System and method for cell-level resolution imaging of the eye by in vivo transmission interference.

Abstract

The present invention relates to a system (1) for cell-level resolution imaging by in vivo full-field transmission interference for an individual's eye (6), and a method (100) for imaging by in vivo full-field transmission interference for an individual's eye (6).

Description

Technical Field

[0001] The present invention relates to in vivo eye imaging.

[0002] More precisely, the present invention relates to a system for in vivo imaging with cellular-level resolution for an individual's eye, and a method for obtaining in vivo cellular-level resolution for an individual's eye.

Background Art

[0003] Optical imaging plays an important role in daily diagnosis regarding eye health. Currently, almost all ophthalmic hospitals are equipped with slit lamp biomicroscopy, fundus cameras, and optical coherence tomography (OCT) devices that provide a whole-eye image. These technologies can address most aspects of the eye condition, but some important diseases such as dry eye (affected by 340 million people) and glaucoma (affected by 80 million people) may remain undiagnosed. To effectively treat these diseases, early and accurate diagnosis is required, but it is difficult to achieve with insufficient resolution and just looking at the whole eye. In principle, by using a higher-resolution diagnostic device capable of evaluating eye health at the cellular level, earlier and more accurate diagnosis, as well as a deeper understanding of the disease, can be achieved. However, mainly due to the limitations of current technologies, such high-resolution imaging is not currently widely available in clinical settings.

[0004] Several attempts have been made to create high-resolution imaging devices for clinical use. Confocal microscopy provides in vivo cellular-level resolution of the anterior segment of the eye, useful for diagnosing several conditions such as dry eye or endothelial dystrophy. However, this technique requires physical contact with the eye. This requirement has hindered its adoption in clinical settings, resulting in a reduced number of patients being imaged. Another class of imaging devices based on adaptive optics achieves cellular-level resolution in the retina, but these devices are complex to positionally match and expensive, making them currently only accessible to a few large clinical centers. [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to propose a solution to this situation. [Means for solving the problem]

[0006] Therefore, the present invention provides a system for cell-level resolution imaging by in vivo full-field transmission interference for an individual eye having an external surface. The eye has an external surface. The system is • An object block configured to position an individual's eye within the imaging plane when the system is operating, having an axis that coincides with the imaging axis, A lighting block comprising a light source coupled to a first optical lens and a polarizing beam splitter, configured to together produce an illumination beam, wherein the illumination beam is substantially collimated and symmetrical with respect to the axis of the object block, and during operation, at least a portion of the illumination beam penetrates the individual's eye, focuses onto the region of the eye's internal reflective layer, and is back-reflected by the internal reflective layer to form a secondary light source, and during operation, the imaging surface is located between the eye's internal reflective layer and its outer surface, and the lighting block and A detection block comprising a tube lens and a two-dimensional acquisition device, wherein the two-dimensional acquisition device comprises a plurality of sensors positioned within the detection plane and is configured to acquire at least one two-dimensional interference signal resulting from the interference between a substantially collimated reference wave obtained by direct retransmission of a first portion of a secondary light source by the eye and a divergent sample wave obtained by scattering a second portion of the secondary light source by a region of a slice of an individual's eye, wherein the detection plane is optically conjugate to the imaging plane, and the slice of the eye is localized on the imaging plane, The system comprises the following: Here, the divergent sample wave has an additional optical phase shift with respect to a substantially collimated reference wave.

[0007] Advantageously, a polarizing beam splitter is configured to polarize the illumination beam and filter out light that would be specularly reflected by the individual's eye during operation.

[0008] In another embodiment, the system, • An object arm (also called an "object block") configured to receive an individual's eye within the imaging plane when the system is operating, having an axis that coincides with the imaging axis, A lighting arm (also called a "lighting block") comprising a light source coupled to a first optical lens and a polarizing beam splitter, configured to together generate an illumination beam, wherein the illumination beam is substantially collimated and symmetrical with respect to the axis of the object arm, and during operation, at least a portion of the illumination beam penetrates the individual's eye, focuses into the region of the eye's internal reflective layer, and is back-reflected by the internal reflective layer to form an incident light wave (corresponding to a secondary light source), and during operation, the imaging plane is located between the eye's internal reflective layer and the eye's outer surface, and the lighting block, A detection arm (also called a "detection block") comprising a tube lens and a two-dimensional acquisition device, wherein the two-dimensional acquisition device comprises a plurality of sensors positioned within the detection surface and is configured to acquire at least one two-dimensional interference signal resulting from the interference between a substantially collimated reference wave obtained by the transmission of an incident light wave and a divergent sample wave obtained by scattering the incident light wave by voxels of a slice of the individual's eye (corresponding to a region of the individual's eye slice in three dimensions, such as a cube or cuboid), and the detection surface is optically conjugate to the imaging surface by an optical system including a polarizing beam splitter, the detection arm and The system comprises the following: Here, the divergent sample wave has an additional phase shift with respect to a substantially collimated reference wave. The polarizing beam splitter is configured to polarize the illumination beam and filter out light that is specularly reflected by the individual's eye during operation.

[0009] These systems function in transmission, in contrast to OCT systems, which function in reflection. Furthermore, the transmission configuration allows for a symmetrical and robust common-path optical design where two light waves (transmitted and sampled) propagate along the same object block (i.e., arm), whereas in OCT systems, two light waves (reflected from the sample and reference mirror) propagate within two separate optical blocks (i.e., arms). Beyond symmetry and robustness, such a common-path design has the advantage of being less sensitive to undesirable eye movements.

[0010] According to another advantageous aspect of the present invention, the system may include, individually or in any possible combination, one or more of the features described in the following embodiments.

[0011] Advantageously, the illumination beam is spatially incoherent light. By using a spatially incoherent light source, speckle in the image can be avoided.

[0012] Advantageously, the internal reflective layer of the eye is the sclera of the individual eye.

[0013] Preferably, the illumination beam has a divergence angle of less than 20 degrees. The divergence angle is measured relative to the axis of the object block (i.e., the arm), and one dimension of the area of ​​the internal reflective layer of the eye is less than 6 millimeters when illuminated by the illumination beam. This limited divergence angle substantially collimates the illumination beam, thereby enabling the acquisition of a high-contrast image.

[0014] Advantageously, the light source is positioned within the light source plane, and the system further includes an aperture positioned within the light source plane, the aperture configured to control the divergence angle of the illumination beam. The aperture allows for control of the degree of collimation of the illumination beam, and consequently, the contrast of the image obtained by the system.

[0015] Advantageously, the system further includes a linear polarizer placed between the polarizing beam splitter and the light source, or between the polarizing beam splitter and the two-dimensional acquisition device. Using a linear polarizer helps to improve the polarization degree of different beams traveling within the system, and thus improve interference contrast.

[0016] In some embodiments, the system further comprises at least one processor and a tunable lens. The tunable lens is positioned between a polarizing beam splitter and a two-dimensional acquisition device, has a controllable focal length, and is configured to be controlled to acquire multiple two-dimensional interference signals and determine multiple working images. Each of the multiple working images corresponds to a separate imaging plane along the imaging axis. The processor is configured to compute a 3D image of the individual's eye based on the multiple working images.

[0017] In some other embodiments, the system further comprises at least one processor (or processing unit) and a variable-focus lens. The variable-focus lens is positioned between a polarizing beam splitter and a two-dimensional acquisition device, has a controllable focal length, and is configured to be controlled to acquire multiple two-dimensional interference signals to determine multiple working images. Each of the multiple working images corresponds to a separate imaging plane along the imaging axis. At least one processor (or processing unit) is configured to compute a 3D image of the individual's eye based on the multiple working images. By having controllable elements such as the variable-focus lens, the position of the imaging plane can be changed. Thus, several images can be acquired along the imaging axis and, consequently, in the depth direction along the depth of the individual's eye during examination.

[0018] In some embodiments, a plurality of predetermined phase differences in the range of -π / 2 to +π / 2 are introduced between a reference wave and a sample wave by controlling the focal length of a tunable lens to acquire a plurality of two-dimensional interference signals in the depth direction within a cross-section of an individual's eye. At least one processor (or processing unit) is further configured to compute a tomographic image based on a linear combination of the two-dimensional interference signals among the plurality of two-dimensional interference signals.

[0019] Advantageously, the system further comprises an optical multiplexing system positioned between a polarizing beam splitter and a two-dimensional acquisition device to acquire multiple two-dimensional interference signals in the depth direction within the cross-section of an individual's eye in a single acquisition, wherein the optical multiplexing system is configured to provide a predetermined phase difference between a reference wave and a sample wave in the range of -π / 2 to +π / 2.

[0020] In some embodiments, The system further comprises a second optical lens positioned within a lighting block (i.e., an arm) or an object block (i.e., an arm), The object block (i.e., the arm) further comprises a third optical lens, · The second optical lens is configured to generate a focused beam from the illumination beam such that the focused beam converges on the rear focal plane of the third lens so that a substantially collimated beam exits from the third optical lens.

[0021] In these embodiments, the system enables obtaining an image of the anterior part of the eye (cornea, lens) during the examination.

[0022] Preferably, the third optical lens is a microscope objective lens, a turret of microscope objective lenses, or a zoom microscope objective lens, and those objective lenses have a numerical aperture of 0.25 or more.

[0023] Advantageously, the system further comprises an optical mask disposed in a plane also referred to as the mask plane between the polarization beam splitter and the two-dimensional acquisition device, and a lens or a lens system that conjugates the rear focal plane of the third lens with the mask plane. The optical mask is configured to partially block a substantial portion of the reference wave while minimizing the reduction of the object signal so as to improve the image contrast. An optical mask means an element that selectively blocks a part of the incident illumination. Typically, the optical mask is a transparent glass plate partially covered with a partially opaque paint or material. The entire optical mask can be made without using glass (see https: / / microcosmos.store / products / oblique-illumination-and-dark-field-filters). Typically, the partially opaque part is located at the center of the light beam to reduce the reference light. By selecting the thickness and type of the mask material, the transparency of the mask and the degree of reference light suppression can be controlled.

[0024] In some embodiments, the system further comprises a mobile platform with a third optical lens attached thereto and at least one processor (or processing unit), the mobile platform being configured to move along the imaging axis to acquire a plurality of two-dimensional interference signals and determine a plurality of working images, each of the plurality of working images corresponding to a separate imaging plane along the axis of the object block (i.e., the arm), and the at least one processor (or processing unit) being configured to calculate a 3D image of a three-dimensional sample based on the plurality of working images when moving the platform along the imaging axis. By having a moving element such as the mobile platform, the imaging plane can be moved along the imaging axis. Thus, along the imaging axis, and thus within the depth of the individual's eye being inspected, several images can be acquired in the depth direction.

[0025] In some embodiments, by moving the mobile platform along the imaging axis so as to acquire a plurality of two-dimensional interference signals in the depth direction inside the cross-section of the individual's eye, a plurality of predetermined phase differences in the range of -π / 2 to +π / 2 are brought about between the reference wave and the sample wave, and the at least one processor (or processing unit) is further configured to calculate a tomographic image based on the linear combination of the two-dimensional interference signals among the plurality of two-dimensional interference signals.

[0026] In some embodiments, the system further comprises a mobile platform to which a tube lens is attached, and at least one processor (or processing unit), wherein the mobile platform is configured to move along the imaging axis to acquire multiple two-dimensional interference signals and determine multiple working images, each of which working images corresponds to a separate imaging plane along the axis of an object block (i.e., an arm), and at least one processor (or processing unit) is configured to compute a 3D image of a three-dimensional sample based on the multiple working images as the platform moves along the imaging axis. By having a mobile element such as a mobile platform, the imaging plane can be moved along the imaging axis. Thus, several images can be acquired along the imaging axis and, consequently, in the depth direction within the depth of the individual's eye during examination.

[0027] In some embodiments, a moving platform is moved along the imaging axis to acquire multiple two-dimensional interference signals in the depth direction within a cross-section of an individual's eye, thereby introducing multiple predetermined phase differences between a reference wave and a sample wave in the range of -π / 2 to +π / 2, and at least one processor (or processing unit) is further configured to compute a tomographic image based on a linear combination of the two-dimensional interference signals among the multiple two-dimensional interference signals.

[0028] In some embodiments, the system further comprises an additional eye imaging system, such as a fundus camera or slit-lamp system, or an additional axial position tracking system, such as an optical coherence tomography device, a macroview camera system, a stereo camera, or a pupil camera, the additional eye imaging system or additional axial position tracking system being integrated by a dichroic mirror positioned within the illumination block (i.e., arm), within the object block (i.e., arm), or between the beam splitter and the two-dimensional acquisition device.

[0029] Another aspect of the present invention is a method for in vivo full-field transmission interference imaging of an individual's eye, (a) Positioning the eye on the imaging plane within the object block of the system for in vivo full-field transmission interference imaging, (b) Illuminating an individual's eye with a substantially collimated, spatially incoherent, and symmetrical illumination beam, such that the illumination beam is focused onto a substantially small area of ​​the eye's internal reflective layer, which is then reflected back by the internal reflective layer to form a secondary light source, and the imaging plane is located between the internal reflective layer and the outer surface of the eye. (c) Acquiring at least one two-dimensional interference signal using a two-dimensional acquisition device comprising a plurality of sensors arranged within the detection surface, At least one two-dimensional interference signal arises from the interference between a reference wave obtained by the direct retransmission of a first portion of the secondary light source by the eye, and a sample wave obtained by the scattering of a second portion of the secondary light source by a region of the slice of the individual's eye. The imaging plane is conjugate to the detection plane, and the eye slices are localized on the imaging plane; at least one two-dimensional interference signal is acquired. (d) At least one first image is obtained from at least one two-dimensional interferometric signal by at least one processor of the system. This includes methods.

[0030] In another embodiment, the method is (a) Positioning the eye on the imaging plane within the object arm (which may also be called the "object block") of the system for in vivo full-field transmission interference imaging, (b) Illuminating an individual's eye with a substantially collimated, spatially incoherent and symmetrical illumination beam, such that the illumination beam is focused onto a substantially small area of ​​the eye's internal reflective layer, which then reflects back through the internal reflective layer to form an incident light wave (corresponding to a secondary light source), (c) Acquiring at least one two-dimensional interference signal using a two-dimensional acquisition device comprising a plurality of sensors arranged within the detection surface, At least one two-dimensional interference signal arises from the interference between a reference wave obtained by the transmission of an incident light wave and a sample wave obtained by scattering the incident light wave by voxels in a slice of the individual's eye (corresponding to a region of the individual's eye slice in three dimensions, such as a cube or rectangular prism). The imaging plane is conjugate to the detection plane, and at least one two-dimensional interference signal is acquired. (d) The system's processing unit acquires at least one first image from at least one two-dimensional interference signal. Includes.

[0031] According to another advantageous aspect of the present invention, the method may include, alone or in any possible combination, one or more of the features described in the following embodiments.

[0032] In some embodiments, in step (d), multiple first images are acquired at separate points in time, and the method is • Calculating the average image, which is a linear combination of multiple first images, • Obtaining a first image valid at a time point after a separate time point using a method for imaging by in vivo full-field transmission interference, • By subtracting the calculated average image from the effective first image, an image with improved contrast can be obtained. It also includes.

[0033] In some embodiments, this method The method further includes training an artificial neural network based on at least one training set of images of a training sample, each training set comprising a first training image and a second training image of the same cross-section of the training sample obtained by averaging different images obtained using a method for in vivo full-field transmission interferometry imaging, and the training is performed as follows: • For each training set, the corresponding first training image is provided to the artificial neural network as input. • To provide a corresponding second training image as the target output of the artificial neural network. Includes, The method further includes obtaining a denoised image as the output of a trained artificial neural network, which is obtained by inputting at least one image acquired in step (d) into the trained artificial neural network.

[0034] In some embodiments, the method further includes segmenting and counting cells and nerves in at least one first image obtained.

[0035] In some embodiments, if the system for in vivo full-field transmission interference imaging further includes an additional position tracking system, in step (d), at least one envelope image is obtained using the system for in vivo full-field transmission interference imaging at at least one time point. The method is • For each of at least one Enface image, obtain at least one corresponding image using an additional location tracking system, • Calculating the axial position of the individual's eye along the imaging axis over time, based on at least one corresponding image acquired using an additional position tracking system. It also includes.

[0036] In some embodiments, in step (c), a set of two-dimensional interference signals is acquired, and the method further comprises using at least one processor (or processing unit) of a system for in vivo full-field transmission interference imaging to compute an image also called a motion image, which represents the temporal variation in intensity between the two-dimensional interference signals during the set of two-dimensional interference signals.

[0037] Furthermore, the preferred and advantageous characteristics associated with the system can also be applied to the method. [Brief explanation of the drawing]

[0038] [Figure 1a] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments also known as retinal imaging embodiments. [Figure 1b] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments also known as retinal imaging embodiments. [Figure 2a] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye in several embodiments, showing the illumination divergence angle of the illumination beam in a retinal imaging embodiment. [Figure 2b] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye in several embodiments, showing the illumination divergence angle of the illumination beam in a corneal imaging embodiment. [Figure 3a] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments also known as corneal imaging embodiments. [Figure 3b] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments also known as corneal imaging embodiments. [Figure 4] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to another corneal imaging embodiment. [Figure 5a] This is a schematic diagram of an example of a system for in vivo full-field transmission interference imaging of an individual's eye, in several embodiments, in which the system includes controllable elements. [Figure 5b] This is a schematic diagram of an example of a system for in vivo full-field transmission interference imaging of an individual's eye, in several embodiments, in which the system includes controllable elements. [Figure 6a] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments, in which the system includes an optical multiplexing system. [Figure 6b]This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments, in which the system includes an optical multiplexing system. [Figure 7] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments, in which the system includes an additional eye imaging system. [Figure 8a] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, in several embodiments, in which the system enables partial suppression of a reference wave. [Figure 8b] This is a schematic diagram of a system for in vivo full-field transmission interference imaging of an individual's eye, in several embodiments, in which the system enables partial suppression of a reference wave. [Figure 9] This figure shows an example flowchart illustrating the key steps of a method for in vivo full-field transmission interference imaging of an individual's eye, according to several embodiments. [Figure 10a] These are images of the internal reflective layers of the human eye, such as retinal and corneal images. [Figure 10b] These are images of the internal reflective layers of the human eye, such as retinal and corneal images. [Figure 10c] These are images of the internal reflective layers of the human eye, such as retinal and corneal images. [Figure 10d] These are images of the internal reflective layers of the human eye, such as retinal and corneal images. [Figure 10e] These are images of the internal reflective layers of the human eye, such as retinal and corneal images. [Figure 10f] These are images of the internal reflective layers of the human eye, such as retinal and corneal images. [Figure 10g] These are images of the internal reflective layers of the human eye, such as retinal and corneal images. [Figure 11a] This figure shows one of two interference images of the corneal stroma. [Figure 11b] This figure shows one of two interference images of the corneal stroma. [Figure 11c]This figure shows an optical cross-section resulting from the difference between two interference images of the corneal stroma. [Modes for carrying out the invention]

[0039] The present invention relates to a system 1 for in vivo imaging of a person's eye at cellular level resolution, and a method for obtaining in vivo cellular level resolution of a person's eye using the system 1.

[0040] More precisely, System 1 is a full-field transmission interferometric imaging system. Figures 1a and 1b illustrate examples of System 1 in several embodiments, also known as retinal imaging embodiments. Here, the entire field is used, as opposed to imaging methods that use scanning systems to produce images. Full-field imaging uses an array of sensors, such as those available in CCD or CMOS cameras. The illumination of a full-field system is typically spatially wider (hundreds of micrometers to several millimeters) than the illumination of a scanning system (typically a few microns).

[0041] As shown in Figure 1, System 1 comprises a lighting block (also called a lighting arm) 2, an object block (also called an arm) 3, and a detection block (also called a detection arm). The detection block includes a two-dimensional acquisition device 4. System 1 also includes at least one processor (or processing unit) 5.

[0042] The object block 3 is intended to receive the individual's eye 6 under examination during function (meaning the object block 3 is configured to position the eye). During function, the individual's eye 6 is positioned along the object block 3 within the imaging plane. The object block 3 has an axis that coincides with the imaging axis d. The axis of the object block 3 and the imaging axis d do not necessarily have to coincide with the axis of the eye 6. For example, the eye 6 may be tilted relative to its axis. As shown in Figure 1, the eye 6 includes an outer surface 6a which can be defined as the outer surface of the cornea C and a back surface 6b formed by the outer surface of the sclera S.

[0043] Illumination block 2 comprises a light source 21 positioned in a plane also called the light source plane, a first optical lens 22, and a polarizing beam splitter 23. The light source 21 is typically selected to emit spatially incoherent light to avoid speckle artifacts present in coherent light sources. For example, the light source 21 is a light-emitting diode (LED) that emits light in the near-infrared (NIR) spectrum at approximately 850 nm. This wavelength is comfortably accepted by the eye because the human eye is less sensitive to this wavelength. NIR light is also beneficial for three reasons: firstly, compared to visible light, NIR light is less absorbed by the choroidal blood vessels beneath the retina, leaving more light available for back illumination; secondly, this wavelength is short enough for imaging at cellular resolution (approximately 1 micron); and thirdly, NIR light is efficiently detected by cameras based on CMOS technology, which is more advanced than the technology of InGaAs cameras currently operating on infrared light.

[0044] The light source 21, the first optical lens 22, and the polarizing beam splitter 23 are coupled together to generate an illumination beam. The first optical lens 22 is selected so that the illumination beam is substantially collimated. Substantially collimated means that the illumination beam has a divergence angle α of 20 degrees or less. As will be further explained, such a divergence angle α of the illumination beam allows the system 1 to generate a substantially small light spot on the internal reflective layer of the eye 6 when working with the individual's eye 6. Figures 2a and 2b illustrate the divergence angle α for different embodiments of the system 1, namely a retinal imaging embodiment and a corneal imaging embodiment, respectively. We take the case where the internal reflective layer of the eye 6 is the posterior sclera S as an example. Non-collimated illumination with a half-angle greater than 20 degrees produces a large illumination spot on the sclera exceeding 6 mm (calculated from the focal length of 17 mm for an unaccommodated eye), while a fully collimated beam is focused by the eye to a minimum spot of less than 1 mm, the size of which depends on the size of the light source 21 and the focal length of the first optical lens 22. In an unaccommodated eye without abnormalities, the minimum spot is located on the retina R overlapping the sclera S. To produce the minimum spot on the sclera S, the first optical lens 22 is slightly shifted along the illumination path to produce a slightly divergent illumination beam (within 0 to 20 degrees) that can be efficiently focused on the sclera S. The 0 to 20 degree range is important, in particular, to ensure high interference contrast in the image, as will be seen later. The illumination beam is symmetric with respect to the axis of object block 3. The polarizing beam splitter 23 is configured to polarize the illumination beam, rejecting copolarized specular reflection from the eye 6 and transmitting the orthogonal polarized beam component after depolarization by the internal reflective layer.

[0045] When System 1 is operational, i.e., when the individual's eye 6 during examination is positioned within the imaging plane of object block 3, the illumination beam illuminates along the direction of object block 3 and penetrates the eye 6 by passing through its outer surface 6a. The light is refracted by the anterior segment of the eye and ultimately forms a focal point on the internal reflective layer of the eye 6. The selection of the internal reflective layer of the eye 6 and the size of the focal point on this layer is determined by the degree of collimation of the illumination beam. For example, the internal reflective layer of the eye 6 can be the retina R, sclera S, cornea C, or iris IR, with the most typical internal reflective layer being the sclera S.

[0046] In some embodiments, System 1 further comprises an aperture positioned within the light source plane. The aperture is configured to control and adjust the divergence angle of the illumination beam. As already mentioned, the degree of collimation of the illumination beam affects the contrast of the image obtained using System 1.

[0047] The degree of collimation can be controlled by a combination of optical elements in system 1, including the first optical lens 22. For example, the desired collimation can be achieved by shifting the first optical lens 22 relative to the light source 21 along the illumination block 2, by changing the focal length of the first optical lens 22, or by changing the aperture size, and thus by changing the effective size of the light source 21. The collimation illumination angle, controlled by the effective light source size and the focal length of the first optical lens 22, is

number

[0048] The required degree of collimation, which depends on the internal reflective layer of interest, is:

number

number

[0049] In another case where the iris IR is an internal reflective layer, the size of the illumination spot is controlled directly by the aperture, rather than by the focal length of the eye.

[0050] At this time, the light is reflected backward by the internal reflective layer of the eye 6, forming a secondary light source (which may also be called an incident light wave) that generates a light wave that propagates from the internal reflective layer toward the outer surface 6a of the eye. As a result, the light from the secondary light source propagates in the opposite direction to the propagation direction of the illumination beam I that propagates from the polarizing beam splitter toward the eye 6 in the object block. The properties of the internal reflective layer of the eye 6 depend on the embodiment and may be, for example, the retina R, sclera S, cornea C, or iris IR, as will be described later.

[0051] A portion of the incident light wave is directly retransmitted by eye 6, forming reference wave I. Another portion of these incident light waves is scattered by the slice of eye 6, forming sample wave SA. Reference wave I and sample wave SA have different divergence angles. Both reference wave I and sample wave SA produce optical interference. More specifically, reference wave I is quasi-collimated. The degree of collimation of reference wave I depends on the size of the focal point on the internal reflective layer of eye 6 and the focal length of eye 6.

number

[0052] While well-known OCT systems generally include four separate blocks arranged in an orthogonal cross shape around a beam splitter, with each block associated with a specific optical path among the illumination path, object (or sample) path, reference path, and detection path, System 1 is more compact with only three blocks. This is due to the fact that the object (or sample) path and reference path are associated with one specific block, object block 3. Another distinguishing feature between System 1 and an OCT system is that the polarizing beam splitter 23 is configured to generate an illumination beam using the light source 21 and the first optical lens 22, rather than separating the sample wave from the reference wave as in an OCT system.

[0053] The difference in divergence angle between the reference wave and the sample wave is a crucial factor in generating high-contrast interference in the image acquired by System 1. The quasi-collimated reference wave I satisfies only a small portion of the numerical aperture (NA) of the acquisition optics, while the divergent sample wave SA satisfies most of the NA. For example, a microscope objective lens with 0.3 NA has a 35-degree acceptance angle, which is lower than the angular distribution of the sample wave SA generated by submicron organelles. Since the sample wave SA is affected by the optical focus of the acquisition optics, it has an additional shift in optical phase known as Gouy phase shift. The phase-shifted reference wave and sample wave produce interference with contrast depending on the value of the phase shift. This phase is controlled by the NA of the acquisition optics and the position of the organelle relative to the imaging plane. The imaging plane coincides with the optical focal plane. In other words, to detect a two-dimensional interference signal close to the slice of eye 6, generated by the interference of a substantially collimated reference wave and a divergent sample wave using a two-dimensional acquisition device, the slice of eye 6 should be within the imaging plane of system 1 (i.e., within the object plane of the two-dimensional acquisition device), and the imaging plane is located between the internal reflective layer and the outer surface 6a of eye 6. The above explains the requirements for a collimated illumination and a small spot on the internal reflective layer; the smaller the focused spot, the more the reference wave is collimated, the less the influence of the reference wave on the Gouy phase shift, the larger the phase shift between the sample wave and the reference wave, and the higher the interference contrast. Typically, the optimal image contrast is obtained when the illumination beam has a divergence angle of about 4 degrees, which corresponds to a spot size of 1 millimeter on the internal reflective layer. The entire beam formed by both the reference wave I and the sample wave SA will also be referred to as the output beam below.

[0054] As shown in Figure 1, the reference wave I and sample wave SA enter the polarizing beam splitter 23. The polarizing beam splitter 23 is also configured to distinguish the transmitted light of interest from the reflected light. More precisely, specularly reflected light from the surface of eye 6 retains its initial polarization and is therefore filtered out (reflected from detection) by the same beam splitter 23 upon passing behind it. On the other hand, light reflected from an internal multi-diffuse reflective layer such as the sclera S is depolarized and then transmitted / refracted through the sample layer, after which this light passes through the polarizing beam splitter 23 and can be detected. Furthermore, the polarization selection of the polarizing beam splitter 23 can be improved by adding a linear polarizer between the polarizing beam splitter 23 and the light source 21, or between the polarizing beam splitter 23 and the two-dimensional acquisition device 4 (for example, the typical polarization extinction ratio of a beam splitter is 1000:1, which worsens for uncollimated light beams, but the extinction ratio of a linear polarizer can reach 100000:1). In fact, after specular reflection, this light is no longer polarized and therefore does not pass through the polarizing beam splitter 23.

[0055] The two-dimensional acquisition device 4 comprises multiple sensors arranged along the imaging axis and positioned within a detection plane. The detection plane is optically conjugate to the imaging plane of object block 3 by an optical system OS comprising a polarizing beam splitter 23 and a tube lens 7. Depending on the embodiment, the optical system OS comprises additional elements which will be further described. When system 1 is operating, light entering from the optical interference between the reference wave I and the sample wave SA enters the multiple sensors that acquire the corresponding two-dimensional interference signal. Hereafter, the expression "two-dimensional interference signal" will be used interchangeably with the expression "interference image".

[0056] The tube lens 7 is part of the optical system OS that conjugates the imaging plane and the detection plane. It is then possible to use system 1 to acquire a two-dimensional interference signal representing the retina R of the individual's eye 6. The tube lens 7 is configured to adjust the optical magnification of the interference image acquired by the two-dimensional acquisition device 4 to optimize the size of the pattern observed in the two-dimensional interference signal acquired by the two-dimensional acquisition device 4. Typically, the pattern size is selected based on the resolution of the optical system OS and the size of the two-dimensional acquisition device 4 to ensure pattern sampling according to the Nyquist signal sampling criterion.

[0057] For example, the two-dimensional acquisition device 4 is a CMOS camera with improved sensitivity to near-infrared light (e.g., having a quantum efficiency of approximately 25% to 50% at 850 nm). The camera frame rate typically allows for image acquisition at at least 80 frames per second to suppress the effects of eye movement of the individual during the examination.

[0058] At least one processor (or processing unit) 5 is configured to process the two-dimensional interference signal acquired by the two-dimensional acquisition device 4.

[0059] In some embodiments, System 1 further includes a linear polarizer positioned between the polarizing beam splitter and the light source, or between the polarizing beam splitter and the two-dimensional acquisition device, or both at those positions. When positioned between the polarizing beam splitter 23 and the light source 21, such a linear polarizer is intended to improve the polarization degree of the illumination beam. Improving the polarization degree of the illumination beam helps to improve the contrast of the interference image obtained using System 1. When the linear polarizer is positioned between the polarizing beam splitter and the two-dimensional acquisition device, the linear polarizer is intended to further eliminate parasitic stray light that is specularly reflected by the individual's eye 6 during examination.

[0060] In some embodiments, also called anterior segment imaging embodiments, the system 1 further comprises a second optical lens 24 and a third optical lens 31, as shown in Figure 3. These embodiments correspond to cases where the system 1 generates an image of the cornea C or lens (LC) of the eye 6. The second optical lens 24 is configured to generate a focused beam that focuses from the illumination beam to the back focal plane P of the third optical lens 31, such that a substantially collimated beam exits the third optical lens 31. The third optical lens 31 is part of an optical system S that conjugates the imaging plane and the detection plane. The second optical lens 24 may also be part of an optical system OS that conjugates the imaging plane and the detection plane, if it is implemented within an object block 3, as shown in Figure 4. In these cases, the system 1 is more compact.

[0061] For example, the third lens 31 is a microscope objective lens, a microscope objective lens turret, or a zoom microscope objective lens, and these objective lenses have an numerical aperture of 0.25 or greater. Typically, if the third lens 31 is a microscope objective lens, it has a magnification of 10x, a numerical aperture of 0.3 NA and a working distance of 18 mm, or a magnification of 20x, a numerical aperture of 0.45 NA and a working distance of 8 mm, or a magnification of 50x, a numerical aperture of 0.65 NA and a working distance of 10 mm. These working distance values ​​are advantageous for the safety of the individual's eye 6 during examination. However, objective lenses with other magnifications, numerical apertures and working distances can also be used. Alternatively, the third lens 31 may be a conventional optical lens.

[0062] An embodiment in which System 1 does not include the third optical lens 31 is also called a retinal imaging embodiment.

[0063] Using System 1, it is also possible to acquire a series of depth-direction interferometric images of several parts of the individual's eye 6 during the examination. For this purpose, in some embodiments, System 1 comprises either a movable element or a controllable element.

[0064] For example, in some embodiments, System 1 includes a tunable lens 9 between a polarizing beam splitter and a two-dimensional acquisition device. More precisely, the tunable lens 9 is part of an optical system OS that conjugates the imaging plane and the detection plane. The tunable lens 9 has a focal length controllable using at least one processor (or processing unit) 5. By tuning the focal length of the tunable lens 9, the imaging plane on the object block 3 is adjusted. Figure 5 shows an example of System 1 with the tunable lens 9.

[0065] In some embodiments, if system 1 includes a third lens 31, the third lens 31 is mounted on a mobile platform 12a controllable using at least one processor (or processing unit) 5. For example, the mobile platform 12a is moved by a piezoelectric motor or a voice coil motor. Moving the mobile platform adjusts the imaging plane on the object block 3. An example of the mobile platform 12a is shown in Figure 5a.

[0066] In other embodiments, the tube lens 7 is mounted on a mobile platform 12b controllable using at least one processor (or processing unit) 5. For example, the mobile platform is moved by a piezoelectric motor or a voice coil motor. Moving the mobile platform 12b adjusts the imaging plane on the object block 3. An example of the mobile platform 12b is shown in Figure 5b.

[0067] Using System 1, it is also possible to acquire several interference images simultaneously, which can be applied to optical tomography as described later. For this purpose, in some embodiments, System 1 includes optical multiplexing systems 10a and 10b positioned between the polarizing beam splitter 23 and the two-dimensional acquisition device 4.

[0068] For example, optical multiplexing systems 10a and 10b include a plurality of additional beam splitters or prisms, or glass plates, that are spatially shifted relative to the polarizing beam splitter 23. For example, as shown in Figure 6a, there are two additional beam splitters that receive a portion of the output beam. The optical path through which the portion of the output beam received by the additional beam splitters travels is different from the optical path through which the portion of the output beam reflected by the two additional beam splitters travels. Thus, the two-dimensional acquisition device acquires two two-dimensional interference signals with different optical phases. The two parts of the two-dimensional acquisition device 4 are conjugate to two different imaging planes. A phase difference close to π between the reference wave and the sample wave is adjusted by changing the defocus of each beam through the beam splitters and optionally the thickness of the glass plates. The dimensions of these components are determined by the dimensions of the two-dimensional acquisition device and the magnification of the optical system OS. The magnification is important because the axial position of the image plane in the sample changes with the magnification squared in the plane of the two-dimensional acquisition device 4. For example, one optimal configuration to support an 11mm sensor and an optical system with 0.3NA and 10x magnification is a 5mm beam splitter, a 5mm mirror, and 5±1mm compensation glass, which allows simultaneous capture of two image planes separated axially by only 10 microns.

[0069] Figure 6b shows another example in which four two-dimensional interference signals with four different optical phase differences are simultaneously acquired by the two-dimensional acquisition device 4. In this case, the optical multiplexing system 10b consists of a combination of four beam splitters and optionally a glass plate. This configuration consists of a first beam splitter that divides the optical beam into two paths. For each path, a beam splitter and a right-angle prism are arranged to divide a portion of the incident beam from the tube lens into four optical beams that are evenly distributed with respect to optical power light. The relative optical phase shift between beams can be adjusted by implementing glass plates of different thicknesses in each optical path.

[0070] In some embodiments, system 1 is coupled to an additional imaging system 11, as shown in Figure 7.

[0071] For example, System 1 further comprises additional eye imaging systems such as a macro camera, stereo camera, pupil camera, fundus camera system, slit lamp system, or optical coherence tomography system. The additional eye imaging system is coupled to System 1 by a dichroic mirror or glass plate 13 located within the illumination block 2, within the object block 3, or between the polarizing beam splitter 23 and the two-dimensional acquisition device 4. Typically, the additional system provides a global field of view of the eye with a larger field of view than the transmission interferometer device 1. This allows for easy positional alignment with the eye and qualitative general health examinations. In addition, such a device allows for recording lateral eye movements during acquisition and allows for feedback for image reconstruction.

[0072] In another example, the additional imaging system 11 is an axial position tracking system. An axial position tracking system is a system that can estimate the axial position of one of the layers of eye 6 relative to system 1. An example of such an axial position tracking system is an optical coherence tomography system. The additional axial position tracking system is coupled to system 1 by a dichroic mirror placed in the illumination block 2, in the object block 3, or between the beam splitter 23 and the two-dimensional acquisition device 4. Additionally, the axial position tracking system can be created using a macro camera or pupil camera, or a stereo camera, through the estimation of defocus in the image.

[0073] In some embodiments, the detection block has a relatively long length. In other words, the distance between the polarizing beam splitter 23 and the two-dimensional acquisition device 4 is relatively long. Examples of these embodiments are shown in Figures 8a and 8b, corresponding to the retinal imaging embodiment and the corneal imaging embodiment, respectively. In these embodiments, the value of the divergence angle γ of the reference wave after transmission through the polarizing beam splitter 23 toward the two-dimensional acquisition device 4 is such that the majority 14 of the reference wave, schematically represented by the dotted rectangle, does not reach the two-dimensional acquisition device 4. Thus, the amount of light from the reference wave in the total signal received by the two-dimensional acquisition device 4 is reduced. The reference wave is typically larger than the sample wave. Therefore, reducing the sample wave balances the optical interferometer and improves contrast. In these embodiments, a typical value for the distance between the polarizing beam splitter 23 and the two-dimensional acquisition device 4 is 250 mm to 700 mm.

[0074] Here, we describe method 100 for in vivo full-field transmission interference imaging of an individual's eye. This method is performed by system 1 as described above. Figure 9 is an example flowchart illustrating method 100.

[0075] In step S0, the individual being examined is positioned such that the eye 6 being examined is positioned (i.e., positioned) on the imaging plane of the object block 3 of system 1.

[0076] In step S1, the eye 6 is illuminated with an illumination beam. The illumination beam penetrates the eye 6 by passing through the outer surface 6a, focuses on the region of the eye's internal reflective layer, and is reflected backward by the eye's internal reflective layer. If System 1 is a system according to a retinal imaging embodiment, the internal reflective layer is the retina R of the eye 6. If System 1 is a system according to a corneal imaging embodiment, the internal reflective layer is either the sclera S of the eye 6 or the iris IR of the eye 6. The backward-reflected light forms a secondary light source (which may also be called an incident light wave) that generates a light wave that propagates from the internal reflective layer toward the outer surface 6a of the eye. As a result, the light from the secondary light source propagates in the opposite direction to the propagation direction of the illumination beam I that propagates from the polarizing beam splitter toward the eye 6 in the object block. As mentioned above, the reference wave is emitted from the eye after the incident light wave has been transmitted through it, and the sample wave is emitted from the eye after the incident light wave has been scattered by a region of the slice of the individual's eye (or a voxel corresponding to a region of the slice of the individual's eye in three dimensions, such as a cube or rectangular prism).

[0077] In step S2, the two-dimensional acquisition device 4 acquires a two-dimensional interference signal, i.e., an interference image.

[0078] Figures 10a, 10b, 10c, 10d, 10e, 10f, and 10g show images obtained using Method 100 of corneal endothelial cells, lens epithelium, lens fibers, corneal nerves, retinal photoreceptors, retinal nerve fibers, and retinal blood vessels, respectively.

[0079] In some embodiments, Method 100 for in vivo full-field transmission interference imaging of an individual's eye 6 allows obtaining a three-dimensional image of the volume of the eye 6 during examination. In these embodiments, the method is carried out by System 1, where System 1 includes a movable component or a controllable component. In the case of a movable component, the movable component is moved to displace the imaging plane along the imaging axis in order to acquire multiple two-dimensional interference signals on different working planes along the imaging axis. In the case of a controllable component, i.e., a component with variable properties, the variable properties are changed to displace the imaging plane along the imaging axis in order to acquire multiple two-dimensional interference signals on different working planes along the imaging axis. In other words, in order to acquire a two-dimensional interference signal close to a slice of eye 6, generated by the interference of a substantially collimated reference wave and a divergent sample wave using a two-dimensional acquisition device, the slice of eye 6 should be within the imaging plane of System 1 (i.e., within the object plane of the two-dimensional acquisition device), and the imaging plane is located between the internal reflective layer and the outer surface 6a of eye 6.

[0080] For example, if system 1 includes a variable-focus lens 9, at least one processor (or processing unit) 5 controls the focal length of the variable-focus lens 9.

[0081] In another example, if system 1 includes a third lens 31 mounted on a moving platform controllable by at least one processor (or processing unit) 5, then at least one processor (or processing unit) 5 controls the movement of the moving platform to change the total focal length of the optical system, thereby moving the imaging plane.

[0082] In another example, if system 1 comprises a tube lens 7 mounted on a moving platform controllable by at least one processor (or processing unit) 5, then at least one processor (or processing unit) 5 controls the movement of the moving platform to change the total focal length of the optical system OS, thereby moving the imaging plane.

[0083] In all these examples, a three-dimensional image of the volume of eye 6 is obtained by collecting all images acquired at different positions on the imaging plane from which a three-dimensional stereoscopic view of the volume is obtained.

[0084] More specifically, the position of the imaging plane can be changed by several millimeters along the imaging axis. During this ongoing focus swipe, a series of images can be acquired, each image captured from a different depth of eye 6. If the focus swipe and image acquisition are fast (several seconds), eye 6 remains almost stationary throughout the entire acquisition. Therefore, only minimal repositioning is required in post-processing to reconstruct a three-dimensional image based on the series of images.

[0085] In some embodiments, Method 100 enables optical tomography. Optical tomography means imaging within a given plane, in other words, within a given tomography, while excluding out-of-focus light. In these embodiments, the Method is carried out by System 1, where System 1 includes a movable or controllable component, or where System 1 includes optical multiplexing systems 10a, 10b.

[0086] In the case of a movable component, the movable component is moved to displace the imaging plane along the imaging axis in order to acquire multiple two-dimensional interference signals on different imaging planes along the imaging axis. In the case of a controllable component, i.e., a component with variable characteristics, the variable characteristics are changed to displace the imaging plane along the imaging axis in order to acquire multiple two-dimensional interference signals on different imaging planes. In the case of optical multiplexing systems 10a and 10b, different two-dimensional interference signals are acquired simultaneously.

[0087] Multiple two-dimensional interference signals are acquired by the two-dimensional acquisition device 4 (or from different beams received via optical multiplexing systems 10a and 10b) for an imaging plane located in depth near a given cross-section corresponding to an optical phase difference in the range of -π / 2 to +π / 2.

[0088] At this time, the change in optical phase difference affects the visible intensity of the light detected by the two-dimensional acquisition device 4. This can be shown using a Gaussian model, which can express the electric fields of transmitted and scattered waves as follows.

[0089]

number

[0090] The two waves interfere with each other, generating an intensity that can be detected by the camera.

[0091]

number

[0092] By combining these terms, which are constant with respect to z, the equation can be rewritten as follows:

[0093]

number

number

number

[0094] By moving the imaging plane, at least two two-dimensional interference signals are acquired (or acquired via an optical multiplexing system), and from the phases of these interference signals, it is possible to remove out-of-focus light through image processing and obtain an optical tomographic image by, for example, the pixel-by-pixel difference between the two acquired interference signals. Figures 11a and 11b show two interference images of the corneal stroma corresponding to two different phases, which, when one is subtracted from the other, give the optical tomography shown in Figure 11c. Bright corneal stroma cells 111 can be observed in the optical tomography of Figure 11c.

[0095] More specifically, by acquiring two-dimensional interference signals for different imaging planes and calculating the difference between the two two-dimensional interference signals, the following image intensities can be obtained.

[0096]

number

[0097] Therefore, from equation [Equation 7], if an object (such as a cell) is located far from the imaging plane, the intensity difference is zero, and the signal is excluded from detection. On the other hand, if the object that scatters light is close to the imaging plane, the intensity obtained by the difference is not zero, but becomes stronger. This process is also called optical tomography or tomography.

[0098] In some embodiments, the method 100 for in vivo full-field transmission interference imaging of an individual's eye 6 allows obtaining a three-dimensional image of the volume of the eye 6 under examination from images obtained by optical tomography as described above. The method includes moving the imaging plane to compute multiple tomographic images sequentially, and then collecting multiple tomographic images.

[0099] The contrast of the interference image acquired in step S4 can be enhanced.

[0100] In fact, for example, prior to step S2, multiple interference images are acquired, each at a different point in time. An image, also called an average image, is calculated by at least one processor (or processing unit) 5. For example, the average image is a linear combination of the multiple interference images. Then, in step S4, an effective image is acquired at a point in time that follows a separate point in time where the contrast should be improved. In step S5, at least one processor (or processing unit) 5 calculates the subtraction of the effective image and the calculated average image. The result of the subtraction is an image with improved contrast, in which static background light has been removed from the scene.

[0101] The signal-to-noise ratio of the interference image acquired in step S2 can be increased.

[0102] In practice, for example, before step S2, there is a step of acquiring at least one training set consisting of images of training samples, where each training set includes a first training image of a cross-section of the training sample and a second training image of the same cross-section of the training sample, acquired as a time-averaged image of several images. The target averaged image typically has a higher signal-to-noise ratio, a larger bit depth (i.e., dynamic range), and less motion artifact. The training images are acquired from an in vivo human eye (cornea, lens, retina). After acquiring at least one training set of images, a step of training an artificial neural network is performed. This step includes providing the corresponding first training image as input to the artificial neural network for a given training set of images, and providing the corresponding second training image as the target output of the artificial neural network. An image-to-image transformation network, typically a U-net network, can be used. The application of this step to the applications of the present invention is unique because structures in images obtained using method 100 can be seen with opposite contrast (phase), i.e., light and dark, depending on the axial position of the tissue relative to system 1. The second training image is typically obtained by first acquiring an image with a reduced camera field of view to increase the acquisition speed and suppress motion artifacts. Typically, an image with a high bit depth (dynamic range) of 12 bits or more is acquired. The second training image is then obtained by increasing the signal-to-noise ratio by averaging the time stack of the acquired single image. The first training image is selected as one of the images from the same stack. Typically, additional Gaussian noise is added to the first image to simulate a low signal-to-noise camera. The trained neural network can then learn a statistical pattern to transform the first image obtained in step (d) of Method 100 into a clean, denoised second image.

[0103] After acquiring the interference image in step S4, the method further includes inputting the acquired interference image into a trained artificial neural network. The output of the trained artificial neural network is a denoised image.

[0104] Method 100 enables several applications.

[0105] For example, Method 100 improves image quality to enable visualization of cells in the interference image acquired in step S4 and counting of the number of cells. In this case, Method 100 includes step S6, performed by at least one processor (or processing unit) 5, after step S4, which segments and counts cells and nerves in the interference image. For example, a U-net neural network can be used for segmentation.

[0106] In another example, Method 100 enables axial tracking of an individual's eye during examination. In this case, the Method is carried out using System 1, which is coupled to an axial position tracking system, such as an optical coherence tomography (OCT) system. The axial position tracking system is coupled to System 1 by a dichroic mirror or glass plate 13 within an illumination block 2, an object block 3, or between a polarizing beam splitter 23 and a two-dimensional acquisition device 4. In this case, the Method further includes, in step S4, acquiring at least one enface image using System 1 at at least one time point in time. The enface image shows a specific layer or cross-section of the tissue being imaged. Then, for each of the at least one enface image, the Method further includes acquiring at least one corresponding image using the OCT device. Finally, the Method further includes calculating the axial position of the individual's eye along the imaging axis over time based on at least one corresponding image acquired using the additional OCT images.

[0107] For example, consider the case where the axial position tracking system is an optical coherence tomography (OCT) system 11. OCT typically refers to spectral region OCT or sweep source OCT, from which cross-sectional images are acquired. Several layers corresponding to the cross-sectional images (such as the corneal surface and retinal nerve fiber layer) are pre-selected and segmented. The positions of the segmented layers relative to the center of the image are then calculated.

[0108] Macro cameras, pupillary cameras, and other devices such as slit-lamp systems can be used as axial position tracking systems. These devices can acquire images that can be processed. Depth or axial position can be obtained by calculating the defocus of the image. Using a stereo camera, depth can be directly extracted from two stereo images.

[0109] In another example, Method 100 enables imaging of cell dynamics in an individual's eye 6. In this case, Method 100 further includes using at least one processor (or processing unit) 5 of System 1 to compute an image, also called a dynamic image. The dynamic image represents the temporal variation in intensity between two-dimensional interferometric signals during a set of two-dimensional interferometric signals. More precisely, the temporal variation is acquired at each pixel. The mean offset background is removed from each image to reveal the temporal intensity variation attributable to moving organelles within each voxel in the imaging plane. These intensity variations are then presented on a color scale, describing the variation characteristics of each pixel, such as the spectral mean and bandwidth of the Fourier transform, as well as the standard deviation of the variation. Encoding these values ​​in an RGB or HSV colormap produces a colorful dynamic image, where brighter colors indicate faster organelle movement.

[0110] Variations are also possible.

[0111] In some variations, System 1 further comprises an optical mask 8. These variations are shown, for example, in Figures 1b and 3b. The optical mask is located in a plane, also called the mask plane, between the polarizing beam splitter 23 and the two-dimensional acquisition device 4. System 1 further comprises a lens or system of lenses that conjugates the back focal plane of a third optical lens 31 with the mask plane. The optical mask 8 is configured to block a portion of the reference wave. As a result, the contrast of the image acquired by the two-dimensional acquisition device 4 is improved due to the balance between the sample wave and the reference wave in the interferometer.

Claims

1. A system (1) for cell-level resolution imaging by in vivo full-field transmission interference on an individual eye (6), wherein the eye (6) has an outer surface (6a), An object block (3) configured to position the individual's eye (6) within the imaging plane when the system (1) is operating, having an axis that coincides with the imaging axis, An illumination block (2) comprising a light source (21) coupled to a first optical lens (22) and a polarizing beam splitter (23) and configured to together generate an illumination beam, wherein the illumination beam is substantially collimated and symmetric with respect to the axis of the object block (3), During operation, at least a portion of the illumination beam penetrates the individual's eye (6), focuses on the region of the internal reflective layer of the eye (6), and is reflected back by the internal reflective layer to form a secondary light source. During operation, the imaging surface is positioned between the internal reflective layer and the outer surface (6a) of the eye (6) and the illumination block (2), A detection block comprising a tube lens (7) and a two-dimensional acquisition device (4), wherein the two-dimensional acquisition device (4) comprises a plurality of sensors arranged in a detection surface and is configured to acquire at least one two-dimensional interference signal resulting from the interference between a substantially collimated reference wave obtained by direct retransmission of a first portion of the secondary light source by the eye (6) and a divergent sample wave obtained by scattering a second portion of the secondary light source by a region of a slice of the individual's eye (6), wherein the detection surface is optically conjugate to the imaging surface, and the slice of the eye is localized on the imaging surface, Equipped with, The divergent sample wave has an additional optical phase shift with respect to the substantially collimated reference wave. System (1).

2. The system (1) according to claim 1, wherein the polarizing beam splitter (23) is configured to polarize the illumination beam and filter out light that is specularly reflected by the individual's eye (6) during operation.

3. The system (1) according to claim 1 or 2, wherein the illumination beam has a divergence angle of less than 20 degrees, the divergence angle is measured with respect to the axis of the object block (3), and one dimension of the region of the internal reflective layer is less than 6 millimeters when illuminated by the illumination beam.

4. The system (1) according to any one of claims 1 to 3, wherein the light source (21) is disposed within the light source surface, and the system (1) further comprises an aperture disposed within the light source surface, the aperture being configured to control the divergence angle of the illumination beam.

5. The system (1) according to any one of claims 1 to 4, further comprising at least one processor (5) and a tunable lens (9) positioned between the polarizing beam splitter (23) and the two-dimensional acquisition device (4), wherein the tunable lens (9) has a controllable focal length and is configured to be controlled to acquire a plurality of two-dimensional interference signals to determine a plurality of working images, each of the plurality of working images corresponding to a separate imaging plane along the imaging axis, and the at least one processor (5) is configured to calculate a 3D image of the individual's eye (6) based on the plurality of working images.

6. The system (1) according to claim 5, wherein the focal length of the tunable lens is controlled to acquire a plurality of two-dimensional interference signals in the depth direction within a cross-section of the eye of the individual, thereby resulting in a plurality of predetermined phase differences between the reference wave and the sample wave in the range of -π / 2 to +π / 2, and the at least one processor (5) is further configured to calculate a tomographic image based on a linear combination of the two-dimensional interference signals among the plurality of two-dimensional interference signals.

7. The system (1) according to any one of claims 1 to 6, further comprising an optical multiplexing system (10a, 10b) positioned between the polarizing beam splitter (23) and the two-dimensional acquisition device (4) to acquire a plurality of two-dimensional interference signals in the depth direction within a cross-section of the individual's eye (6) in a single acquisition, wherein the optical multiplexing system (10a, 10b) is configured to provide a predetermined phase difference between the reference wave and the sample wave in the range of -π / 2 to +π / 2.

8. The lighting block (2) or the object block (3) further comprises a second optical lens (24) disposed within it. - The object block (3) further comprises a third optical lens (31), The system (1) according to any one of claims 1 to 7, wherein the second optical lens (24) is configured to generate a focused beam from the illumination beam, and the focused beam is focused on the back focal plane of the third optical lens (31) such that a substantially collimated beam exits the third optical lens (31).

9. The system (1) according to claim 8, wherein the third optical lens (31) is a microscope objective lens, a turret for a microscope objective lens, or a zoom microscope objective lens, and the microscope objective lens has a numerical aperture of 0.25 or more.

10. The system (1) according to claim 8 or 9, further comprising: an optical mask (8) disposed in a plane also called the mask plane between the polarizing beam splitter (23) and the two-dimensional acquisition device (4); and a lens or lens system conjugating the back focal plane of the third optical lens (31) with the mask plane, wherein the optical mask (8) is configured to partially block a portion of the reference wave to improve image contrast.

11. The system (1) according to any one of claims 8 to 10, further comprising a mobile platform to which the third optical lens (31) is attached, and at least one processor (5), wherein the mobile platform is configured to move along the imaging axis to acquire a plurality of two-dimensional interference signals and determine a plurality of working images, each of the plurality of working images corresponding to a separate imaging plane along the axis of the object block (3), and the at least one processor (5) is configured to calculate a 3D image of the individual's eye (6) based on the plurality of working images as the mobile platform moves along the imaging axis.

12. The system (1) according to claim 11, wherein the moving platform is moved along the imaging axis to acquire a plurality of two-dimensional interference signals in the depth direction within a cross-section of the eye of the individual, thereby introducing a plurality of predetermined phase differences between the reference wave and the sample wave in the range of -π / 2 to +π / 2, and the at least one processor (5) is further configured to calculate a tomographic image based on a linear combination of the two-dimensional interference signals among the plurality of two-dimensional interference signals.

13. The system according to any one of claims 1 to 12, further comprising an additional eye imaging system such as a fundus camera system or a slit lamp system, or an additional position tracking system such as a macroview camera system, a stereo camera, or a pupil camera, wherein the additional eye imaging system or the additional position tracking system is integrated by a dichroic mirror positioned within the illumination block (2), within the object block (3), or between the polarizing beam splitter (23) and the two-dimensional acquisition device (4).

14. A method (100) for in vivo full-field transmission interference imaging of an individual eye (6), wherein the eye (6) has an outer surface (6a), (a) Positioning the eye (6) on the imaging plane within the object block of the system (1) for in vivo full-field transmission interference imaging, (b) Illuminating the individual's eye (6) with a substantially collimated, spatially incoherent and symmetric illumination beam, such that the illumination beam is focused onto a substantially small area of ​​the internal reflective layer of the eye (6), and is back-reflected by the internal reflective layer to form a secondary light source, and the imaging surface is located between the internal reflective layer and the outer surface of the eye. (c) Acquiring at least one two-dimensional interference signal using a two-dimensional acquisition device (4) which has multiple sensors arranged within the detection surface, The at least one two-dimensional interference signal arises from the interference between a reference wave obtained by direct retransmission of the first portion of the secondary light source and a sample wave obtained by scattering of the second portion of the secondary light source by a region of the slice of the individual's eye (6), The imaging plane is conjugate to the detection plane, and the slice of the eye is localized on the imaging plane, thereby acquiring at least one two-dimensional interference signal. (d) At least one first image is obtained from the at least one two-dimensional interference signal by at least one processor (5) of the system (1), Method (100), including the method (100).

15. The method according to claim 14 (100), wherein the system for imaging by in vivo full-field transmission interference is the system described in any one of claims 1 to 13.

16. If the system for in vivo full-field transmission interference imaging further comprises an additional position tracking system, the system is the system according to claim 13, wherein in step (d), at least one envelope image is obtained using the system for in vivo full-field transmission interference imaging at at least one time point in time. - For each of the at least one Enface image, at least one corresponding image is obtained using the additional location tracking system, - Calculate the axial position of the individual's eye (6) along the imaging axis over time based on the at least one corresponding image acquired using the additional position tracking system. The method according to claim 14, further comprising (100).

17. In step (c), a set of two-dimensional interference signals is acquired. The method according to any one of claims 14 to 16 (100), further comprising using the at least one processor (5) of the system (1) for in vivo full-field transmission interferometry imaging to compute an image also called a motion image, wherein the motion image represents the temporal variation in intensity between the two-dimensional interferometry signals during a set of two-dimensional interferometry signals.

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