Systems and methods for determining cross-linking distribution in cornea and / or structural characteristics of cornea

Abstract

To provide illumination and imaging techniques to determine a corneal thickness.SOLUTION: In a corneal measurement system, an optical element 112 focuses an excitation light on an area of corneal tissue at a selected depth. In response, a fluorescing agent applied to a cornea 2 generates a fluorescence emission. An aperture of a pinhole structure selectively transmits the fluorescence emission from the area of corneal tissue at the selected depth. A detector captures the selected fluorescence emission transmitted by the aperture and communicates information relating to a measurement of the selected fluorescence emission captured by the detector. A controller 120 receives the information from the detector and determines a measurement of the fluorescing agent in the area of corneal tissue at the selected depth. The system may include a scan mechanism that causes the optical element to scan the cornea at a plurality of depths, and the controller may determine a measurement of the fluorescing agent in the cornea as a function of depth.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 444,910, filed January 11, 2017, and U.S. Provisional Patent Application No. 62 / 573,440, filed October 17, 2017, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Field The present disclosure relates to systems and methods for ophthalmic treatment or surgery, and more particularly to systems and methods for determining the distribution of a cross-linking agent in the cornea and / or determining structural characteristics of the cornea, such as corneal thickness.

[0003] 2. Description of Related Art Crosslinking procedures can be used to treat eyes affected by disorders such as keratoconus. Specifically, keratoconus is a degenerative eye disorder in which structural changes within the cornea weaken the cornea and cause it to change into an abnormal, conical shape. Crosslinking procedures can strengthen and stabilize the area weakened by keratoconus and prevent undesirable shape changes.

[0004] Cross-linking procedures can also be used after surgical procedures such as laser in situ keratomileusis (LASIK). For example, a complication known as post-LASIK ectasia can occur due to thinning and weakening of the cornea caused by LASIK surgery. In post-LASIK ectasia, the cornea undergoes progressive steepening (bulging). Therefore, cross-linking procedures can strengthen and stabilize the structure of the cornea after LASIK surgery and prevent post-LASIK ectasia.

[0005] Crosslinking procedures can also be used to induce refractive index changes in the cornea to correct disorders such as myopia, hyperopia, nearsightedness, hyperopia, astigmatism, irregular astigmatism, and presbyopia. Summary of the Invention [Means for solving the problem]

[0006] overview According to aspects of the present disclosure, systems and methods use illumination and imaging techniques to determine the distribution of cross-linking agents in the cornea. Additionally or alternatively, the systems and methods use illumination and imaging techniques to determine structural characteristics of the cornea, such as corneal thickness.

[0007] According to one embodiment, a corneal measurement system includes a light source configured to emit excitation light that causes a fluorescent agent, e.g., a cross-linking agent, applied to the cornea to fluoresce. The system includes an optical element configured to receive the excitation light from the light source and to focus the excitation light on a region of corneal tissue at a selected depth of the cornea. The fluorescent agent in the cornea fluoresces in response to the excitation light. The system includes a pinhole structure including an aperture. The pinhole structure is configured to receive the fluorescent emission from the fluorescent agent in the cornea. The aperture is configured to selectively transmit the fluorescent emission from the region of corneal tissue at the selected depth. The system includes a detector configured to capture the selected fluorescent emission transmitted by the aperture and to communicate information related to a measurement of the selected fluorescent emission captured by the detector. The system includes a controller communicatively connected to the detector and configured to receive information from the detector and, based on the information, determine a measurement of the fluorescent agent in the region of corneal tissue at the selected depth.

[0008] In some cases, the measurement system may include a scanning mechanism configured to cause the optical element to scan the cornea at multiple depths and focus excitation light on a respective region of corneal tissue at each depth. For each depth, (i) an aperture in the pinhole structure is configured to selectively transmit fluorescent emission from the respective region of corneal tissue, and (ii) a detector is configured to capture the selected fluorescent emission transmitted by the aperture and communicate information related to a measurement value of the selected fluorescent emission captured by the detector. The controller is configured to receive information from the detector for each depth and, based on the information about the multiple depths, determine a measurement value of the fluorescent agent in the cornea as a function of depth. The multiple depths may range from the anterior surface of the cornea to the posterior surface of the cornea, and the controller may be further configured to determine at least one of a position of the posterior surface, a distance between the anterior and posterior surfaces, or a position of an interface between sections of the cornea based on the information about the multiple depths.

[0009] According to another embodiment, a corneal measurement system includes a light source configured to emit incident light. The system includes an optical element positioned to receive the incident light from the light source and configured to focus the incident light on regions of corneal tissue at selected depths of the cornea. The regions of corneal tissue reflect the incident light. The system includes a scanning mechanism configured to cause the optical element to scan the cornea at multiple depths and focus excitation light on respective regions of corneal tissue at each depth. The multiple depths range from the anterior surface of the cornea to the posterior surface of the cornea. The system includes a pinhole structure including an aperture. The pinhole structure is positioned to receive reflected light from each region of corneal tissue for each depth. The aperture is configured to selectively transmit reflected light from each region of corneal tissue for each depth. The system includes a detector positioned to capture selected reflected light transmitted by the aperture for each depth, the detector configured to communicate information regarding a measurement value of the selected reflected light captured by the detector for each depth. The system includes a controller communicatively connected to the detector and configured to receive information from the detector for each depth and determine at least one of the position of the posterior surface, the distance between the anterior and posterior surfaces, or the position of the substratum corneum interface based on the information for the plurality of depths.

[0010] According to yet another embodiment, a corneal measurement system includes a light source configured to emit excitation light that causes a fluorescent agent applied to the cornea to emit fluorescent light. The system includes at least one optical element positioned to receive the excitation light from the light source and configured to deliver the excitation light. The excitation light penetrates multiple depths of the cornea. The fluorescent agent in the cornea emits fluorescent light in response to the excitation light. The system includes a detector positioned to capture an image of the fluorescent emission from the cornea. The system includes a controller communicatively connected to the detector and configured to receive the image from the detector, scan the image to measure the fluorescent emission at multiple depths, and determine a measurement of the fluorescent agent in the cornea as a function of depth based on the measurements of the fluorescent emission at the multiple depths. The excitation light may further spread in at least one lateral direction as it penetrates multiple depths of the cornea, and the controller may further be configured to scan the image to measure fluorescence emission along at least one lateral direction at the multiple depths, and to determine a measurement of the fluorescent agent in the cornea as a function of depth and lateral position based on the measurements of fluorescence emission along at least one transverse direction at the multiple depths. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 illustrates an example of a system for delivering a cross-linking agent and photoactivating light to the cornea of ​​an eye to cause cross-linking of corneal collagen, according to an embodiment of the present disclosure. [Figure 2A] FIG. 2A shows a photochemical kinetic reaction diagram involving riboflavin and photoactivating light (e.g., ultraviolet A (UVA) light) applied during a corneal cross-linking procedure, according to an embodiment of the present disclosure. [Figure 2B] FIG. 2B shows a diagram of parameters that can affect the photochemical kinetic reaction shown in FIG. 2A. [Figure 3] FIG. 3 shows an example of a system for measuring fluorescence associated with the distribution of a photosensitizer, such as riboflavin, in the eye, according to an embodiment of the present disclosure. [Figure 4]FIG. 4 shows another example of a system for measuring fluorescence associated with the distribution of a photosensitizer, such as riboflavin, in the eye, according to an embodiment of the present disclosure. [Figure 5] FIG. 5 illustrates yet another example of a system for measuring fluorescence associated with the distribution of a photosensitizer, such as riboflavin, in the eye, according to an embodiment of the present disclosure. [Figure 6] FIG. 6 shows a further example of a system for measuring fluorescence associated with the distribution of a photosensitizer, such as riboflavin, in the eye, according to an embodiment of the present disclosure. [Figure 7] FIG. 7 shows yet a further example of a system for measuring fluorescence associated with the distribution of a photosensitizer, such as riboflavin, in the eye, according to an embodiment of the present disclosure. [Figure 8] FIG. 8 shows an additional example of a system for measuring fluorescence associated with the distribution of a photosensitizer, such as riboflavin, in the eye, according to an embodiment of the present disclosure. [Figure 9] FIG. 9 illustrates an example approach for measuring corneal thickness according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0012] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will herein be described in detail. It is to be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit of the disclosure.

[0013] explanation 1 illustrates an example of a treatment system 100 for causing collagen cross-linking in a cornea 2 of an eye 1. The treatment system 100 includes an applicator 132 for applying a cross-linking agent 130 to the cornea 2. In an example embodiment, the applicator 132 may be an eye dropper, syringe, or the like that applies the photosensitizer 130 as droplets to the cornea 2. Examples of systems and methods for applying a cross-linking agent are described in U.S. Patent Application No. 15 / 486,778, filed April 13, 2017, and entitled "Systems and Methods for Delivering Drugs to an Eye," the entire contents of which are incorporated herein by reference.

[0014] The cross-linking agent 130 may be provided in a formulation that allows the cross-linking agent 130 to migrate through the corneal epithelium 2a to the underlying region in the corneal stroma 2b. Alternatively, the corneal epithelium 2a may be removed or otherwise incised to allow the cross-linking agent 130 to be applied more directly to the underlying tissue.

[0015] The treatment system 100 includes an illumination system comprising a light source 110 and optical elements 112 for directing light to the cornea 2. The light photoactivates a cross-linking agent 130, resulting in cross-linking activity in the cornea 2. For example, the cross-linking agent may include riboflavin, and the photoactivating light may include ultraviolet A (UVA) light (e.g., approximately 365 nm). Alternatively, the photoactivating light may include another wavelength, such as a visible wavelength (e.g., approximately 452 nm). As described further below, corneal cross-linking improves corneal strength by creating chemical bonds within the corneal tissue according to a photochemical kinetic reaction system. For example, riboflavin and photoactivating light may be applied to stabilize and / or strengthen corneal tissue to address conditions such as keratoconus or post-LASIK keratectasia.

[0016] Treatment system 100 includes one or more controllers 120 that control aspects of system 100, including light source 110 and / or optical element 112. In implementation, cornea 2 can be more broadly treated with cross-linking agent 130 (e.g., using an eye dropper, syringe, etc.), and photoactivating light from light source 110 can be selectively directed to the treated area of ​​cornea 2 according to a particular pattern.

[0017] The optical element 112 may include one or more mirrors or lenses for directing and focusing the photoactivating light emitted by the light source 110 in a specific pattern on the cornea 2. The optical element 112 may further include filters for partially blocking wavelengths of light emitted by the light source 110 and for selecting specific wavelengths of light to be directed to the cornea 2 for photoactivating the cross-linking agent 130. Furthermore, the optical element 112 may include one or more beam splitters for splitting the beam of light emitted by the light source 110 and may include one or more heat sinks for absorbing the light emitted by the light source 110. The optical element 112 may also accurately and precisely focus the photoactivating light to a specific focal plane within the cornea 2, for example, at a specific depth in the underlying region 2b where cross-linking activity is desired.

[0018] Furthermore, the specific regime of photoactivating light can be adjusted to achieve a desired degree of cross-linking in selected regions of the cornea 2. One or more controllers 120 can be used to control the operation of light source 110 and / or optical element 112 to precisely deliver photoactivating light according to any combination of wavelength, bandwidth, intensity, power, location, depth of penetration, and / or duration of treatment (duration of exposure cycle, duration of dark cycle, and ratio of duration of exposure cycle to duration of dark cycle).

[0019] For example, the parameters of photoactivation of the cross-linking agent 130 can be adjusted to reduce the amount of time required to achieve the desired cross-linking. In an exemplary implementation, this time can be reduced from minutes to seconds. 2 Although photoactivating light of 5 mW / cm can be applied in some configurations, higher doses of photoactivating light, e.g., multiples of 5 mW / cm, may be used. 2 can be applied to reduce the time required to achieve the desired cross-linking. The total dose of energy absorbed by the cornea 2 can be described as the effective dose, which is the amount of energy absorbed through an area of ​​the corneal epithelium 2a. For example, the effective dose for an area of ​​the corneal surface 2A can be, for example, 5 J / cm 2 , or 20 J / cm 2 or 30J / cm 2 The described effective doses can be delivered from a single application of energy or from repeated applications of energy.

[0020] The optical element 112 of the treatment system 100 may include a digital micromirror device (DMD) to spatially and temporally modulate the application of photoactivating light. Using DMD technology, photoactivating light from the light source 110 is projected in a precise spatial pattern created by a matrix of microscopic mirrors arranged on a semiconductor chip. Each mirror represents one or more pixels in the projected light pattern. Using a DMD, topography-guided crosslinking can be performed. Several different spatial and temporal irradiance and dose profiles can be used to control the DMD according to the topography. These spatial and temporal dose profiles can be created using continuous wave illumination, but they can also be modulated via pulsed illumination by pulsing the illumination source under varying frequency and duty cycle regimes. Alternatively, the DMD can be modulated with different frequencies and duty cycles for each pixel, providing ultimate flexibility using continuous wave illumination. Alternatively, both pulsed illumination and a combination of modulated DMD frequencies and duty cycles can be combined. This allows for specific amounts of spatially defined corneal crosslinking. This spatially defined cross-linking may be combined with dosimetry, interferometry, optical coherence tomography (OCT), corneal topography, etc. for pre-treatment planning and / or real-time monitoring and adjustment of corneal cross-linking during treatment. Aspects of dosimetry systems are described in further detail below. Additionally, pre-clinical patient information may be combined with finite element biomechanical computer modeling to generate patient-specific pre-treatment plans.

[0021] To control aspects of the delivery of photoactivating light, embodiments may also use aspects of multiphoton excitation microscopy. Specifically, rather than delivering a single photon of a specific wavelength to the cornea 2, the treatment system 100 may deliver multiple photons of longer wavelengths, i.e., lower energy, that combine to initiate crosslinking. Advantageously, longer wavelengths are less scattered within the cornea 2 than shorter wavelengths, allowing longer wavelengths of light to penetrate the cornea 2 more efficiently than shorter wavelengths. The shadowing effect of incident radiation at greater depths within the cornea is also reduced compared to conventional short-wavelength radiation, because light is absorbed much less by the photosensitizer at longer wavelengths. This allows for enhanced control over depth-specific crosslinking. For example, some embodiments may use two-photon radiation, where each photon has approximately half the energy required to excite molecules in the crosslinking agent 130 to initiate the photochemical kinetic reactions described further below. When the crosslinking agent molecule absorbs both photons simultaneously, the molecule absorbs enough energy to release reactive radicals in the corneal tissue. Embodiments may also utilize lower energy photons, such that the crosslinking agent molecule must simultaneously absorb, for example, three, four, or five photons to release reactive radicals. Because the likelihood of nearly simultaneous absorption of multiple photons is low, a high flux of excitation photons may be required, and a high flux may be transmitted through the femtosecond laser.

[0022] Many conditions and parameters affect the cross-linking of corneal collagen with the cross-linking agent 130. For example, the irradiance and dose of photoactivating light affect the amount and rate of cross-linking.

[0023] UVA light may be applied continuously (continuous wave (CW)) or as pulsed light, and this choice has an effect on the amount, rate, and extent of cross-linking, particularly when the cross-linking agent 130 is riboflavin. When UVA light is applied as pulsed light, the duration of the exposure cycle, the duration of the dark cycle, and the ratio of the duration of the exposure cycle to the duration of the dark cycle have an effect on the resulting corneal hardening. Pulsed light irradiation can be used to produce more or less hardening of corneal tissue than can be achieved with continuous wave irradiation for the same amount or dose of delivered energy. Light pulses of appropriate length and frequency can be used to achieve more optimal chemical amplification. For pulsed light treatment, the on / off duty cycle can be from about 1000 / 1 to about 1 / 1000, and the irradiance can be an average irradiance of about 1 mW / cm. 2 ~Approx. 1000mW / cm 2 and the pulse repetition rate may be about 0.01 Hz to about 1000 Hz or about 1000 Hz to about 100,000 Hz.

[0024] The treatment system 100 can generate pulsed light using a DMD by electronically turning the light source 110 on and off and / or by using a mechanical or optoelectronic (e.g., Pockels cell) shutter, mechanical chopper, or rotating aperture. The pixel-specific adjustability of the DMD and subsequent stiffening based on the adjusted frequency, duty cycle, irradiance, and dose delivered to the cornea can impart complex biomechanical stiffness patterns to the cornea, allowing for various amounts of refractive correction. These refractive corrections can include combinations of corrections for myopia, hyperopia, astigmatism, irregular astigmatism, presbyopia, and complex corneal refractive surfaces caused by ocular diseases such as keratoconus, pellucid marginal disease, post-LASIK corneal ectasia, and other conditions of corneal biomechanical alteration / degeneration. A particular advantage of the DMD system and method is that it enables randomized, asynchronous pulse topography patterning that produces aperiodic, uniform-appearing illumination that eliminates the possibility of inducing photosensitive epileptic seizures or flicker dizziness for pulse frequencies between 2 Hz and 84 Hz.

[0025] While the example embodiment may use a stepped on / off pulsed light function, it is understood that other functions for applying light to the cornea can be used to achieve similar effects. For example, light may be applied to the cornea according to a sinusoidal function, a sawtooth function, or other composite function or curve, or any combination of functions or curves. In fact, it is understood that the function may be substantially stepped, where there may be a more gradual transition between on / off values. Furthermore, it is understood that the irradiance does not necessarily decrease to a value of zero during the off cycle, and may be greater than zero during the off cycle. Desired effects can be achieved by applying light to the cornea according to a curve in which the irradiance varies between two or more values.

[0026] Examples of systems and methods for delivering photoactivating light are described, for example, in U.S. Patent Application Publication No. 2011 / 0237999, filed March 18, 2011, and entitled "Systems and Methods for Applying and Monitoring Eye Therapy," U.S. Patent Application Publication No. 2012 / 0215155, filed April 3, 2012, and entitled "Systems and Methods for Applying and Monitoring Eye Therapy," and U.S. Patent Application Publication No. 2013 / 0245536, filed March 15, 2013, and entitled "Systems and Methods for Corneal Cross-Linking with Pulsed Light," the entire contents of which are incorporated herein by reference.

[0027] The addition of oxygen also affects the amount of corneal stiffening. Human tissue has a very low O2 content compared to the atmosphere. However, the rate of crosslinking in the cornea is related to the O2 concentration at the time of photoactivating light irradiation. Therefore, it may be advantageous to actively increase or decrease the O2 concentration during irradiation to control the rate of crosslinking until the desired amount of crosslinking is achieved. Oxygen can be applied during the crosslinking procedure in a number of different ways. One approach involves supersaturating riboflavin with O2. Thus, when riboflavin is applied to the eye, a higher concentration of O2 is delivered directly into the cornea along with the riboflavin, thereby influencing the O2-mediated reaction when the riboflavin is exposed to photoactivating light. Another approach involves maintaining a steady state of O2 (at a selected concentration) at the surface of the cornea, exposing the cornea to a selected amount of O2, which then enters the cornea. 1 , for example, treatment system 100 also includes an oxygen source 140 and, optionally, an oxygen delivery device 142 that delivers a selected concentration of oxygen to cornea 2. Examples of systems and methods for applying oxygen during cross-linking procedures are described, for example, in U.S. Pat. No. 8,574,277, filed October 21, 2010, and entitled "Eye Therapy," and U.S. Patent Application Publication No. 2013 / 0060187, filed October 31, 2012, and entitled "Systems and Methods for Corneal Cross-Linking with Pulsed Light," the entire contents of which are incorporated herein by reference. Additionally, examples of mask devices for delivering concentrations of oxygen and photoactivating light in ocular treatments are described in U.S. Provisional Patent Application Publication No. 2017 / 0156926, filed December 3, 2016, and entitled "Systems and Methods for Treating an Eye with a Mask Device," the entire contents of which are incorporated herein by reference. For example, a mask may be placed over the eye to create a consistent and known concentration of oxygen on the surface.

[0028] When riboflavin absorbs radiant energy, specifically light, it undergoes photoactivation. There are two photochemical kinetic pathways for photoactivating riboflavin: Type I and Type II. Some of the reactions involved in both the Type I and Type II mechanisms are as follows:

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[0029] In the reactions described herein, Rf represents ground state riboflavin. * 1 represents riboflavin in the excited singlet state. * 3 represents riboflavin in the triplet excited state. ·- is the reduced radical anion form of riboflavin. · is the radical form of riboflavin. RfH2 is the reduced form of riboflavin. DH is the substrate. DH ·+ is the intermediate radical cation. · is a radical. D ox is the oxidized form of the substrate.

[0030] Riboflavin is in its triplet excited state Rf as shown in reactions (r1) to (r3). * 3. Triplet excited state Rf * From 3, riboflavin generally reacts further according to either a Type I or Type II mechanism. In the Type I mechanism, a substrate reacts with excited-state riboflavin to generate a radical or radical ion by hydrogen atom or electron transfer, respectively. In the Type II mechanism, excited-state riboflavin reacts with oxygen to form singlet molecular oxygen. The singlet molecular oxygen then acts in tissues to generate additional cross-linking bonds.

[0031] The oxygen concentration in the cornea is regulated by UVA irradiance and temperature and rapidly decreases at the onset of UVA exposure. By utilizing pulsed light with a specific duty cycle, frequency, and irradiance, inputs from both Type I and Type II photochemical kinetic mechanisms can be used to achieve greater photochemical efficiency. Furthermore, the use of pulsed light allows for control of the rate of the reaction involving riboflavin. The rate of the reaction can be increased or decreased as needed by controlling one of the following parameters: irradiance, dose, on / off duty cycle, riboflavin concentration, immersion time, and others. Furthermore, additional components that affect the rate of reaction and cross-linking may be added to the cornea.

[0032] If UVA radiation is stopped immediately after oxygen depletion, the oxygen concentration will begin to increase (replenish). Excess oxygen can be harmful in the corneal cross-linking process because oxygen can inhibit free radical photopolymerization reactions by interacting with radical species to form chain-terminating peroxide molecules. To achieve a more optimal oxygen regeneration rate, pulse rate, exposure dose, and other parameters can be adjusted. Calculating and adjusting the oxygen regeneration rate is another example of adjusting reaction parameters to achieve the desired amount of corneal hardening.

[0033] Various chemical reactions can deplete the oxygen content throughout the cornea, except in very thin corneal layers where oxygen diffusion can keep up with the rate of reaction. This diffusion-controlled zone will gradually move deeper into the cornea as the reactive capacity of the stroma to take up oxygen decreases.

[0034] As the irradiation dose increases, riboflavin is reversibly or irreversibly reduced (inactivated) and / or photodegraded to greater extents. Photon optimization can be achieved by returning reduced riboflavin to ground-state riboflavin in a Type I reaction. The rate at which reduced riboflavin returns to ground state in a Type I reaction is determined by numerous factors. These factors include, but are not limited to, the on / off duty cycle of the pulsed light treatment, the pulse repetition rate frequency, the irradiation dose, and the dose. Furthermore, riboflavin concentration, immersion time, and the addition of other agents, including oxidizing agents, affect the rate of oxygen uptake. These and other parameters, including the duty cycle, pulse repetition rate frequency, irradiation dose, and dose, can be selected to achieve more optimal photon efficiency and efficiently utilize both Type I and Type II photochemical kinetic mechanisms for riboflavin photosensitization. Furthermore, these parameters can be selected to achieve more optimal chemical amplification effects.

[0035] However, in addition to the above photochemical kinetic reactions (r1)-(r8), the present inventors have identified the following photochemical kinetic reactions (r9)-(r26) that also occur during the photoactivation of riboflavin:

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[0036] Figure 2A shows the photochemical kinetic diagrams provided for the above reactions (r1) to (r26). This diagram summarizes the photochemical transformation of riboflavin (Rf) under UVA photoactivating light and its interaction with various donors (DH) via electron transfer. As shown, (A) reactions (r6) to (r8) are carried out through the presence of singlet oxygen (Type II mechanism); (B) reactions (r4) to (r17) are carried out without the use of oxygen (Type I mechanism); and (C) reactions (r13) to (r17) are carried out through the use of peroxide (HO), superoxide (O), and hydroxylase (H2O). - ), and hydroxyl radicals ( ·Cross-linking activity occurs through the presence of hydroxyl groups (OH).

[0037] As shown in Figure 2A, we also identified that higher cross-linking activity results from reactions involving peroxide, superoxide, and hydroxyl radicals. Lower cross-linking activity results from reactions involving singlet oxygen and from non-oxygen reactions. Several models based on reactions (r1) through (r26) can explain the level of cross-linking activity generated by each reaction. For example, if singlet oxygen plays a smaller role in generating cross-linking activity, the model can be simplified by treating the cross-linking activity resulting from singlet oxygen as a constant.

[0038] As shown in reactions (r1) to (r3), all reactions are Rf3 * Starting from reaction (r10), through chemical reaction with ground state Rf, and reaction (r9), through inactivation by interaction with water, Rf3 * Quenching of occurs.

[0039] As noted above, excess oxygen can be detrimental to the corneal cross-linking process. As shown in Figure 2A, when a system becomes photon-limited and oxygen-rich, cross-linking can be disrupted from further reactions involving superoxide, peroxide, and hydroxyl radicals. In fact, in some cases, excess oxygen can result in net cross-linking disruption relative to cross-linking occurrence.

[0040] As noted above, a wide variety of factors influence the rate of the cross-linking reaction and the amount of biomechanical stiffness achieved by cross-linking. Many of these factors are interrelated, so that changes in one factor can have unexpected effects on another. However, the photochemical kinetic reactions (r1) through (r26) identified above provide a more comprehensive model for understanding the relationships between different factors in the cross-linking process. Thus, systems and methods can adjust various parameters for the cross-linking process according to this photochemical kinetic cross-linking model, thereby providing a unified description of oxygen dynamics and cross-linking activity. The model can be used to evaluate expected outcomes based on various combinations of treatment parameters and identify combinations of treatment parameters that provide desired results. For example, parameters may include, but are not limited to, the concentration and / or immersion time of the applied cross-linking agent; the dose, wavelength, irradiance, duration, and / or on / off duty cycle of the photoactivating light; the oxygenation state of the tissue; and / or the presence of additional agents and solutions.

[0041] As shown in FIG. 2B, the behavior of the reaction system can be affected by various parameters. For example, the irradiance of the photoactivating light delivered to the system determines the Rf3 * The photoactivation light affects the photons available in the system to generate riboflavin. Furthermore, delivering more oxygen to the system drives the oxygen-based reaction. Meanwhile, pulsing the photoactivating light affects the ability to convert reduced riboflavin back to ground state riboflavin by allowing additional time for oxygen diffusion. Of course, other parameters can be varied to control the reaction system.

[0042] Further aspects of the photochemical kinetic reactions provided in Reactions (r1) through (r26) are described in U.S. Patent Application Publication No. 2016 / 0310319, filed April 27, 2016, and entitled "Systems and Methods for Cross-Linking Treatments of an Eye," the entire contents of which are incorporated herein by reference.

[0043] When light of a particular wavelength is applied to a cross-linking agent, such as riboflavin, the light can excite the cross-linking agent and cause it to fluoresce. Thus, the excitation light can be used to cause the cross-linking agent in corneal tissue to fluoresce and determine how the cross-linking agent is distributed in the corneal tissue. When an image of the cornea is taken during application of the excitation light, for example, the intensity (magnitude) of the fluorescence can be measured to determine the amount of cross-linking agent taken up by the corneal tissue, i.e., the dose. Using these principles, a dosimetry system can determine the presence and distribution of a cross-linking agent in the cornea by capturing one or more images of the fluorescence from the cross-linking agent in response to the excitation light. Aspects of such systems are described, for example, in U.S. Patent No. 9,020,580, issued April 28, 2015, and entitled "Systems and Methods for Monitoring Time-Based Photo Active Agent Delivery or Photo Active Marker Presence," and U.S. Patent Application Publication No. 2016 / 0338588, filed May 23, 2016, and entitled "Systems and Methods for Monitoring Cross-Linking Activity for Corneal Treatments," the entire contents of which are incorporated herein by reference. Specifically, U.S. Patent No. 9,020,580 discloses an example dosimetry system that uses a modified Scheimpflug configuration, while U.S. Patent Application Publication No. 2016 / 0338588 discloses the use of hyperspectral imaging to analyze fluorescence.

[0044] Currently available cross-linking treatment systems do not indicate whether sufficient riboflavin is present in the corneal stroma prior to initiating the cross-linking treatment. This can increase surgical variability and lead to suboptimal clinical outcomes. Advantageously, aspects of the present disclosure address this issue by providing a quantitative, depth-resolved measurement of riboflavin concentration that can be compared to pre-defined target values ​​known to provide effective cross-linking activity.

[0045] According to aspects of the present disclosure, embodiments specifically configure a confocal fluorescence microscope to measure riboflavin distribution in the corneal stroma as a function of depth. Thus, the embodiments provide an indication of whether sufficient riboflavin is present at a given corneal stroma depth to proceed with corneal cross-linking. The embodiments may be integrated with the cross-linking treatment systems described herein or may be standalone measurement systems. The embodiments are also suitable for measuring fluorescence induced by photoactivating UV irradiation applied during the cross-linking treatment, thereby enabling the progress of cross-linking activity to be measured in real time.

[0046] According to aspects of the present invention, the systems and methods can achieve one or more of the following: 1. Measure the time evolution of the depth profile of riboflavin in the corneal stroma at selected sites in order to initiate the cross-linking procedure at a more effective time. 2. Determine the three-dimensional (3D) distribution of riboflavin in the corneal stroma as a function of time. 3. Measure the depth profile of cross-linking concentration at the treated site to keep the treatment procedure under control. 4. Reconstruct the 3D distribution of cross-linking concentrations after treatment. 5. Locate the opacified area in the corneal stroma.

[0047] To achieve the foregoing, the systems and methods may employ aspects of one or more of the following techniques: 1. Fluorescence microscopy. 2. Confocal microscopy. 3. Scheimpflug photography. 4. 3D reconstruction, image deconvolution, image registration, and other image processing techniques.

[0048] Existing confocal fluorescence microscopes are not suitable for commercial use on living human corneas because they require fluid immersion / contact objectives, are highly complex and expensive, and use high laser intensities. Advantageously, aspects of the present disclosure address this issue by simplifying the measurement system so that it is optimized to provide an indication of the presence of sufficient riboflavin.

[0049] To illustrate aspects of the present disclosure, FIG. 3 shows an example of a system 300 for measuring the concentration of an exogenous cross-linking agent, such as riboflavin, applied to the cornea 2 as a function of depth. The system 300 includes a laser or LED light source 302 that emits light to excite the cross-linking agent in the cornea 2. For example, the light source 302 can emit UV excitation light, e.g., having a wavelength of 365 nm. Alternatively, the light source 302 can emit blue excitation light, e.g., having a wavelength of 458 nm; in this case, the light source 302 may optionally use white light in combination with a blue light narrow-band filter.

[0050] As shown in FIG. 3 , excitation light from a light source 302 is directed through an opening in a light source pinhole structure 304 and a collimating lens 306. In some cases, the light source 302 may be fiber-coupled. The excitation light is then directed to a dichroic filter (or mirror) 308. Depending on the wavelength of the excitation light, the dichroic filter 308 reflects the excitation light toward a lens 310, e.g., an objective lens or similar optical element. The lens 310 focuses the excitation light onto a region of the cornea 2 at a given depth along the z-axis. The transverse (xy-plane) resolution of the excitation light can be greater than 10 μm, e.g., from about 10 μm to about 200 μm. In contrast, a typical confocal microscope requires a transverse resolution of less than 10 μm.

[0051] In response to the excitation light, the cross-linking agent in the cornea 2 fluoresces. For example, riboflavin in the cornea 2 can emit green fluorescence. The fluorescent emission travels through lens 310 to dichroic filter 308. In contrast to reflecting light having the wavelength of the excitation light, dichroic filter 308 passes light having the fluorescence wavelength to color filter 312. Color filter 312 transmits the fluorescent emission to lens 314, e.g., an imaging lens, while blocking residual excitation light and / or other light not having the wavelength of the fluorescent emission. Lens 314 focuses the fluorescent emission onto detector 318 through the opening of detector pinhole 316. Detector pinhole structure 316 prevents light originating above or below a given corneal depth from reaching detector 318. In other words, detector pinhole structure 316 prevents out-of-focus light from reaching detector 318. The pinhole structure 316 may be configured to allow for a lower resolution along the z-axis of greater than 10 μm, for example, from about 10 μm to about 100 μm. In contrast, conventional confocal microscopes require a resolution of less than 10 μm. Typically, the system 300 can provide a working distance in air of greater than 10 mm and does not require fluid immersion or physical contact with the eye.

[0052] Detector 318 may be a photodiode, a photomultiplier tube, or a camera. The image plane of detector 318 is parallel to the lens plane of lens 314 and the region of excited corneal tissue along the xy plane. This configuration ensures that the region of excited corneal tissue is uniformly focused on detector 318. Similarly, detector pinhole structure 316 is parallel to the image plane of detector 318.

[0053] The detector 318 is used to quantify the amount, e.g., intensity, of fluorescence emitted from the excited region of the cornea at a given depth. The system 300 can be operated in stages to deliver excitation light to various respective depths of the cornea 2 and detect the amount of fluorescence from the cross-linking agent at each depth. As shown in FIG. 3 , the system 300 includes a scanning mechanism 320 that causes the system 300 to scan the cornea 2 at various depths along the z-axis. For example, the scanning mechanism 320 can be a mechanical or electromechanical device that moves or manipulates the lens 310 and / or other elements of the system 300 to adjust the delivery of excitation light to other depths. According to one implementation, the system 300 can be operated to first excite a region at the anterior surface 2a of the cornea, and then subsequently excite regions at a series of depths below the anterior surface 2a up to at least about 200 μm into the corneal stroma 2b. The distance between two consecutive depths for delivery of excitation light can range from about 10 μm to about 100 μm.

[0054] The amount of fluorescence detected at a given depth indicates the amount of cross-linking agent at that depth. Thus, system 300 can be used to determine whether a sufficient amount of cross-linking agent is present to initiate the cross-linking procedure. A certain amount of cross-linking agent may be required to exceed a predetermined threshold at a given depth or series of depths before the cross-linking procedure can proceed.

[0055] The time series signals from detector 318 may be sampled frequently and synchronized with a scanning mechanism 310 that varies the scan depth by moving one or more elements of system 300. Information from detector 318 can be used to reconstruct a riboflavin concentration curve along the z-axis. For example, the time series signals from detector 318 can be processed to calculate (1) the location of the posterior surface 2c of the cornea 2, (2) the riboflavin concentration as a function of depth into the cornea 2, (3) the anterior surface 2a of the cornea, (4) the distance between the posterior surface 2c and the anterior surface 2a, and (5) the location of a subcorneal interface, such as the epithelial-stromal interface.

[0056] System 300 may include auxiliary optics, such as an imaging camera and alignment lasers, to assist in aligning system 300 to the desired x, y, z position to begin a scan. Additionally, several scans may be averaged together to increase the signal-to-noise ratio.

[0057] Additionally, data processing algorithms can be used to detect incomplete scans. For example, when a scan captures fluorescent emissions from the anterior surface of the cornea 2, the information from detector 318 will show a sudden increase in the fluorescent signal followed by a gradual decrease in the fluorescent signal as the scan moves from the anterior surface 2a deeper into the corneal stroma 2b. If the information from detector 318 does not show a sudden increase in the fluorescent signal, the scan can be considered incomplete, perhaps because it did not capture the anterior surface 2a.

[0058] System 300 can provide other useful information for improving aspects of the cross-linking procedure. For example, system 300 can be used periodically or continuously to monitor the amount of fluorescence emitted by the cross-linking agent as the cross-linking procedure progresses. Specifically, repeated scans over time can indicate when cross-linking activity has progressed to a desired stromal depth. Furthermore, the position of the posterior surface of the cornea as detected by system 300 can be used to adjust the treatment plane of the cross-linking procedure system.

[0059] Generally, the embodiment includes an illumination path and an imaging path. The illumination path directs point illumination to the cornea 2, and the imaging path collects fluorescent emission resulting from excitation by the point illumination. Each illumination and imaging path can use an on-axis or off-axis configuration. The system 300 shown in FIG. 3 is an on-axis optical system. The system 300 uses an on-axis illumination path 300a, in which the region of excited corneal tissue along the xy plane is perpendicular to the illumination path 300a for the excitation light. The system 300 also uses an on-axis imaging path 300b, in which the region of excited corneal tissue along the xy plane is perpendicular to the imaging path 300b for the fluorescent emission.

[0060] In embodiments in which both the illumination path and the imaging path are on-axis, the illumination path and the imaging path can be on either the reflection side or the transmission side of a dichroic mirror. As shown in FIG. 3 , for example, illumination path 300a is on the reflection side of dichroic filter 308, while imaging path 300b is on the transmission side of dichroic filter 308. Specifically, excitation light from light source 302 is directed to dichroic filter 308, which reflects the excitation light, e.g., 90°, toward cornea 2 based on the wavelength of the excitation light. Dichroic filter 308 passes fluorescent emission toward detector 318 based on the wavelength of the fluorescent emission.

[0061] 4, on the other hand, illustrates an example of a system 400 similar to system 300, but including an illumination path 400a disposed on the transmission side of a dichroic mirror 408 and an imaging path 400b disposed on the reflection side of the dichroic mirror 408. Specifically, excitation light from light source 302 is directed to dichroic filter 408, which passes the excitation light toward cornea 2 based on the wavelength of the excitation light. Meanwhile, dichroic filter 408 reflects the fluorescent emission toward detector 318, e.g., by 90°, based on the wavelength of the fluorescent emission.

[0062] In contrast to the above-described examples of systems 300 and 400, FIG. 5 illustrates an example of a system 500 that uses an on-axis illumination path 500a and an off-axis imaging path 500b. A light source 502 emits light to excite a cross-linking agent in the cornea 2. The excitation light from the light source 502 is directed through an opening in the light source's pinhole structure 504 to a lens 510, such as an objective lens or similar optical element. The lens 510 focuses the excitation light onto a region of the cornea 2 along the xy plane at a given depth along the z-axis. As shown in FIG. 5, the region of excited corneal tissue along the xy plane is perpendicular to the illumination path 500a for the excitation light. Therefore, the illumination path 500b is considered to be on-axis.

[0063] In response to the excitation light, the cross-linking agent in the cornea 2 emits fluorescence. The fluorescent emission travels through lens 514 to color filter 512. Lens 514 focuses the fluorescent emission onto detector 518. Color filter 512 blocks residual excitation light and / or other light that does not have the wavelength of the fluorescent emission. The fluorescent emission passes through an opening in the detector's pinhole structure 516a (solid line) or 516b (dashed line), thereby preventing light originating above or below a given corneal depth from reaching detector 318. Detector 518 may be a photodiode, photomultiplier tube, or camera. Detector 518 can be used to quantify the amount, e.g., intensity, of fluorescence emitted from an excited region of the cornea at a given depth.

[0064] The region of excited corneal tissue along the xy plane is not perpendicular to the imaging path 500b for fluorescence emission. In other words, the imaging path 500b extends from the xy plane at a given depth at an angle that is not equal to 90°. Therefore, the imaging path 500b is considered to be off-axis.

[0065] According to one embodiment, the image plane of detector 518 is parallel to the lens plane of lens 514. As shown in Figure 4, the detector pinhole structure 516a is correspondingly parallel to the image plane and the lens plane, i.e., perpendicular to imaging path 500b.

[0066] According to another embodiment, the image plane of detector 518 is not parallel to the lens plane of lens 514. Instead, detector 518 and lens 514 are arranged such that the image plane and lens plane are in a Scheimpflug configuration. In this configuration, the excited region of the corneal tissue along the xy plane is more uniformly focused at detector 518, even though imaging path 500b is not perpendicular to the excited region of the corneal tissue. Detector pinhole structure 516b is correspondingly angled relative to the image plane and lens plane, e.g., parallel to the xy plane, to prevent light originating above or below a given corneal depth from reaching detector 518.

[0067] 6 shows an example of a system 600 that uses an off-axis illumination path 600a and an off-axis imaging path 600b. A light source 602 emits light to excite a cross-linking agent in the cornea 2. The excitation light from the light source 602 passes through an opening in the light source's pinhole structure 604a (solid line) or 604b (dashed line) and is then directed to a lens 610, such as an objective lens or similar optical element. The lens 610 focuses the excitation light onto a region of the cornea 2 along the xy plane at a given depth along the z axis.

[0068] The region of excited corneal tissue along the xy plane is not perpendicular to the illumination path 600a for the excitation light. In other words, the illumination path 600a extends into the xy plane at a given depth at an angle that is not equal to 90°. Therefore, the illumination path 600a is considered to be off-axis.

[0069] In response to the excitation light, the cross-linking agent in the cornea 2 emits fluorescence. The fluorescent emission travels through a lens 614 to a color filter 612. The lens 614 focuses the fluorescent emission onto a detector 618. The color filter 612 blocks residual excitation light and / or other light that does not have the wavelength of the fluorescent emission. The fluorescent emission passes through an opening in the detector's pinhole structure 616a (solid line) or 616b (dashed line), thereby preventing light originating from above or below a given corneal depth from reaching the detector 618. The detector 618 may be a photodiode, a photomultiplier tube, or a camera. The detector 618 can be used to quantify the amount, e.g., intensity, of fluorescence emitted from an excited region of the cornea at a given depth.

[0070] The region of excited corneal tissue along the xy plane is not perpendicular to the imaging path 600b for fluorescence emission. In other words, the imaging path 600b extends from the xy plane at a given depth at an angle that is not equal to 90°. Therefore, the imaging path 600b is considered to be off-axis.

[0071] According to one embodiment, the image plane of detector 618 is parallel to the lens plane of lens 614. As shown in Figure 6, the detector pinhole structure 616a is also parallel to the image plane and the lens plane, and is perpendicular to imaging path 600b. Correspondingly, the source pinhole structure 604a is also perpendicular to illumination path 600a.

[0072] According to another embodiment, the image plane of detector 618 is not parallel to the lens plane of lens 614. Instead, detector 618 and lens 614 are positioned such that the image plane and lens plane are in a Scheimpflug configuration. In this configuration, the excited region of corneal tissue along the xy plane is more uniformly focused at detector 618, even though imaging path 600b is not perpendicular to the excited region of corneal tissue. Detector pinhole structure 616b is angled relative to the image plane and lens plane and is not perpendicular to imaging path 600b. Correspondingly, light source 602 and lens 610 are positioned such that the light source plane and lens plane are in a Scheimpflug configuration. Furthermore, light source pinhole structure 604b is angled relative to the light source plane and lens plane and is not perpendicular to illumination path 600a. In this configuration, excitation light is more uniformly focused to the excited region of corneal tissue along the xy plane. The source pinhole structure 604b and the detector pinhole structure 616b may be parallel to the xy plane.

[0073] Although the above example system may use a pinhole aperture to create a single-point illumination, a pinhole array or slit illumination may alternatively be used. When the example system is incorporated into a cross-linking treatment system as described above, slit illumination can be generated via a DMD. While slit illumination may be used for illumination, a pinhole aperture or vertical slit in the imaging path may be sufficient to collect fluorescent emission.

[0074] For example, as shown in Figure 6, if the illumination path uses a Scheimpflug configuration, the dot array can be created using a mask, grating, acousto-optical component, diffractive optical element (DOE), etc. The dot array is aligned along the z-axis and has a separation greater than the axial resolution of the imaging path.

[0075] In the example systems 500, 600, fluorescent emissions are collected from a smaller corneal area defined by the overlap between the illumination and imaging paths. Thus, a smaller volume of corneal tissue can be interrogated by the systems 500, 600. Advantageously, this allows for relaxed optical requirements or constraints on the pinhole configuration and the numerical aperture of the respective lenses, which in turn allows for a larger working distance between the systems 500, 600 and the subject.

[0076] Relative movement, i.e., scanning, between the eye and example systems 300, 400, 500, or 600 allows the system to generate and measure fluorescent emissions at various depths along the z-axis. Example systems may scan only along the z-axis, rather than along the x- and y-axes. Scanning along the z-axis can be achieved according to various approaches. According to one approach, aspects of the system can be actively moved along the z-axis, while the subject's eye remains at fixed x, y, and z positions. The entire system or specific components can be moved along the z-axis by motors or the like.

[0077] According to another approach, the system can scan along the z-axis by actively moving the subject's eyes at high frequency: In this approach, the subject's head can be placed in a chair that can be moved up and down along the z-axis over a small range at high frequency, while the system remains in a fixed x, y, z position.

[0078] To illustrate further aspects of the present disclosure, FIG. 7 shows an example of a system 700 for detecting fluorescence associated with the distribution of a cross-linking agent, e.g., riboflavin, in the cornea 2. The system 700 includes an illumination path 700a that projects a slit light pattern 10 onto tissue, e.g., the corneal stroma, of the cornea 2 via a slit lamp. For example, to generate the light pattern 10, the system may use a light source 702 that directs excitation light to at least a collector lens 704, a slit 706, and an objective lens 708 configured according to the principles of Kohler illumination. As shown in FIG. 7 , the light source 702 may include a filament, and using the Kohler configuration, the filament is imaged onto the objective lens 708, while the slit 706 is imaged at the cornea 2.

[0079] The x-, y-, and z-axes shown in FIG. 7 define an object space corresponding to the light pattern 10 received by the cornea 2. The light pattern 10 extends from front to back along the xz plane. The cross-linking agent exposed to the light pattern 10 at the cornea 2 is excited and emits fluorescence. The system 700 includes an imaging path 700b, which includes at least a Scheimpflug lens 710. The Scheimpflug lens 710 transmits the fluorescent emission to a detector 712. A magnified image 10' of the pattern 10 is produced in the image plane of the detector 712. The x', y', and z' axes shown in FIG. 7 define an image space corresponding to the image 10'. A bandpass filter may be used in the imaging path to attenuate the excitation light and other stray light.

[0080] In one embodiment, system 700 uses a long, movable slit aperture 714a that can be scanned along the z' axis in the image plane. The aperture 714a is scanned across an array of pixels, e.g., a CCD array, that correspond to fluorescent emissions. Scanning the aperture along the z' axis over an appropriate range creates a fluorescent intensity profile at the cornea 2. This profile is recorded in the z direction (direction of corneal depth) in object space and then averaged in the x direction over the slit length (mapped to object space).

[0081] The example system 700 provides rapid acquisition of depth profiles due to the relatively large aperture size. However, the profiles are not resolved laterally, i.e., along the x-axis. If a particular spatial resolution in the lateral direction is desired, an alternative embodiment may use a lenticular array of pinhole apertures 714b rather than a slit aperture 714a. A single scan of the pinhole aperture array 714b along the z' axis produces a two-dimensional profile of fluorescence intensity that is spatially resolved in both the z direction (direction of corneal depth) and the x direction (lateral direction). The aperture size (width of the slit aperture, diameter of the pinhole aperture) may be selected to balance acquisition speed and spatial resolution.

[0082] FIG. 8 shows another example embodiment of a system 800 for acquiring a three-dimensional profile of fluorescence emission due to the response of a cross-linking agent in the cornea 2 to excitation light. The system 800 includes an illumination path 800a for providing excitation light. The illumination path 800a can be fabricated, for example, according to any of the above-described embodiments. To capture the fluorescence emission, the system 800 includes a first imaging path 800b and a second imaging path 800c. The system 800 rotates the illumination path 800a and the first and second imaging paths 800b, 800c 180 degrees around the eye to create a data set of images, which provides a three-dimensional profile. The three-dimensional profile indicates the presence of the cross-linking agent in a volume of corneal tissue. This can include several steps, for example, as follows: 1. Deconvolution - removes the blurring effect associated with finite aperture sizes. 2. Correct for the variable magnification of the Scheimpflug lens across the field of view. 3. Refractive correction - removes image distortion caused by refraction of light at the anterior surface of the cornea. 4. Smoothing - removes high spatial frequency components in the image due to shot noise (a small number of photons per pixel) and diffraction patterns. 5. Correct for eye movements during image recording. Suitable calibration procedures can be developed for image distortion correction.

[0083] According to aspects of the invention, embodiments may include a two-channel design including an illumination and detection channel, for example as shown in FIG.

[0084] An embodiment may include an illumination channel that provides a collimated slit or pixel beam at the stroma, for example as shown in FIG.

[0085] An implementation may include an eye-tracking channel.

[0086] An embodiment may include an illumination channel that is part of the eye-tracking optics (eg, tracking the corneal apex using Purkinje images).

[0087] An embodiment may include an illumination channel that may include one of the following: a commercially available or OEM slit lamp, or pixel projection optics.

[0088] Embodiments may include an illumination beam that acts as a source of stray light propagating in various directions. Physical mechanisms of stray light generation may include riboflavin fluorescence, cross-linking fluorescence, and / or bulk scattering in the corneal stroma.

[0089] An embodiment may include a detection channel that picks up stray light that moves light rays through a high numerical aperture (NA) lens to the image plane, for example, as shown in Figure 7. The object plane of the lens (conjugated to the image plane) is aligned with the illumination beam at the corneal stroma using the Scheimpflug principle, for example, as shown in Figure 7. Using the Scheimpflug principle, it is possible to image an illuminated slit or other pattern between the anterior and posterior surfaces of the cornea at a convenient angle, for example, 45°, as shown in Figure 7.

[0090] The thickness of the optical slices contributing to the image can be significantly reduced by using high NA lenses and the confocal principle. The latter principle involves scanning a small aperture across the image plane while recording the image. In some embodiments, a CCD array may be used, in which pixels are switched on in a specific order until the entire area is covered. The pixel switching order must be selected to minimize its (electrical or optical) crosstalk.

[0091] According to a further aspect of the present disclosure, embodiments can be used to provide a high-resolution, non-contact corneal pachymeter. As described above, embodiments can quantify the amount, e.g., intensity, of fluorescence emitted by a cross-linking agent in the cornea at various depths in response to excitation light. For example, as shown in FIG. 3 , system 300 can scan cornea 2 at various depths along the z-axis to measure the concentration of cross-linking agent as a function of corneal depth. Such a scan can be sufficient to assess the entire thickness of the cornea. Specifically, the scan can extend from at least the interface between the ambient air and the cornea (i.e., the anterior surface of the cornea) to the interface between the endothelium and the anterior chamber (i.e., the posterior surface of the cornea). As also described above, when the detector of an embodiment captures fluorescent emission from the anterior surface of the cornea, the fluorescence signal increases sharply, followed by a gradual decrease as the scan moves deeper from the anterior surface into the corneal stroma. When the scan reaches the interface between the endothelium and the anterior chamber (the posterior surface), the fluorescence signal abruptly decreases to zero. By measuring the distance between the depths at which a sudden increase (signal peak) and a sudden decrease (to zero) in the fluorescent signal are detected, the thickness of the cornea can be determined. Thus, in addition to providing information about the presence of a cross-linking agent in the cornea, embodiments can provide a measurement of corneal thickness, which is typically required before a cross-linking procedure to ensure endothelial safety.

[0092] To reduce corneal thickness measurement noise and obtain more accurate measurements, multiple scans may be taken at various transverse locations across the cornea (e.g., at various (x,y) locations as shown in FIG. 3) and through the corneal thickness (e.g., along the z-axis as shown in FIG. 3). Measurements obtained from these multiple scans may then be averaged. For example, to assess central and / or peripheral corneal thickness, a pachymetry map may be created that covers a predetermined or user-selected set of transverse locations. For example, a pachymetry map may be created over a section relevant to keratoconus.

[0093] Using aspects of confocal fluorescence microscopy, embodiments can achieve high-resolution (e.g., less than 100 μm) and rapid (e.g., less than 1 second) measurements of corneal thickness. Furthermore, such measurements can be made without physical contact with the cornea, thereby enhancing subject safety and comfort. Furthermore, this approach to corneal thickness measurement is insensitive to variations in corneal density, hydration state, and refractive properties. This approach is also insensitive to the presence of opacities sometimes associated with the eye, particularly in diseased eyes. Thus, this approach can provide a more accurate measurement of corneal thickness, as opposed to, for example, ultrasound-based approaches that rely on a single nominal velocity of sound to measure corneal thickness.

[0094] Rather than detecting a signal based on fluorescence emission from a cross-linking agent in the cornea in response to excitation light, another implementation of the above embodiment can determine corneal thickness by detecting a signal based on incident light reflected from the cornea where no cross-linking agent is present. The reflected light from the scan through the cornea provides a signal indicative of the presence of an anterior corneal surface and a posterior corneal surface. Specifically, signal spikes or other signal changes can characterize the anterior and posterior corneal surfaces. Advantageously, this another implementation can provide a reliable measurement of corneal thickness regardless of the presence of any cross-linking agent in the cornea.

[0095] In summary, FIG. 9 generally illustrates an example approach 900 for measuring corneal thickness via an embodiment using aspects of a confocal microscope. In act 902, light is directed to the cornea through various corneal depths (z) via a configured confocal microscope system for one or more locations (x, y) across the cornea. In act 904, fluorescence emission from a cross-linking agent in the cornea or reflected light from the cornea in the absence of a cross-linking agent is detected from various corneal depths. In act 906, a first signal associated with the anterior surface of the cornea and a corresponding first depth are detected from the fluorescence emission / reflected light. In act 908, a second signal associated with the posterior surface of the cornea and a corresponding second depth are detected from the fluorescence emission / reflected light. In act 910, the corneal thickness is determined from the distance between the first depth and the second depth. In optional act 912, if there is more than one position (x,y) across the cornea, the corneal thickness obtained from the more than one position is averaged to obtain a more accurate measurement.

[0096] As noted above, according to some aspects of the present disclosure, some or all of the steps of the procedures described above and illustrated may be automated or led under the control of a controller (e.g., controller 120). Generally, the controller may be implemented as a combination of hardware and software elements. Hardware aspects may include a combination of operatively connected hardware components, including a microprocessor, logic circuitry, communication / networking ports, digital filters, memory, or logic circuitry. The controller may be adapted to perform operations specified by computer-executable code, which may be stored on a computer-readable medium.

[0097] As noted above, the controller may be a programmable processing device that executes software or stored instructions, such as a conventional external computer or an on-board field programmable gate array (FPGA) or digital signal processor (DSP). Generally, the physical processor and / or machine used by the embodiments of the present disclosure for any processing or evaluation may include one or more networked or non-networked general-purpose computer systems, microprocessors, field programmable gate arrays (FPGAs), digital signal processors (DSPs), microcontrollers, etc., programmed according to the teachings of the exemplary embodiments of the present disclosure, as will be understood by those skilled in the computer and software arts. The physical processor and / or machine may be externally networked with the image capture device or may be embedded so as to reside within the image capture device. As will be understood by those skilled in the software arts, appropriate software can be readily created by a programmer of ordinary skill based on the teachings of the exemplary embodiments. Furthermore, as will be understood by those skilled in the electrical arts, the devices and subsystems of the exemplary embodiments can be implemented by creating application-specific integrated circuits or by interconnecting an appropriate network of conventional component circuits. Thus, example implementations are not limited to any specific combination of hardware circuitry and / or software.

[0098] Example embodiments of the present disclosure stored on any one or combination of computer-readable media may include software for controlling the devices and subsystems of the example embodiments, for driving the devices and subsystems of the example embodiments, for interacting with the devices and subsystems of the example embodiments with a human user, and the like. Such software may include, but is not limited to, device drivers, firmware, operating systems, development tools, application software, and the like. Such computer-readable media may also include computer program products of example embodiments of the present disclosure for performing all or a portion (if the processing is distributed) of the processing performed in execution. Computer code devices of example embodiments of the present disclosure may include any suitable interpretable or executable code mechanism, including, but not limited to, scripts, interpretable programs, dynamic link libraries (DLLs), Java classes and applets, fully executable programs, and the like. Furthermore, portions of the processing of example embodiments of the present disclosure may be distributed for better performance, reliability, cost, and the like.

[0099] Common forms of computer-readable media may include, for example, a floppy disk, a flexible disk, a hard disk, magnetic tape, any other suitable magnetic media, a CD-ROM, a CDRW, a DVD, any other suitable optical media, punch cards, paper tape, optical mark sheets, any other suitable physical media with a pattern of holes or other optically recognizable indicia, RAM, PROM, EPROM, FLASH-EPROM, any other suitable memory chip or cartridge, a carrier wave, or any other suitable medium from which a computer can read.

[0100] While the present disclosure has been described with reference to one or more specific embodiments, those skilled in the art will recognize that many modifications can be made to the present disclosure without departing from the spirit and scope of the present disclosure. Each of these embodiments and obvious variations thereof is considered to be within the spirit and scope of the present disclosure. It is also contemplated that further embodiments according to aspects of the present disclosure may be combined with any number of features of any of the embodiments described herein.

Claims

1. 1. A corneal measurement system comprising: a light source configured to emit excitation light that causes a fluorescent agent applied to the cornea to fluoresce; at least one optical element positioned to receive the excitation light from the light source and configured to focus the excitation light onto a region of corneal tissue at a selected depth of the cornea, wherein the fluorescent agent in the cornea produces the fluorescent emission in response to the excitation light; a pinhole structure including an opening positioned to receive the fluorescent emission from the fluorescent agent at the cornea, the opening configured to selectively transmit the fluorescent emission from a region of the corneal tissue at the selected depth; a detector positioned to capture selected fluorescent emissions transmitted by the aperture, the detector configured to communicate information regarding a measurement of the selected fluorescent emissions captured by the detector; a controller communicatively connected to the detector and configured to receive the information from the detector and determine, based on the information, a measurement of the fluorescent agent in the region of the corneal tissue at the selected depth; A system including:

2. 2. The measurement system of claim 1, wherein the measurements of the fluorescent agent are determined according to a resolution of better than 10 μm along an axis corresponding to a depth of the corneal tissue and / or along a plane transverse to said axis.

3. a scanning mechanism configured to cause the at least one optical element to scan the cornea at a plurality of depths and to focus the excitation light on a respective region of corneal tissue at each depth; For each depth, (i) the openings of the pinhole structure are configured to selectively transmit the fluorescent emissions from respective regions of the corneal tissue; and (ii) the detector is configured to capture the selected fluorescent emissions transmitted by the aperture and to communicate information regarding a measurement of the selected fluorescent emissions captured by the detector; and 2. The measurement system of claim 1, wherein the controller is configured to receive the information from the detector for each depth and determine a measurement of the fluorescent agent in the cornea as a function of depth based on the information for the plurality of depths.

4. 3. The measurement system of claim 2, wherein the plurality of depths range from an anterior surface of the cornea to a posterior surface of the cornea, and the controller is further configured to determine at least one of a position of the posterior surface, a distance between the anterior and posterior surfaces, or a position of an interface between sections of the cornea based on the information about the plurality of depths.

5. 4. The measurement system of claim 3, wherein the controller evaluates changes in the measurements of the fluorescent agent in the cornea across the multiple depths to determine at least one of the position of the posterior surface, the distance between the anterior and posterior surfaces, or the position of an interface between sections of the cornea, the changes corresponding to structural features of the cornea.

6. 3. The measurement system of claim 2, wherein the controller is further configured to detect an incomplete scan of the cornea by evaluating changes in the measurements of the fluorescent agent in the cornea across the multiple depths, the changes corresponding to structural features of the cornea.

7. 3. The measurement system of claim 2, wherein the scanning mechanism is configured to cause the at least one optical element to scan the cornea more than once at the plurality of depths, and the controller is further configured to determine the amount of the fluorescent agent in the cornea as a function of depth based on information from the more than one scan.

8. 3. The measurement system of claim 2, wherein the plurality of depths extend from the anterior surface of the cornea to at least about 200 μm into the cornea, and adjacent depths in the plurality of depths are spaced apart by about 10 μm to about 100 μm.

9. the scanning mechanism is further configured to operate the at least one optical element to scan the cornea at a plurality of transverse positions across each depth and to focus the excitation light onto a respective region of corneal tissue at each transverse position at each depth; For each transverse position at each depth, (i) the openings of the pinhole structure are configured to selectively transmit the fluorescent emissions from respective regions of the corneal tissue; and (ii) the detector is configured to capture the selected fluorescent emissions transmitted by the aperture and to communicate information regarding a measurement of the selected fluorescent emissions captured by the detector; and 3. The measurement system of claim 2, wherein the controller is configured to receive the information from the detector for each transverse position and determine measurements of the fluorescent agent in the cornea as a function of transverse position and depth based on the information for the multiple transverse positions at the multiple depths.

10. 9. The measurement system of claim 8, wherein the plurality of depths range from an anterior surface of the cornea to a posterior surface of the cornea, and the controller is further configured to determine at least one of a position of the posterior surface, a distance between the anterior and posterior surfaces, or a position of an interface between sections of the cornea based on the information about the plurality of transverse positions at the plurality of depths.

11. 2. The measurement system of claim 1, wherein the fluorescent agent is a cross-linking agent, and the controller is further configured to communicate that a measurement value of the cross-linking agent in the cornea at the selected depth of the cornea meets a threshold value for initiating a cross-linking procedure of the cornea.

12. 1. A corneal measurement system comprising: a light source configured to emit incident light; at least one optical element positioned to receive the incident light from the light source and configured to focus the incident light onto an area of ​​corneal tissue at a selected depth of the cornea, the area of ​​corneal tissue reflecting the incident light; a scanning mechanism configured to cause the at least one optical element to scan the cornea at a plurality of depths and to focus the incident light on a respective region of corneal tissue at each depth, the plurality of depths ranging from an anterior surface of the cornea to a posterior surface of the cornea; a pinhole structure including an opening positioned to receive reflected light from a respective region of the corneal tissue at each depth, the opening of the pinhole structure configured to selectively transmit the reflected light from a respective region of the corneal tissue at each depth; a detector positioned to capture selected reflected light transmitted by the opening for each depth, the detector configured to communicate information regarding a measurement of the selected reflected light captured by the detector for each depth; a controller communicatively connected to the detector and configured to receive the information from the detector for each depth and determine at least one of a position of the posterior surface, a distance between the anterior surface and the posterior surface, or a position of a substratum corneum interface based on the information for the plurality of depths; A system including:

13. 13. The measurement system of claim 12, wherein the measurements of the selected reflected light are determined according to a resolution of better than 10 μm along an axis corresponding to a depth of the corneal tissue and / or along a plane transverse to said axis.

14. 13. The measurement system of claim 12, wherein the controller evaluates changes in the measurements of the reflected light across the multiple depths to determine at least one of the position of the posterior surface, the distance between the anterior and posterior surfaces, or the position of an interface between sections of the cornea, the changes corresponding to structural features of the cornea.

15. 13. The measurement system of claim 12, wherein the controller is further configured to detect an incomplete scan of the cornea by evaluating changes in the measurements of the reflected light at the cornea across the multiple depths, the changes corresponding to structural features of the cornea.

16. 13. The measurement system of claim 12, wherein the scanning mechanism is configured to cause the measurement system to scan the cornea more than once at multiple depths, and the controller is further configured to determine at least one of the position of the posterior surface, the distance between the anterior surface and the posterior surface, or the position of the substratum corneum interface based on information from the more than one scan.

17. the scanning mechanism is further configured to cause the at least one optical element to scan the cornea at a plurality of transverse positions along each depth and to focus the incident light onto a respective region of corneal tissue at each transverse position at each depth; For each transverse position along each depth, (i) the openings of the pinhole structure are configured to selectively transmit the reflected light from respective regions of the corneal tissue; and (ii) the detector is configured to capture the selected reflected light transmitted by the aperture and to communicate information regarding a measurement of the selected reflected light captured by the detector; and 13. The measurement system of claim 12, wherein the controller is configured to receive the information from the detector for each transverse position and determine at least one of the position of the posterior surface, the distance between the anterior surface and the posterior surface, or the position of the substratum corneum interface based on the information for multiple transverse positions at the multiple depths.

18. 1. A corneal measurement system comprising: a light source configured to emit excitation light that causes a fluorescent agent applied to the cornea to fluoresce; at least one optical element positioned to receive the excitation light from the light source and configured to deliver the excitation light, wherein the excitation light penetrates multiple depths of the cornea and the fluorescent agent in the cornea produces the fluorescent emission in response to the excitation light; a detector positioned to capture an image of the fluorescent emission from the cornea; a controller communicatively connected to the detector and configured to receive the image from the detector, scan the image to measure the fluorescent emission at the plurality of depths, and determine the measurement of the fluorescent agent in the cornea as a function of depth based on the measurements of the fluorescent emission at the plurality of depths; A system including:

19. The measurement system of claim 18 , wherein the controller scans the image with a slit aperture that scans an array of pixels along the multiple depths.

20. 20. The measurement system of claim 18, wherein the excitation light further spreads in at least one lateral direction as it penetrates the multiple depths of the cornea, and the controller is further configured to scan the image to measure the fluorescence emission along the at least one lateral direction at the multiple depths, and to determine measurements of the fluorescent agent in the cornea as a function of depth and lateral position based on the measurements of the fluorescence emission along at least one transverse direction at the multiple depths, and wherein the controller scans the image with a lenticular array of pinhole apertures that scans an array of lateral pixels along the multiple depths through the cornea.

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