CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application No. 63/376,210, filed September 19, 2022, entitled “Systems, Methods, and Devices for All-Optic Biomechanical Imaging with Motion Tracking,” which is hereby incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01EY028666 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

FIELD

The present disclosure relates generally to biomechanical imaging of eyes, and more particularly, to detection of one or more conditions of an eye using Brillouin spectroscopy, for example, subclinical keratoconus.

BACKGROUND

Providing approximately 70% refractive power of the human eye, the cornea serves the primary role to clearly focus light on the retina. As corneal refractive power derives from its delicate aspherical profile, to maintain corneal shape under ocular forces, such as in-plane tension and intraocular pressure (IOP), the cornea has evolved to an intricate lattice of hundreds of lamellas intertwined by collagen fibers and proteoglycans to provide sufficient mechanical strength. Deterioration of corneal biomechanics is the root cause of corneal ectatic disorders, such as keratoconus (KC) and postoperative ectasia after laser vision correction procedures (e.g., laser in situ keratomileusis (LASIK)). In these conditions, the mechanical balance between intraocular forces and corneal resistance is disrupted, resulting in corneal thinning and warpage that causes dramatic alterations in corneal refractive power.

Corneal biomechanical properties could function as a primary clinical data to evaluate reduced corneal elastic modulus (i.e., the measure of tissue’s resistance to deformation under an applied force), and thus help identify patients at risk of developing ectasia before morphologic parameters, such as thickness and curvature, are altered. However, in vivo biomechanical measurements have proven difficult to obtain and have demonstrated significant limitations in early detection of decreased corneal modulus. Moreover, while clinically manifest keratoconus has highly recognizable morphological features identifiable using a variety of available technologies, differentiation of subclinical keratoconus from normal corneas has proven challenging. Multiple strategies have been investigated, from single device metrics to combined device protocols using artificial intelligence (Al). The majority of these strategies, however, have primarily utilized morphologic features rather than biomechanical parameters.

Clinically available corneal biomechanical measurement devices include the Ocular Response Analyzer (ORA) and the Corneal Visualization Scheimpflug Technology (Corvis-ST). In multiple studies, the ORA has shown limited utility in discriminating early or subclinical keratoconus from normal populations. While initial reports investigating the ability of Corvis- ST for the identification of keratoconus and subclinical keratoconus were encouraging, the technology has ultimately not performed well without additional morphologic metrics. Even when combining Scheimpflug morphologic and Corvis biomechanical metrics, this strategy has proven limited in differentiating the subtlest forms of subclinical disease.

Unlike the above noted technologies, Brillouin microscopy can probe the longitudinal modulus locally, with three-dimensional, micron-scale resolution, and without tissue deformation. However, commercially-available Brillouin ophthalmic instruments have been limited in both spatial and mechanical accuracy. Spatially, since 10-20 seconds are required per axial scan at a single point on the cornea, the measurement is prone to significant artifacts due to patient’s eye movement. Mechanically, the measurement by commercially-available Brillouin ophthalmic instructions relies on calibration using materials of known Brillouin properties, which can be impacted by environmental conditions.

Embodiments of the disclosed subject matter may address one or more of the abovenoted problems and disadvantages, among other things.

SUMMARY

Embodiments of the disclosed subject matter provide systems and methods employing Brillouin spectroscopy measurements to determine that a patient’s eye exhibits subclinical keratoconus, which may otherwise be difficult or impossible to detect with existing ophthalmic instruments. In some embodiments, the Brillouin spectroscopy measurements can yield a Brillouin frequency shift, or other Brillouin reading, that provides a quantitative estimate of the longitudinal modulus at the measurement point in the patient’s eye. In some embodiments, these Brillouin readings can be used to calculate one or metrics indicative of the likelihood of subclinical keratoconus. In some embodiments, the Brillouin readings can be compiled into a two-dimensional or three-dimensional map, for example, for display to a clinician or physician.

In some embodiments, the system can exhibit enhanced mechanical and spatial accuracy over commercially-available Brillouin imaging systems. In some embodiments, a Brillouin spectroscopy modality of the system can be calibrated to an atomic absorption line, for example, via an elemental gas cell, such as a rubidium (Rb) vapor cell. In some embodiments, the system can employ three-dimensional tracking of the patient’s eye, the location of interrogation by a Brillouin modality, or both. The tracking can be performed by one or more optical imaging modalities. For example, an interferometric or ranging system (e.g., an optical coherence tomography system) can track depth of a Brillouin measurement point in the patient’s eye. Alternatively or additionally, an enface imaging system (e.g., a single lens imaging system) can track lateral location of the Brillouin measurement point with respect to the patient’s eye and/or movement of the patient’s eye.

In one or more embodiments, a method for detection of subclinical keratoconus can comprise, for each of a plurality of lateral measurement points with respect to a patient’ s eye, obtaining a plurality of Brillouin spectroscopy measurements at different depths along an axial direction for the respective lateral measurement point. The method can further comprise, for each lateral measurement point, determining a Brillouin reading using some or all of the plurality of Brillouin spectroscopy measurements for the respective lateral measurement point. The method can also comprise calculating one or more metrics based at least in part on the Brillouin readings for the determined plurality of lateral measurement points. The method can further comprise determining that the patient’s eye exhibits subclinical keratoconus based at least in part on the calculated one or more metrics.

In one or more embodiments, a method for detection of subclinical keratoconus can comprise obtaining a plurality of Brillouin spectroscopy measurements for different measurement points with respect to a patient’s eye. The method can also comprise using one or more optical modalities to track locations of the respective measurement points and/or the patient’s eye in three-dimensions during the obtaining. The method can further comprise assigning each Brillouin spectroscopy measurement to a particular lateral location and depth with respect to or within the patient’s eye based on the tracking. The method can also comprise determining a Brillouin reading for each lateral location using Brillouin spectroscopy measurements assigned to the respective lateral location but at different depths. The method can further comprise calculating one or more metrics based at least in part on the determined Brillouin readings, and determining that the patient’s eye exhibits subclinical keratoconus based at least in part on the one or more metrics.

In one or more embodiments, a system can comprise a Brillouin spectroscopy modality, one or more optical imaging modalities, and a controller. The Brillouin spectroscopy modality can be configured to obtain Brillouin spectroscopy measurements for different measurement points with respect to a patient’s eye. The one or more optical imaging modalities can be configured to track locations of the respective measurement points and/or the patient’s eye in three-dimensions. The controller can comprise one or more processors and computer-readable storage media storing instructions that, when executed by the one or more processors, cause the controller to determine that the patient’s eye exhibits subclinical keratoconus based at least in part on the obtained Brillouin spectroscopy measurements.

In one or more embodiments, a system can comprise one or more processors and computer-readable storage media. The computer-readable storage media can store instructions that, when executed by the one or more processors, cause the system to determine that a patient’s eye exhibits subclinical keratoconus based on Brillouin spectroscopy measurements of the patient’s eye.

Any of the various innovations of this disclosure can be used in combination or separately. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will hereinafter be described with reference to the accompanying drawings, which have not necessarily been drawn to scale. Where applicable, some elements may be simplified or otherwise not illustrated in order to assist in the illustration and description of underlying features. Throughout the figures, like reference numerals denote like elements.

FIG. 1A is a simplified schematic diagram of a system for detection of subclinical keratoconus in a patient’s eye, according to one or more embodiments of the disclosed subject matter.

FIGS. 1B-1C illustrate detection aspects in front and cross-sectional views of a patient’s eye, according to one or more embodiments of the disclosed subject matter.

FIG. ID depicts a generalized example of a computing environment in which the disclosed technologies may be implemented.

FIG. 2A illustrates an arrangement of optical components for a Brillouin spectroscopy modality, according to one or more embodiments of the disclosed subject matter.

FIG. 2B illustrates aspects of an eye examination system employing a Brillouin spectroscopy modality, according to one or more embodiments of the disclosed subject matter. FIG. 2C illustrates a temperature-controlled gas cell for use in a Brillouin spectroscopy modality, according to one or more embodiments of the disclosed subject matter.

FIG. 2D is a graph reflecting a calibration approach for correlating pixel position to Brillouin shift, according to one or more embodiments of the disclosed subject matter.

FIG. 2E illustrates an adjustable arrangement of optical components for a Brillouin spectroscopy, according to one or more embodiments of the disclosed subject matter.

FIG. 2F illustrates angle matching between a cylindrical lens and an entrance slot of a virtually imaged phase array etalon of a Brillouin spectroscopy modality, according to one or more embodiments of the disclosed subject matter.

FIG. 2G illustrates aspects of collimator selection for beam adjustment to increase signal intensity in a Brillouin spectroscopy modality, according to one or more embodiments of the disclosed subject matter.

FIG. 3 is a simplified schematic diagram of a subclinical keratoconus detection system, according to one or more embodiments of the disclosed subject matter.

FIG. 4A is a process flow diagram of a method for detection of subclinical keratoconus by creating a two-dimensional map from Brillouin spectroscopy measurements, according to one or more embodiments of the disclosed subject matter.

FIG. 4B is a process flow diagram of another method for detection of subclinical keratoconus by creating a two-dimensional map from Brillouin spectroscopy measurements, according to one or more embodiments of the disclosed subject matter.

FIG. 4C is a process flow diagram of a method for detection of subclinical keratoconus by creating a three-dimensional map from Brillouin spectroscopy measurements, according to one or more embodiments of the disclosed subject matter.

FIG. 4D shows aspects of a Brillouin reading determination for a Brillouin depth profile, according to one or more embodiments of the disclosed subject matter.

FIG. 5A is a graph of Brillouin shift of water versus time for a continuous acquisition over 10 seconds and with an exposure time of 0.05 seconds.

FIG. 5B is a graph of Brillouin shift of water versus time for acquisition over 2000 seconds and with an exposure time of 0.05 seconds and an acquisition interval of 1 second.

FIG. 5C is a graph illustrating distance determination by measuring the position of the reflection peak from the anterior corneal surface.

FIG. 5D is a graph illustrating measurement errors and standard deviations of measured axial displacements compared to known axial displacements of a translation stage. FIG. 5E is a graph illustrating measurement errors and standard deviations of measured lateral displacements compared to known lateral displacements of a translation stage.

FIGS. 6A-6B are graphs of lateral eye movement versus time when a patient breathes regularly and when the patient holds their breath, respectively.

FIGS. 6C-6D are graphs of axial displacement of the cornea versus time when a patient breathes regularly and when the patient holds their breath, respectively.

FIGS. 6E-6F are graphs illustrating correction of Brillouin profile measurements by pairing with coordinates measured by optical coherence tomography when a patient breathes regularly and when the patient holds their breath, respectively.

FIG. 7A illustrates an exemplary two-dimensional Brillouin map of a normal cornea obtained using the disclosed detection system.

FIG. 7B illustrates representative Brillouin profiles at the center and periphery of the normal cornea obtained using the disclosed detection system.

FIGS. 8A-8B illustrates a two-dimensional Brillouin map and corresponding OCT cross- sectional image.

FIGS. 9A-9D illustrate raw, binarization, edge, and Brillouin on OCT profiles in an exemplary process for relating Brillouin readings to OCT depth measurements.

FIGS. 10A-10B are graphs of a polynomial 3 orders fit and Fourier 3 orders fit, respectively, to a depth profile of measured Brillouin shifts in an eye.

FIG. IOC is a graph of Brillouin shift versus detector pixel number for reconstructing an anterior layer of the measured cornea.

FIGS. 11A-11C illustrate aspects of reconstructing another layer of the measured cornea using an erosion approach.

FIGS. 12A-12D are box plots comparing control eyes and subclinical keratoconus eyes for Brillouin readings of mean Brillouin shift for a plateau region, a mean Brillouin shift for a 150-pm anterior region, a minimum Brillouin shift for the plateau region, and a minimum Brillouin shift for the 150-pm anterior region, respectively.

FIG. 13 is a graph of receiver operating characteristic (ROC) curves for control eyes versus subclinical keratoconus eyes for the disclosed Brillouin spectroscopy system and conventional Scheimpflug metrics.

DETAILED DESCRIPTION

General Considerations

For purposes of this description, certain aspects, advantages, and novel features of the embodiments of this disclosure are described herein. The disclosed methods and systems should not be construed as being limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with one another. The methods and systems are not limited to any specific aspect or feature or combination thereof, nor do the disclosed embodiments require that any one or more specific advantages be present, or problems be solved. The technologies from any embodiment or example can be combined with the technologies described in any one or more of the other embodiments or examples. In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the disclosed technology.

Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods. Additionally, the description sometimes uses terms like “provide” or “achieve” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms may vary depending on the particular implementation and are readily discernible by one skilled in the art.

The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person skilled in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods, as known to those skilled in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about,” “substantially,” or “approximately” is recited. Whenever “substantially,” “approximately,” “about,” or similar language is explicitly used in combination with a specific value, variations up to and including 10% of that value are intended, unless explicitly stated otherwise. Directions and other relative references may be used to facilitate discussion of the drawings and principles herein but are not intended to be limiting. For example, certain terms may be used such as “inner,” “outer,” “upper,” “lower,” “top,” “bottom,” “interior,” “exterior,” “left,” right,” “front,” “back,” “rear,” and the like. Such terms are used, where applicable, to provide some clarity of description when dealing with relative relationships, particularly with respect to the illustrated embodiments. Such terms are not, however, intended to imply absolute relationships, positions, and/or orientations. For example, with respect to an object, an “upper” part can become a “lower” part simply by turning the object over. Nevertheless, it is still the same part, and the object remains the same.

As used herein, “comprising” means “including,” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise.

Although there are alternatives for various components, parameters, operating conditions, etc. set forth herein, that does not mean that those alternatives are necessarily equivalent and/or perform equally well. Nor does it mean that the alternatives are listed in a preferred order, unless stated otherwise. Unless stated otherwise, any of the groups defined below can be substituted or unsubstituted.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one skilled in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Features of the presently disclosed subject matter will be apparent from the following detailed description and the appended claims.

Introduction

Disclosed herein are systems and methods employing Brillouin spectroscopy measurements to evaluate a biomechanical properties of an eye of a patient (e.g., a human or animal) and/or to provide detection (e.g., early detection) of a progressive eye disease, for example, subclinical keratoconus. Spontaneous Brillouin scattering arises from the interaction between incident light and acoustic phonons generated by inherent density /pres sure fluctuation, which results in a frequency shift of the scattered light. The relation between the Brillouin shift, AVB, and the longitudinal modulus, M of the interrogated material can be expressed as: where z is the probing laser wavelength, n is the corresponding refractive index, V is the speed of sound, p is the density. Although refractive index and density vary spatially in cornea, the ratio pin ² remains approximately constant according to the Gladstone-Dale relation, which makes the measured Brillouin shift proportional to the longitudinal modulus. Longitudinal modulus determined by Brillouin spectroscopy is different from the Young’s modulus of the interrogated material. A universal conversion from the longitudinal modulus to the Young’s modulus has not been established because of frequency-dependent modulus properties and near incompressibility of biological tissues. However, for certain tissues such as the cornea, a strong correlation between the two moduli has been observed, and an increase (or decrease) of the longitudinal modulus as measured by Brillouin spectroscopy can be interpreted as an indication of increase (or decrease) in Young’s modulus. In some embodiments, the Brillouin shift can be directly measured via a Brillouin spectroscopy modality and can be used to determine a Brillouin reading for one or more measurement locations (e.g., within or with respect to the patient’s eye). Alternatively or additionally, in some embodiments, the Brillouin reading can be and/or determined from one or more measured Brillouin shifts, one or more measured Brillouin widths, one or more measured Brillouin signal strengths, or any combination of the foregoing.

In some embodiments, a system 100 for evaluating biomechanical properties of a sample 102 (e.g., a patient’s eye) can have a sampling interface 104, a Brillouin modality 106, and one or more optical imaging modalities 108, for example, as shown in FIG. 1A. In some embodiments, the sampling interface 104 can be constructed to interact with the sample 102, for example, by providing one or more features that align and/or maintain a position of the sample 102 with respect to a focal point of the system 100. For example, the sampling interface 104 can include a chin rest (e.g., that accepts a chin of a human patient during interrogation), a bite bar (e.g., that the human patient can bite during interrogation), a forehead rest (e.g., that abuts the forehead of the human patient during interrogation), or any other feature that aligns or positions the sample 102 or a portion thereof. In some embodiments, the sampling interface 104 can include one or more optical components (e.g., polarizers, beam splitters, lenses, mirrors, couplers, filters, etc.) that route light from the different modalities 106, 108 to the sample 102 and light from the sample 102 to the different modalities 106, 108.

In some embodiments, the one or more optical imaging modalities 108 can track the location of a measurement point (e.g., focal point) with respect to sample 102 and/or movement of the sample 102 (e.g., eye) in three-dimensions. In the illustrated example of FIG. 1A, the one or more optical imaging modalities 108 comprises a pair of modalities, in particular, a first modality 110 configured to measure depth (e.g., along axial direction 118) and a second modality 112 configured to measure lateral locations (e.g., in a plane perpendicular to axial direction 118, for example, a plane tangential to a curvature of the front of the cornea). In some embodiments, the first modality 110 comprises an interferometric or ranging system, such as but not limited to an optical coherence tomography (OCT) system. For example, the first modality 110 can be configured to measure the depth 120 along axial direction 118 with an accuracy of 20 pm or less (e.g., about 3 pm). In some embodiments, the second modality 112 comprises an optical imaging system, such as but not limited to a single lens imaging system. For example, the second modality can be configured to measure lateral locations with an accuracy of 50 pm or less (e.g., about 10 pm).

In some embodiments, the Brillouin modality 106 can illuminate the sample 102 with interrogating light via sampling interface 104 and can detect the Brillouin scattered light emanating from the sample 102. In some embodiments, the Brillouin modality 106 can have a detection arm similar to detection arm 200 of FIG. 2 A and/or a configuration similar to setup 230 of FIG. 2B, described in further detail below. Alternatively, in some embodiments, the Brillouin modality 106 can have a configuration (or aspects thereof) as described in “Noncontact three-dimensional mapping of intracellular hydro-mechanical properties by Brillouin microscopy,” Nature Methods, December 2015, 12(12): pp. 1132-34, which is hereby incorporated by reference herein. Alternatively or additionally, in some embodiments, the Brillouin modality 106 can have a configuration (or aspects thereof) as described in any of U.S. Patent No. 11,408,770, issued August 9, 2022 and entitled “Brillouin imaging devices, and systems and methods employing such devices,” U.S. Patent No. 11,143,555, issued November 12, 2021 and entitled “Methods and devices for reducing spectral noise and spectrometry systems employing such devices,” U.S. Patent No. 11,060,912, issued July 13, 2021 and entitled “Multi-stage parallel spectroscopy systems and methods,” U.S. Patent No. 10,989,591, issued April 27, 2021 and entitled “Methods and arrangements to enhance optical signals within aberrated or scattering samples,” and U.S. Publication No. 2022/0042908, published February 10, 2022 and entitled “Full-field Brillouin microscopy systems and methods,” each of which is incorporated herein by reference.

In the illustrated example of FIG. 1A, the system 100 includes a control system 114 operatively coupled to and configured to control operation of the Brillouin modality, the first modality 110, and the second modality 112, for example, to synchronize operation thereof and/or to combine together data from the separate modalities, as described in further detail hereinbelow. Alternatively or additionally, in some embodiments, control system 114 can control operation of the modalities 106-112 to acquire data, and a separate system (not shown) can process the data (e.g., to determine Brillouin readings, create two-dimensional or three- dimensional maps, calculating one or more metrics, diagnosing or otherwise characterizing the sample, etc.) received from control system 114 (e.g., independent of the data acquisition) or directly from the modalities 106-112 (e.g., without routing through control system 114). For example the separate system can be a separate controller, server, image processor, or data processing device that is local to or remote from system 100.

The system 100 can sequentially interrogate multiple points within and/or with respect to sample 102, for example, by moving the sample 102 and/or focal point. For example, the sample 102 can be mounted on or supported by a one-dimensional, two-dimensional, or three- dimensional translation stage for moving the sample 102 with respect to sampling interface 104. Alternatively or additionally, the sampling interface 104, or a portion or portions thereof, can be mounted on or supported by a one-dimensional, two-dimensional, or three-dimensional translation storage for moving the focal point defined by the sampling interface 104 with respect to the sample 102. Alternatively or additionally, the sampling interface 104 can include one or more adjustable optical components to move the focal point in one-dimension, two-dimensions, or three-dimensions, for example, movable mirrors and/or movable lenses that redirect a light path and/or change a location of the focal point for interrogating and detected light. In some embodiments, control system 114 can control movement of the sample 102 and/or focal point, for example, by sending signals to the respective translation stage. Alternatively or additionally, positioning of the focal point with respect to the sample 102 can be manually controlled, for example, based on operator input to a user interface 116 (e.g., via a joystick, via on-screen selection of a point on a displayed image of a sample, etc.).

For example, when the sample 102 is a patient’s eye 130 as shown in FIGS. 1B-1C, the focal point can be moved to one of a plurality of lateral locations 132 (e.g., at a respective x-y location with respect to a front of the cornea) as shown in FIG. IB, and to one of a plurality of depths 134 (e.g., at a respective z location with respect to the front of the cornea) as shown in FIG. 1C. After interrogating at the measurement location and acquiring the resulting Brillouin scattered light, the focal point can be moved (e.g., via movement of the eye 130 or by adjusting the sampling interface) to the next of the plurality of lateral locations 132 and/or depths 134. Although particular locations and depths are illustrated in FIGS. 1B-1C, these locations and depths are exemplary for purposes of illustration. Other locations and/or depths (predetermined or otherwise) are also possible according to one or more embodiments. Moreover, although the locations and depths are illustrated in a regular arrangement (e.g., aligned to the x-y-z ordinate system, equal step size, etc.) in FIGS. 1B-1C, other arrangements are also possible according to one or more embodiments.

In some embodiments, system 100 can create a map of biomechanical information (e.g., Brillouin readings) of the sample 102 based on the measurements obtained by Brillouin modality and the tracking information from first and second modalities 110, 112. In some embodiments, a two-dimensional map can be created based on a mean, median, or mode of measured Brillouin shifts, Brillouin widths, Brillouin signal strengths, or combinations thereof, for some or all of the different depths in a selected depth range (e.g., along axial direction 118). Alternatively or additionally, in some embodiments, a three-dimensional map can be created. Values for points between the measured locations in the two-dimensional or three-dimensional map can be provided via interpolation, median filtering, or any other data processing technique. In some embodiments, the created map can be displayed to an operator of system 100, for example, via user interface 116. Alternatively, in some embodiments, the map can be retained in the form of data, for example, without displaying a visual graphic. In some embodiments, control system 114 or a separate data processing system (not shown) can create the map.

In some embodiments, system 100 can calculate one or more metrics based on the Brillouin readings and can use the metric(s) to provide a diagnosis or determine a condition (e.g., presence or status of a disease) of the sample 102, for example, a determination that a patient’s eye exhibits subclinical keratoconus. In some embodiments, the metric(s) can be determined based on the created map, for example, a mean, median, or mode of values in the map, a minimum of values in the map, a spatial standard deviation of values in the map, and/or Zemike function coefficients from a Zemike fit to the map. In some embodiments, a determination that the patient’s eye exhibits subclinical keratoconus is made in response to the metric(s) being outside of a predetermined range, for example, a Brillouin shift being less than a predetermined value. In some embodiments, control system 114 or a separate data processing system (not shown) can create the one or more metric and/or make the diagnosis or determination based on the metric(s).

Brillouin Spectroscopy Systems

In some embodiments, the Brillouin modality can be a double-stage virtually-imaged phase array (VIPA) Brillouin light scattering spectrometer, a detection arm of which is illustrated in FIG. 2A. The detection arm 200 can include a first cylindrical lens 204, which receives scattered light 202 from the sample. A first VIPA etalon 206 can be disposed at the focal plane of the first cylindrical lens 204. An output from the first VIPA etalon 206 is focused by first spherical lens 208 onto a mask 210 with a horizontal slit, and a second spherical lens 212 focuses the output from mask 210 onto a second cylindrical lens 214. A second cylindrical lens 214, which has an orientation orthogonal to the first cylindrical lens 204, can be disposed between the second spherical lens 212 and a second VIPA etalon 216, which has an orientation orthogonal to the first VIPA etalon 206. In each stage of the detection arm 200, the VIPA etalon 206, 216 produces a spectrally dispersed pattern in the focal planes of the respective lenses 208, 218 placed just after the etalons. The spectrally dispersed pattern is the Fourier transform of the electromagnetic field at the output of each VIPA. In a double-stage VIPA spectrometer, the two spectral dispersion stages are cascaded orthogonally to each other and the planes of the respective spectrally dispersed patterns are conjugated. The image of the spectrally dispersed pattern is projected by spherical lens 218 onto a two-dimensional array of pixels of detector 220 (e.g., CCD camera).

FIG. 2B illustrates an exemplary setup 230 for a Brillouin light scattering spectrometer. A collimated light beam can be generated by a collimating lens 242 set in front of laser 244. In some embodiments, the laser 244 can be locked to an absorption line of atomic gas cell 234. The collimated light beam can be directed from collimating lens 242 through a polarizer 240 (e.g., a linear polarizer), thereby resulting a polarized light beam that is then reflected by polarization beam splitter 238 toward the sample. In the illustrated example, the reflected light from polarization beam splitter 238 can pass through a quarter- wave plate 246 to form the interrogating light beam. The interrogating light beam is Brillouin scattered by the sample, and the backward Brillouin scattered light is collected by the microscope optics (e.g., optics of the sampling interface) and passes through the same quarter-wave plate 246, such that the polarization of the collected light allows it to pass through the polarization beam splitter 238 en route to the Brillouin detection arm 200 via reflecting element 236 (e.g., mirror) and coupling lens 232 (e.g., fiber collimator).

In some embodiments, an atomic gas cell 234 can be disposed along the optical path between the polarization beam splitter 238 and the Brillouin detection arm 200. The gas cell 234 can have one or more absorption lines (e.g., wavelengths) to remove stray interrogating light (e.g., having a wavelength of the laser 244) prior to detection by Brillouin detection arm 200. In some embodiments, the gas cell 234 can exhibit a removal ratio of 10 ⁴ or less (e.g., an absorption ratio greater than 10’ ³ , for example, about 10“ ⁷ ) with respect to the laser wavelength. For example, the atomic gas cell 234 can be a rubidium vapor cell. In the illustrated example, the gas cell 234 is disposed between the reflecting element 236 and coupling lens 232, but other locations along the optical path between the polarization beam splitter 238 and detection arm 200 are also possible. Although FIGS. 2A-2B illustrate particular optical components and arrangements thereof for a Brillouin modality, fewer or different optical components and/or different arrangements of components are also possible according to one or more embodiments. Accordingly, embodiments of the disclosed subject matter are not limited to the details of FIGS. 2A-2B.

When using a gas cell to absorb the interrogating light from the laser 244, the removal ratio can be proportional to temperature. In some embodiments, removal of the undesirable interrogating via the gas cell can be increased by heating the gas cell to a temperature greater than room temperature, for example, about 90 °C. In some embodiments, the gas cell, or at least the vapor contained therein, can be at a substantially uniform temperature, for example, by appropriate configuration of a heating element (e.g. heating tape), insulation (e.g., foam box), or combinations thereof. The substantially uniform heating can avoid, or at least reduce, the size of the reflection signal of the interrogating light, and/or avoid, or at least reduce, precipitation of the vapor within the gas cell. For example, FIG. 2C illustrates an exemplary configuration 250 for uniformly heated gas cell. A glass housing 254 of the gas cell has windows 256, 258 on opposite ends thereof and an elemental vapor (e.g., rubidium) within an interior volume 252 between the windows 256, 258. The glass housing 254 can also have a sealed port 260, which was used to introduce the vapor into the interior volume 252. An insulating enclosure 262 (e.g., foam box) can be provided around the glass housing 254, with openings for each of the windows 256, 258 and sealed port 260. One or more heating elements 264 (e.g., heat tape) can be provided, for example, as part of insulating enclosure 262 or between insulating enclosure 262 and gas cell 254. The heating element(s) 264 can thus uniformly heat the vapor within volume 252, while the optical path for the Brillouin detection arm can pass through the openings in the enclosure 262, the windows 256, 258, and the vapor within volume 252.

In conventional Brillouin spectroscopy systems, two different materials (e.g., water and polystyrene) are used with known Brillouin shifts and linear fitting to estimate frequencies along a dispersion axis. After obtaining a ratio of GHz/pixel, the Brillouin shift of an unknown material can be calculated by counting pixels between its Stokes and Anti-Stokes peaks. The accuracy of this calibration depends heavily on the accuracy of the known Brillouin shifts. However, the Brillouin shifts of the standard materials can vary with time due to changes in temperature (e.g., room or ambient temperature variations). For example, the Brillouin shift of water changes 7.4 MHz/°C, while the Brillouin shift of polystyrene suffers a nonlinear decrease when the temperature increases. To overcome this drawback and improve calibration accuracy, embodiments of the disclosed subject matter use an atomic gas vapor (e.g., rubidium), which can offer highly conserved frequency standard at its narrow absorption lines. Since water occupies 78% of the cornea by volume (and thus it has a Brillouin shift in the same spectral region as the cornea and since the speed of sound in water at different temperatures has been thoroughly examined, 2ater can be used as the calibration sample. However, other calibration samples are also possible according to one or more contemplated embodiments.

For example, using a Rb vapor cell, three locked laser frequencies (e.g., vi=384229.1497 GHz, V2=VI-1.1678 GHZ, V3=VI+2.9607 GHZ) can be used to excite the Brillouin signal of water. Alternatively or additionally, polynomial fitting can be used instead of linear fitting to calculate frequencies along the dispersion axis. After the calculation, the water can be excited by another two locked laser frequencies (e.g., V4=VI+31.7 MHz, vs=vi+92.0 MHz) to verify the accuracy. For each excitation frequency, multiple spectra (e.g., one-hundred) can be acquired and an average obtained. Pixel positions of these frequency components can be extracted using Lorentzian-envelop fitting. The relation between the pixel position and the frequency shift is shown in Fig. 2(b). The frequency of Stokes at vj, (S(vs)) was set to be the largest because the dispersion order of Stokes was higher. The excitation laser frequencies for Stokes were on the left of S(v3), while those for Anti-Stokes (AS) were on the right of AS(v2).

The relation between the frequency, v, and the pixel position, x, can be given as: where ai is the coefficient. Based on the known frequency differences, Avi-2 = 1.1678 GHz and Avi-3 = 2.9607 GHz, the coefficients ai to <24 can be calculated through the positions of the Stokes and Anti-Stokes. In theory, the difference between S(v3) and S(vi) does not equal Avi-3 precisely because of the change of excitation frequency, according to Eqn. (1). As the speed of sound is much smaller than that of light, the error introduced by this approximation was limited to tens of kHz, which can be ignored. To determine ao, the Brillouin shift of water with known temperature can be assigned to AS(vi) because vi would be the excitation frequency in the in vivo test. According to Eqn. (2), ao could be calculated once a\ to <24 are determined. If the free spectral range (FSR) of the spectrometer is not known (and thus the frequency difference between S(v2) and AS(v3) could not be determined), the system may not be able to directly use polynomial fitting to find the coefficients. After getting coefficients o to <24, the Brillouin shift of an unknown material, AvB_unknown, can be calculated via: where S and AS were Stokes and Anti-Stokes frequencies, respectively, of this material calculated by Eqn. (2), and AvB_water is the Brillouin shift of water. Brillouin shift of water could be assigned to either S(vi) or AS(vi). In some embodiments, averaging can be used in Eqn. (3) to avoid, or at least reduce the influence of, noise in calculating the unknown frequency.

To verify the accuracy of the calculated coefficients in Eqn. (2), the laser can be locked to V4 and V5 sequentially to generate test frequencies, marked as squares in FIG. 2D. As V4 = vi + 31.7 MHz and vs = vi + 92.0 MHz, comparing the calculated results from Eqn. (2) with the well- defined 31.7 MHz and 92.0 MHz showed that the errors and the standard deviations of the Stokes and Anti-Stokes excited by V4 were 3.04 ± 6.51 MHz and 3.50 ± 6.37 MHz, while those excited by V5 were 0.31 ± 7.02 MHz and 4.12 ± 6.61 MHz. As the frequency accuracy only related to fitting accuracy, change of the Brillouin shift did not influence this accuracy. Therefore, taking the largest error of 4.12 MHz into account, the relative accuracy in the 5.7 GHz region (typical of cornea values) can be better than 0.07%.

To convert pixel distance to frequency, the system can be calibrated, for example, before use on each patient. For example, the use of different gas cell absorption lines can provide an accuracy of several MHz if there is no drift during the following measurement (e.g., about 20 minutes for an eye of the patient). However, due to slow mechanical drift of the mirror mounts in the spectrometer, the zero-frequency set by calibration can vary over time. This change can introduce a frequency error of 170 MHz if there is only one pixel drift. Even though the calibration can also satisfy a zero-drift requirement, increasing the speed of interrogation (e.g., to less than 1 min) can help minimize the impact of any drift, as well as help guarantee accuracy of several MHz for the frequencies for verification (e.g., v4 and v5). To overcome the influence of drift, in some embodiments, a differential method can be used to calculate Brillouin shifts of the cornea. For example, instead of using absolute pixel positions, the distance between the Stokes and Anti-Stokes can be used. As the Stokes and Anti-Stokes share the same zero point, the use of distance between them cancelled out the influence of any drift. To convert the absolute coordinates determined during calibration to relative distances, after determining the calibration coefficients, distances of one-hundred pairs of Stokes and Anti-Stokes (e.g., representing from 5 GHz to 8 GHz) can be calculated. A relation can then be derived between the distance and the frequency by polynomial fitting.

In some embodiments, the focal point of the Brillouin modality can be moved to different lateral points across the cornea, as well as at different depths (e.g., axial scan at each lateral point). However, such movement can occupy a significant part of the total assessment time for a patient. For example, the time required to measure 40 points with respect to a patient’s eye may take around 20 minutes, but the time spent on actually acquiring data may only be about 3 minutes (e.g., about 200 second if each axial scan takes about 5 seconds). The remaining time may be a function of user input (e.g., manual moving the focal point via joystick manipulation) and system mechanics (e.g., how quickly the focal point can be moved). During movement between lateral points, interrogating light for the Brilloin modality is note required because it does not provide any position information. However, since the interrogating light may be the brightest of the light sources employed in the system (e.g., ~ 7 mW) and since the patient can see the interrogating light during the movement, the patient’s eyes may get tired. Accordingly, in some embodiments, the interrogating light for the Brillouin modality can be selectively blocked and/or turned off. For example, in some embodiments, a programmable shutter can be provided (e.g., as part of the laser for the Brillouin modality or within the sampling interface), and the shutter can be controlled to only allow the interrogating light to reach the patient’s eye when a Brillouin scan is being performed and to block the interrogating light at other times (e.g., during movement between lateral measurement points).

In conventional Brillouin modalities, fixed lens mounts were used for cylindrical lenses in the detection arm (e.g., cylindrical lenses 204, 214). Since each VIPA in the detection arm relies on multi-reflection between two parallel flat surfaces to separate different frequencies spatially, the focused beam line after the respective cylindrical lens should be substantially parallel to the entrance slot of the VIPA. For example, FIG. 2F shows a first VIPA 206 with an initial focused beam line 280 from the first cylindrical lenses 204 that is slightly misaligned with the entrance slot 274, for example, due to manufacturing error in the lens mount. Because of the angle mismatch, the focused beam line 280 cannot be coupled with the VIPA 206 efficiently, which can reduce the system throughput. In some embodiments, one or more of the optical components can be mounted on rotational stages so as to better align the optical beam path with the entrance slot of each VIPA, thereby maximizing, or at least improving throughput, and avoiding, or at least reducing, beam aberration.

For example, FIG. 2E illustrates a detection arm 270 where both cylindrical lenses 204, 214 are mounted on respective rotational stages 272, 276. In some embodiments, by rotating the orientation of the cylindrical lens before VIPA 206 via the respective rotational stage, the focused beam line 280 can be rotated in direction 282 to better match the entrance slot 274, as shown by final beam line 284 in FIG. 2F. In some embodiments, an optimal orientation of the cylindrical lens with respect to the VIPA can be confirmed by monitoring an output power of VIPA 206 with a power meter, for example, by rotating the cylindrical lens until a local power maximum is obtained. Once the orientation of the first cylindrical lens 204 is set, the second cylindrical lens 214 can be rotated to an orientation aligned with entrance slot 278, for example, where an output beam from VIPA 216 attains a pure circle, which can be confirmed by a beam analyzer or a camera. With the introduction of the additional rotational degrees of freedom, the Brillouin modality can reach a maximum, or at least improved, throughput regardless of lens mount quality. In some embodiments, a controller for the Brillouin modality or for a system including the Brillouin modality can control rotation of the cylindrical lenses to achieve optimal, or at least improved, alignment.

In some embodiments, a coupling lens providing input to the detection arm of the Brillouin modality can be selected to improve signal intensity. For example, Brillouin signals of the aqueous humor can be closer than those of water, and thus the Brillouin modality for examining the eye of a patient can employ a smaller fiber collimator in order to focus light onto the diffracted orders. For example, the graph 286 in FIG. 2G reflects use of a larger fiber collimator selected to get a strong Brillouin signal 292 from water, which fiber collimator had a broader diffractive envelop 288. In contrast, in some embodiments, the use of a smaller fiber collimator led to a narrower diffractive envelop 296 and energy redistribution, as shown in graph 294 in FIG. 2G. As a result, weak scattering could be tightly focused on the signal of interest 290, for example, the Brillouin signals from the aqueous humor and the Brillouin signals from the cornea, which are between those of the aqueous humor. In addition to improving signal strength, the smaller fiber collimator can also increase measurement speed.

In some embodiments, the Brillouin modality can interact with the patient via a sampling interface, for example, a human interface. In some embodiments, the sampling interface may include one or more features that maintain a position relationship between the Brillouin modality (and the corresponding optical tracking modalities) and the patient and/or isolate the Brillouin modality from movement or vibrations. In some embodiments, a shock-absorbing and/or vibration-dampening polymer (e.g., a polyurethane, such as Sorbothane®) can be used as a passive isolation solution for the human interface, for example, to isolate the human interface from vibrations from the floor. For example, polyurethane mounting feet can be placed under the base of the human interface (e.g., a slit lamp frame). With damped vibrations, the standard deviation of OCT tracking performed on an enucleated pig eye mounted on the human interface were 1 pm smaller.

In some embodiments, the measurements by the Brillouin and optical tracking modalities can be synchronized. In some embodiments, a controller of the system can send a trigger signal to a scanning stage, the Brillouin modality, the depth-tracking modality, and the lateral-position- tracking modality. These four functional parts may then work according to their own timing crystals. Alternatively or additionally, synchronization can include acquiring intensity information and the time of the acquisition for each of the Brillion, depth-tracking, and lateral- position-tracking modalities. In post-processing, Brillouin shifts can be combined with position information at the same time period to enable position correction.

Alternatively or additionally, in some embodiments, synchronization can employ separate hardware to process external triggers of the different functional parts. For example, in some embodiments, the exposure time of a Brillouin spectrum may be different than that of the depth-tracking modality (e.g., 50 ms for the Brillouin spectrum versus 0.5 ms for an OCT spectrum). Due to different time bases in the Brillouin modality and the depth-tracking modality, some Brillouin spectra can contain more depth-tracking spectra while other Brillouin spectra contain fewer. To avoid this issue, external triggers of different functional parts can be connected to a field programmable gate array (FPGA)-based control board. By using phase lock loops (PLLs) inside the FPGA, synchronized triggers with different frequencies can be sent to the modalities simultaneously.

In some embodiments, a scan depth for the Brillouin modality can be greater than a thickness of the cornea, for example, to ensure a scan through the cornea regardless of patient movement. For example, even though the cornea is only about 500 pm thick, a scan range is 1.5 mm with a step size of 15 pm can be used. Based on the ratio between the thickness of the cornea and the scan range, a large number of redundant points may be generated during each scan. In some embodiments, all of the measured points for each scan can be processed without distinguishing these redundant or futile points. Alternatively, in some embodiments, these redundant or futile points can be removed to facilitate processing, for example, by reducing processing time. For example, a controller of the system (or a separate data processing device) can evaluate the metrics of fit quality and signal-to-noise ratio (SNR) of each Brillouin spectrum, which metrics can be used to remove points in air, since scans in air do not generate meaningful Brillouin signals. Alternatively or additionally, Brillouin shifts can be used to remove points in the aqueous humor. For example, with respect to Brillouin shifts, the cornea has a much larger value than that of the aqueous humor (e.g., 5.70 GHz vs 5.25 GHz). By filtering points based on their Brillouin shift values, the redundant points in the aqueous humor can be excluded. In some embodiments, the removal of points from processing can help reduce processing time, for example, from 3 days down to 1 day.

Subclinical Keratoconus Detection Systems

Referring to FIG. 3, a system 300 for determining a patient’s eye 304 exhibits subclinical keratoconus is shown. In the illustrated example, the system 300 includes a patient interface 302, a Brillouin modality for corneal modulus measurement, an optical coherence tomography (OCT) device for axial tracking, and a single-lens imaging system 316 for in-plane pupil tracking. In some embodiments, the components of the patient interface 302 can be mounted on a breadboard sitting on an ophthalmic slit lamp frame, which can be moved in three dimensions, for example, via a joystick. To provide spatially-resolved Brillouin shifts across the cornea, the Brillouin modality had a confocal microscopy configuration. The light source 346 of the Brillouin modality was a continuous wave laser centered at 780 nm and locked to an absorption line of gas cell 322 (e.g., rubidium vapor cell). The locking not only stabilized the frequency of light source 346 but also enabled reflection reduction via the gas cell 322 before coupling scattered light into the Brillouin spectrometer 338 via objective lens 324.

In the illustrated example, two Bragg filters 344 (e.g., with corresponding reflecting members 342, for example, mirrors) were placed along the optical path after the light source 346 to suppress amplified spontaneous emission (ASE), thereby further enhancing frequency performance. After filtering by Bragg filters 344, the free space laser light was coupled into a polarization-maintaining fiber 340 and directed to the patient interface 302. In the human interface 302, after passing through a polarizer 334, the output beam from fiber collimator 336 can be enlarged by a telescope made up by two lenses 332 (e.g.,/= 20 mm) and 330 (e.g.,/= 75 mm), for example, to get an effective 0.1 numeric aperture (NA) on lens 306 (e.g.,/= 50 mm) via reflecting element 328 (e.g., mirror), half-wave plate 326, polarization beam splitter (PBS) 318, dichroic mirror 310, and quarter-wave plate 308. In some embodiments, lens 306 can be mounted on a translational stage (not shown) to enable axial scanning through the corneal depth.

To detect the frequency shift, AVB, the scattered light from the eye 304 can be collected by the lens 306, passed through quarter-wave plate 308, reflected by dichroic mirror 310, and then redirected by PBS 318 and reflecting element 320 (e.g., mirror) to pass through gas cell 322 to remove the unshifted frequency. Objective lens 324 can be used to couple the light exiting gas cell 322 into a fiber connected to Brillouin spectrometer 338. In some embodiments, the Brillouin spectrometer can use a pair of 15-GHz VIPA etalons as the core dispersion components to distinguish Brillouin shifts from the excitation laser frequency. As the VIPA played a similar role as a grating, a configuration conceptually similar to blazing can be used (e.g., input beam diameter = 1.31 mm,/= 200 mm for the lenses before and after the VIPA) to maximize, or at least increase, intensity of Brillouin scattering according to the dispersion law of a VIPA. In some embodiments, the exposure time of the spectrometer 338 can be set to about 0.05 seconds. In some embodiments, to track axial movement of the measured point, the sampling arm of a frequency-domain OCT can share the same optical path with the light source 346 through dichroic mirror 310. In some embodiments, the light source 348 of the OCT can be a superluminescent diode (SLD) with a bandwidth of 50 nm and a central wavelength of about 840 nm. For example, the OCT can have an axial resolution of 6.2 pm. In some embodiments, the power of the OCT light source 348 can be equally split into reference and sampling arms by fiber coupler 350. For example, the collimated beam diameters at the outputs of the fiber coupler 350 can be about 1.31 mm. The reference arm of the OCT can include a polarization controller 352, a fiber coupler 354, a neutral density filter 356, a lens 358, and a reflecting element 360 (e.g., mirror), and the sampling arm of the OCT can include a polarization controller 314, a fiber coupler 312, dichroic mirror 310, quarter-wave plate 308, and lens 306. In some embodiments, the focal lengths of lens 306 and the corresponding lens 358 can be the same, for example, about 50 mm (e.g., corresponding to a transverse resolution of about 40.8 pm and a depth of focus (DOF) of about 3.1 mm).

In some embodiments, after focusing light from OCT light source 348 onto the cornea of eye 304, the scattered light can be interfered with the reflection from the reference arm and analyzed by a separate spectrometer. For example, the spectrometer can be formed by a first achromatic lens 362 (e.g.,/= 45 mm), a volume phase holographic grating (VPHG) 364 (e.g., 1800 Ip/mm), a second achromatic lens 366 (e.g.,/= 100 mm), and a line camera 368. Within the spectrometer, incident light can be diffracted by the VPHG 364 and imaged onto line camera 368 via the second achromatic lens. An exposure time of the OCT can be set based on the Brillouin exposure time, for example, to reduce data amount. For example, when employing an 0.05 s Brillouin exposure time, the exposure time of the OCT can be set at 0.5 ms, even though the OCT could work much faster than that. The sensitivity at such an exposure time can be about 116 dB, and the 10-dB sensitivity roll-off distance can be about 2 mm.

Besides potential axial movement during an A-scan, patients’ eyes could also exhibit lateral movement. In some embodiments, an imaging system 316 can be placed next to the objective lens 306 to monitor this lateral movement, for example, by tracking the pupil of the eye 304. In some embodiments, imaging system 316 can have its own light source, for example, a light-emitting diode (LED) having a central wavelength of about 970 nm. For example, the illumination power on the whole eye 304 can be about 2 mW, and the exposure time of the camera of imaging system 316 can be about 20 ms. In some embodiments, a long-pass filter can be provided before the camera of imaging system 316, for example, to totally reject light from the Brillouin light source 346 and/or keeping at least part of the light from the OCT light source 348, for example, for use in annotating measured points. In some embodiments, a ratio of distance/pixel can be calculated by imaging a standard grid on the focal plane, and a pupil tracking algorithm can employ binarization and edge detection.

The combination of Brillouin modality with one or more optical modalities for tracking can be realized in many different configurations, and the system 300 of FIG. 3 represents only one of such configurations. However, embodiments of the disclosed subject matter are not limited to the configuration specifically described for system 300. Rather, other configurations for combining Brillouin and optical tracking modalities are also possible according to one or more contemplated embodiments.

Subclinical Keratoconus Detection Methods

FIG. 4A illustrates aspects of a method 400 for determining if a patient’s eye exhibits subclinical keratoconus. The method 400 can initiate at process block 402, where one or more Brillouin spectroscopy measurements can be obtained for a measurement point with respect to a patient’s eye (e.g., within the cornea, within the aqueous humor, or adjacent to a front of the cornea). In some embodiments, the obtaining of process block 402 can include concurrently tracking lateral location and depth of the measurement point and/or movement of the patient’s eye (e.g., via one or more optical imaging modalities), and correlating the Brillouin spectroscopy measurement with a corresponding location with respect to a patient’s eye. For example, in some embodiments, the system 300 of FIG. 3 can be used to direct light to and collect scattered Brillouin light from the patient’s eye to perform a Brillouin spectroscopy measurement, as well as to provide tracking.

The method 400 can proceed to decision block 404, where it is determined if additional depths should be interrogated. For example, in some embodiments, Brillouin spectroscopy measurements are obtained for different depths (e.g., an axial scan) with respect to a patient’s eye at a same lateral measurement point. If additional depths are desired, the method 400 can proceed to process block 406, where the focal point for the Brillouin modality is advanced to the next depth, for example, by moving one or both of the focal point and the eye with respect to each other along an axial direction. For example, the step size between adjacent depths in an axial scan can be about 15 pm. After moving to the next depth, the method 400 can proceed from process block 406 to process block 402, where Brillouin spectroscopy measurement(s) are obtained at the new depth.

If no additional depths are desired at decision block 404 (e.g., an axial scan has been completed), the method 400 can proceed to process block 408, where a Brillouin reading can be determined for the lateral measurement point using the Brillouin spectroscopy measurement(s) from the different depths. In some embodiments, the Brillouin reading can be a selected one of the Brillouin shifts measured by the axial scan or a mean, median, or mode of the Brillouin shifts measured by the axial scan. Alternatively or additionally, in some embodiments, the Brillouin reading can comprise a selected one of Brillouin widths or signal strengths measured by the axial scan, or a mean, median, or mode of the Brillouin widths or signal strengths measured by the axial scan. In some embodiments, the Brillouin readings can be determined by a controller of the measurement system (e.g., system 300, or control system 114 of system 100), or a separate data processing system. In the illustrated example of FIG. 4A, the determination of a Brillouin reading occurs after an axial scan for the lateral measurement point but before moving to a next lateral measurement point; however, in some embodiments, the determination of a Brillouin reading can occur at different times, for example, concurrent with moving to a next lateral measurement point (e.g., at a same time as process block 412), concurrent with a obtaining further Brillouin spectroscopy measurements (e.g., at a same time as a repetition of process block 402), or after all Brillouin spectroscopy measurements have been obtained (e.g., after decision block 410).

In some embodiments, the determination of process block 408 can include selecting a subset of depths from the axial scan for use in generating the Brillouin reading. For example, the Brillouin reading can be determined in process block 408 as an average Brillouin shift for the respective lateral measurement point using data only for depths less than or equal to 150 pm from a front of a cornea of the patient’s eye. Alternatively, in some embodiments, the Brillouin reading can be determined in process block 408 as an average Brillouin shift for the respective lateral measurement point using data only for depths within an anterior plateau region of a cornea of the patient’s eye. Alternatively or additionally, in some embodiments, the Brillouin reading can be determined in process block 408 by removing at least some of the Brillouin spectroscopy measurements based on a quality-of-fit metric, a signal-to-noise ratio, or a value of the Brillouin spectroscopy measurement. For example, a Brillouin spectroscopy measurement can be removed from further processing when the measured Brillouin shift is less than or equal to a predetermined threshold (e.g., 5.5 GHz x (780/1), where is a wavelength of interrogating light of the Brillouin spectroscopy measurement in nanometers).

The method 400 can proceed to decision block 410, where it is determined if additional lateral points should be interrogated. For example, in some embodiments, Brillouin spectrometry measurements are obtained at different lateral positions with respect to a patient’s eye. For example, in some embodiments, about 40 points are measured at different lateral locations with respect to the eye. If additional measurement points are desired, the method 400 can proceed to process block 412, where the focal point for the Brillouin modality is advanced to the next lateral measurement point, for example, by laterally moving one or both of the focal point and the eye with respect to each other (e.g., across a front surface of the cornea). In some embodiments, a next lateral measurement point can be instructed by an operator of the system (e.g., via joystick or manual selection on a displayed image of the eye). After moving to the next lateral measurement point, the method can return to process block 402, where Brillouin spectroscopy measurement(s) are obtained at the new lateral measurement point.

If no additional measurement points are desired at decision block 410 (e.g., all lateral measurement points have been evaluated), the method 400 can proceed to optional process block 414, where a two-dimensional map can be created based on the Brillouin readings. For example, the two-dimensional map can be created by spatially distributing the determined Brillouin readings based on the respective lateral measurement points thereof. Values of the map between measurement points can be determined using interpolation, median filtering, and/or any other image or data processing technique. In some embodiments, the two- dimensional map can be graphically displayed, for example, to an operator via a user interface of the measurement system, to a clinician or physician via electronic display or printout, etc. Alternatively, in some embodiments, the two-dimensional map can be retained in the form of data, e.g., without any corresponding visual display.

The method 400 can proceed to decision block 416, where it is determined if other nonBrillouin maps are desired. In some embodiments, the created two-dimensional map of Brillouin readings can be combined with or supplemented by information from one or more nonBrillouin maps, for example, to enhance detection of subclinical keratoconus. If non-Brillouin maps are desired at decision block 416, the method 400 can proceed to process block 418, where such non-Brillouin measurements of the eye can be obtained. For example, in some embodiments, the non-Brillouin measurements can comprise pachymetry, tomography, topography, etc. In some embodiments, the non-Brillouin measurements can be obtained using one or more conventional systems, for example, separate from system 100 or system 300.

After obtaining the non-Brillouin measurements in process block 418 or if non-Brillouin measurements were not desired at decision block 416, the method 400 can proceed to process block 420, where one or more metrics can be calculated based at least in part on the determined Brillouin readings, for example, the created two-dimensional map. In some embodiments, the one or more metrics can comprise a mean, median, or mode of values (e.g., Brillouin readings and interpolated values, for example, Brillouin shift) within the two-dimensional map. Alternatively or additionally, in some embodiments, the one or more metrics can comprise a spatial standard deviation of values within the two-dimensional map. Alternatively or additionally, in some embodiments, the one or more metrics can comprise a minimum of values within the two-dimensional map. Alternatively or additionally, in some embodiments, the one or more metrics can comprise Zemick function coefficients. In some embodiments, the two- dimensional map can be created and/or the metric(s) calculated by a controller of the measurement system (e.g., system 300, or control system 114 of system 100), or a separate data processing system.

The method 400 can proceed to process block 422, where a determination that the eye exhibits subclinical keratoconus can be made responsive to the calculated one or more metrics. For example, a determination of subclinical keratoconus can be made when the metric is outside a predetermined range or less than a predetermined threshold. In some embodiments, the determination of subclinical keratoconus can be made by a controller of the measurement system (e.g., system 300, or control system 114 of system 100), or a separate data processing system. Alternatively, in some embodiments, the determination of subclinical keratoconus is made by an operator of the measurement system or a separate data processing system, or by a clinician or physician that receives the calculated metric(s) and/or the maps.

For example, in some embodiments, a determination of subclinical keratoconus can be made when a calculated metric is less than 5.5 GHz x (780/1), where is a wavelength of interrogating light of the Brillouin spectroscopy measurement in nanometers. Alternatively, in some embodiments, at least one of change in slope, cornea curvature, derivative of the slope, or full- width half-maximum based at least in part on Zernike function coefficients can be determined, and a determination of subclinical can be made responsively thereto.

Alternatively, in some embodiments, the Brillouin readings can be determined for an anterior plateau region 472 of the cornea, as shown in the Brillouin depth profile 470 of FIG. 4D. In some embodiments, to objectively identify the plateau region 472, the depth profile 470 can be fit with a first linear fit 474 of low shift change per depth (the “plateau” region) and a second linear fit 476 of high shift change per depth (the “slope” region). The intersection 478 of these two linear fits 474, 476 marked a posterior end of the plateau region 472. The anterior end of the plateau region 472 can be determined by the tail of the second linear fit 476 (e.g., the slope region) and a thickness of the cornea as measured by one of the optical tracking modalities (e.g., OCT). In some embodiments, the metric(s) can comprise a mean Brillouin shift for the two-dimensional map, and the subclinical keratoconus can be determined when the mean Brillouin shift is less than 5.696 GHz. Alternatively or additionally, the metric(s) can comprise a minimum Brillouin shift in the two-dimensional map, and the subclinical keratoconus can be determined when the minimum Brillouin shift is less than 5.681 GHz. Alternatively or additionally, the metric(s) can comprise a spatial standard deviation for the two-dimensional map, and the subclinical keratoconus can be determined when the spatial standard deviation is O.O18 + O.OO3 GHz.

Alternatively, in some embodiments, the Brillouin readings can be determined for a 150- pm anterior region 482 of the cornea, as shown in the Brillouin depth profile 470 of FIG. 4D. As compared to the anterior plateau region 472, the 150-pm anterior region 482 may be more straightforward to evaluate and more universal in terms of depth extent across corneas of different morphological and mechanical characteristics. In some embodiments, the metric(s) can comprise a mean Brillouin shift for the two-dimensional map, and the subclinical keratoconus can be determined when the mean Brillouin shift is less than 5.704 GHz. Alternatively or additionally, the metric(s) can comprise a minimum Brillouin shift in the two-dimensional map, and the subclinical keratoconus can be determined when the minimum Brillouin shift is less than 5.686 GHz. Alternatively or additionally, the metric(s) can comprise a spatial standard deviation for the two-dimensional map, and the subclinical keratoconus can be determined when the spatial standard deviation is 0.018 + 0.003 GHz.

Although blocks 402-422 of method 400 have been described as being performed once, in some embodiments, multiple repetitions of a particular process block may be employed before proceeding to the next decision block or process block. In addition, although blocks 402- 422 of method 400 have been separately illustrated and described, in some embodiments, process blocks may be combined and performed together (simultaneously or sequentially). Moreover, although FIG. 4A illustrates a particular order for blocks 402-422, embodiments of the disclosed subject matter are not limited thereto. Indeed, in certain embodiments, the blocks may occur in a different order than illustrated or simultaneously with other blocks.

In some embodiments, method 400 can include steps or other aspects not specifically illustrated in FIG. 4A. Alternatively or additionally, in some embodiments, method 400 may comprise only some of blocks 402-422 of FIG. 4A. For example, in some embodiments, method 400 can include some or all of blocks 408, 414, 420, and 422 (e.g., with or without obtaining non-Brillouin measurements 418), e.g., to perform a data processing method for previously obtained Brillouin spectroscopy measurements. Alternatively or additionally, in some embodiments, method 400 can include some or all of blocks 402, 406, 412 (e.g., with or without Brillouin reading determination 408), e.g., to perform a data acquisition method that obtains Brillouin spectroscopy measurements for subsequent processing. FIG. 4B illustrates aspects of another method 430 for determining whether a patient’s eye exhibits subclinical keratoconus. The method 430 can initiate a process block 432, where one or more Brillouin spectroscopy measurements can be obtained for a measurement point with respect to a patient’s eye (e.g., within the cornea, within the aqueous humor, or adjacent to a front of the cornea). At process block 434, the location of the measurement point and/or the patient’s eye can be tracked in three-dimensions, for example, using one or more optical imaging modalities. For example, in some embodiments, the system 300 of FIG. 3 can be used to direct light to and collect scattered Brillouin light from the patient’ s eye to perform the Brillouin spectroscopy measurement of process block 432 as well as to provide the tracking of process block 434.

The method 430 can proceed to process block 436, where the obtained Brillouin spectroscopy measurement can be assigned to a lateral location and depth based on the tracking of process block 434. In some embodiments, the assignments of lateral location and depth can be made by a controller of the measurement system (e.g., system 300, or control system 114 of system 100) or by a separate data processing system. The method 430 can proceed to decision block 438, wherein it is determined if additional measurement points are desired. For example, in some embodiments, Brillouin spectroscopy measurements can be obtained at different depths (e.g., axial scan) and/or a different lateral locations, so as to obtain a three-dimensional distribution of biomechanical data. If additional measurement points are desired, the method 430 can proceed to process block 440, where the focal point for the Brillouin modality is advanced to the next measurement point, for example, by moving one or both of the focal point and the eye with respect to each other along lateral and/or axial directions. In some embodiments, the movement can be to a different depth while maintaining a same lateral location, to a different lateral location while maintaining a same depth, or to a different lateral location and different depth. After moving to the next measurement point, the method 430 can proceed from process block 440 to process block 432, where Brillouin spectroscopy measurement(s) are obtained at the new measurement point.

If no additional measurement points are desired at decision block 438, the method 430 can proceed to process block 442, where a Brillouin reading can be determined for the lateral measurement point using the Brillouin spectroscopy measurement(s) from the different depths, for example, in a manner similar to that described for process block 408 in FIG. 4A. The method 430 can proceed to optional process block 444, where a two-dimensional map can be created based on the Brillouin readings, for example, in a manner similar to that described for process block 414 in FIG. 4A. The method 430 can proceed to decision block 446, where it is determined if other non-Brillouin maps are desired. If non-Brillouin maps are desired at decision block 446, the method 430 can proceed to process block 448, where such non-Brillouin measurements of the eye can be obtained, for example, in a manner similar to that of process block 418 in FIG. 4A.

After obtaining the non-Brillouin measurements in process block 448 or if non-Brillouin measurements were not desired at decision block 446, the method 430 can proceed to process block 450, where one or more metrics can be calculated based at least in part on the determined Brillouin readings, for example, in a manner similar to that of process block 420 in FIG. 4A. The method 400 can proceed to process block 452, where a determination that the eye exhibits subclinical keratoconus can be made responsive to the calculated one or more metrics, for example, in a manner similar to that of process block 452 in FIG. 4A.

Although blocks 432-452 of method 430 have been described as being performed once, in some embodiments, multiple repetitions of a particular process block may be employed before proceeding to the next decision block or process block. In addition, although blocks 432- 452 of method 430 have been separately illustrated and described, in some embodiments, process blocks may be combined and performed together (simultaneously or sequentially). For example, the obtaining of Brillouin spectroscopy measurements of process block 432 and the tracking of process block 434 can occur simultaneously in some embodiments. Moreover, although FIG. 4B illustrates a particular order for blocks 432-452, embodiments of the disclosed subject matter are not limited thereto. Indeed, in certain embodiments, the blocks may occur in a different order than illustrated or simultaneously with other blocks.

In some embodiments, method 430 can include steps or other aspects not specifically illustrated in FIG. 4B. Alternatively or additionally, in some embodiments, method 430 may comprise only some of blocks 432-452 of FIG. 4B. For example, in some embodiments, method 430 can include some or all of blocks 436, 442, 444, 450, and 452 (e.g., with or without obtaining non-Brillouin measurements 448), e.g., to perform a data processing method for previously obtained Brillouin spectroscopy measurements. Alternatively or additionally, in some embodiments, method 430 can include some or all of blocks 432, 434, 436, and 440 (e.g., with or without Brillouin reading determination 442), e.g., to perform a data acquisition method that obtains Brillouin spectroscopy measurements for subsequent processing.

FIG. 4C illustrates aspects of another method 460 for determining a patient’s eye exhibits subclinical keratoconus. Similar to method 430 of FIG. 4B, method 460 can include blocks 432-440 and 446-448. However, instead of determining Brillouin readings in process block 442 and creating a two-dimensional map in optional process block 444 of FIG. 4B, method 460 includes optional process block 462, where a three-dimensional map can be created based on the Brillouin spectroscopy measurements. For example, the three-dimensional map can be created by spatially distributing the determined Brillouin readings based on the respective lateral locations and depths of the measurement points. Values of the map between measurement points can be determined using interpolation, median filtering, and/or any other image or data processing technique. In some embodiments, the three-dimensional map can be graphically displayed, for example, to an operator via a user interface of the measurement system, to a clinician or physician via electronic display or printout, etc. Alternatively, in some embodiments, the three-dimensional map can be retained in the form of data, e.g., without any corresponding visual display.

The method 460 can also include process block 464, where a determination that the eye exhibits subclinical keratoconus can be made responsive to the creation of the three-dimensional map. In some embodiments, one or more metrics (e.g., similar to those described above for the two-dimensional map) can be calculated. Alternatively, in some embodiments, the determination of subclinical keratoconus can be made without any calculation of metric separate from the three-dimensional map. In some embodiments, the determination of subclinical keratoconus can be made by a controller of the measurement system (e.g., system 300, or control system 114 of system 100), or a separate data processing system. Alternatively, in some embodiments, the determination of subclinical keratoconus is made by an operator of the measurement system or a separate data processing system, or by a clinician or physician that receives the calculated metric(s) and/or the maps.

Although blocks 432-440, 446-448, and 462-464 of method 460 have been described as being performed once, in some embodiments, multiple repetitions of a particular process block may be employed before proceeding to the next decision block or process block. In addition, although blocks 432-440, 446-448, and 462-464 of method 460 have been separately illustrated and described, in some embodiments, process blocks may be combined and performed together (simultaneously or sequentially). Moreover, although FIG. 4C illustrates a particular order for blocks 432-440, 446-448, and 462-464, embodiments of the disclosed subject matter are not limited thereto. Indeed, in certain embodiments, the blocks may occur in a different order than illustrated or simultaneously with other blocks. In some embodiments, method 460 can include steps or other aspects not specifically illustrated in FIG. 4C. Alternatively or additionally, in some embodiments, method 460 may comprise only some of blocks 432-440, 446-448, and 462- 464 of FIG. 4C. Computer Implementation

FIG. ID depicts a generalized example of a suitable computing environment 131 in which the described innovations may be implemented, such as but not limited to aspects of control system 114, separate data processing system, method 400, method 430, and/or method 460. The computing environment 131 is not intended to suggest any limitation as to scope of use or functionality, as the innovations may be implemented in diverse general-purpose or special-purpose computing systems. For example, the computing environment 131 can be any of a variety of computing devices (e.g., desktop computer, laptop computer, server computer, tablet computer, etc.).

With reference to FIG. ID, the computing environment 131 includes one or more processing units 135, 137 and memory 139, 141. In FIG. ID, this basic configuration 151 is included within a dashed line. The processing units 135, 137 execute computer-executable instructions. A processing unit can be a central processing unit (CPU), processor in an application-specific integrated circuit (ASIC), or any other type of processor (e.g., hardware processors, graphics processing units (GPUs), virtual processors, etc.). In a multi-processing system, multiple processing units execute computer-executable instructions to increase processing power. For example, FIG. ID shows a central processing unit 135 as well as a graphics processing unit or co-processing unit 137. The tangible memory 139, 141 may be volatile memory (e.g., registers, cache, RAM), non-volatile memory (e.g., ROM, EEPROM, flash memory, etc.), or some combination of the two, accessible by the processing unit(s). The memory 139, 141 stores software 133 implementing one or more innovations described herein, in the form of computer-executable instructions suitable for execution by the processing unit(s).

A computing system may have additional features. For example, the computing environment 131 includes storage 161, one or more input devices 171, one or more output devices 181, and one or more communication connections 191. An interconnection mechanism (not shown) such as a bus, controller, or network interconnects the components of the computing environment 131. Typically, operating system software (not shown) provides an operating environment for other software executing in the computing environment 131, and coordinates activities of the components of the computing environment 131.

The tangible storage 161 may be removable or non-removable, and includes magnetic disks, magnetic tapes or cassettes, CD-ROMs, DVDs, or any other medium which can be used to store information in a non-transitory way, and which can be accessed within the computing environment 131. The storage 161 can store instructions for the software 133 implementing one or more innovations described herein. The input device(s) 171 may be a touch input device such as a keyboard, mouse, pen, or trackball, a voice input device, a scanning device, or another device that provides input to the computing environment 131. The output device(s) 181 may be a display, printer, speaker, CD- writer, or another device that provides output from computing environment 131.

The communication connection(s) 191 enable communication over a communication medium to another computing entity. The communication medium conveys information such as computer-executable instructions, audio or video input or output, or other data in a modulated data signal. A modulated data signal is a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media can use an electrical, optical, radio-frequency (RF), or another carrier.

Any of the disclosed methods can be implemented as computer-executable instructions stored on one or more computer-readable storage media (e.g., one or more optical media discs, volatile memory components (such as DRAM or SRAM), or non-volatile memory components (such as flash memory or hard drives)) and executed on a computer (e.g., any commercially available computer, including smart phones or other mobile devices that include computing hardware). The term computer-readable storage media does not include communication connections, such as signals and carrier waves. Any of the computer-executable instructions for implementing the disclosed techniques as well as any data created and used during implementation of the disclosed embodiments can be stored on one or more computer-readable storage media. The computer-executable instructions can be part of, for example, a dedicated software application or a software application that is accessed or downloaded via a web browser or other software application (such as a remote computing application). Such software can be executed, for example, on a single local computer (e.g., any suitable commercially available computer) or in a network environment (e.g., via the Internet, a wide-area network, a local-area network, a client-server network (such as a cloud computing network), or any other such network) using one or more network computers.

For clarity, only certain selected aspects of the software-based implementations are described. Other details that are well known in the art are omitted. For example, it should be understood that the disclosed technology is not limited to any specific computer language or program. For instance, aspects of the disclosed technology can be implemented by software written in C++, Java™, Python®, and/or any other suitable computer language. Likewise, the disclosed technology is not limited to any particular computer or type of hardware. Certain details of suitable computers and hardware are well known and need not be set forth in detail in this disclosure.

It should also be well understood that any functionality described herein can be performed, at least in part, by one or more hardware logic components, instead of software. For example, and without limitation, illustrative types of hardware logic components that can be used include Field-programmable Gate Arrays (FPGAs), Program- specific Integrated Circuits (ASICs), Program- specific Standard Products (ASSPs), System-on-a-chip systems (SOCs), Complex Programmable Logic Devices (CPLDs), etc.

Furthermore, any of the software-based embodiments (comprising, for example, computer-executable instructions for causing a computer to perform any of the disclosed methods) can be uploaded, downloaded, or remotely accessed through a suitable communication means. Such suitable communication means include, for example, the Internet, the World Wide Web, an intranet, software applications, cable (including fiber optic cable), magnetic communications, electromagnetic communications (including RF, microwave, and infrared communications), electronic communications, or other such communication means. In any of the above-described examples and embodiments, provision of a request (e.g., data request), indication (e.g., data signal), instruction (e.g., control signal), or any other communication between systems, components, devices, etc. can be by generation and transmission of an appropriate electrical signal by wired or wireless connections.

Fabricated Examples and Experimental Results

A system having the configuration of FIG. 3 was constructed and used for evaluating eyes of various test subjects. The stability of the Brillouin spectrometer within the system was optimized and evaluated. Even though the laser frequency was locked to Rb absorption line, mechanical drift inside the Brillouin spectrometer could also lead to instability. In the shortterm, the spectrometer can be assumed to be stable. To evaluate the long-term stability, standard deviations of a 10-second and a 2000-second running were compared. As shown in FIGS. 5A- 5B, the standard deviation of the Brillouin shift of water was maintained, for example, at about 6.9 MHz (0.12% relatively) during the 2000-second test, thus indicating that the Brillouin spectrometer was stable during these periods.

The tracking accuracy was tested using porcine eyes to simulate clinical imaging scenarios. To test the accuracy of axial tracking in clinic, a porcine eye was mounted on a translational stage to move along 1 mm range at a step size of 100 pm. At each position, 100 frames were acquired and averaged. The measured position of the porcine eye was determined by the position of the reflection peak from its anterior surface, as shown in FIG. 5C. In FIG. 5D, the errors and the standard deviations of the distances measured by the optical coherence tomography (OCT) system were plotted along with the known distances read from the translational stage. Within the 1 mm movement range, the maximum error was about 3 pm. Even though the frequency domain OCT could reach an accuracy of tens of nanometers when using mirrors as targets on both arms, 3 pm was achieved because the porcine eye had a much lower scattering ratio, which was close to clinical scenarios.

Like the test of axial accuracy, the test of lateral accuracy was also based on tracking the movement of a porcine eye, thereby imitating an in vivo situation. The porcine eye was mounted on a translational stage, and the stage was subsequently moved horizontally. The movement range was 9 mm (from -4.5 mm to 4.5 mm) at a step size of 0.5 mm. At each position, 100 frames were acquired and averaged. Measured distances were calculated by counting pixel changes of the center of the pupil and multiplying them by the ratio of distance/pixel. The error and standard deviation at each position is shown in FIG. 5E. As shown, the error between the tracking algorithm and known displacements was within about 10 pm.

After individual tests of the Brillouin spectrometer and the 3D tracking, the system was configured for in vivo measurements. To operate in clinical settings, the in vivo system included a human interface adapted from an ophthalmic slit lamp frame, which allowed human-operated motion in three dimensions via a joystick. During the measurement, the patient was asked to sit in front of the human interface, rest their head on a chinrest and maintain their position by biting a fixed bite bar. The bite bar was enclosed by an inside-out individual-use plastic bag for sanitation. A green LED bead was placed right above the objective lens to serve as a fixation target for the patient. To operate all functional parts of the system simultaneously (e.g., Brillouin spectrometer, OCT system, enface imaging system), a software interface was used, and a universal clock was used for synchronization.

In a pupil tracking window, a small white dot near the center of the pupil identified the location of the Brillouin laser dot , while the large white dot at the edge of the pupil was the reflected image of the 970 nm lamp of the enface imaging system. The pupil tracking window also included overlays of concentric red circles having diameters of 2, 5, 7, 8, and 9 mm. When picking a point of interest to measure, the operator moved the cursor to the laser dot and clicked, after which an A-scan would start. After each scan, the measured point was marked as a green dot in the pupil tracking window. Even though the thickness of human cornea is about 500-600 pm, the scanning range was set to 1.8 mm at a step size of 15 pm to account for unpredictable patient movement. The start point of a scan was determined by the OCT system. During each scan, the acquired Brillouin spectra were fitted using the Lorentzian function to calculate Brillouin shifts. The Brillouin profile window showed these roughly calculated Brillouin shifts along the depth without position correction to give an idea of the scan quality. Fine data processing was separately conducted after initial data acquisition.

Table 1 below lists the specifications of the experimental system for detection of subclinical keratoconus via in vivo measurements. The optimized configuration in the Brillouin spectrometer enabled a much shorter exposure time than the traditional Brillouin systems (e.g., 0.05 s versus 0.2 s) with similar frequency stability and only a slight increase in laser power (e.g., 7 mW versus 5 mW), which was still well below safety limits.

Table 1: Operational details of experimental system for detection of subclinical keratoconus

To verify the tracking performance and use tracking information to compensate motion errors of measured Brillouin shifts, the center of the pupil was scanned twice under different breathing conditions. The center of the pupil was determined via the pupil tracking by the en face imaging system. The patient was asked to hold their breath during the first scan and breathe regularly during the second scan. During each scan, Brillouin shifts, axial movement, and lateral movement were recorded simultaneously. The lateral moving patterns (e.g., x-y displacement) for breathing regularly and held breath are shown in FIGS. 6A-6B, respectively. No obvious difference could be ascertained between FIGS. 6A-6B. The in -plane eye movement stayed within a range of approximately 40 pm during the scan regardless of the breathing condition, suggesting that the use of the green LED target for patient fixation is effective at limiting lateral movement of the patient’s eye. Moreover, since the OCT system exhibits a transverse resolution of about 40.8 pm, the eye in-plane movement could be ignored.

In contrast to the stable patterns of in-plane movement, the axial movement showed a clear difference between breathing conditions, as shown in FIGS. 6C-6D. In particular, when the patient breathed regularly, a derivative oscillation was superimposed on a continuous movement away from the objective lens, as shown in FIG. 6C. The total drift was about 200 pm, modulated by a period of 2-3 seconds. However, when the patient held their breath, only a 200 pm continuous drift was observed, as shown in FIG. 6D.

Even though the motion pattern was influenced by breathing, the axial tracking indicated that the patient tended to move hundreds of micrometers during an A-scan. Since the corneal thickness if about 500-600 pm, this patient movement could lead to distortion of the measured Brillouin distribution from the cornea to the aqueous humor. To correct such distortion and recover the Brillouin profile, Brillouin shifts were paired with their axial coordinates measured by the OCT. By adjusting the Brillouin shifts according to their axial coordinates, the corrected Brillouin profiles showed a smooth transition from the cornea to the aqueous, as shown in FIGS. 6E-6F. In FIGS. 6E-6F, the cornea is marked with a shadow, while the aqueous humor is the tail part with a Brillouin shift of about 5.24 GHz. In the situation of breath-holding, corneal thickness was corrected to around 500 pm (instead of the original 700 pm), as shown in FIG. 6F. For the more complicated motion pattern caused by regular breathing, the corrected Brillouin profile in FIG. 6E shared a same shape as the corrected one in FIG. 6F, indicating the efficacy of tracking-based correction. Moreover, the same Brillouin shift of the aqueous humor in different breathing cases served as a last calibration check, proving the feasibility of using the aqueous humor as a baseline.

After validating the efficacy of 3D tracking in Brillouin profile correction, the in vivo setup was used to plot a map of modulus distribution across the cornea. The right eye of a patient with a normal cornea was measured. As the Brillouin shift is also sensitive to the temperature, the room temperature was kept at 23 °C. During the exam, twenty-nine (29) A- scans were performed throughout the whole cornea. Since two-dimensional (2-D) geometrical maps are commonly employed in conventional ophthalmological diagnosis, 2-D Brillouin maps were also created, for example, to resemble clinical maps. To do so, the system took advantage of the relatively flat Brillouin shifts in the anterior corneal region, as shown in FIGS. 6E-6F. Therefore, the mean value of the plateau part of a Brillouin profile was used to represent the Brillouin shift at that measured point. The end point of the plateau was determined by finding the intersection of the flat and the steep slopes using linear fitting. To connect scattered measured points, 2-D interpolation was used.

This plateau averaging strategy was feasible because in-plane eye movement was shown to be smaller than the transverse resolution of the OCT, for example, within 40 pm. If the inplane eye movement was larger, the results may not be marked as 2-D points on the cornea. Instead, a three-dimensional (3-D) map could be used. When conducting plateau averaging in a recovered Brillouin profile, it appears that reaccommodating was not necessary in some cases, such as the case shown in FIG. 6F, because the values in the plateau region were the same without reaccommodating. However, without correction, unpredictable irregular Brillouin profiles would make it difficult to select the plateau region.

A Brillouin map of a normal cornea is shown in FIG. 7A, which coordinate origin was set at the center of the pupil. In this map, there is a softer region within the <7 = 4 mm circle, where Brillouin shifts were about 5.72 GHz. The periphery (e.g., outside the 7 = 4 mm circle) shows a larger Brillouin shift, varying from 5.74 to 5.76 GHz. The superior part was about 0.02 GHz stiffer than the inferior. To further investigate the change of Brillouin shifts along the depth and have a better understanding of the variance of modulus across the cornea, two representative Brillouin profiles at the center (X=-0.40 mm, Y=-0.03 mm) and the periphery (X=-1.80 mm, Y=2.65 mm) were plotted in FIG. 7B after correction by tracking information. The positive X coordinate means the point is on the right of the origin, while the negative Y coordinate means the point is on top of the origin. The corrected Brillouin profiles suggested cornea thicknesses similar to that measured by the commercial instrument. The central cornea had lower modulus than the periphery, and the modulus decreased from the anterior to the posterior, as shown in FIG. 7B.

Besides the in-plane biomechanical distribution, a cross-section map along the depth may also be useful, as it can reveal the variation of biomechanics among different layers. Unlike the in-plane analysis, averaging is not used when analyzing depth-dependent changes. Instead, for example, corneas can be classified by selecting representative points from different regions and comparing their Brillouin profiles. In FIG. 7B, Brillouin profiles at the center and the periphery from a same cornea are overlapped for comparison, showing that intuitive differences exist in the maximum value, corneal thickness at the measured point, the length of the plateau region, the slope of the plateau region, and the slope at the posterior edge of the cornea. A derivative difference exists in the area of the corneal region. To calculate this parameter, polynomial fitting was applied to the Brillouin profile to get a function between the Brillouin shift,/, and the depth position, z. The area Brillouin frequency, farea, can be expressed as: where zo is the thickness of the measured point. Even though the example in FIG. 7B comes from the same cornea, the above-noted parameters can also be used to compare different corneas.

The disclosed system (also referred to herein as the Brillouin ophthalmic instrument) shows promising results from patients in the clinic for mapping focal, depth-dependent biomechanical properties of the cornea. Given the expectation that relevant corneal abnormalities are focal in nature in their earliest manifestations, this feature makes Brillouin microscopy a leading candidate for corneal biomechanical measurement. When bringing Brillouin microscopy into the clinic, patients’ comfort is a factor, the improvement of which can translate into more reliable and efficient measurements than existing instruments. The measurement time of the disclosed system was shortened three-fold by adopting an optimal VIPA configuration, for example, reducing the acquisition time of a Brillouin spectrum at a measurement location from 0.2 seconds to 0.05 seconds, thus allowing an A-scan at multiple depths to be completed within 5 seconds. For a Brillouin map built from 30-40 A-scans, the total measurement time was less than 20 minutes.

In addition to decreased acquisition time and improved patient comfort, the disclosed system achieved much improved spatial accuracy. Even though each A-scan only took 5 seconds, in vivo tests showed that inevitable patient movement still introduced an error of hundreds of micrometers, which was comparable to the corneal thickness. To overcome this motion artifact, precise three-dimensional tracking was performed along with Brillouin detection. For example, axial tracking via OCT had an accuracy of about 3 pm, and lateral tracking via a single lens imaging system had an accuracy of about 10 pm, which tracking accuracies allowed successful reconstruction of disordered Brillouin shifts. The three- dimensional tracking also allowed patients to blink or even sit back to relax between adjacent A- scans.

Combining OCT with Brillouin also allowed the disclosed system to take advantage of thickness measurements by the OCT, for example, to determine the corneal region from a prolonged scanning range. Even though only the cornea and the following aqueous humor were shown in the depth-dependent results (e.g., FIG. 7B), the actual scanning range was 1.8 mm to allow space for patient movement. Thus, scans were started before the focal point reached the cornea. With the help of the thickness measured by the OCT, the starting point of the cornea was determined by stepping back from the end of the clear posterior corneal edge with a step size of the thickness. Without this OCT-guided approach, noisy Brillouin shifts could be included in plateau averaging or meaningful Brillouin shifts could be eliminated.

The disclosed system also exhibits an improved frequency accuracy as compared to prior Brillouin spectroscopy systems. For example, the use of standard Rb absorption frequencies allowed the disclosed system to have certified MHz-level accuracy. Tests showed that the absolute accuracy was better than 4.12 MHz, corresponding to a relative accuracy of 0.07% in the 5.7 GHz corneal region. If converting to longitudinal modulus, the resulting accuracy was better than 0.14%. Also, the stability results confirmed that the disclosed system could maintain this accuracy over 2000 seconds, longer than the measurement for each patient. Such improved accuracy and stability can allow for the identification of MHz level changes in post-LASIK or subtle keratoconus, so that corneal biomechanical alterations can be revealed at early stages.

In FIG. 7A, the Brillouin map of the cornea has the origin set at the center of the pupil. The map of FIG. 7A shows a softer region within the d = 5 mm circle, whose Brillouin shifts were about 5.72 GHz. The periphery outside the <7 = 5 mm circle shows a little bit larger Brillouin shift, varying from 5.74 to 5.76 GHz. The superior part was about 0.02 GHz stiffer than the inferior. Instead of or in addition to using absolute values to quantify measured corneas, Zemike polynomials fit to the two-dimensional map of FIG. 7A can be used, for example, by comparing coefficients of different polynomials. Since the interpolated Brilloin map is not a pure circle, when applying Zernike fitting, the center <7 = 5 mm region can be filtered out from the original map first. Then, Zemike fitting can be applied to the filtered-out center <7 = 5 mm region. Zernike functions, are a product of the Zernike radial polynomials and sine- and cosine-functions: where n = 0,1,2, ... is the degree of the function, while m = -n to n, with (n-m) being even, is the order. Different Zemike functions represent different properties. For instance, Z shows the average shift of the whole region, Zj relates to tilt in x, and Z ₂ relates to astigmatism. As deterioration of the cornea shows a decrease of Brillouin shifts in the weak region, the combination of Zemike functions can exhibit the average change, the tilting direction, the divergence, and a series of properties. Comparing the coefficients can provide a measure of whether the cornea is normal or not (e.g., exhibiting subclinical keratoconus).

Instead of or in addition to using absolute values and/or Zernike polynomial to quantify measured corneas, biomechanical profiles can be used, for example, by comparing biomechanical profiles along vertical and/or horizontal lines can be compared. For example, since the Zemike fitting sacrifices some measurement points to generate the round region, profiles along the vertical line and the horizontal line can be used to take advantage of measurement points at the periphery of the cornea. As comeal deterioration from keratoconus or refractive surgeries mainly occurs at the center and the periphery is less impacted, the change of slope from the superior to the inferior can be used to distinguish abnormal corneas. Curvature, derivative, and full width at half maximum can be used to quantify the change of the profiles.

Although two-dimensional Brillouin maps have been created by performing an average of comeal Brillouin depth profiles, embodiments of the disclosed subject matter are not limited thereto. Rather, the use of two-dimensional Brillouin maps was chosen for convenience, for example, to compare with pachymetry or topography maps, and to provide a quick visual impression of Brillouin-based biomechanical distribution. Alternatively or additionally, in some embodiments, three-dimensional Brillouin maps can be created in used in the evaluation of subclinical keratoconus. Indeed, the Brillouin spectroscopy measurements acquired by the disclosed system contains reliable three-dimensional information that can allow extraction of additional data, such as but not limited to strength decrease from the anterior to the posterior and depth-dependent change of the same cornea before and after surgeries.

For example, when trying to get an overall understanding of the cornea, localized profile comparisons may be limited in the information it can provide. Thus, in some embodiments, a distribution map along depth can be used. In contrast to the two-dimensional Brillouin map that has numerous points for two-dimensional interpolation, only points along a designated axis were used when building a cross-section map. For instance, to build the cross-section map along the middle of the cornea, only the points around X = 1 mm were used, as shown in FIG. 8A. For example, points having the same (or substantially the same) X coordinates can be used. In the illustrated example of FIGS. 8A-8B, points within ±0.25 mm around X = 1 mm were used due to limitations in the accuracy of the joystick. After obtaining coordinates in the XY plane, the coordinates in the Z plane were also obtained. Due to the use of the manual joystick, relative depth differences among the selected points were unknown, and an ancillary OCT image was acquired along the X = 1 mm line, as shown in FIG. 8B. To fill the blank among the scanned points, an erosion-dilation tomographic algorithm was developed to reconstruct the cross-section image. This algorithm was designed based on the fact that lamellas are piled layer by layer within the cornea, so that within each layer the change of Brillouin shifts should be substantially continuous.

The algorithm included five steps:

1) Allocate the Brillouin profiles to the OCT image according to their Y positions.

2) Use the known Brillouin values on the anterior surface and polynomial fitting to estimate Brillouin distribution across this surface.

3) Erode this surface and repeat Step 2) on the new layer. This iteration reconstructs the cornea from the anterior surface.

4) Due to the change of corneal curvature, Steps 2) and 3) are applied to the posterior surface. Opposite to the erosion used to create a new layer, dilation is used when building a new layer from the posterior side.

5) Average the two reconstructed images to create the cross-section map.

To build the relation between the Brillouin and OCT coordinates, the apex in the OCT map, which is also the origin of the Brillouin map in FIG. 8A, was located. The raw OCT map is shown in FIG. 9A. The raw OCT map was binarized by Canny edge detection, resulting in the binarization map of FIG. 9B. The processed map of FIG. 9C was generated by removing small particles based on continuous pixels they contain. In FIG. 9C, three edges are evident - the anterior edge of the cornea, the posterior edge of the epithelium, and the posterior edge of the cornea. To determine the apex of the anterior edge, polynomial fitting was applied to the anterior curve. The apex was located at the zero point of its first derivative. After setting the origin, measured Brillouin profiles were assigned to their corresponding positions read from their Y values in FIG. 9A. Uimited by the scanning range of OCT, the most inferior point in Fig. FIG. 9A is out of the OCT range, so that only the remaining five points are used when reconstructing the cross-section map. As shown in FIG. 9D, the five vertical lines represent the five measured depth-dependent Brillouin profiles. Since the Brillouin profile and the OCT map have different sampling rates, resampling was performed on the Brillouin profiles to make the Brillouin profile start from the anterior edge and end at the posterior edge.

The corneal region in a Brillouin profile was filtered out based on the thickness measured by the OCT. Even though linear interpolation can be used to resample the corneal profile, the interpolated signal is not smooth because of intermittent original points, which violates the condition that the biomechanical change should be continuous from the anterior to the posterior. Thus, curve fitting, instead of interpolation, was used to resample the profile. A three-order polynomial or three-order Fourier fitting were adopted according to the shape of the profile, as shown in FIGS. 10A-10B, respectively. Then, pixel positions along the vertical lines in FIG. 9D were applied to the fitting function to yield the corresponding Brillouin shifts. Once the Brillouin shifts were redistributed through the cornea, reconstruction was conducted layer by layer. To build the first anterior layer, the first values of the five Brillouin profiles were plotted along their horizontal pixel positions, as shown in FIG. IOC. To estimate the variation across this layer, polynomial fitting was applied to these five points and the reconstructed Brillouin shifts of this layer were plotted.

Similar to the process of building the first layer, the second layer was built based on the five discrete Brillouin values on the second layer. Because of the varying corneal curvature from anterior to the posterior, the values on the second layer may not be the second values of the five Brillouin profiles. To determine the positions of the second layer, a morphological algorithm - erosion - was applied to the OCT map in FIG. 11 A, which highlights the corneal region of FIG. 9C. The aqueous humor was regarded as the cornea when reconstructing from the anterior, as shown in FIG. 1 IB, for example, to avoid erosion from the posterior edge disrupting layer determination. The erosion function peels off one layer when applied once. For example, the erosion was applied to FIG. 1 IB seventy times, resulting in the pattern shown in FIG. 11C. To build the second layer, the erosion was applied once, and the corresponding Brillouin values on the new layer were used for polynomial fitting and reconstructing the second layer. By repeating this process through the cornea, a cross-section map built from the anterior can be assembled.

The same procedure was repeated from the posterior edge to build another cross-section map because of the varying corneal curvature. The only difference is that dilation was used to replace erosion when determining layer positions. Starting from the posterior edge, dilation creates a new layer on top when being conducted once. The final cross-section map was built by averaging the two maps built from the anterior and the posterior. This final cross-section map reflected a biomechanical decrease from the anterior to the posterior, as well as a center part of the cornea being softer than its periphery.

The disclosed system (also referred to as motion-tracking (MT) Brillouin microscope) was used to evaluate eyes of patients for subclinical keratoconus. All patients also underwent a complete ocular examination that included best corrected distance visual acuity (CDVA), manifest refraction, and slit lamp biomicroscopy, as well as corneal imaging performed using Scheimpflug tomography (Pentacam HR; Oculus Optikgerate GmbH, Germany) and anterior segment optical coherence tomography (AS-OCT, Avanti RTVue XR version 2018.1.1.63; Optovue). Study inclusion criteria included patients aged 21 to 55 years that belonged to one of two groups as determined by routine clinical exam and corneal imaging: 1) patients with bilaterally normal corneas, who served as the control group, and 2) patients with subclinical keratoconus (SKC) in one or both eyes, with only one eye included per patient. For all control patients, both eyes were considered normal based on the KISA% index classification system (i.e. < 60%), IS-value <1.4, and a BAD-D score < 1.65, with both values derived directly from the Pentacam data output.

The SKC cohort was comprised of patients found to have highly asymmetric keratoconus with clinical disease in the fellow eye (7 eyes from 7 patients) or were determined to be keratoconus suspects in both eyes but did not meet criteria for clinical disease in either eye (8 eyes from 8 patients). In patients with highly asymmetric keratoconus, one eye had clinically- evident disease, with reduced spectacle-corrected visual acuity, corneal thinning appreciable on slit lamp examination, KISA% index > 60%, IS-value >3.0, and BAD-D < 3.0, with both values derived directly from the Pentacam data output. This eye was not included in analysis. The clinically-uninvolved eye had no clinical evidence of disease, no physical findings on slit-lamp examination, 20/20 or better CDVA, KISA% index < 60%, IS value <1.6, and BAD-D < 2.6. This eye was included in data analysis. In bilateral keratoconus suspect patients, there was no clinical evidence of disease, no physical findings on slit-lamp examination, and 20/20 or better CDVA in either eye. Patients in this group all had suspicious imaging findings, including asymmetric anterior curvature as assessed using a 0.5D fixed scale, but all had KISA% index < 60%, IS value <1.6, and BAD-D < 2.6. In these patients, where both eyes would qualify for study analysis, the eye with the lower BAD-D score was included for data analysis. Exclusion criteria included inadequate corneal imaging or corneal scarring that could impact MT Brillouin imaging.

For each patient, age, sex, eye, manifest refraction sphere and cylinder, manifest refraction spherical equivalent (MRSE), and CDVA were recorded to assess the comparability of the groups in these characteristics. Maximum anterior keratometry (K Max), thinnest corneal thickness (TCT), Inferior-superior (IS) value (as automatically calculated by the Pentacam device), index of area variance (ISV), index of vertical asymmetry (IVA), keratoconus index (KI), central keratoconus index (CKI), index of height asymmetry (IHA), index of height decentration (IHD), Ambrosio’s relational thickness (ART max), and the BAD-D score were also collected for each patient. Using the disclosed system, measurements were made to assess the properties of a single point in the cornea by performing high-resolution spectral analysis to measure the frequency shift AVB at that point. The system performs a motorized Z-scan (~5-second) to provide depth information at each lateral location chosen by an operator. For each cornea, approximately forty (40) locations were measured within an 8 mm diameter circle, with typical imaging time of 20 minutes or less per patient. Even though three-dimensional information was acquired during the Brillouin scan, two-dimensional Brillouin maps were created to be comparative with the two- dimensional geometrical maps typically used in clinical evaluations.

Two criteria were applied to extract representative two-dimensional information from the three-dimensional scans, in particular, using values for either a plateau region or an anterior region of a certain depth (e.g., 150 pm depth from the front of the cornea). Since typical depth profiles of corneas present a characteristic “plateau” region in the anterior portion of the cornea, which also represents the stiffest region of the normal cornea, the average value of such anterior plateau was used to represent the Brillouin shift at each measured point in the two-dimensional map. To objectively identify the plateau region, each depth profile was fit with two sequential linear fits of low (the “plateau” region) and high (the “slope” region) shift change per depth. The intersection of these two lines served as an indication of the end of the plateau. The start point was determined by the tail of the slope region and the thickness measured by the OCT (see Supplementary Figure: Plateau Region Determination). The anterior plateau region from which the local Brillouin shift value was obtained, represented as a percentage of total corneal thickness, varied slightly between groups (Controls = 41.4 ± 4.1%, SKC = 39.6 ± 3.5%). The results showed these two averaging modalities were equivalent in the information conveyed with respect to subclinical keratoconus determination.

For both depth averaging procedures, once the value at one lateral location was determined, a two-dimensional interpolation routine was used to connect scattered measured points to create the two-dimensional Brillouin maps. Analysis focused on a more localized corneal region including the central 2 mm values, all values from the 2-5 mm region, and inferior values from the 5-7 mm region. Metrics extracted from these Brillouin maps included (but are not limited to) Mean Brillouin Shift (Mean) that represents the average of values in the map created by the two-dimensional interpolation, Minimum Brillouin Shift (Min) that represents the lowest (e.g., softest, weakest) Brillouin shift measured, and Spatial Standard Deviation (Spatial St. Dev).

For this power analysis, a standard deviation of 0.015 GHz was used, which was based on the mean modulus metric to produce enough power for the less sensitive of the two metrics. A difference of subclinical cases with respect to the control group was estimated as ~16.3MHz. Therefore, if the true difference in the experimental and control means is 16.3MHz, fifteen experimental subjects and fifteen control subjects were sufficient to reject the null hypothesis that the population means of the experimental and control groups are equal with probability (power) = 0.8. In the analysis plan, a Type I error probability associated with this test of this null hypothesis of 0.05 was used. Statistical analysis was performed using Microsoft Excel and SPSS (IBM, Armonk, New York, USA) statistical programs. For each of the MT Brillouin and Scheimpflug metrics evaluated, Student’s t-test was used to compare Control and SKC groups. For reporting purposes, in the statistical analysis, the significance threshold was lowered to 0.00213 to account for the Bonferroni correction of multiple t-test comparisons and keep the family-wise error to 0.05. Receiver operating characteristic (ROC) curves were generated to compare the ability of various MT Brillouin and Scheimpflug metrics to distinguish between Controls and SKC eyes. These analyses were performed using GraphPad Prism version 10.0.0 software.

There were 30 eyes total included in this analysis, with 15 eyes from 15 patients imaged in each group. There were no significant differences in age, sex, refraction, CDVA, K Max, or KISA% index between groups. Only thinnest corneal thickness (TCT) was significantly different, with SKC eyes being thinner by more than 30 |im on average. Table 2 shows the Scheimpflug metric data for each group. Among the evaluated metrics, IS Value, IVA, KI, and IHD were significantly different between groups, while ISV, CKI, IHA, Art Max, and BAD-D were not.

Table 3 shows the comparative Brillouin shift data for each population. There were significant differences between groups for Mean and Min Brillouin shift for both the plateau region and the anterior 150 micron measurements. The spatial variations in Brillouin shift across the central cornea in the Control group were small, demonstrated by a low spatial standard deviation (SSD), on the order of instrument precision, while focal minimum values representing the softest/weakest portions of the cornea were clear in all SKC group maps. Group differences are shown in FIGS. 12A-12D, which illustrate the range of values for each group. As shown in FIGS. 12A-12D, there is a clear separation of Mean and Min MT Brillouin shift values between groups, as demonstrated by a highly statistically significant difference in all metrics analyzed. Table 2: Scheimpflug Metric Comparison

Table 3: Brillouin Modulus Value Comparison

Receiver operating characteristic (ROC) curves were generated to evaluate the ability of MT Brillouin imaging to distinguish between Controls and SKC eyes. As shown in FIG. 13, Mean and Min Brillouin shift values from both the Plateau and A 150 regions completely separated the Control and SKC populations, while the best performing Scheimpflug metrics (KI, IS Value, and IVA) had an AUROC of 0.88 to 0.91. Metrics derived from the anterior portion of the corneal stroma by the disclosed system were able to identify discrete focal corneal biomechanical alterations in all subclinical keratoconic eyes, which were not otherwise present in any control eyes. There were significant group differences in Mean and Minimum MT Brillouin shift values as measured both in the plateau and anterior 150 micron regions, and these metrics accurately distinguished all subclinical keratoconic eyes from normal controls, including in multiple eyes where Scheimpflug metrics did not.

The term “subclinical” was chosen for this study, despite the lack of consensus surrounding the definition of subclinical KC. There are a variety of proposed criteria for categorization of eyes as subclinical based on the presence of “normal” topography, including a KISA% score < 60, K Max < 47.0D, or IS value < 1.45. Further classification has been reported for eyes with “normal topography and tomography” that have utilized the BAD-D metric <1.45 or <1.6. For the data obtained using the disclosed system, all subclinical eyes have a KISA% index < 60, 14/15 (93.3%) having a KISA% index < 35, 13/15 (86.7%) having a K max < 47.0 D, 12/15 (80%) having an IS Value < 1.45, and 10/15 (66.7%) having a BAD-D < 1.45. However, these conventional metrics tend to be of limited value in the identification of subclinical KC. For example, all eyes in both the Control and SKC cohorts had a KISA% in the normal range (<60), KISA% was not significantly different between controls and SKC eyes, and KISA% poorly differentiated groups (AUROC 0.7). Additionally, the BAD-D exhibited limited performance, with no significant difference in mean BAD-D between groups and a low AUROC (0.73). Thus, the disclosed system can identify SKC in eyes of patients that may otherwise be undetectable using currently available systems.

Both Mean and Minimum MT Brillouin shift values, corresponding to the softest focal value in the cornea, fully differentiated SKC and Control groups. There were no material differences in the efficacy of values derived from direct calculation of the plateau region and the automated anterior 150 microns. In addition, the MB Brillouin metrics obtained by the disclosed system could differentiate the two groups without combined analysis of any morphologic metrics. In contrast, recent studies have evaluated combining morphologic and/or biomechanical data from various devices and incorporating artificial intelligence (Al) algorithms to further improve the efficacy of SKC differentiation. However, such strategies have achieved varying degrees of success, with AUROCs for the Pentacam Random Forest (PRFI) ranging from 0.65 to 0.87, and a novel Al-enhanced TBI achieving an AUROC of 0.945, albeit in a population with less strict criteria for subclinical KC classification using “relatively normal front surface curvature” with some eyes having an IS value >4 and/or K max >55D. Of note, none of the aforementioned strategies fully differentiated the subclinical and normal control populations. Additional Examples of the Disclosed Technology

In view of the above-described implementations of the disclosed subject matter, this application discloses the additional examples in the clauses enumerated below. It should be noted that one feature of a clause in isolation, or more than one feature of the clause taken in combination, and, optionally, in combination with one or more features of one or more further clauses are further examples also falling within the disclosure of this application.

Clause 1. A method for detection of subclinical keratoconus, the method comprising:

(a) obtaining, for each of a plurality of lateral measurement points with respect to a patient’s eye, a plurality of Brillouin spectroscopy measurements at different depths along an axial direction for the respective lateral measurement point;

(b) determining, for each lateral measurement point, a Brillouin reading using some or all of the plurality of Brillouin spectroscopy measurements from (a) for the respective lateral measurement point;

(c) calculating one or more metrics based at least in part on the Brillouin readings for the plurality of lateral measurement points determined in (b); and

(d) determining that the patient’s eye exhibits subclinical keratoconus based at least in part on the one or more metrics calculated in (c).

Clause 2. A method for detection of subclinical keratoconus, the method comprising: obtaining a plurality of Brillouin spectroscopy measurements for different measurement points with respect to a patient’s eye; tracking, using one or more optical modalities during the obtaining, locations of the respective measurement points and/or the patient’s eye in three-dimensions; assigning, based on the tracking, each Brillouin spectroscopy measurement to a particular lateral location and depth with respect to or within the patient’s eye; determining, for each lateral location, a Brillouin reading using Brillouin spectroscopy measurements assigned to the respective lateral location but at different depths; calculating one or more metrics based at least in part on the determined Brillouin readings; and determining that the patient’s eye exhibits subclinical keratoconus based at least in part on the one or more metrics.

Clause 3. The method of any clause or example herein, in particular, any one of Clauses 1- 2, wherein the Brillouin reading comprises: a mean, median, or mode of measured Brillouin shifts for some or all of the different depths in a selected depth range; a mean, median, or mode of measured Brillouin widths for some or all of the different depths in a selected depth range; a mean, median, or mode of measured Brillouin signal strengths for some or all of the different depths in a selected depth range; or any combination of the above.

Clause 4. The method of any clause or example herein, in particular, any one of Clauses 1- 3, wherein: the calculating the one or more metrics comprises creating a two-dimensional map based on the determined Brillouin readings; and the calculated one or more metrics is based at least in part on the two-dimensional map.

Clause 5. The method of any clause or example herein, in particular, Clause 4, wherein the creating the two-dimensional map comprises interpolating values for points between the plurality of lateral measurement points or the lateral locations in the two-dimensional map.

Clause 6. The method of any clause or example herein, in particular, any one of Clauses 4-

5, wherein the calculated one or more metrics comprises a mean, median, or mode of values in the two-dimensional map created by the interpolating.

Clause 7. The method of any clause or example herein, in particular, any one of Clauses 4-

6, wherein the calculated one or more metrics comprises a spatial standard deviation of values in two-dimensional map created by the interpolating.

Clause 8. The method of any clause or example herein, in particular, any one of Clauses 4-

7, wherein the creating the two-dimensional map comprises median filtering.

Clause 9. The method of any clause or example herein, in particular, any one of Clauses 1-

8, wherein the calculated one or more metrics comprises a minimum of the determined Brillouin readings.

Clause 10. The method of any clause or example herein, in particular, any one of Clauses 1-

9, further comprising, during the obtaining of (a), using one or more optical imaging modalities to track, in three-dimensions, a location of the respective Brillouin spectroscopy measurement and/or the patient’s eye. Clause 11. The method of any clause or example herein, in particular, any one of Clauses 2 or 10, wherein the one or more optical imaging modalities includes an interferometric or ranging system that measures the depth along the axial direction of the respective measurement point.

Clause 12. The method of any clause or example herein, in particular, Clause 11, wherein the interferometric or ranging system is an optical coherence tomography (OCT) system.

Clause 13. The method of any clause or example herein, in particular, any one of Clauses 11-12, wherein the depth measurements by the interferometric or ranging system have an accuracy of 20 pm or less.

Clause 14. The method of any clause or example herein, in particular, any one of Clauses 11-13, wherein the depth measurements by the interferometric or ranging system have an accuracy of 5 pm or less.

Clause 15. The method of any clause or example herein, in particular, any one of Clauses 2- 14, wherein the one or more optical imaging modalities comprises an imaging system that measures displacement of the patient’ s eye in a lateral direction, for example, one or more directions in a lateral plane.

Clause 16. The method of any clause or example herein, in particular, Clause 15, wherein the displacement measurements by the imaging system have an accuracy of 50 pm or less.

Clause 17. The method of any clause or example herein, in particular, Clause 15, wherein the displacement measurements by the imaging system have an accuracy of 10 pm or less.

Clause 18. The method of any clause or example herein, in particular, any one of Clauses 1-

17, wherein, in the determining of (b), the average Brillouin shift for the respective lateral measurement point is formed using the Brillouin spectroscopy measurements only from depths along the axial direction less than or equal to 150 pm from a front of a cornea of the patient’s eye.

Clause 19. The method of any clause or example herein, in particular, any one of Clauses 1-

18, wherein the determining that the patient’s eye exhibits subclinical keratoconus comprises comparing the calculated one or more metrics to a respective predetermined range, and the subclinical keratoconus is determined in response to at least one of the one or more metrics being outside of the respective predetermined range.

Clause 20. The method of any clause or example herein, in particular, any one of Clauses 1-

19, wherein the Brillouin reading is formed using the Brillouin spectroscopy measurements only from one or more limited depth regions along the axial direction as measured from a front of a cornea of the patient’s eye.

Clause 21. The method of any clause or example herein, in particular, Clause 20, wherein the one or more limited depth regions is a single depth region having depths of less than or equal to 150 pm as measured from the front of the cornea.

Clause 22. The method of any clause or example herein, in particular, Clause 21, wherein: the one or more metrics comprises a mean Brillouin shift for the two-dimensional map, and the subclinical keratoconus is determined when the mean Brillouin shift is less than 5.704 GHz; the one or more metrics comprises a minimum Brillouin shift in the two-dimensional map, and the subclinical keratoconus is determined when the minimum Brillouin shift is less than 5.686 GHz; the one or more metrics comprises a spatial standard deviation for the two-dimensional map, and the subclinical keratoconus is determined when the spatial standard deviation is 0.018 ± 0.003 GHz; or any combination of the above.

Clause 23. The method of any clause or example herein, in particular, any one of Clauses 21-22, further comprising measuring the depth along the axial direction of the respective measurement point using optical coherence tomography.

Clause 24. The method of any clause or example herein, in particular, Clause 20, wherein the Brillouin reading is formed using the Brillouin spectroscopy measurements only from depths along the axial direction within an anterior plateau region of a cornea of the patient’s eye.

Clause 25. The method of any clause or example herein, in particular, Clause 25, wherein the determining the Brillouin reading further comprises: measuring a thickness of the cornea at the respective measurement point; assembling the plurality of Brillouin spectroscopy measurements at the different depths into a depth profile; determining a first linear fit for Brillouin spectroscopy measurements for a substantially flat portion at an anterior end of the depth profile; determining a second linear fit for Brillouin spectroscopy measurements for a sloped portion adjacent to the anterior end of the depth profile; identifying an end of the anterior plateau region as a depth corresponding to an intersection between the first and second linear fits; and identifying a start of the anterior plateau region based on the measured thickness of the cornea and a posterior end of the sloped portion.

Clause 26. The method of any clause or example herein, in particular, any one of Clauses 24-25, wherein: the one or more metrics comprises a mean Brillouin shift for the two-dimensional map, and the subclinical keratoconus is determined when the mean Brillouin shift is less than 5.696 GHz; the one or more metrics comprises a minimum Brillouin shift in the two-dimensional map, and the subclinical keratoconus is determined when the minimum Brillouin shift is less than 5.681 GHz; the one or more metrics comprises a spatial standard deviation for the two-dimensional map, and the subclinical keratoconus is determined when the spatial standard deviation is 0.018 ± 0.003 GHz; or any combination of the above.

Clause 27. The method of any clause or example herein, in particular, any one of Clauses 24-26, wherein a thickness of the cornea is measured using optical coherence tomography.

Clause 28. The method of any clause or example herein, in particular, any one of Clauses 1- 27, wherein the obtaining the plurality of Brillouin spectroscopy measurements comprises using a laser locked to a first frequency to illuminate the patient’s eye and using a Brillouin spectrometer to detect light from the patient’s eye, the Brillouin spectrometer comprising a filter that removes light having the first frequency.

Clause 29. The method of any clause or example herein, in particular, Clause 28, wherein the filter is a gas cell, and the laser is locked to an absorption line of the gas cell.

Clause 30. The method of any clause or example herein, in particular, Clause 29, wherein the gas cell is a rubidium vapor cell.

Clause 31. The method of any clause or example herein, in particular, any one of Clauses 29-30, further comprising, during the obtaining of (a), heating the gas cell to have a substantially uniform, elevated temperature therein.

Clause 32. The method of any clause or example herein, in particular, any one of Clauses 29-31, wherein the gas cell exhibits a removal ratio of 10 ⁴ or less. Clause 33. The method of any clause or example herein, in particular, any one of Clauses 28-32, wherein an intensity of light at the first frequency after the filter is no more than 10’ ³ than light at the first frequency before the filter.

Clause 34. The method of any clause or example herein, in particular, any one of Clauses 1- 33, wherein the obtaining the plurality of spectroscopy measurements comprises, for each depth along the axial direction, correlating a distance between a pixel position at which a Stokes peak is detected and a pixel position at which an Anti-Stokes peak is detected to a measured Brillouin shift for the respective depth.

Clause 35. The method of any clause or example herein, in particular, Clause 34, wherein the distance between the pixel positions is correlated to the measured Brillouin shift based on a predetermined polynomial fit.

Clause 36. The method of any clause or example herein, in particular, any one of Clauses 1- 35, wherein the determining the Brillouin reading comprises removing at least some of the Brillouin spectroscopy measurements from use in creating the Brillouin reading based on a quality-of-fit metric, a signal-to-noise ratio, or a value of the Brillouin spectroscopy measurement.

Clause 37. The method of any clause or example herein, in particular, Clause 36, wherein a Brillouin spectroscopy measurement is removed when a measured Brillouin shift is less than or equal to a predetermined threshold.

Clause 38. The method of any clause or example herein, in particular, Clause 37, wherein the predetermined threshold is 5.5 GHz times (780/1), and is a wavelength of interrogating light of the Brillouin spectroscopy measurement in nanometers.

Clause 39. The method of any clause or example herein, in particular, any one of Clauses 1-

38, wherein, for each depth along the axial direction, a time to obtain the respective Brillouin spectroscopy measurement is less than 0.2 seconds.

Clause 40. The method of any clause or example herein, in particular, any one of Clauses 1-

39, wherein, for each depth along the axial direction, the time to obtain the respective Brillouin spectroscopy measurement is about 0.05 seconds.

Clause 41. The method of any clause or example herein, in particular, any one of Clause 4-

40, wherein the calculating the one or more metrics comprises applying a Zemike fit to the two- dimensional map, and the one or more metrics comprises Zernike function coefficients. Clause 42. The method of any clause or example herein, in particular, Clause 41, wherein the determining that the patient’s eye exhibits keratoconus comprises determining at least one of change in slope, cornea curvature, derivative of the slope, or full- width half-maximum based at least in part on the Zernike function coefficients.

Clause 43. The method of any clause or example herein, in particular, any one of Clauses 1- 42, wherein the calculating the one or more metrics is based on the determined Brillouin readings and information obtained from one or more other testing modalities of the patient’s eye.

Clause 44. The method of any clause or example herein, in particular, Clause 43, wherein the information obtained from the one or more other testing modalities comprise information obtained from pachymetry of the patient’s eye and/or from tomography of the patient’s eye.

Clause 45. A system comprising: a Brillouin spectroscopy modality configured to obtain Brillouin spectroscopy measurements for different measurement points with respect to a patient’s eye; one or more optical imaging modalities configured to track locations of the respective measurement points and/or the patient’s eye in three-dimensions; and a controller comprising one or more processors and computer-readable storage media storing instructions that, when executed by the one or more processors, cause the controller to determine that the patient’s eye exhibits subclinical keratoconus based at least in part on the obtained Brillouin spectroscopy measurements.

Clause 46. The system of any clause or example herein, in particular, Clause 45, wherein the controller is operatively coupled to and configured to control operation of the Brillouin spectroscopy modality and the one or more optical imaging modalities.

Clause 47. The system of any clause or example herein, in particular, any one of Clauses 45- 46, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to: obtain, for each of a plurality of lateral measurement points, a plurality of Brillouin spectroscopy measurements at different depths along an axial direction for the respective lateral measurement point; determine, for each lateral measurement point, a Brillouin reading using some or all of the plurality of Brillouin spectroscopy measurements for the respective lateral measurement point; calculate one or more metrics based at least in part on the determined Brillouin readings for the plurality of lateral measurement points; and determine that the patient’s eye exhibits subclinical keratoconus based at least in part on the calculated one or more metrics.

Clause 48. The system of any clause or example herein, in particular, any one of Clauses 45- 46, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to: obtain a plurality of Brillouin spectroscopy measurements for different measurement points with respect to the patient’s eye; track locations of the respective measurement points and/or the patient’s eye during the obtaining; assign each Brillouin spectroscopy measurement to a particular lateral location and depth with respect to or within the patient’s eye; determine a Brillouin reading for each lateral location using Brillouin spectroscopy measurements assigned to the respective lateral location but at different depths; calculate one or more metrics based at least in part on the determined Brillouin readings; and determine that the patient’s eye exhibits subclinical keratoconus based at least in part on the one or more metrics.

Clause 49. The system of any clause or example herein, in particular, any one of Clauses 47-

48, wherein the Brillouin reading comprises: a mean, median, or mode of measured Brillouin shifts for some or all of the different depths in a selected depth range; a mean, median, or mode of measured Brillouin widths for some or all of the different depths in a selected depth range; a mean, median, or mode of measured Brillouin signal strengths for some or all of the different depths in a selected depth range; or any combination of the above.

Clause 50. The system of any clause or example herein, in particular, any one of Clauses 47-

49, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to create a two-dimensional map based on the determined Brillouin readings, and the calculated one or more metrics is based at least in part on the two-dimensional map.

Clause 51. The system of any clause or example herein, in particular, Clause 51, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to create the two-dimensional map by interpolating values for points between the plurality of lateral measurement points or the lateral locations.

Clause 52. The system of any clause or example herein, in particular, any one of Clauses 50-

51, wherein the calculated one or more metrics comprises a mean, median, or mode of values in the two-dimensional map created by the interpolating, a spatial standard deviation of values in two-dimensional map created by the interpolating, or any combination of the foregoing.

Clause 53. The system of any clause or example herein, in particular, any one of Clauses SO-

52, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to create the two-dimensional map by using median filtering.

Clause 54. The system of any clause or example herein, in particular, any one of Clauses 47-

53, wherein the calculated one or more metrics comprises a minimum of the determined Brillouin readings.

Clause 55. The system of any clause or example herein, in particular, any one of Clauses 45-

54, wherein the one or more optical imaging modalities comprises an interferometric or ranging system configured to measure depth along an axial direction of the respective measurement point.

Clause 56. The system of any clause or example herein, in particular, Clause 55, wherein the interferometric or ranging system is an optical coherence tomography (OCT) system.

Clause 57. The system of any clause or example herein, in particular, any one of Clauses 55-

56, wherein the interferometric or ranging system is configured to have an accuracy for depth measurements of 20 pm or less, for example, 5 pm or less.

Clause 58. The system of any clause or example herein, in particular, any one of Clauses 45-

57, wherein the one or more optical imaging modalities comprises an imaging system configured to measure displacement of the patient’s eye in a lateral direction.

Clause 59. The system of any clause or example herein, in particular, Clause 58, wherein the imaging system is configured to have an accuracy for displacement measurements of 50 pm or less, for example, 10 pm or less.

Clause 60. The system of any clause or example herein, in particular, any one of Clauses 58- 59, wherein the imaging system is a single lens imaging system. Clause 61. The system of any clause or example herein, in particular, any one of Clauses 47-

60, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to determine that the patient’s eye exhibits subclinical keratoconus when the calculated one or more metrics is outside of a respective predetermined range.

Clause 62. The system of any clause or example herein, in particular, any one of Clauses 47-

61, wherein the computer-readable storage media stores additional instructions that, when executed by the one or more processors, further cause the system to select a subset of depths for determining the respective Brillouin reading, the subset being an anterior region of a predetermined depth or an anterior plateau region of a cornea of the patient’s eye.

Clause 63. The system of any clause or example herein, in particular, any one of Clauses 45-

62, wherein the Brillouin spectroscopy modality comprises: a laser locked to a first frequency and configured to illuminate the patient’s eye; and a filter disposed along an optical path between the patient’s eye and a detection arm, the filter being configured to remove 184ight having the first frequency.

Clause 64. The system of any clause or example herein, in particular, Clause 63, wherein the filter is a gas cell, and the laser is locked to an absorption line of the gas cell.

Clause 65. The system of any clause or example herein, in particular, Clause 64, wherein the gas cell is a rubidium vapor cell.

Clause 66. The system of any clause or example herein, in particular, any one of Clauses 64-

65, further comprising: an insulated housing containing the gas cell therein; a heater configured to heat the gas cell to have a substantially uniform, elevated temperature therein; or any combination of the above.

Clause 67. The system of any clause or example herein, in particular, any one of Clauses 64-

66, wherein the gas cell exhibits a removal ratio of 10 ⁴ or less.

Clause 68. The system of any clause or example herein, in particular, any one of Clauses 63-

67, wherein the filter is constructed such that an intensity of light at the first frequency after the filter is no more than 10’ ³ than light at the first frequency before the filter.

Clause 69. The system of any clause or example herein, in particular, any one of Clauses 45-

68, further comprising: a shutter disposed along an optical path between a laser of the Brillouin spectroscopy modality and the patient’s eye, the laser being configured to illuminate the patient’s eye when the shutter is open, wherein the computer-readable storage media stores additional instructions that, when executed by the one or more processors, further cause the shutter to open while obtaining the Brillouin spectroscopy measurements and to close when moving between measurement points.

Clause 70. The system any clause or example herein, in particular, any one of Clauses 45-69, wherein: the Brillouin spectroscopy modality has a detection arm comprising a virtually imaged phase array (VIPA) etalon having an entrance slot, a cylindrical lens, and a rotational stage upon which the cylindrical lens is mounted, and the computer-readable storage media stores additional instructions that, when executed by the one or more processors, further cause the controller to change, via the rotational stage, an orientation of the cylindrical lens so as to focus a beam line onto the entrance slot of the VIPA etalon.

Clause 71. A system comprising: one or more processors; and computer-readable storage media storing instructions that, when executed by the one or more processors, cause the system to determine that a patient’s eye exhibits subclinical keratoconus in response to Brillouin spectroscopy measurements of the patient’s eye.

Clause 72. The system of any clause or example herein, in particular, Clause 71, wherein the system is operatively coupled to and configured to control operation of a Brillouin spectroscopy modality, one or more optical imaging modalities, or any combination of the foregoing.

Clause 73. The system of any clause or example herein, in particular, any one of Clauses 71- 72, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to: determine, for each of a plurality of lateral measurement points with respect to the patient’s eye, a Brillouin reading using some or all of a plurality of Brillouin spectroscopy measurements obtained at different depths along an axial direction for the respective lateral measurement point; calculate one or more metrics based at least in part on the determined Brillouin readings for the plurality of lateral measurement points; and determine that the patient’s eye exhibits subclinical keratoconus based at least in part on the calculated one or more metrics.

Clause 74. The system of any clause or example herein, in particular, any one of Clauses 71-

73, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to: assign each of a plurality of Brillouin spectroscopy measurements obtained for different measurement points with respect to the patient’s eye to a particular lateral location and depth with respect to or within the patient’s eye based on tracked locations of the respective measurement points and/or the patient’s eye; determine a Brillouin reading for each lateral location using the Brillouin spectroscopy measurements assigned to the respective lateral location but at different depths; calculate one or more metrics based at least in part on the determined Brillouin readings; and determine that the patient’s eye exhibits subclinical keratoconus based at least in part on the one or more metrics.

Clause 75. The system of any clause or example herein, in particular, any one of Clauses 73-

74, wherein the Brillouin reading comprises: a mean, median, or mode of measured Brillouin shifts for some or all of the different depths in a selected depth range; a mean, median, or mode of measured Brillouin widths for some or all of the different depths in a selected depth range; a mean, median, or mode of measured Brillouin signal strengths for some or all of the different depths in a selected depth range; or any combination of the above.

Clause 76. The system of any clause or example herein, in particular, any one of Clauses 73-

75, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to create a two-dimensional map based on the determined Brillouin readings, and the calculated one or more metrics is based at least in part on the two-dimensional map.

Clause 77. The system of any clause or example herein, in particular, Clause 76, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to create the two-dimensional map by interpolating values for points between the plurality of lateral measurement points or the lateral locations. Clause 78. The system of any clause or example herein, in particular, any one of Clauses 76-

77, wherein the calculated one or more metrics comprises a mean, median, or mode of values in the two-dimensional map created by the interpolating, a spatial standard deviation of values in two-dimensional map created by the interpolating, or any combination of the foregoing.

Clause 79. The system of any clause or example herein, in particular, any one of Clauses 76-

78, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to create the two-dimensional map by using median filtering.

Clause 80. The system of any clause or example herein, in particular, any one of Clauses 73-

79, wherein the calculated one or more metrics comprises a minimum of the determined Brillouin readings.

Clause 81. The system of any clause or example herein, in particular, any one of Clauses 73-

80, wherein the computer-readable storage media stores instructions that, when executed by the one or more processors, cause the system to determine that the patient’s eye exhibits subclinical keratoconus when the calculated one or more metrics is outside of a respective predetermined range.

Clause 82. The system of any clause or example herein, in particular, any one of Clauses 73-

81, wherein the computer-readable storage media stores additional instructions that, when executed by the one or more processors, further cause the system to select a subset of depths for determining the respective Brillouin reading, the subset being an anterior region of a predetermined depth or an anterior plateau region of a cornea of the patient’s eye.

Clause 83. The method of any clause or example herein, in particular, Clause 82, wherein: the subset is the anterior region having depths of less than or equal to 150 pm as measured from the front of the cornea; the one or more metrics comprises a mean Brillouin shift for the two-dimensional map, and the subclinical keratoconus is determined when the mean Brillouin shift is less than 5.704 GHz; the one or more metrics comprises a minimum Brillouin shift in the two-dimensional map, and the subclinical keratoconus is determined when the minimum Brillouin shift is less than 5.686 GHz; the one or more metrics comprises a spatial standard deviation for the two-dimensional map, and the subclinical keratoconus is determined when the spatial standard deviation is 0.018 ± 0.003 GHz; or any combination of the above.

Clause 84. The method of any clause or example herein, in particular, Clause 82, wherein: the subset is the anterior plateau region; the one or more metrics comprises a mean Brillouin shift for the two-dimensional map, and the subclinical keratoconus is determined when the mean Brillouin shift is less than 5.696 GHz; the one or more metrics comprises a minimum Brillouin shift in the two-dimensional map, and the subclinical keratoconus is determined when the minimum Brillouin shift is less than 5.681 GHz; the one or more metrics comprises a spatial standard deviation for the two-dimensional map, and the subclinical keratoconus is determined when the spatial standard deviation is 0.018 ± 0.003 GHz; or any combination of the above.

Conclusion

Although the discussion above mentions “imaging,” the production of an actual image is not strictly necessary. Indeed, the mentions of “imaging” are intended to include the acquisition of data via the Brillouin and/or optical tracking modalities where an image may not be produced. For example, the Brillouin modality may produce graphical results of the Brillouin signatures, or produce values (or a graphical display of values) corresponding to the measured physical properties of the sample. Similarly, the optical tracking modalities may produce data or other information used in the processing of the Brillouin data without an actual image being produced. Accordingly, the use of the term “imaging” herein is intended to include such scenarios and should not be understood as limiting.

Although particular optical components and configuration have been illustrated in the figures and discussed in detail herein, embodiments of the disclosed subject matter are not limited thereto. Indeed, one of ordinary skill in the art will readily appreciate that different optical components or configurations can be selected and/or optical components added to provide the same effect. In practical implementations, embodiments may include additional optical components, fewer optical components, or other variations beyond those illustrated, for example, additional reflecting elements to manipulate the beam path to fit a particular microscope geometry. Accordingly, embodiments of the disclosed subject matter are not limited to the particular optical configurations specifically illustrated and described herein.

Any of the features illustrated or described herein, for example, with respect to FIGS. 1A-13 and Clauses 1-84, can be combined with any other feature illustrated or described herein, for example, with respect to FIGS. 1A-13 and Clauses 1-84 to provide systems, devices, structures, methods, and embodiments not otherwise illustrated or specifically described herein. All features described herein are independent of one another and, except where structurally impossible, can be used in combination with any other feature described herein. In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the disclosed technology. Rather, the scope is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.