Collagen is an abundant protein in animals. The mechanical properties and structural stability of collagen based tissues, such as corneal tissue, can be influenced by increasing collagen cross-links (CXL), in the form of intra- or inter-molecule chemical bonds.
An aspect of the invention provides a method of altering curvature of a cornea. The method includes receiving one or more measurements of topography of a cornea, calculating a pattern defining locations and amounts of cross-linking required to achieve a desired level of vision correction, wherein the amounts of cross-linking are, at least in part, a function of a number of overlapping treatment layers having different z depths at a given coordinate, and controlling a light source to apply light energy pulses to the cornea to cross-link collagen in accordance with the pattern.
This aspect of the invention can have a variety of embodiments. In some embodiments, the method may include receiving one or more measurements of thickness of the cornea, wherein the amounts of cross-linking are also, at least in part, a function of the thickness of the cornea.
In some embodiments, multiple treatment layers are defined based on their respective z depths measured relative to the surface of the cornea, and a subset of the defined treatment layers is treated to achieve a desired effect in the cornea. In some embodiments, the subset of the defined treatment layers includes layers that are adjacent to each other in the z direction. In some embodiments, the subset of the defined layers includes layers that are not adjacent to each other but are instead spaced apart to achieve a desired effect in the cornea.
In some embodiments, the light energy pulses are applied in the absence of an exogenous photosensitizer.
In some embodiments, the light energy pulses ionize water molecules within the cornea to generate reactive oxygen species.
In some embodiments, the light energy pulses have a wavelength that is not absorbed by amino acids in collagen.
In some embodiments, the light energy pulses have a wavelength that is absorbed by amino acids in collagen.
In some embodiments, the method further includes applying an exogenous photosensitizer to the cornea before controlling the light source.
In some embodiments, the exogenous photosensitizer is riboflavin.
In some embodiments, the light source is a laser.
In some embodiments, the laser is a femtosecond laser.
In some embodiments, the light energy pulses have an average power output between about 10 mW and about 100 mW.
In some embodiments, the light energy pulses have a pulse energy between about 0.1 nJ and about 10 nJ.
In some embodiments, the light energy pulses have a wavelength between about 600 nm and about 1600 nm.
Another aspect of the invention provides a system for treating a cornea. The system includes a light source configured to project light energy pulses onto at least a portion of a cornea and a controller programmed to calculate the pattern and control the light source in accordance with any of embodiments above.
Another aspect of the invention provides a system for adapting a laser system for treating a cornea. The system includes laser modification optics adapted and configured to adjust laser output of the laser system and a controller programmed to calculate the pattern and control the laser modification optics as the light source in accordance with any of the embodiments above.
Another aspect of the invention provides a method of treating a cornea. The method includes controlling a light source to apply light energy pulses to a single corneal layer selected from the group consisting of: an anterior corneal layer and a posterior corneal layer. The light energy pulses: are below an optical breakdown threshold for the cornea; and ionize water molecules within the treated corneal layer to generate reactive oxygen species that cross-link collagen within the single corneal layer.
This aspect of the invention can have a variety of embodiments. The anterior corneal layer can extend between an anterior surface of the cornea and about 200 microns from the anterior surface. The posterior corneal layer can extend between a posterior surface of the cornea and about 200 microns from the posterior surface.
Another aspect of the invention provides a method of treating a cornea. The method includes controlling a light source to apply light energy pulses to at least a corneal stroma layer of a cornea. The light energy pulses: are below an optical breakdown threshold for the cornea; and ionize water molecules within the treated corneal stromal layer to generate reactive oxygen species that cross-link collagen within the cornea.
These aspects can have a variety of embodiments. The light source can be a laser. The laser can be a femtosecond laser.
The light energy pulses can have an average power output between about 10 mW and about 100 mW. The light energy pulses can have a pulse energy between about 0.1 nJ and about 10 nJ. The light energy pulses can have a wavelength between about 600 nm and about 1600 nm. The light energy pulses can have a wavelength that is not absorbed by amino acids in collagen.
The light energy pulses can be applied in a pattern. The pattern can extend across a center of an iris posterior to the cornea. The pattern can surround, but not extend across a center of an iris posterior to the cornea.
The method can treat keratoconus or alter curvature of the cornea.
Another aspect of the invention provides a system for treating a cornea. The system includes: a light source configured to project light energy pulses onto at least a portion of a cornea; and a controller programmed to control the light source in accordance with any of the methods described herein.
Another aspect of the invention provides a system for adapting a laser system for treating a cornea. The system includes: laser modification optics adapted and configured to adjust laser output of the laser system; and a controller programmed to control the laser modification optics as the light source in accordance with any of the methods described herein.
Another aspect of the invention provides a method of treating a cornea where the method includes flattening the cornea with a material that transmits light, generating pulses with a tunable femtosecond laser system, focusing the generated pulses on a focal volme at a specific depth within the cornea as measured from a surface of the eye, moving the focal volume at the specific depth to define a treatment pattern, and repeating the focusing and moving steps at multiple different depths. This aspect can have multiple embodiments described below. It is understood that each embodiment below can be combined with all of the other embodiments of this aspect.
In some embodiments, the focusing is achieved by using an aspheric lens.
In some embodiments, the moving of the focus point takes place at 30 mm/s in a direction parallel with the material used to flatten the cornea.
In some embodiments, adjacent ones of the multiple different depths are separated by 50 μm.
In some embodiments, the flattening the cornea includes pressing a glass coverslip against the cornea.
In some embodiments, the generating pulses is performed with a temporal pulse width of 140 fs at 80 MHz repetition rate with central wavelength set to 1060 nm.
In some embodiments, the treatment pattern is a zig-zag pattern.
For a fuller understanding of the nature and desired objects of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawing figures wherein like reference characters denote corresponding parts throughout the several views.
The instant invention is most clearly understood with reference to the following definitions. As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
As used in the specification and claims, the terms “comprises,” “comprising,” “containing,” “having,” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like.
Unless specifically stated or obvious from context, the term “or,” as used herein, is understood to be inclusive.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).
Embodiments of the invention provide methods, computer-readable media, and systems for treating a cornea by applying light to one or more corneal layer(s) to cross-link collagen. The cross-linking is performed to achieve a desired level of vision correction and/or the desired level of stiffening of the cornea. The amount of cross-linking is, at least in part, a function of a number of overlapping treatment layers having different z depths at a given coordinate. A treatment layer is a selected depth, measured from the posterior or anterior surface of the cornea, at which treatment light (such as a laser) is focused. It will be understood that the aperture used to focus the treatment light allows definition of such layers within the cornea.
Referring now to
Exemplary Therapies
The methods and systems can be used to treat various corneal disorders including keratoconus, myopia, hyperopia, stigmatism, irregular astigmatism, and other ectatic diseases (e.g., those that result from a weakened corneal stroma). The methods and systems can also be used in refractive surgery, e.g., to modify corneal curvature or correct irregular surfaces and higher-order optical aberrations.
Exemplary Irradiation Parameters
As described in International Publication No. WO 2017/070637 and U.S. Patent Application Publication Nos. 2018/0193188 and 2018/0221201, corneal cross-linking can be achieved without the need for exogenous photosensitizers such as riboflavin by ionizing water within corneal tissue to generate reactive oxygen species that cross-link collagen strands. Cross-linking can be achieved over a broad range of wavelengths including those that are not absorbed by amino acids within collagen strands. For example, the laser wavelength can be in the range from about 250 nm to about 1600 nm. In some embodiments, the laser wavelength can be in the range from about 250 nm to about 1600 nm, but excluding wavelengths between 260-290 nm, 520-580 nm, 780-870 nm, and 1040-1160 nm.
By controlling pulse energy to be below the optical breakdown threshold of collagen (about 1.0×1013 W cm-2), the mechanical properties of the collagen can be modified without modifying the refractive index of the collagen. For example, the curvature of the cornea can be modified to change the refractive power of the cornea.
Ionization can be created within tissue using a laser emission that is absorbed by the tissue. For example, the laser emission can be based on ultrashort laser pulses. As used herein, the phrase “ultrashort laser pulses” includes emissions in the femtosecond, picosecond, and nanosecond ranges. Nonlinear absorption of laser emissions can occur, in part, due to the highly compressed nature of the light pulses, allowing treatments of the interior of a transparent dielectric, such as corneal tissue, without affecting the surface layer. In some embodiments, a tunable femtosecond laser system (e.g., Coherent, Chameleon Ultra II, Santa Clara, Calif.) may be employed to generate laser pulses with temporal pulse width of 140 fs (femtoseconds) at 80 MHz repetition rate, and with central wavelength set to 1060 nm. The laser may be coupled through a single mode fiber cable (e.g., P1-980A-FC-1 single mode fiber patch cable, Thorlabs, Newton, N.J.) through an optical setup as described below.
The ultrashort laser pulse can induce low-density plasma that ionizes water molecules within the tissue, while still operating below the energy level required for optical breakdown. Optical breakdown is the effect of an ultrafast laser focused in the interior of collagen-rich tissue, where photoionization triggers non-linear absorption. Continued supply of incoming photons leads to the buildup of free electrons, further leading to avalanche ionization, which enhances the growth of free electron density resulting in formation of plasma. As contrasted from the low-density plasma, high-density, opaque plasma strongly absorbs laser energy through free carrier absorption. The high-density plasma expands rapidly, creating a shockwave that propagates into surrounding material, creating optical breakdown.
Collagen cross-linking can be safely induced when the laser is operated below optical breakdown level in the so-called “low-density plasma” regime. For example, the laser emission, as defined by its wavelength, temporal pulse width, and pulse energy, as well as the numerical aperture of the scanning objective and the scanning speed should be high enough to induce ionization of water molecules in the collagen rich tissue, but below optical breakdown level. Further, such ionization can be induced in the cornea without reducing the transparency of the cornea.
Without being bound by theory, the ionization can cause the formation of reactive oxygen products, such as singlet oxygen, OH-, and H202, which, in turn, can interact with collagen and increase cross-linking in the fibrils, as shown in
In certain aspects, the presently disclosed subject matter provides methods of inducing such ionization. The methods can be used in the treatment of various ectatic diseases or during refractive surgery. The methods can include modifying the corneal curvature by inducing selective corneal cross-linking.
Exemplary Corneal Layers
Referring now to
The treatment can be carried out on multiple treatment layers that are adjacent to each other, or on treatment layers that are not adjacent to each other. For example, treatment can be carried out starting at the anterior surface of the cornea and proceeding to additional layers below, toward the posterior of the cornea. Not all adjacent layers might be treated, and some layers may be skipped to achieve a desired physical effect in the cornea. For example, only layers at or near the anterior surface of the cornea may be treated, or only layers at or near the posterior surface of the cornea may be treated, or both of the above sets of layers, leaving the central layers of the cornea untreated.
Exemplary Cross-Linking Patterns
Light energy pulses can be applied in a variety of patterns to produce a desired corneal treatment. For example, the curvature of the cornea can be modified to change the refractive power of the cornea. The applied pattern can be a custom-generated pattern based on imaging of a particular subject's cornea. However, and without being bound by theory, Applicant describes general principles of cross-linking patterns below.
Corneal curvature can be flattened to reduce the optical power of the cornea by cross-linking in a solid pattern that extends over the center of an iris posterior to the cornea. Corneal curvature can be steepened to increase the optical power of the cornea by cross-linking in a pattern surrounding, but not extending over the center of an iris posterior to the cornea. For example, an un-cross-linked region over the center of the iris can have a cross-sectional dimension of about 4 mm.
Although a square and an annular pattern are depicted in
Additionally, cross-linking within a pattern can be produced using various sub-patterns within the outline of the pattern. For example, cross-linking can be performed in rows and/or columns that begin and break at the borders of the pattern. In some embodiments, cross-linking can wrap in a zigzag or serpentine manner from row-to-row. In still other embodiments, the pattern can be a matrix of cross-linked dots (e.g., in a rectangular grid or close-packed pattern). In still other embodiments, cross-linking can occur in lines that follow the pattern. For example, the pulses can form an annulus or spiral.
Also, cross-linking can be performed in multiple overlapping planes within a corneal layer. For example, a plurality of planes (e.g., 2, 3, 4, 5, and the like) can be cross-linked at a depth offset of about 25 μm, about 50 μm, and the like.
Without being bound by theory, it is believed that a substantially linear refractive power change based on number of treatment layers will be achieved until saturation is reached due to the finite thickness of the cornea. In an embodiment, an 8 diopter refractive power change was achieved by using 15 treatment planes.
As used herein, “planes” (or “layers”) can either include the classical geometrical definition as a flat, two-dimensional surface or can refer to a treatment surface having a defined depth from a curved surface or a treatment layer. (In some embodiments, the application of a cover slip to the cornea will flatten or substantially flatten the normally curved cornea.)
Depths can be determined by measuring thickness of the cornea with a pachymeter, then focusing the light energy pulses on desired locations within the cornea.
Exemplary Systems
As shown in
The objective 302 can be a scanning objective with a large numerical aperture. The large numerical aperture (NA) allows the objective 302 to focus diffuse light to a small area. A laser 304 supplies the light (e.g., laser light) to the objective 302. In one embodiment, the NA is 0.4. In another embodiment, the numerical aperture is 0.6, with a long working distance. However, the NA could be varied together with the pulse energy to achieve similar effect in a different control volume. Without being bound by theory, Applicant believes that NAs below 0.4, between about 0.4 and about 0.95, above 0.95, and above 1 would be capable of creating low-density plasma without causing optical breakdown.
In an embodiment, one or more optical filters 306 can be interspersed between the laser 304 and the objective 302.
The laser 304 can be a femtosecond laser that outputs laser light. In some embodiments, the laser light has a single frequency, and in other embodiments includes multiple frequencies. Embodiments can use any wavelength including multiple or continuous spectra covering a wide range of wavelengths. In embodiments, radiation at frequencies that may harm tissue or reduce the locality of the generation of reactive species are minimized or eliminated. Radiation that may be directly absorbed by the collagen can be minimized or eliminated, e.g., through filters. In an embodiment, the frequency or frequencies of the laser 304 are outside of the ultraviolet range. In embodiments, the frequency or frequencies of the laser 304 are in the infrared frequency band. The laser 304 receives control input from controls 308, which can be implemented on a stand-alone processing device, e.g., a computer executing software, or as embedded circuitry of the system.
Generation of such short pulses can be achieved with the technique of passive mode locking. The laser 304 can be any suitable laser type, including bulk lasers, fiber lasers, dye lasers, semiconductor lasers, and other types of lasers. In an embodiment, the laser operates in the infrared frequency range. In other embodiments, the lasers may cover a wide range of spectra domain. In embodiments, the disclosed subject matter can be implemented as an add-on system to a femtosecond laser system, such as used in certain Lasik systems.
In particular embodiments, the laser can be a Nd:Glass femtosecond laser. In embodiments, the laser wavelength can be in the range from about 250 nm to about 1600 nm. In embodiments, the femtosecond laser can have a temporal pulse width of from about 20 fs to about 26 ps (picoseconds). In embodiments, the pulse energy is from about 0.1 nJ to 100 nJ, 0.1 nJ to about 50 μJ, 0.1 nJ to about 10 μJ, from about 0.5 nJ to 50 nJ, or from about 1 nJ to 10 nJ. In embodiments, the femtosecond laser can be a Spirit® femtosecond laser in combination with a Spirit-NOPA® amplifier (Spectra-Physics, Santa Clara, Calif.).
As further shown in
Referring still to
Referring to
In embodiments, multiple beams can be provided by splitting a laser beam to multiple scanning objectives. For example, a laser head can include multiple scanning objectives bundled together, as shown in
Implementation in Computer-Readable Media and/or Hardware
The methods described herein can be readily implemented, in whole or in part, in software that can be stored in computer-readable media for execution by a computer processor. For example, the computer-readable media can be volatile memory (e.g., random access memory and the like) non-volatile memory (e.g., read-only memory, hard disks, floppy disks, magnetic tape, optical discs, paper tape, punch cards, and the like).
Additionally or alternatively, the methods described herein can be implemented in computer hardware such as an application-specific integrated circuit (ASIC).
A total of 60 fresh pig eyes were used for the study. Fifteen of these eyes underwent corneal flattening, and the treated eyes were paired with 10 control eyes. Thirteen eyes underwent laser irradiation to induce post-treatment steepening; these eyes were also paired with 10 control eyes. The remaining 12 eyes were used for a separate control study, to evaluate the effects of the experimental setup.
For the flattening treatment (
The initially large change in refractive power is due to a combination of the effects of the treatment itself and experimental conditions, which include temporary flattening of the cornea with a coverslip to ensure even volumetric exposure of the stroma to laser irradiation. The coverslip has an effect analogous to that of orthokeratology (ortho-K), a temporary reshaping of the cornea used to reduce refractive errors, and the duration of the effect is similar to that of an ortho-K procedure.
Once the coverslip effect wears off, the adjusted curvature remains stable throughout the rest of the 24-hour period. By contrast, laser treatment of the peripheral zone of the cornea leads to its steepening (
Ex vivo rabbit eyes for the experiments were delivered to the lab as intact rabbit heads from a local abattoir (La Granja Live Poultry Corporation, New York, N.Y.) within an hour after being euthanized. Eyes were isolated, rinsed with Dulbecco's phosphate-buffered saline (DPBS, 1×, Sigma-Aldrich), inspected for presence of defects and gradually brought to room temperature in a humidified chamber. Defective samples were discarded. After removing excess tissue, the eye globe was mounted onto a custom-built eye holder (
A Nd:Glass femtosecond laser oscillator system (HIGH Q LASER™, Spectra-Physics) was applied to generate laser pulses with temporal pulse width of 99 fs and 52.06 MHz repetition rate at 1060 nm wavelength. A ZEISS® PLAN-NEOFLUAR® 40×/0.6 objective lens was employed to focus the beam, and the average pulse energy and photon energy produced by the proposed lasing system are 60 mW and 1.1696 eV respectively after the objective lens. The laser beam was motorized by Z825B motors (Thorlabs) through a 3-dimensional PT1 translation stage (Thorlabs). Schematic diagram shows a femtosecond laser optical system set up in
In any of the disclosed embodiments, the lasing trajectory may follow a zig-zag pattern within the treatment layer, with the focal volume moving in the horizontal plane at 30 mm/s. Schematic diagrams of treatment paths are shown in
Confocal Laser Scanning Microscopy (CLSM)
Referring now to
The images show no evidence of negative effects of the applied femtosecond laser treatment on the cellular component of the rabbit cornea. In the cases of the anterior and the posterior application of the treatment, CLSM shows no significant differences in the morphology or cellular density of the stromal keratocytes and endothelium layers comparing to the untreated control. These preliminary results could contribute to the evidence of safe application of the tested laser irradiation for vision corrections.
Two-Photon Fluorescence (TPF) Microscopy
Isolated untreated control and laser treated corneal samples were cut into 2 mm2 blocks by a customized slicer and mounted by 50% glycerol in PBS in a 3 mm Petri dish filled with PBS solution. TPF was conducted by a two-photon microscope (Bruker) with MAI TAI™ DEEPSEE™ Ti: Sapphire laser (Spectra Physics) as the excitation source. A 40×/0.8 NA water immersion objective (Olympus) was applied to collect the fluorescence signal. The signal was registered with two different photomultiplier tubes, one in the red (580-620 nm) and one in the green (480-570 nm) wavelength regime. Excitation wavelengths used were 826 nm to excite collagen matrix
Results
Due to the nature of the delivery of concentrated nonlinear laser energy, the alteration of refractive power is spatially resolved and, thus, controllable. This may be particularly applied for the treatment of selective volumetric regions of corneal tissue that yields macroscopic changes in overall corneal curvature, which can be utilized for selectively treatment of myopia, hyperopia, stigmatism and irregular astigmatism. In order to show the spatial resolution of the proposed treatment, two treatment patterns were employed in this study. The anterior treatment pattern utilized the treatment from the superficial surface to the central cornea, and the posterior treatment pattern applied the treatment from the central cornea to the endothelium layer.
A total of 47 eyes were applied in this study. 20 eyes were subjected to anterior treatment, whereas 8 eyes were exposed to the posterior treatment. Treated samples were properly paired with untreated control eyes. The remaining 8 eyes were used as untreated control eyes to evaluate the experimental setup.
For the anterior treatment pattern, initially a steep change in corneal curvature, corresponding to an approximate 7.1% change in the overall refractive power (about 3.5 diopters on average), is followed by a partial recovery. The major curvature change occurs within 8 hours from the treatment, after which the corneal refractive power stabilizes at about 94.5% of its initial refractive power before the treatment (about 2.7 diopters on average). The relative significant change of corneal refractive power is further evident by the paired untreated control eyes, which showed approximately no change of refractive power over the 24 hours characterization period (
Two-photon autofluorescence (TPF) identifies fibrillar collagen in response to near-infrared laser light excitation. Thus, TPF imaging is employed to evaluate the laser induced CxLs in the cornea. The collagen extracellular structural differences among anterior treated, posterior treated, and untreated control eyes are presented in
Histological analysis of H&E-stained sections (
Treatment of the posterior stroma provides similar change in corneal curvature to that seen in treatment of the anterior stroma. This is unexpected due to differences in architecture of these two corneal segments, and it goes against conventional wisdom of ophthalmologists. The ability to achieve changes in eye refractive power through treatment of the corneal stroma also allows for treatment to be extended throughout the corneal thickness to treat more severe cases of myopia.
Working Example 3
Referring now to
Referring now to
Mechanical Properties
Mechanical properties of corneas were tested before and after treatment at varying layers. Inflation tests provide information about mechanical properties of the cornea.
Although preferred embodiments of the invention have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the following claims.
The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated herein in their entireties by reference.
This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/773,000, filed Nov. 29, 2018. The entire content of this application is hereby incorporated by reference herein.
Filing Document | Filing Date | Country | Kind |
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PCT/US2019/063320 | 11/26/2019 | WO | 00 |
Number | Date | Country | |
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62773000 | Nov 2018 | US |