the beam scanning mechanism scans uses a physical rotation of a mirror.40QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 25. The handheld OCT imaging probe of claim 21, wherein the beam scanning mechanism scans uses translation of the optical fiber tip.

26. The handheld OCT imaging probe of claim 21, wherein using the handheld OCT imaging probe is configured such that images obtained from the first optical assembly and the second optical assembly are registered and merged to create a single image characterizing full-thickness properties of the structure.

27. The handheld OCT imaging probe of claim 21, wherein the first arm and the second arm are configured to be controlled with a force such that a specific pressure is applied to the nerve.

28. The handheld OCT imaging probe of claim 21, wherein the first arm and the second arm each contain calibrating structures that are used for at least one of alignment of PS-OCT imaging data, or calibration of the y location of the imaging beam during beam scanning.

29. The handheld OCT imaging probe of claim 21, wherein the handheld OCT imaging is configured to interrogate the first arm and second arm via an optical switch.

30. The handheld OCT imaging probe of claim 21, wherein the handheld OCT imaging probe is configured to interrogate the first arm and the second arm via delay encoding.

31. The handheld OCT imaging probe of claim 21, wherein a multi-channel receiver is used to capture OCT data from the first arm and the second arm.

32. The handheld OCT imaging probe of claim 21, wherein the handheld OCT imaging probe is connected to an OCT console.

33. The handheld OCT imaging probe of claim 21, wherein the handheld OCT imaging probe is configured to provide measurement of a structure.

34. The handheld OCT imaging probe of claim 33, wherein the structure is a nerve, and the measurements comprise at least one of fascicular architecture, myelination, perfusion, or scarring.

35. The handheld OCT imaging probe of claim 21, wherein the handheld OCT imaging probe is configured to measure a duration of an applied pressure, or a metric derived from the duration, and generate signals to the user based on sound or display indicators.41QB1125141.049611100748731.1MGH 2025-208-02Quarles 125141.04961 36. The intraoperative imaging probe of claim 1, wherein the intraoperative imaging probe is handheld.

37. A method of imaging a sample using an intraoperative imaging probe, the method comprising:providing the intraoperative imaging probe comprising a housing, a first arm extending from the housing comprising a first proximal end and a first distal end, wherein a first optical assembly is disposed within the first arm, and a second arm extending from the housing comprising a second proximal end and a second distal end, wherein a second optical assembly is disposed within the second arm;positioning the sample between the first distal end and the second distal end; performing dual-sided optical coherence tomography (OCT) imaging using the first optical assembly and the second optical assembly to collect images of the sample;scanning imaging beams from the first and second optical assemblies using a beam scanning mechanism; andadjusting a distance between the first arm and the second arm using an adjustment mechanism,wherein the distance between the first arm and the second arm is determined based on a distance between the first distal end and the second distal end.

38. The method of claim 37, further comprising calibrating the imaging using a first calibration element disposed in the first arm and a second calibration element disposed in the second arm.

39. The method of claim 38, wherein the first calibration element and the second calibration element comprise at least one of glass or silicon.

40. The method of claim 38, wherein the first calibration element and the second calibration element comprise periodic markers.

41. The method of claim 38, wherein the first calibration element and the second calibration element comprise birefringent structures.42QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 42. The method of claim 37, wherein scanning imaging beams comprises scanning in at least two axes relative to the axes of the proximal end to distal end of the respective arm.

43. The method of claim 37, wherein scanning imaging beams comprises physically rotating a mirror.

44. The method of claim 37, wherein scanning imaging beams comprises translating the tip of an optical fiber or waveguide.

45. The method of claim 37, further comprising rapidly switching an OCT signal between the first arm and the second arm using an optical switch such that imaging from the first and second optical assemblies is effectively simultaneous.

46. The method of claim 37, wherein collecting images comprises using delay encoding.

47. The method of claim 37, wherein performing dual-sided OCT imaging comprises acquiring polarization-sensitive OCT data.

48. The method of claim 37, wherein performing dual-sided OCT imaging comprises acquiring angiographic OCT data.

49. The method of claim 37, wherein scanning imaging beams comprises generating three-dimensional volumes of approximately 2 mm in a first axis and up to 10 mm in a second axis.

50. The method of claim 37, wherein collecting images comprises achieving transverse resolutions less than 25 pm across all scan angles.

51. The method of claim 37, wherein adjusting the distance comprises using a pullback actuator, force adjustor, and force transducer.

52. The method of claim 37, wherein the intraoperative imaging probe is handheld.

53. The method of claim 37, further comprising connecting the intraoperative imaging probe to an OCT console.

54. The method of claim 37, further comprising registering and merging images obtained from the first optical assembly and the second optical assembly to create a single image characterizing full-thickness properties of the sample.

55. The method of claim 37, wherein the sample is a peripheral nerve.43QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 56. The method of claim 55, further comprising imaging at least one of fascicular architecture, myelination, perfusion, or scarring of the peripheral nerve.

57. A method of imaging a structure using a handheld optical coherence tomography (OCT) imaging device, the method comprising:providing the handheld OCT imaging device comprising a housing, a first arm extending from the housing comprising a first proximal end and a first distal end, wherein a first optical assembly is disposed within the first arm, and a second arm extending from the housing comprising a second proximal end and a second distal end, wherein a second optical assembly is disposed within the second arm;articulating the first arm and second arm to apply pressure to the structure;scanning imaging beams from the first and second optical assemblies using a beam scanning mechanism; andadjusting a pressure applied to the structure using an adjustment mechanism, wherein adjusting the pressure comprises adjusting a distance between the first distal end and the second distal end.

58. The method of claim 57, wherein scanning imaging beams comprises scanning in a y-direction sufficient to image a full-length of the structure.

59. The method of claim 57, wherein scanning imaging beams comprises scanning in the x dimension.

60. The method of claim 57, wherein scanning imaging beams comprises physically rotating a mirror.

61. The method of claim 57, wherein scanning imaging beams comprises translating an optical fiber tip.

62. The method of claim 57, further comprising registering and merging images obtained from the first optical assembly and the second optical assembly to create a single image characterizing full-thickness properties of the structure.

63. The method of claim 57, further comprising controlling the first arm and the second arm with a force such that a specific pressure is applied to the structure.44QB\125141.04961\100748731.1MGH 2025-208-02Quarles 125141.04961 64. The method of claim 57, further comprising using calibrating structures in the first arm and the second arm for at least one of alignment of PS-OCT imaging data, or calibration of the y location of the imaging beam during beam scanning.

65. The method of claim 57, further comprising interrogating the first arm and second arm via an optical switch.

66. The method of claim 57, further comprising interrogating the first arm and the second arm via delay encoding.

67. The method of claim 57, further comprising capturing OCT data from the first arm and the second arm using a multi-channel receiver.

68. The method of claim 57, further comprising connecting the handheld OCT imaging device to an OCT console.

69. The method of claim 57, further comprising measuring the structure.

70. The method of claim 69, wherein the structure is a nerve, and measuring comprises determining at least one of fascicular architecture, myelination, perfusion, or scarring.

71. The method of claim 57, further comprising measuring a duration of an applied pressure, or a metric derived from the duration, and generating signals to a user based on sound or display indicators.

72. The method of claim 37, wherein the intraoperative imaging probe is handheld.45QB\125141.04961\100748731.1

Images