Methods and uses for scleral optical imaging for detection of disease

Abstract

The present invention relates to a method of diagnosing and / or prognosing a disease associated with an altered structure in a posterior segment of the eye wherein the method comprises analyzing an image of the altered structure of the posterior segment of the eye relative to a reference obtained by a scleral optical imaging (TOI) device wherein the altered structure is indicative of the presence and / or progression of the disease in a subject.

Description

[0001] Diseases associated with changes in the posterior segment of the eye, such as glaucoma, age-related macular degeneration (AMD) and diabetic retinopathy, are the main causes of visual impairment worldwide. For example, it is estimated that 196 million people will be affected by age-related macular degeneration (AMD) in 2020. The posterior segment of the eye includes the posterior two-thirds of the eye, including the vitreous humor, retina, choroid and optic nerve. Among them, changes in the retina, especially changes in retinal neurons and retinal pigment epithelium (RPE) are usually associated with many posterior segment diseases.

[0002] The retina is the ten-layer membrane of light-sensitive neural tissue of the eye closest to the vitreous body. The function of the retina is to convert received light stimuli into nerve impulses and send them to the visual center of the brain via the optic nerve. The retinal pigment epithelium (RPE) is the single layer of pigmented retinal cells closest to the sclera.

[0003] Although RPE cells are located in the outside of sensory neuroretina, they play some key roles, such as light absorption, epithelial transport and maintaining visual cycle. Some RPE cell morphological characteristics, i.e. cell density, adjacent cell number (number of neighbors), eccentricity and form factor, are assumed to be different according to cell maturation and condition. Some other studies have reported the RPE cell loss caused by eye diseases and aging.

[0004] Several diagnostic imaging modalities allow for in vivo assessment of the human eye (e.g. optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), and fundus autofluorescence), these methods do not allow for the diagnosis of retinal diseases in their early stages, as small changes in RPE cell morphology cannot be detected. Furthermore, in vivo imaging of the RPE layer at the single-cell level is challenging due to several factors, namely low contrast between adjacent cells, motion artifacts, nonlinearity of the retinal layers, and difficulty in focal identification of the image. Instruments used in ophthalmology clinics for routine fundus examinations are unable to observe small changes in cell morphology that are present in the early stages of the disease degenerative process.

[0005] Transscleral optical imaging (TOI) disclosed in 2017 is a novel non-invasive in vivo high-resolution imaging method for the posterior structure (particularly retina) of the eye. The use of both adaptive optics and oblique illumination enhances the contrast of macroscopic and microscopic posterior segment structures (such as tissue structure, vasculature and RPE cells). The resulting excellent imaging resolution can achieve very high resolution, including to the cell level, such as distinguishing the cell membrane of a single RPE cell.

[0006] The applicant described in WO / 2017 / 195163A1 discloses a method for imaging tissue of an eye, the method comprising the steps of providing oblique illumination to the eye through a plurality of light emitting regions of a light delivery device, the plurality of light emitting regions being independently controllable and arranged to direct light toward at least one of the retina and the iris of the eye; generating an output beam from light backscattered from at least one of the retina and the iris by the oblique illumination; capturing the output beam with an imaging system to provide an image sequence of the fundus of the eye; and retrieving phase and absorption contrast images from the image sequence of the fundus, wherein the image sequence of the fundus of the capturing step is obtained by sequentially turning on one or more of the plurality of light emitting regions at a time in the step of providing the oblique illumination. In other words, the method for oblique illumination, including transscleral illumination and translid illumination, allows dark field and phase gradient techniques by using the scattering properties of the fundus. Oblique illumination, such as transscleral oblique flood illumination, increases the contrast of many biological structures that make up the retinal layers, and combined with adaptive optics high-resolution imaging, enables the observation of cells that play a key role in disease-related degenerative processes. Obtaining high-resolution images at the cellular level will enable new views of retinal structure and a better understanding of degenerative retinal disease processes.

[0007] In WO2020 / 121243A1, WO2021 / 058367A1 and WO2021 / 191331A1, the applicant discloses other developments and elements of the TOI device. In WO2020 / 121243A1, a TOI system with trans-scleral / trans-eyelid illumination of the fundus is disclosed. The TOI system includes multiple emission areas; each of the emission areas is configured to be independently controllable and directed toward the sclera of a predetermined eye to be measured, thereby providing trans-scleral oblique illumination of the fundus; an active eye aberration correction system; and an imaging system configured to create multiple fundus images on multiple imaging sensors. In WO2021 / 191331, the light delivery device is combined with optical coherence tomography (OCT) imaging.

[0008] In vivo observation of the human retina at the cellular level is crucial for detecting structural alterations before irreversible vision loss occurs, tracking the time course of retinal diseases, and evaluating and monitoring the early effects of treatments. Despite remarkable advances in optical coherence tomography (OCT) and adaptive optics, in vivo imaging of several retinal cells remains elusive.

[0009] Laforest T. et al. "Transscleral Optical Phase Imaging of the Human Retina-TOPI" https: / / arxiv.org / abs / 1905.06877A transscleral optical imaging (TOI) device is disclosed that allows imaging of retinal cells with high contrast, high resolution, and within acquisition times suitable for clinical use. TOI relies on high-angle oblique illumination of the retina combined with adaptive optics to enhance phase contrast of transparent cells.

[0010] Given the lack of methods in the art that provide accurate detection of early signs of disease-related structural changes in the posterior segment of the eye, there is a significant and unmet need in the art for methods that can generate high-resolution images of the posterior segment of the eye to allow analysis of disease states.

[0011] Therefore, the present invention provides a new method for cellular resolution imaging of the posterior segment of the eye for early diagnosis, prognosis and treatment susceptibility of diseases associated with structural alterations in the posterior segment of the eye.

[0012] Therefore, the technical problem to be solved by the present invention is to provide a method for early detection of changes in the posterior segment structure of the eye, which enables early diagnosis, prognosis and treatment of diseases related to changes in the posterior segment structure of the eye.

[0013] Therefore, the present invention relates to the following:

[0014] 1. A method for diagnosing and / or prognosing a disease associated with an altered structure in the posterior segment of an eye, wherein the method comprises analyzing an image of the altered structure of the posterior segment of the eye obtained by transscleral optical imaging (TOI), wherein the altered structure indicates the presence and / or progression of the disease in a subject.

[0015] 2. A method of treating a disease associated with an altered structure in the posterior segment of the eye, wherein the method comprises the steps of:

[0016] a) analyzing the image of the altered structure of the posterior segment of the eye obtained by the TOI, wherein the presence of the altered structure indicates the presence of the disease or the progression of the disease in the subject; and

[0017] b) administering to said subject identified as having or developing a disease according to step (a) an appropriate treatment for said disease.

[0018] 3. A method according to item 1 or 2, wherein the altered structure is determined relative to a reference, the reference being a TOI image of the posterior segment, the TOI image being obtained from a subject of a similar condition known not to have the disease or known not to be at risk of developing the disease.

[0019] 4. A method for

[0020] (i) evaluating the therapeutic effect of a treatment directed against a disease associated with altered structures in the posterior segment of the eye in a subject, or

[0021] (ii) a method of determining a subject's compliance with a prescribed treatment for a disease associated with an altered structure in the posterior segment of the eye,

[0022] The method comprises analyzing a first image and a second image of the posterior segment of the eye of the subject obtained by TOI,

[0023] (a) wherein the first image is acquired before the treatment or before the second image;

[0024] (b) wherein the second image is to be obtained after treatment or after the first image;

[0025] (c) wherein the analysis of the first image and the second image is an analysis and comparison of the structure of the posterior segment in (a) and (b);

[0026] Wherein maintenance or reduction of the altered structure between (a) and (b) indicates that the treatment has a therapeutic effect or that the subject is compliant with the treatment.

[0027] 5. A method according to item 4, wherein the altered structure is a structure that is altered relative to a structure determined from an analysis of a TOI image of the posterior segment, the TOI image being obtained from a subject of a similar condition who is known not to have the disease or who is known not to be at risk of developing the disease.

[0028] 6. The method of claim 4, wherein the maintenance of the changed structure between (a) and (b) is determined by

[0029] (d) determining that the altered structure is substantially unchanged between (a) and (b) according to item 4; or

[0030] (e) determining that any further change or progression of the altered structure between (a) and (b) according to claim 4 is not as severe or advanced as the change or progression between a set of reference first TOI images and a set of reference second TOI images of the posterior segment, the set of reference first TOI images and the set of reference second TOI images being obtained from subjects with similar conditions known to have the disease and the subjects not being treated for the disease, wherein the reference first images and the reference second images are obtained or are to be obtained at the same interval as the first images and the second images according to (a) and (b) according to item 4.

[0031] 7. A method according to any one of items 4 to 6, wherein the second image is to be obtained at least 2 days and no more than 730 days after the first image.

[0032] 8. The method according to any one of items 1 to 7, wherein the disease associated with the altered structure in the posterior segment of the eye is uveitis, glaucoma, macular edema, macular hole, macular pucker, diabetic macular edema, diabetic retinopathy, diabetic eye disease, retinopathy, age-related macular degeneration (AMD), wet AMD, dry AMD, early AMD, intermediate AMD, central serous chorioretinopathy, scleritis, optic nerve degeneration, geographic atrophy, choroidopathy, ocular sarcoidosis, optic neuritis, choroidal neovascularization, retinitis pigmentosa, retinal tear, Stargardt disease, disease), eye cancer, retinitis, corneal ulcer, cataract, viral infection (such as cytomegalovirus, herpes simplex, herpes zoster), fungal infection (such as histoplasmosis), parasitic infection (such as toxocariasis, toxocariasis), bacterial infection (such as tuberculosis, syphilis), sarcoidosis, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinal vascular disease, Vogt-Koyanagi-Harada syndrome, Behcet's disease disease), idiopathic retinal vasculitis, Vogt-Koyanagi-Harada syndrome, acute posterior multifocal squamous pigment epitheliopathy (APMPPE), ocular pseudohistoplasmosis syndrome (POHS), birdshot retinochoroidopathy, multiple sclerosis, sympathetic ophthalmia, punctate inner choroidopathy, pars planitis, iridocyclitis, diabetic retinopathy, retinopathy of prematurity (ROP), ischemic vascular disease, hereditary retinal dystrophy, retinal detachment, abnormal angiogenesis, retinal angiomatous proliferation (RAP), intraretinal microvascular abnormalities, epiretinal neovascularization, choroidal angiogenesis, choroidal vasculopathy stroke, hypertension, diabetes, cardiovascular disease, prematurity, and papilledema.

[0033] 9. A method according to any one of items 1 to 8, wherein the image of the posterior segment of the eye is an image of the choroid, choriocapillaris, Bruch's membrane, retinal choroidal tissue, neural retinal tissue, nerve fiber layer, retinal pigment epithelium (RPE), photoreceptors, ganglion cell layer, retinal vasculature, subretinal space, retina, macula, lamina cribrosa, optic disc or optic nerve.

[0034] 10. The method according to any one of items 1 to 9, wherein the altered structure is an alteration of tissue structure.

[0035] 11. The method according to item 10, wherein the change in tissue structure is a change in cell pattern, cell density, cell size, cell distribution or cell reflectivity.

[0036] 12. The method according to item 10, wherein the change in tissue structure is a change in tissue reflectivity, which is a change in low-reflection areas, high-reflection areas, low-reflection areas within high-reflection areas, or any combination thereof.

[0037] 13. A method according to any one of items 1 to 12, wherein the image is an image of the RPE.

[0038] 14. A method according to any one of items 1 to 12, wherein the image is an image of the choriocapillaris.

[0039] 15. A method according to any one of items 1 to 12, wherein the image is an image of the nerve fiber layer, optic disc and / or retinal vasculature.

[0040] 16. A method according to item 13, wherein the disease is AMD and the altered structure is a change in RPE cell density, RPE cell size, hyperreflective areas, hyporeflective areas and / or hyporeflective areas within hyperreflective areas.

[0041] 17. The method according to item 13 or 14, wherein the disease is central serous chorioretinopathy and the altered structures are changes in the RPE and choriocapillaris.

[0042] 18. A method according to item 15, wherein the disease is glaucoma and the changed structure is a change in the nerve fiber layer (including but not limited to a change in the orientation of the nerve fiber bundle, a change in the thickness of the nerve fiber bundle, a change in the size of the nerve fiber bundle) or a change in the morphology of the optic disc (including but not limited to a change in the optic nerve head, a change in the physiological cup, a change in the cup-disc ratio, a change in the lamina cribrosa).

[0043] 19. The method according to item 15, wherein the disease is diabetic retinopathy and the altered structure is an alteration of the retinal vasculature.

[0044] 20. The method according to any one of items 1 to 12, wherein the altered structure is indicative of geographic atrophy, drusen, reticular pseudodrusen, neovasculature and / or retinal pigment epithelial degeneration.

[0045] 21. Use of an image of a subject's eye obtained by transscleral optical imaging (TOI) for the diagnosis and / or prognosis of a disease associated with an altered structure in the posterior segment of the eye, wherein the use comprises analyzing the image for the altered structure, wherein the altered structure is indicative of the presence and / or progression of the disease in the subject.

[0046] 22. Use of an image of a subject's eye obtained by TOI for treating a disease associated with an altered structure in the posterior segment of the eye, wherein the use comprises the steps of:

[0047] a) analyzing said image for altered structures, wherein the presence of altered structures indicates the presence of said disease or the progression of said disease in said subject; and

[0048] b) administering to said subject identified as having or developing a disease according to step (a) an appropriate treatment for said disease.

[0049] 23. The use according to item 21 or 22, wherein the altered structure is determined relative to a reference TOI image of the posterior segment, the reference TOI image being obtained from a subject of a similar condition known not to suffer from the disease or known not to be at risk of developing the disease.

[0050] 24. A first image and a second image of the subject's eye obtained by transscleral optical imaging (TOI) are used to

[0051] (i) evaluating the therapeutic effect of a treatment directed against a disease associated with altered structures in the posterior segment of the eye in a subject, or

[0052] (ii) use for determining compliance of a subject with a prescribed treatment for a disease associated with altered structures in the posterior segment of the eye,

[0053] The use comprises analyzing the first image and the second image,

[0054] (a) wherein the first image is acquired before the treatment or before the second image;

[0055] (b) wherein the second image is to be obtained after treatment or after the first image;

[0056] (c) wherein the analysis of the first image and the second image is an analysis and comparison of the structure of the posterior segment in (a) and (b);

[0057] Wherein maintenance or reduction of the altered structure between (a) and (b) indicates that the treatment has a therapeutic effect or that the subject is compliant with the treatment.

[0058] 25. The use according to item 24, wherein the altered structure is a structure altered relative to a structure determined from an analysis of a TOI image of the posterior segment, the TOI image being obtained from a subject of a similar condition known not to have the disease or known not to be at risk of developing the disease.

[0059] 26. The use according to item 24, wherein the maintenance of the altered structure between (a) and (b) is determined by

[0060] (d) determining that the altered structure is substantially unchanged between (a) and (b) according to item 24; or

[0061] (e) determining that any further change or progression of the altered structure between (a) and (b) according to item 24 is not as severe or advanced as the change or progression between a set of reference first TOI images and a set of reference second TOI images of the posterior segment, wherein the set of reference first TOI images and the set of reference second TOI images are obtained from subjects with similar conditions known to have the disease and the subjects have not been treated for the disease, wherein the reference first images and the reference second images are obtained or are to be obtained at the same interval as the first images and the second images according to (a) and (b) according to item 24.

[0062] 27. The use according to any one of items 24 to 26, wherein the second image is to be obtained at least 2 days and not more than 730 days after the first image.

[0063] 28. The use according to any one of items 21 to 27, wherein the disease associated with the altered structure in the posterior segment of the eye is uveitis, glaucoma, macular edema, macular hole, macular pucker, diabetic macular edema, diabetic retinopathy, diabetic eye disease, retinopathy, age-related macular degeneration (AMD), wet AMD, dry AMD, early AMD, intermediate AMD, central serous chorioretinopathy, scleritis, optic nerve degeneration, geographic atrophy, choroidopathy, ocular sarcoidosis, optic neuritis, choroidal neovascularization, retinitis pigmentosa, retinal tear, Stargardt's disease, eye cancer, retinitis, corneal ulcer, cataract, viral infection (such as cytomegalovirus, herpes simplex, herpes zoster), fungal infection (such as histoplasmosis), parasitic infection (such as toxocariasis, toxocariasis), bacterial infection (such as tuberculosis, syphilis) , sarcoidosis, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinal vasculopathy, Vogt-Koyanagi-Harada syndrome, Behcet's disease, idiopathic retinal vasculitis, Vogt-Koyanagi-Harada syndrome, acute posterior multifocal squamous pigment epitheliopathy (APMPPE), ocular pseudohistoplasmosis syndrome (POHS), birdshot retinochoroidopathy, multiple sclerosis, sympathetic ophthalmia, punctate inner choroidopathy, pars planitis, iridocyclitis diabetic retinopathy, retinopathy of prematurity (ROP), ischemic vascular disease, hereditary retinal dystrophy, retinal detachment, abnormal angiogenesis, retinal angiomatous proliferation (RAP), intraretinal microvascular abnormalities, preretinal neovascularization, choroidal angiogenesis, choroidal vasculopathy stroke, hypertension, diabetes, cardiovascular disease, prematurity, and papilledema.

[0064] 29. The use according to any one of items 21 to 28, wherein the image of the posterior segment of the eye is an image of the choroid, choriocapillaris, Bruch's membrane, retinal choroidal tissue, neuroretinal tissue, nerve fiber layer, retinal pigment epithelium (RPE), photoreceptors, ganglion cell layer, retinal vasculature, subretinal space, retina, macula, lamina cribrosa, optic disc or optic nerve.

[0065] 30. The use according to any one of items 21 to 29, wherein the altered structure is an alteration of tissue structure.

[0066] 31. The use according to item 30, wherein the change in tissue structure is a change in cell morphology, cell density, cell size, cell distribution or cell reflectivity.

[0067] 32. The use according to item 31, wherein the change in tissue structure is a change in tissue reflectivity, which is a change in low reflective areas, high reflective areas, low reflective areas within high reflective areas, or any combination thereof.

[0068] 33. The use according to any one of items 21 to 32, wherein the image is an image of the RPE.

[0069] 34. The use according to any one of items 21 to 32, wherein the image is an image of the choriocapillaris.

[0070] 35. The use according to any one of items 21 to 32, wherein the image is an image of the nerve fiber layer, the optic disc and / or the retinal vasculature.

[0071] 36. The use according to item 33, wherein the disease is AMD and the altered structure is a change in RPE cell density, RPE cell size, hyperreflective areas, hyporeflective areas and / or hyporeflective areas within hyperreflective areas.

[0072] 37. The use according to item 33 or 34, wherein the disease is central serous chorioretinopathy and the altered structures are changes in the RPE and choriocapillaris.

[0073] 38. The use according to item 35, wherein the disease is glaucoma and the changed structure is a change in the nerve fiber layer (including but not limited to a change in the orientation of the nerve fiber bundle, a change in the thickness of the nerve fiber bundle, a change in the size of the nerve fiber bundle) or a change in the morphology of the optic disc (including but not limited to a change in the optic nerve head, a change in the physiological cup, a change in the cup-disc ratio, a change in the lamina cribrosa).

[0074] 39. The use according to item 35, wherein the disease is diabetic retinopathy and the altered structure is an alteration of the retinal vasculature.

[0075] 40. The use according to any one of items 21 to 32, wherein the altered structure is indicative of geographic atrophy, drusen, reticular pseudodrusen, neovasculature and / or retinal pigment epithelial degeneration.

[0076] Thus, the present invention provides a highly accurate method for detecting changes in the structure of the posterior segment of the eye for significantly improving the diagnosis, prognosis, monitoring and treatment of diseases associated with said structural changes.

[0077] According to one embodiment of the present invention, the present invention provides a method for diagnosing and / or prognosing a disease associated with an altered structure in the posterior segment of the eye, wherein the method comprises analyzing an image of the altered structure of the posterior segment of the eye obtained by transscleral optical imaging (TOI), wherein the altered structure indicates the presence and / or progression of the disease in the subject.

[0078] In another embodiment of the present invention, the present invention provides a method for treating a disease associated with an altered structure in the posterior segment of the eye, wherein the method comprises the following steps: a) analyzing an image of the altered structure of the posterior segment of the eye obtained by TOI, wherein the presence of the altered structure indicates the presence of a disease or the development of a disease in a subject; and b) administering an appropriate treatment for the disease to the subject identified as having or developing the disease according to step (a).

[0079] In certain embodiments, the method of the present invention may refer to a method wherein the altered structure is determined relative to a reference, which is a TOI image of the posterior segment obtained from a subject of a similar condition known not to have the disease or known not to be at risk of developing the disease.

[0080] The methods described herein involve analysis of a TOI image of an eye for determining altered structures. One of skill in the art is aware of the anatomy of the posterior segment of the eye under disease-free conditions (anatomy in a normal eye), and can therefore empirically determine the presence or absence of altered structures in a TOI image, for example, by methods including, but not limited to, visual inspection. However, altered structures can also be determined by comparison with a reference. As used herein, the term "reference" refers to a predetermined or known structure of the posterior segment of the eye. Deviations in an image of a subject compared to a reference determine changes in the structure, which can, for example, indicate the presence of a disease state, the progression of a disease state, or a predisposition to the development of a disease state. In certain embodiments, a "reference" as used herein is a reference TOI image from a subject known not to have a disease or known not to be at risk of developing a disease. In other embodiments, if it is desired to monitor the progression of a disease or compliance with therapy, the reference can be a TOI image or a set of TOI images obtained from a reference subject known to have a disease or known to be at a predisposition to develop a disease, and the subject is untreated for such a disease. In an analysis according to the method relative to a reference of a set of images obtained from untreated subjects, the altered structure is considered to be maintained if changes in the altered structure in a set of images from the analyzed subject do not progress as in the reference images or do not advance as in the reference images.

[0081] The altered structure of the posterior segment of the eye, whether determined empirically (i.e., without comparison to a reference) or relative to a reference, can be an altered macroscopic or microscopic structure. Non-limiting examples of macroscopic structures include vasculature (such as, but not limited to, retinal vasculature), where the altered structure can include, but is not limited to, altered size, altered vessel density, or altered vessel morphology. The altered structure can also be microscopic, such as altered intracellular or extracellular changes. Preferably, the altered structure determined is an altered cellular structure of tissue of the posterior segment of the eye. Non-limiting changes in cellular structure can include changes in cell density, cell size, and / or cell morphology.

[0082] The microscopic change of structure need not be limited to the change due to the change of any specific cell or cell group itself, but can be attributed to the change caused by their structure or phenotype or dependent on the change of their structure or phenotype.The microscopic change of this structure includes the change of low-reflection or high-reflection area.Therefore, the structure of change can include the change in low-reflection area, such as but not limited to the change (increase or decrease) of density, concentration, grouping or pattern of low-reflection area.The structure of change can also include the change of high-reflection area, such as but not limited to the change (increase or decrease) of density, concentration, grouping or pattern of high-reflection area.The change of structure can also include the change of both high-reflection area and low-reflection area as described in this paragraph or elsewhere herein.The change of structure can also include the change (appearance, disappearance, increase of concentration / density or reduction of concentration / density) of the area with high-reflection area and low-reflection area, such as low-reflection area (referred to as low-reflection area surrounded by high-reflection halo in the art) in high-reflection area.

[0083] In case of determining the altered structure relative to a reference, it is not necessary to determine the reference each time. The reference may be based, for example, on a TOI image already obtained from the subject undergoing analysis, but at an earlier point in time, including before the therapeutic intervention. The reference image may additionally or alternatively be based on a standard TOI image, for example an image obtained from an unrelated subject known not to suffer from the disease in question or known not to be at risk of developing the disease in question. The reference may also be the result of normalization of a large number of images. In this case, both the normalization of the reference image and the analysis of the subject images may be performed by a machine learning tool, such as a computer with appropriate image analysis software.

[0084] It will be appreciated that the structure of the posterior segment of the eye, both macroscopically and microscopically, depends on a variety of factors, such as the age and sex of the subject, whether they are undergoing medical therapy (e.g., being treated with therapeutic drugs that may or may not be related to the disease under analysis), and / or their lifestyle habits (e.g., whether they are a smoker, whether they drink alcohol, their fitness level, etc.). Thus, where the reference is a standard image, the reference image may be obtained from a source or group of sources that are in similar conditions to the subject, such as a source or group of sources that have similar physical characteristics to the subject and have similar lifestyle standards. To take into account the potential variation, average structural properties may be developed from a large number of sources that are known not to have the disease or are known not to be at risk of having the disease to use as a reference.

[0085] The "TOI device" developed by the inventors of the present application refers to a device for ophthalmic illumination of the fundus using a light delivery device having multiple light sources; wherein each light source is configured to be independently controllable and directed toward the sclera of the eye, thereby providing transscleral oblique illumination of the fundus; an active eye aberration correction system; and an imaging system configured to create multiple fundus images on multiple imaging sensors. Light transmitted through the sclera produces oblique illumination of the posterior retina; which is then imaged using a transpupillary AO full-field camera system. The TOI device provides dark field imaging, high resolution imaging, and large field of view (FOV) imaging.

[0086] The inventors have found that the TOI device advantageously provides cellular resolution label-free high contrast images of the posterior segment of the eye, particularly the retinal layers of a large FOV, without the disadvantages of long exposure times. Compared to trans-pupillary illumination, oblique illumination including trans-scleral or trans-eyelid (e.g., trans-scleral flood illumination) of the retina as used in the TOI greatly increases the signal-to-noise ratio (SNR) of many retinal structures.

[0087] The TOI device as used herein uses an aberration correction method. Correction of optical aberrations is performed in real time with, but not limited to, an adaptive optical closed loop that includes a trans-pupillary detection light source, a wavefront sensor, and a wavefront corrector capable of spatially shaping the wavefront of light that produces a frontal image. The TOI device combines trans-pupillary (transpupil / transpupillary) illumination and trans-scleral illumination to benefit from the advantages of both types of illumination.

[0088] The term "transscleral" means through the sclera or white of the eye. As used herein, the term "sclera" refers to the white of the eye, which is the opaque, fibrous, protective outer layer of the human eye containing primarily collagen and some elastic fibers. The sclera is a connective tissue composed primarily of white collagen fibers. The sclera is located behind the choroid and continues forward, where it becomes transparent above the iris and pupil and is called the cornea.

[0089] As used herein, the term "diagnosis" means confirming the presence or nature of a pathological condition. For purposes of the present invention, diagnosis means confirming the presence of an altered structure of the posterior segment of the eye. The altered structure may refer to changes in the anterior hyaloid membrane, vitreous humor, retina, choroid, and / or optic nerve.

[0090] As used herein, the term "prognosis" refers to a prediction of the likely course or outcome of a disease or the likelihood of recovery from a disease. As will be appreciated by those skilled in the art, although a prediction is preferred, it need not be correct for 100% of the subjects to be diagnosed or evaluated. However, the term requires that a statistically significant portion of the subjects can be identified as having an increased probability of having a given outcome.

[0091] The term "treatment" of a disorder or disease as used herein is well known in the art. "Treatment" of a disorder or disease means that a disorder or disease is suspected or has been diagnosed in a patient / subject. Patients / subjects suspected of having a disorder or disease generally show specific clinical and / or pathological symptoms, and technicians can easily attribute them to specific pathological conditions (i.e., diagnosing a disorder or disease). "Treatment" of a disorder or disease can, for example, lead to the cessation of the progression of a disorder or disease (e.g., the symptoms have not worsened) or the delay of the progression of a disorder or disease (in the case where the cessation of progression is only of a transient nature). "Treatment" of a disorder or disease can also lead to a partial response (e.g., symptom improvement) or a complete response (e.g., symptom disappearance) of a subject / patient suffering from the disorder or disease. Therefore, "treatment" of a disorder or disease can also refer to the improvement of a disorder or disease, which can, for example, lead to the cessation of the progression of a disorder or disease or the delay of the progression of a disorder or disease. This partial or complete response may recur afterwards. It should be understood that a subject / patient can experience a wide range of responses to treatment (such as the exemplary responses described above herein). Treatment of a disorder or disease may include, inter alia, curative treatment (preferably resulting in a complete response and ultimate cure of the disorder or disease) and palliative treatment (including symptomatic relief).

[0092] The term "posterior segment" or grammatical variations thereof refers to the portion of the eye behind the lens or ora serrata. This portion consists of the posterior 2 / 3 of the eye including the anterior hyaloid membrane and all of the following optical structures behind it: vitreous humor, retina, choroid, and optic nerve. As used herein, "posterior segment disease" or "diseases associated with altered structures of the posterior segment of the eye" or grammatical variations thereof refer to diseases that affect the posterior segment of the eye. Posterior segment diseases include, but are not limited to, uveitis, glaucoma, macular edema, macular hole, macular pucker, diabetic macular edema, diabetic retinopathy, diabetic eye disease, retinopathy, age-related macular degeneration (AMD), wet AMD, dry AMD, early AMD, intermediate AMD, central serous chorioretinopathy, scleritis, optic nerve degeneration, geographic atrophy, choroidopathy, ocular sarcoidosis, optic neuritis, choroidal neovascularization, retinitis pigmentosa, retinal tear, Stargardt's disease, eye cancer, retinitis, corneal ulcer, cataract, viral infections (such as cytomegalovirus, herpes simplex, herpes zoster), fungal infections (such as histoplasmosis), parasitic infections (such as toxocariasis, toxocariasis), bacterial infections (such as tuberculosis, syphilis), sarcoidosis, retinal vein occlusion, retinal central vein occlusion, branch retinal vein occlusion, retinal vascular disease, Vogt-Koyanagi-Harada syndrome, Behcet's disease, idiopathic retinal vasculitis, Vogt-Koyanagi-Harada syndrome, acute posterior multifocal squamous pigment epitheliopathy (APMPPE), ocular pseudohistoplasmosis syndrome (POHS), birdshot retinochoroidopathy, multiple sclerosis, sympathetic ophthalmia, punctate inner choroidopathy, pars planitis, iridocyclitis, diabetic retinopathy, retinopathy of prematurity (ROP), ischemic vascular disease, hereditary retinal dystrophy, retinal detachment, abnormal angiogenesis, retinal angiomatous proliferation (RAP), intraretinal microvascular abnormalities, preretinal neovascularization, choroidal angiogenesis, choroidal vasculopathy stroke, hypertension, diabetes, cardiovascular disease, prematurity, and papilledema.

[0093] As used herein, the term "subject" refers to mammals, including non-primates (e.g., camels, donkeys, zebras, cows, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In certain embodiments, the subject is suffering from or susceptible to a disease characterized by changes in the posterior segment of the eye, and is preferably a human.

[0094] In certain embodiments of the present invention, the method relates to a method for: (i) evaluating the therapeutic effect of a treatment for a disease associated with an altered structure in the posterior segment of the eye in a subject, or (ii) determining a subject's compliance with a prescribed treatment for a disease associated with an altered structure in the posterior segment of the eye, the method comprising analyzing a first image and a second image of the posterior segment of the eye of the subject obtained by a TOI, (a) wherein the first image is to be obtained before the treatment or before the second image; (b) wherein the second image is to be obtained after the treatment or after the first image; (c) wherein the analysis of the first image and the second image is an analysis and comparison of the structure of the posterior segment in (a) and (b); wherein maintenance or reduction of the altered structure between (a) and (b) indicates that the treatment has a therapeutic effect or that the subject is compliant with the treatment.

[0095] Furthermore, the methods of the present invention may relate to methods wherein the efficacy of a treatment in a subject or the subject's compliance with a prescribed treatment is determined by analyzing the altered structure relative to the structure determined from analysis of TOI images of the posterior segment obtained from subjects of similar condition known not to have the disease or known not to be at risk of developing the disease.

[0096] In another embodiment of the present invention, the method relates to a method wherein determining maintenance of the altered structure between (a) and (b) is (d) determining wherein the altered structure is substantially unchanged between (a) and (b) of the aforementioned method; or (e) determining wherein any further change or progression of the altered structure between (a) and (b) of the aforementioned method is not as severe or advanced as the change or progression between a set of reference first TOI images and a set of reference second TOI images of the latter segment, the set of reference first TOI images and the set of reference second TOI images being obtained from subjects with similar conditions known to have a disease and the subjects not being treated for the disease, wherein the reference first image and the reference second image are obtained or to be obtained at the same interval as the first image and the second image according to (a) and (b) of the aforementioned method.

[0097] In certain embodiments of the invention, the method relates to a method wherein the second image is to be obtained at least 2 days and not more than 730 days after said first image.

[0098] The inventors of the present invention have found that by analyzing the first image and the second image of the back of the eye, an assessment of the effectiveness of a treatment or a subject's compliance with a given therapy can be accurately predicted when the images are obtained at least 2 days and no more than 730 days apart. Thus, the advanced images obtained by the TOI device provide a means and method for rapid analysis of early detection, intervention, and prognosis of disease states. This is essential in improving treatment selection, treatment compliance, and disease outcomes.

[0099] In one embodiment of the invention, the method relates to a method wherein the disease associated with the altered structure in the posterior segment of the eye is uveitis, glaucoma, macular edema, macular hole, macular pucker, diabetic macular edema, diabetic retinopathy, diabetic eye disease, retinopathy, age-related macular degeneration (AMD), wet AMD, dry AMD, early AMD, intermediate AMD, central serous chorioretinopathy, scleritis, optic nerve degeneration, geographic atrophy, choroidopathy, ocular sarcoidosis, optic neuritis, choroidal neovascularization, retinitis pigmentosa, retinal tear, Stargardt's disease, eye cancer, retinitis, corneal ulcer, cataract, viral infection (such as cytomegalovirus, herpes simplex, herpes zoster), fungal infection (such as histoplasmosis), parasitic infection (such as toxocariasis, toxocariasis), bacterial infection (such as tuberculosis, syphilis ), sarcoidosis, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinal vasculopathy, Vogt-Koyanagi-Harada syndrome, Behcet's disease, idiopathic retinal vasculitis, Vogt-Koyanagi-Harada syndrome, acute posterior multifocal squamous pigment epitheliopathy (APMPPE), ocular pseudohistoplasmosis syndrome (POHS), birdshot retinochoroidopathy, multiple sclerosis, sympathetic ophthalmia, punctate inner choroidopathy, pars planitis, iridocyclitis diabetic retinopathy, retinopathy of prematurity (ROP), ischemic vascular disease, hereditary retinal dystrophy, retinal detachment, abnormal angiogenesis, retinal angiomatous proliferation (RAP), intraretinal microvascular abnormalities, preretinal neovascularization, choroidal angiogenesis, choroidal vasculopathy stroke, hypertension, diabetes, cardiovascular disease, prematurity, and papilledema.

[0100] In a preferred embodiment of the present invention, the method relates to a method wherein the image of the posterior segment of the eye is an image of the choroid, choriocapillaris, Bruch's membrane, retinal choroidal tissue, neural retinal tissue, nerve fiber layer, retinal pigment epithelium (RPE), photoreceptors, ganglion cell layer, retinal vasculature, subretinal space, retina, macula, lamina cribrosa, optic disc or optic nerve.

[0101] In a preferred embodiment of the present invention the method relates to a method wherein the altered structure is an alteration of tissue structure.

[0102] In certain embodiments of the invention, the method relates to a method wherein the change in tissue structure is a change in cell morphology, cell density, cell size, cell distribution or cell reflectivity.

[0103] The present inventors have discovered that, in particular, examination of structural changes in the cellular structure of the posterior segment of the eye provides an accurate means for early detection of disease and monitoring of the therapeutic effect of a given treatment for said disease.

[0104] By analyzing cellular structure to identify changes in cell morphology, density, size, distribution, and reflectivity, the inventors discovered that these specific features can be used to accurately assess disease states.

[0105] In another preferred embodiment of the present invention, the method relates to a method wherein the change in tissue structure is a change in tissue reflectivity, and the change in tissue reflectivity is a change in low reflective areas, high reflective areas, low reflective areas within high reflective areas, or any combination thereof.

[0106] Changes in cellular reflectivity, particularly changes in low reflective areas, high reflective areas, and / or low reflective areas within high reflective areas, can be used to provide key information about morphological changes occurring in tissue structures. Therefore, examination of these features is particularly advantageous for evaluation of the posterior segment of the eye.

[0107] In a preferred embodiment of the invention the method relates to a method wherein said image is an image of the RPE.

[0108] The inventor of the present invention surprisingly found that, by analyzing the image of RPE at the cell level, some cell morphology characteristics can be identified as the early indicators of disease onset.The slight change that occurs in the RPE layer, i.e. the change of cell density, adjacent cell number, eccentricity and form factor can not be checked by the currently available method in this area.This is due to the difficulty of the focus identification of low contrast, motion artifact, retinal layer nonlinearity and image between the adjacent cells.Therefore, method of the present invention provides the specific index that can be used for early detection of disease and prognosis.

[0109] In another preferred embodiment of the present invention, the image is an image of the choriocapillaris.

[0110] In another embodiment of the invention, the image is an image of the nerve fiber layer, optic disc and / or retinal vasculature.

[0111] In another embodiment of the invention, the method relates to a method wherein the disease is AMD and the altered structure is an alteration in RPE cell density, RPE cell size, hyperreflective areas, hyporeflective areas and / or hyporeflective areas within hyperreflective areas.

[0112] The transition from intermediate to advanced AMD can be delayed and possibly prevented by taking a specific high-dose formulation of antioxidants and zinc. Studies have shown that daily supplementation, including: vitamin C (500 mg); vitamin E 400 IU; beta-carotene (15 mg); zinc (as zinc oxide) (80 mg); and copper (as copper oxide) (2 mg), reduced the risk of progression from intermediate to advanced AMD by 25% and reduced the risk of vision loss by 19%. ( www.amd.org There are currently four FDA-approved treatments for wet AMD: laser surgery, photodynamic therapy (PDT), and drugs Pegaptanib sodium and Lucentis T< Ranibizumab is injected intravitreally. Laser, PDT, and pegaptanib can slow the rate of vision loss and / or stop vision loss. Eyetech Pharmaceuticals Inc. and Pfizer Inc.) is approved for the treatment of wet AMD and is a PEGylated oligonucleotide aptamer targeting VEGF. Ranibizumab (Lucentis TM , Genentech / Novartis) is an antibody fragment that targets VEGF and was recently approved by the FDA for the treatment of wet AMD. Laser surgery attempts to destroy fragile, leaky blood vessels using high-energy beams.

[0113] In another embodiment of the invention the method relates to a method wherein said disease is central serous chorioretinopathy and said altered structures are alterations of the RPE and choriocapillaris.

[0114] CSCR is usually a self-limited disease that resolves spontaneously within 3-4 months with an overall good visual outcome. However, relapses are observed in up to 50% of patients within the first year. Current treatments include photodynamic therapy, oral aldosterone antagonism, and subthreshold multifocal laser. A number of new treatments are also being investigated, including anti-vascular endothelial growth factor therapy.

[0115] In one embodiment of the invention, the method relates to a method wherein the disease is glaucoma and the altered structure is a change in the nerve fiber layer (including but not limited to a change in the orientation of the nerve fiber bundles, a change in the thickness of the nerve fiber bundles, a change in the size of the nerve fiber bundles) or a change in the morphology of the optic disc (including but not limited to a change in the optic nerve head, a change in the physiological cup, a change in the cup-disc ratio, a change in the lamina cribrosa).

[0116] Glaucoma has been simply defined as a destructive process of ocular tissue caused by a sustained increase in intraocular pressure (IOP) above its normal physiological limit. Although several etiologies may be involved in the glaucoma complex, the absolute determining factor in treatment selection is the amount of primary and / or induced changes in iridocorneal intracorneal pressure. Current therapies include drugs or surgery aimed at reducing this pressure, although the pathophysiological mechanism by which elevated IOP in glaucoma leads to neuronal damage is unknown. Four types of drugs can be used to attempt to medically suppress elevated IOP: (1) inhibitors of aqueous humor formation (such as carbonic anhydrase inhibitors, β-adrenergic blockers, and α2-adrenergic receptor agonists); (2) miotic agents (such as parasympathomimetics, including cholinergics and anticholinesterase inhibitors); (3) uveoscleral outflow enhancers; and (4) hyperosmolar agents (which produce an osmotic pressure gradient across the blood / aqueous humor barrier within the ciliary epithelium). A fifth class of drugs, neuroprotectants, have emerged as an important supplement to medical therapy and include, for example, NOS inhibitors, excitatory amino acid antagonists, glutamate receptor antagonists, apoptosis inhibitors, and calcium channel blockers.

[0117] In one embodiment of the invention the invention relates to a method wherein the disease is diabetic retinopathy and the altered structure is an alteration of the retinal vasculature.

[0118] In certain embodiments of the invention, the method relates to a method wherein the altered structure is indicative of geographic atrophy, drusen, reticular pseudodrusen, neovasculature and / or retinal pigment epithelial degeneration.

[0119] As used herein, the term "geographic atrophy" refers to late-stage dry AMD. Geographic atrophy is characterized by "islands" of atrophied photoreceptor cells. It is believed that the complement alternative pathway may play a role in the pathogenesis of AMD.

[0120] The term "drusen" as used herein refers to yellowish deposits located deep within the RPE, inside Bruch's membrane.

[0121] The method of the present invention also provides the use of an image of a subject's eye obtained by TOI in treating a disease associated with an altered structure in the posterior segment of the eye, wherein the use comprises the steps of: a) analyzing the altered structure of the image, wherein the presence of the altered structure indicates the presence of a disease or the development of a disease in the subject; and b) administering an appropriate treatment for the disease to the subject identified as having or developing a disease according to step (a).

[0122] In certain embodiments of the invention, the use of the invention relates to a use wherein the altered structure is determined relative to a reference TOI image of the posterior segment obtained from a subject of a similar condition known not to suffer from the disease or known not to be at risk of developing the disease.

[0123] In another embodiment of the present invention, the present invention provides the use of a first image and a second image of a subject's eye obtained by transscleral optical imaging (TOI), which is used to (i) evaluate the therapeutic effect of a treatment for a disease associated with an altered structure in the posterior segment of the eye in the subject, or (ii) determine the subject's compliance with a prescribed treatment for a disease associated with an altered structure in the posterior segment of the eye, the use comprising analyzing the first image and the second image, (a) wherein the first image is to be obtained before the treatment or before the second image; (b) wherein the second image is to be obtained after the treatment or after the first image; (c) wherein the analysis of the first image and the second image is an analysis and comparison of the structure of the posterior segment in (a) and (b); wherein the maintenance or reduction of the altered structure between (a) and (b) indicates that the treatment has a therapeutic effect or that the subject complies with the treatment.

[0124] Furthermore, in one embodiment, the use of a first image and a second image, wherein the altered structure is a structure altered relative to a structure determined from analysis of a TOI image of the posterior segment obtained from a subject of a similar condition known not to have the disease or known not to be at risk of developing the disease.

[0125] In another embodiment of the present invention, the aforementioned use relates to such use, wherein determining the maintenance of the altered structure between (a) and (b) is (d) determining wherein the altered structure is substantially unchanged between (a) and (b) of the aforementioned use; or (e) determining wherein any further change or progression of the altered structure between (a) and (b) of the aforementioned use is not as severe or advanced as the change or progression between a set of reference first TOI images and a set of reference second TOI images of the posterior segment, the set of reference first TOI images and the set of reference second TOI images being obtained from subjects with similar conditions known to suffer from a disease and the subjects not being treated for the disease, wherein the reference first image and the reference second image are obtained or to be obtained at the same interval as the first image and the second image according to (a) and (b) of the aforementioned use.

[0126] In a preferred embodiment of the invention, the use according to the invention relates to the use wherein the disease associated with the altered structure in the posterior segment of the eye is uveitis, glaucoma, macular edema, macular hole, macular pucker, diabetic macular edema, diabetic retinopathy, diabetic eye disease, retinopathy, age-related macular degeneration (AMD), wet AMD, dry AMD, early AMD, intermediate AMD, central serous chorioretinopathy, scleritis, optic nerve degeneration, geographic atrophy, choroidopathy, ocular sarcoidosis, optic neuritis, choroidal neovascularization, retinitis pigmentosa, retinal tear, Stargardt's disease, eye cancer, retinitis, corneal ulcer, cataract, viral infection (such as cytomegalovirus, herpes simplex, herpes zoster), fungal infection (such as histoplasmosis), parasitic infection (such as toxocariasis, toxocariasis), bacterial infection (such as tuberculosis , syphilis), sarcoidosis, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinal vasculopathy, Vogt-Koyanagi-Harada syndrome, Behçet's disease, idiopathic retinal vasculitis, Vogt-Koyanagi-Harada syndrome, acute posterior multifocal squamous pigment epitheliopathy (APMPPE), ocular pseudohistoplasmosis syndrome (POHS), birdshot retinochoroidopathy, multiple sclerosis, sympathetic ophthalmia, punctate inner choroidopathy, pars planitis, iridocyclitis diabetic retinopathy, retinopathy of prematurity (ROP), ischemic vascular disease, hereditary retinal dystrophy, retinal detachment, abnormal angiogenesis, retinal angiomatous proliferation (RAP), intraretinal microvascular abnormalities, preretinal neovascularization, choroidal angiogenesis, choroidal vasculopathy stroke, hypertension, diabetes, cardiovascular disease, prematurity, and papilledema.

[0127] In certain embodiments of the invention, the invention relates to such use, wherein the image of the posterior segment of the eye is an image of the choroid, choriocapillaris, Bruch's membrane, retinal choroidal tissue, neural retinal tissue, nerve fiber layer, retinal pigment epithelium (RPE), photoreceptors, ganglion cell layer, retinal vasculature, subretinal space, retina, macula, lamina cribrosa, optic disc or optic nerve.

[0128] In certain embodiments of the present invention, the invention relates to uses wherein the altered structure is an alteration of tissue structure.

[0129] In certain embodiments of the present invention, the invention relates to uses wherein the change in tissue structure is a change in cell morphology, cell density, cell size, cell distribution or cell reflectivity.

[0130] In certain embodiments of the present invention, the invention relates to uses wherein the change in tissue structure is a change in cell morphology, cell density, cell size, cell distribution or cell reflectivity.

[0131] In certain embodiments of the present invention, the present invention relates to such a use, wherein the change in tissue structure is a change in tissue reflectivity, and the change in tissue reflectivity is a change in low reflective areas, high reflective areas, low reflective areas within high reflective areas, or any combination thereof.

[0132] In a preferred embodiment of the present invention, the present invention relates to the use of an image, wherein said image is an image of the RPE.

[0133] In another preferred embodiment of the present invention, the present invention relates to the use of an image, wherein the image is an image of the choriocapillaris.

[0134] In certain embodiments of the invention, the invention relates to the use of an image, wherein the image is an image of the nerve fiber layer, the optic disc and / or the retinal vasculature.

[0135] In certain embodiments of the invention, the invention relates to a use wherein the disease is AMD and the altered structure is an alteration in RPE cell density, RPE cell size, hyperreflective areas, hyporeflective areas and / or hyporeflective areas within hyperreflective areas.

[0136] In certain embodiments of the invention, the invention relates to the use wherein the disease is central serous chorioretinopathy and the altered structures are alterations of the RPE and choriocapillaris.

[0137] In certain embodiments of the invention, the invention relates to such use, wherein the disease is glaucoma and the altered structure is a change in the nerve fiber layer (including but not limited to a change in the orientation of the nerve fiber bundles, a change in the thickness of the nerve fiber bundles, a change in the size of the nerve fiber bundles) or a change in the morphology of the optic disc (including but not limited to a change in the optic nerve head, a change in the physiological cup, a change in the cup-disc ratio, a change in the lamina cribrosa).

[0138] In certain embodiments of the invention, the invention relates to the use wherein the disease is diabetic retinopathy and the altered structure is an alteration of the retinal vasculature.

[0139] In certain embodiments of the invention, the invention relates to a use wherein the altered structure is indicative of geographic atrophy, drusen, reticular pseudodrusen, neovasculature and / or retinal pigment epithelial degeneration.

[0140] Figure 1 Examples of images of the different degrees of non-neovascular AMD observed and comparison with SD OCT.

[0141] Figure 2 Comparison between images taken with SD OCT, autofluorescence and color and findings in TOI images of AMD patients with focal geographic atrophy. The TOI images show a reticular choroidal pattern with dark spots (or hyporeflective areas) of variable size associated with degenerative remnants of the RPE.

[0142] Figure 3 A) A 5°×5° area (5.4° eccentricity) acquired within the fovea and macula. B) In vivo RPE image of the contralateral normal eye of a CSCR patient (temporal, below the fovea). Absorption of incident infrared (830 nm) light by melanin in the RPE results in a hyporeflective core region on the TOI.

[0143] Figure 4 TOI of the fovea in the case of neurosensory detachment (middle right); low-resolution image with hyperreflective spots and hyperreflective content of pigment epithelial detachment on OCT (far right). FAF (far left) shows hyperautofluorescence, while IR (middle right) shows hyporeflective areas in the detached area with central high reflectivity.

[0144] Figure 5 TOI depicts the RPE mosaic pattern of the change in the upper half (center), and the corresponding inner choroid changes (right) on OCT, and maintains RPE anatomical structure in the lower half (center), and normal corresponding OCT (left). These regions show changes on infrared imaging, confirming the anatomical position of pathology.

[0145] Figure 6 FAF (far left) shows a descending tract with high and low AF, while IR (middle left) shows areas of high reflectivity. TOI (middle right) shows multiple high reflective foci corresponding to high reflective deposits on OCT (far right, between vertical lines). High reflective spots represent areas of increased reflectivity secondary to the disease process.

[0146] Figure 7 Note the loss of the central hyporeflective area in FAF (left) with hyperreflectivity on IR (center), secondary to neurosensory detachment. TOI (center right) demonstrates multiple hyporeflective areas (foci) suggestive of scotoma with inner choroidal changes in the corresponding OCT.

[0147] Figure 8 AMD patient with drusen. Confocal TOI image (center) shows drusen with dark center and white halo (low reflective area within high reflective area). Not all such TOI appearances are visible on OCT en face or b-scans. Retinal blood vessels run through the image, hiding deeper structures.

[0148] Fig. 9 AMD patient with few middle drusen.Confocal TOI image shows a large number of small dark spots, partly with white halo (low reflection area in high reflection area), most of which can not be clearly identified on OCT.However, there are many small RPE changes on OCT b-scan, which may transform visible deposits on TOI.The background color on TOI shows choroidal blood vessels and choriocapillaris.

[0149] Fig.10 AMD patients suffering from drusen are well visible on OCT frontal images.Confocal TOI shows a large amount of irregularly distributed dark spots, and part has a white halo (low reflection area in the high reflection area).Some of them are similar to the typical hexagonal pattern (yellow circle) of normal RPE cells and are arranged.In addition, choroidal blood vessels and retinal blood vessels are all visible.

[0150] Fig.11 AMD patient with small drusen. Confocal TOI shows dark pots with white halos (low reflective areas within high reflective areas) corresponding to drusen. More dark spots (low reflective areas) are visible on the TOI that do not correspond to the OCT image and may be too small to be visible on OCT.

[0151] General methods and materials

[0152] TOI Image Acquisition

[0153] TOI relies on high-angle oblique illumination of the retina combined with a flood-illuminated adaptive optics fundus camera to enhance cellular contrast and correct for ocular aberrations. Transscleral illumination of the retina is performed using two near-infrared light-emitting diodes located on the nasal and temporal sides of the eye. The acquired images include a field of view of at least 4° × 4°.

[0154] The RPE layer images obtained by a single TOI are characterized by a low signal-to-noise ratio (SNR). Therefore, before image analysis, the SNR is increased by acquiring several raw images that are subsequently registered and averaged into a single TOI image. The final TOI-RPE image is output with digital sampling between 0.73 μm and 1.5 μm per pixel.

[0155] Image Processing

[0156] The RPE image processing and analysis that highly automated TOI obtains is divided into four stages.First, the image is normalized in terms of contrast / attenuation, inhomogeneity and noise of the RPE layer, and any out-of-focus (OoF) region is discarded.Second, the shadow of the retinal vasculature present in the innermost (closest to the vitreous body) retinal layer is detected and removed from the final image.Third, separate detection and segmentation of interested cells or structures.Finally, the fourth and last step includes characterizing the interested layer with general and single structure.

[0157] Image filtering and normalization

[0158] In order to adjust for the inhomogeneity of the background layer of interest, background removal is applied. The background removal may include, for example but not limited to, a flat field correction with a two-dimensional Gaussian smoothing kernel.

[0159] For example, for the RPE layer, in order to prevent filtering out the basic RPE morphology in the spatial and frequency domains, the filter sizes, thresholds and values ​​implemented throughout the image processing and analysis methods were obtained experimentally and based on previously published literature in the assessment of the ex vivo and in vivo morphology of RPE cells.

[0160] Detection and removal of blood vessels

[0161] As an example, blood vessel detection is performed by using the following method. Other methods based on machine learning can be applied.

[0162] The detection of blood vessels is performed by using four previously acquired images ( / i| lpГ.Gauss, deH, and Dist). Each image is subjected to subroutine A (SubA). SubA starts with square shaping of the image, and its quadtree decomposition (QuaD) returns a sparse matrix that is subsequently reconstructed as a block map. The QuaD threshold is applied with 3*SD of the image. QuaD is a common method in several fields (including image processing), used from multi-resolution decomposition and analysis to compression and machine learning. Applying QuaD to RPE cell seg-culture is a new method developed specifically for this project. QuaD square blocks of >8 pixels and <10% of the original image size are included in subsequent image processing. After inversion (image complementation), morphological filtering (erosion with a disk-shaped element of 4 pixel radius) is used, followed by dilation with the same disk-shaped element, discarding the obtained square block mapping, the small and interconnected structure at its outer boundary. Finally, the last step of SubA is to reshape the resulting mask to the original image size.

[0163] The OoF mask obtained during the image filtering and normalization stage was summed with the binary average of SubA(Bhpf)-SubA(Causs), SubA(deH) and SubA(Dist) to form the vessel-OoF mask (VOoF). The VOoF mask was used to eliminate infravascular RPE cells from further image processing.

[0164] Cell detection

[0165] Cell center detection is based on the method proposed by Khamidakh et al. (Ann Biomed Eng. 2016; 44: 3408-20), which is hereafter referred to as subroutine B (SubB). In the case where the distance between adjacent cell centers is < 10 pixels, individual cells are detected as identical cells. We apply SubB to Z? hpf (i.e., to contrast-limited adaptive histogram equalization Z? hpf) and to high-pass filtered (|kernel of original image size) / i| lpГ Again, cell centers within <10 pixels were merged. Finally, cell centers that were <10 pixels away from the image border were removed to prevent the inclusion of non-fully imaged cells in the image analysis.

[0166] Cell membrane segmentation

[0167] Method Example 1

[0168] The detection of the cell membrane can be performed using local minimum detection for detecting the center of low-reflection areas or high-reflection areas. Then, a region growing algorithm is applied to define the region of interest.

[0169] Method Example 2

[0170] The detection of the cell membrane at the single cell level starts with convolving the Z?hpf with a disc-like structuring element (radius 4 pixels). The resulting image blur removes any possible local salt and pepper noise that may have appeared during the transformation from Fourier to the spatial domain. The image is then convolved with a star-shaped mask (size 7 pixels). The convolution enhances local vertical, horizontal and diagonal edges in the image. The final hlter is a 7×7 pixel Mexican hat. Using these three filtering stages and subsequent zero crossings in the spatial domain, a binary mask representing the cell membrane is developed. Finally, the mask is skeletonized and cleaned of sporadic branches while discarding single pixels. The inverted mask is convolved with a disc-like structuring element (radius 4 pixels) and re-inverted. This procedure improves the separation of cells and prevents their possible overlap.

[0171] Data analysis

[0172] Consider the example of RPE cells :

[0173] Cells whose area or center overlapped with the VOoF mask were discarded from the cell property analysis.

[0174] The image obtained using the cell mask previously generated and the original TOI was used to assess the morphology and neighboring characteristics of a single RPE cell. The MATFAB regionprops function is used to obtain the basic morphological characteristics (area, center of mass and gravity, eccentricity, density, intensity and circularity) of RPE cells. In addition, the characteristics assessed include the CV (CMDcv) of the RPE cell membrane, the number of adjacent cells and the cell density of the RPE layer. In order to reduce the possible risk of assessment bias, the RPE cells next to the VOoF mask are discarded from many neighbor evaluations. Descriptive analysis has been carried out for each image.

[0175] Normality of variables was assessed using the Shapiro-Wilk test (p>0.10) and histogram skewness (skewness—0.5-0.5).

[0176] In a computer equipped with MATFAB (version R2019, with Bioinformatics Toolbox TM 、FinancialToolbox TM and Statistics and Machine Teaming Toolbox TMThe image processing pipeline and underlying algorithms as well as data management were developed and tested on a DEFF workstation (DEFF XPS139380, Windows 10, 64-bit, 2 1.80GHz, 16.0GB RAM). Image registration was performed using ImageJ1.52 with modified macros with plugins TurboReg and Template Matching. For boxplot generation and statistical analysis, we used R studio 1.2.1335 with gmodels, el071, readxl, and xlsx packages.

[0177] TOI of Central Serous Chorioretinopathy (CSCR)

[0178] Patients with a clinical diagnosis of CSCR (clear optical medium and good fixation) were recruited. For each patient, BCVA, refractive error spherical equivalent and axial length measurements, fundus autofluorescence (FAF), infrared imaging (IR), and SD optical coherence tomography (OCT) were performed in both eyes.

[0179] TOI was successfully performed in 12 patients (21 CSCR eyes and 1 normal fellow eye) with a mean age of 43.3 ± 4 years. The RPE structure or mosaic pattern appears as a fine network of cells with low-reflective (core) areas within high-reflective areas (borders). Figure 3 B)

[0180] Eyes with active CSCR (N=7) showed poor resolution in areas with retinal detachment on the TOI ( Figure 4 ). The presence of subretinal fluid in the path of light scattered from the RPE results in reduced resolution of RPE monolayer imaging.

[0181] Eyes with resolved CSCR (N=14) showed an altered RPE mosaic pattern on the TOI ( Figure 5 ). Changes in the scattering surfaces (RPE and choroid) secondary to CSCR lead to an altered mosaic pattern on the TOI.

[0182] Highly reflective spots on the TOI were observed in all 21 eyes, corresponding to highly reflective outer retinal deposits ( Figure 4 ) or RPE detachment with highly reflective content on OCT ( Figure 6 ).

[0183] TOI images also revealed scotomas (hyporeflective foci) in 19 of 21 eyes. They were associated with normal surrounding RPE, hyperreflective structures or areas ( Figure 7 ).

[0184] TOI for non-neovascular age-related macular degeneration

[0185] Patients with non-neovascular AMD (clear optical media and well-fixed) were recruited. For each patient, SD-OCT, autofluorescence, color and infrared fundus imaging, and TOI were performed in one or both eyes. Images obtained by TOI were compared with conventional imaging including OCT en face and OCT b-scans ( Figures 8 to 11 ).

[0186] 31 eyes of 25 AMD patients (average age 71.8 years, 56% women) are included.The notable feature on the TOI image is the reticular or cellular pattern of choriocapillaris, and the presence of other irregularly distributed low-reflection areas.These low-reflection areas, whose size varies from 1 times to about 15 times of the average RPE cell size, are located on the low-reflectivity area of ​​choriocapillaris, and are particularly present in the border area around the atrophic zone and in the area where visible RPE changes.The subgroup of these cells, showing bright halo (low-reflection area in the high-reflection area) around them, is well correlated with the drusen-like deformation of RPE system on SD-OCT.

[0187] TOI performed in 25 AMD patients revealed different patterns of altered organization in the RPE layer. Therefore, TOI is an important tool for evaluating retinal diseases such as AMD.

Claims

1. A method for diagnosing and / or prognosing a disease associated with an altered structure in the posterior segment of an eye, wherein the method comprises analyzing an image of the altered structure of the posterior segment of the eye obtained by transscleral optical imaging (TOI), wherein the altered structure indicates the presence and / or progression of the disease in a subject.

2. A method of treating a disease associated with an altered structure in the posterior segment of the eye, wherein the method The following steps are involved: a) analyzing the image of the altered structure of the posterior segment of the eye obtained by the TOI, wherein the presence of the altered structure indicates the presence of the disease or the progression of the disease in the subject; and b) administering to said subject identified as having or developing a disease according to step (a) an appropriate treatment for said disease.

3. The method of claim 1 or 2, wherein the altered structure is determined relative to a reference, the reference being a TOI image of the posterior segment obtained from a subject of a similar condition known not to have the disease or known not to be at risk of developing the disease.

4. A method for (i) evaluating the therapeutic effect of a treatment directed against a disease associated with an altered structure in the posterior segment of the eye in a subject, or (ii) a method of determining a subject's compliance with a prescribed treatment for a disease associated with an altered structure in the posterior segment of the eye, The method comprises analyzing a first image and a second image of the posterior segment of the eye of the subject obtained by TOI, (a) wherein the first image is acquired before the treatment or before the second image; (b) wherein the second image is to be obtained after treatment or after the first image; (c) wherein the analysis of the first image and the second image is an analysis and comparison of the structure of the posterior segment in (a) and (b); Wherein maintenance or reduction of the altered structure between (a) and (b) indicates that the treatment has a therapeutic effect or that the subject is compliant with the treatment.

5. The method of claim 4, wherein the altered structure is a structure that is altered relative to a structure determined from analysis of a TOI image of the posterior segment obtained from a subject of a similar condition known not to have the disease or known not to be at risk of developing the disease.

6. The method of claim 4, wherein the maintenance of the changed structure between (a) and (b) is determined by (d) determining that the altered structure is substantially unchanged between (a) and (b) according to claim 4; or (e) determining that any further change or progression of the altered structure between (a) and (b) according to claim 4 is not as severe or advanced as the change or progression between a set of reference first TOI images and a set of reference second TOI images of the posterior segment, the set of reference first TOI images and the set of reference second TOI images being obtained from subjects with similar conditions known to have the disease and the subjects not being treated for the disease, wherein the reference first images and the reference second images are obtained or to be obtained at the same interval as the first images and the second images according to (a) and (b) according to claim 4.

7. The method according to any one of claims 4 to 6, wherein the second image is to be obtained at least 2 days and not more than 730 days after the first image.

8. The method of any one of claims 1 to 7, wherein the disease associated with altered structure in the posterior segment of the eye is uveitis, glaucoma, macular edema, macular hole, macular pucker, diabetic macular edema, diabetic retinopathy, diabetic eye disease, retinopathy, age-related macular degeneration (AMD), wet AMD, dry AMD, early AMD, intermediate AMD, central serous chorioretinopathy, scleritis, optic nerve degeneration, geographic atrophy, choroidopathy, ocular sarcoidosis, optic neuritis, choroidal neovascularization, retinitis pigmentosa, retinal tear, Stargardt's disease, eye cancer, retinitis, corneal ulcer, cataract, viral infection (such as cytomegalovirus, herpes simplex, herpes zoster), fungal infection (such as histoplasmosis), parasitic infection (such as toxocariasis, toxocariasis), bacterial infection (such as tuberculosis, syphilis), sarcoidosis, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinal vasculopathy, Vogt-Koyanagi-Harada syndrome, Behcet's disease, idiopathic retinal vasculitis, Vogt-Koyanagi-Harada syndrome, acute posterior multifocal squamous pigment epitheliopathy (APMPPE), ocular pseudohistoplasmosis syndrome (POHS), birdshot retinochoroidopathy, multiple sclerosis, sympathetic ophthalmia, punctate inner choroidopathy, pars planitis, iridocyclitis diabetic retinopathy, retinopathy of prematurity (ROP), ischemic vascular disease, hereditary retinal dystrophy, retinal detachment, abnormal angiogenesis, retinal angiomatous proliferation (RAP), intraretinal microvascular abnormalities, preretinal neovascularization, choroidal angiogenesis, choroidal vasculopathy stroke, hypertension, diabetes, cardiovascular disease, prematurity, and papilledema.

9. The method of any one of claims 1 to 8, wherein the image of the posterior segment of the eye is an image of the choroid, choriocapillaris, Bruch's membrane, retinal choroidal tissue, neural retinal tissue, nerve fiber layer, retinal pigment epithelium (RPE), photoreceptors, ganglion cell layer, retinal vasculature, subretinal space, retina, macula, lamina cribrosa, optic disc, or optic nerve.

10. The method according to any one of claims 1 to 9, wherein the altered structure is an alteration in tissue structure.

11. The method of claim 10, wherein the change in tissue structure is a change in cell morphology, cell density, cell size, cell distribution or cell reflectivity.

12. The method of claim 10, wherein the change in tissue structure is a change in tissue reflectivity, which is a change in low reflective areas, high reflective areas, low reflective areas within high reflective areas, or any combination thereof.

13. The method according to any one of claims 1 to 12, wherein the image is an image of the RPE.

14. The method according to any one of claims 1 to 12, wherein the image is an image of the choriocapillaris.

15. The method of any one of claims 1 to 12, wherein the image is an image of the nerve fiber layer, the optic disc and / or the retinal vasculature.

16. according to the method for claim 13, the structure that wherein said disease is AMD and said change is the change of the low-reflection area in RPE cell density, RPE cell size, high-reflection area, low-reflection area and / or the high-reflection area.

17. The method of claim 13 or 14, wherein the disease is central serous chorioretinopathy and the altered structures are changes in the RPE and the choriocapillaris.

18. The method of claim 15, wherein the disease is glaucoma and the altered structure is a change in the nerve fiber layer (including but not limited to a change in the orientation of the nerve fiber bundle, a change in the thickness of the nerve fiber bundle, a change in the size of the nerve fiber bundle) or a change in the morphology of the optic disc (including but not limited to a change in the optic nerve head, a change in the physiological cup, a change in the cup-disc ratio, a change in the lamina cribrosa).

19. The method of claim 15, wherein the disease is diabetic retinopathy and the altered structure is an alteration of the retinal vasculature.

20. The method of any one of claims 1 to 12, wherein the altered structure is indicative of geographic atrophy, drusen, reticular pseudodrusen, neovasculature, and / or retinal pigment epithelial degeneration.

21. Use of an image of a subject's eye obtained by transscleral optical imaging (TOI) for the diagnosis and / or prognosis of a disease associated with an altered structure in the posterior segment of the eye, wherein the use comprises analyzing the image for the altered structure, wherein the altered structure is indicative of the presence and / or progression of the disease in the subject.

22. Use of an image of the eye of a subject obtained by TOI in treating a disease associated with an altered structure in the posterior segment of the eye, wherein said use The following steps are involved: a) analyzing said image for altered structures, wherein the presence of altered structures is indicative of said presence of said disease or said progression of said disease in said subject; and b) administering to said subject identified as having or developing a disease according to step (a) an appropriate treatment for said disease.

23. The use according to claim 21 or 22, wherein the altered structure is determined relative to a reference TOI image of the posterior segment, the reference TOI image being obtained from a similarly situated subject known not to suffer from the disease or known not to be at risk of developing the disease.

24. A first image and a second image of the eye of a subject obtained by transscleral optical imaging (TOI) for use in (i) evaluating the efficacy of a treatment directed against a disease associated with an altered structure in the posterior segment of the eye in a subject, or (ii) use for determining compliance of a subject with a prescribed treatment for a disease associated with an altered structure in the posterior segment of the eye, The use comprises analyzing the first image and the second image, (a) wherein the first image is acquired before the treatment or before the second image; (b) wherein the second image is to be obtained after treatment or after the first image; (c) wherein the analysis of the first image and the second image is an analysis and comparison of the structure of the posterior segment in (a) and (b); Wherein said maintenance or reduction of the altered structure between (a) and (b) indicates that said treatment has a therapeutic effect or that said subject is compliant with said treatment.

25. The use according to claim 24, wherein the altered structure is a structure that is altered relative to a structure determined from analysis of a TOI image of the posterior segment obtained from a subject of a similar condition known not to have the disease or known not to be at risk of developing the disease.

26. The use of claim 24, wherein the maintenance of the altered structure between (a) and (b) is determined by (d) determining that the altered structure is substantially unchanged between (a) and (b) according to claim 24; or (e) determining that any further change or progression of the altered structure between (a) and (b) according to claim 24 is not as severe or advanced as the change or progression between a set of reference first TOI images and a set of reference second TOI images of the posterior segment, the set of reference first TOI images and the set of reference second TOI images being obtained from subjects with similar conditions known to have the disease and the subjects not being treated for the disease, wherein the reference first images and the reference second images are obtained or to be obtained at the same interval as the first images and the second images according to (a) and (b) according to claims 24.

27. Use according to any one of claims 24 to 26, wherein the second image is to be obtained at least 2 days and not more than 730 days after the first image.

28. The method of claim 21 , wherein the disease associated with an altered structure in the posterior segment of the eye is uveitis, glaucoma, macular edema, macular hole, macular pucker, diabetic macular edema, diabetic retinopathy, diabetic eye disease, retinopathy, age-related macular degeneration (AMD), wet AMD, dry AMD, early AMD, intermediate AMD, central serous chorioretinopathy, scleritis, optic nerve degeneration, geographic atrophy, choroidopathy, ocular sarcoidosis, optic neuritis, choroidal neovascularization, retinitis pigmentosa, retinal tear, Stargardt's disease, eye cancer, retinitis, corneal ulcer, cataract, viral infection (such as cytomegalovirus, herpes simplex, herpes zoster), fungal infection (such as histoplasmosis), parasitic infection (such as toxocariasis, toxocariasis), bacterial infection (such as tuberculosis, syphilis ), sarcoidosis, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinal vasculopathy, Vogt-Koyanagi-Harada syndrome, Behcet's disease, idiopathic retinal vasculitis, Vogt-Koyanagi-Harada syndrome, acute posterior multifocal squamous pigment epitheliopathy (APMPPE), ocular pseudohistoplasmosis syndrome (POHS), birdshot retinochoroidopathy, multiple sclerosis, sympathetic ophthalmia, punctate inner choroidopathy, pars planitis, iridocyclitis diabetic retinopathy, retinopathy of prematurity (ROP), ischemic vascular disease, hereditary retinal dystrophy, retinal detachment, abnormal angiogenesis, retinal angiomatous proliferation (RAP), intraretinal microvascular abnormalities, preretinal neovascularization, choroidal angiogenesis, choroidal vasculopathy stroke, hypertension, diabetes, cardiovascular disease, prematurity, and papilledema.

29. The use according to any one of claims 21 to 28, wherein the image of the posterior segment of the eye is an image of the choroid, choriocapillaris, Bruch's membrane, retinal choroidal tissue, neuroretinal tissue, nerve fiber layer, retinal pigment epithelium (RPE), photoreceptors, ganglion cell layer, retinal vasculature, subretinal space, retina, macula, lamina cribrosa, optic disc or optic nerve.

30. The use according to any one of claims 21 to 29, wherein the altered structure is an alteration of the tissue structure.

31. The use according to claim 30, wherein the change in tissue structure is a change in cell morphology, cell density, cell size, cell distribution or cell reflectivity.

32. The use according to claim 31, wherein the change in tissue structure is a change in tissue reflectivity, which is a change in low reflective areas, high reflective areas, low reflective areas within high reflective areas, or any combination thereof.

33. The use according to any one of claims 21 to 32, wherein the image is an image of the RPE.

34. The use according to any one of claims 21 to 32, wherein the image is an image of the choriocapillaris.

35. The use according to any one of claims 21 to 32, wherein the image is an image of the nerve fiber layer, the optic disc and / or the retinal vasculature.

36. according to the described purposes of claim 33, the structure that wherein said disease is AMD and said change is the change of the low-reflection area in RPE cell density, RPE cell size, high-reflection area, low-reflection area and / or the high-reflection area.

37. Use according to claim 33 or 34, wherein the disease is central serous chorioretinopathy and the altered structures are changes in the RPE and the choriocapillaris.

38. The use according to claim 35, wherein the disease is glaucoma and the altered structure is a change in the nerve fiber layer (including but not limited to a change in the orientation of the nerve fiber bundle, a change in the thickness of the nerve fiber bundle, a change in the size of the nerve fiber bundle) or a change in the morphology of the optic disc (including but not limited to a change in the optic nerve head, a change in the physiological cup, a change in the cup-disc ratio, a change in the lamina cribrosa).

39. The use according to claim 35, wherein the disease is diabetic retinopathy and the altered structure is an alteration of the retinal vasculature.

40. The use according to any one of claims 21 to 32, wherein the altered structure is indicative of geographic atrophy, drusen, reticular pseudodrusen, neovasculature and / or retinal pigment epithelial degeneration.

Images