Method and system for optically characterizing a scattering bulk medium

Abstract

The present invention relates to a method for evaluating the optical properties of a sample. This method includes the steps of placing a sample within the field of view of a first microscope objective lens disposed in the objective arm of an interferometer, and generating, by an illumination device, a first plurality of N in incident light waves, and for each incident light wave of a specific wavefront, obtaining a second plurality of N ω interference signals resulting from the interference between the wave backscattered by the sample and a reference wave (according to one example, the interference signals are obtained for different N ω frequencies), determining a multi-color reflection matrix including all the interference signals obtained for the N in incident light waves and the N ω frequencies, numerically determining a focused volume reflection matrix based on the multi-color reflection matrix, and determining at least one map having physical parameters of the sample based on this first focused volume reflection matrix.

Description

Technical Field

[0001] This specification relates to a method and system for optically characterizing a scattering bulk medium. This specification is particularly applicable to, but not limited to, biomedical imaging for characterizing biological tissue.

Background Art

[0002] The optical characterization of a scattering bulk medium such as biological tissue is a research field at the boundary between physics and biology. Its purpose is to image the structures that make up biological tissue with a resolution limited by diffraction, that is, on the order of micrometers of the size of the light wavelength. The general principle is as follows. Light waves illuminate the sample, and then the light scattered by the structures in the medium is collected by a set of lenses to form a magnified image of the tissue, for example, in a camera. Among generally known microscopes, transmission microscopes are common because of their simplicity in design and use. However, they have limitations, especially in in-vivo imaging. This is because it is necessary to place the light source and the camera on the opposite side of the sample. Therefore, a reflection approach is preferred, especially in applications including microscopic in-vivo imaging.

[0003] In the case of a thin sample (L < 50 μm), the wave backscattered by the sample is faithful to the reflectivity of the object. Only the numerical aperture of the optical system determines the axial and lateral resolution of the microscope image. However, in the case of deep tissue imaging, the phenomenon of multiple scattering becomes a limiting factor. Specifically, a biological sample is a heterogeneous medium, and its optical refractive index varies on various spatial scales. These refractive index inhomogeneities have an adverse effect on the propagation of light waves (aberration), not only reducing the contrast and resolution of the image, but also limiting the number of useful photons that can be collected (multiple scattering). Since the contribution of single scattering decreases exponentially with depth, photons with aberration, and the scattering mean free path l sIt becomes impossible to distinguish the contribution of multiply scattered photons from samples with characteristic distances exceeding (usually 50 μm to 100 μm) in the tissue.

[0004] To overcome this problem, two types of microscopes, confocal microscopes in the early 1960s and interference microscopes in the early 1990s, were developed. Confocal microscopes improve the resolution and penetration depth of conventional microscopes (usually 100 μm to 300 μm) by spatially filtering scattered photons. Interference microscopes, especially OCT (optical coherence tomography), add temporal filtering to the spatial filtering of confocal microscopes. This can further improve the achievable performance up to several l s (i.e., about 100 μm to 500 μm). However, despite the spatio-temporal windowing of photons, the performance of interference microscopes is still limited by aberrations and multiple scattering in terms of resolution and penetration depth.

[0005] Therefore, several l sBeyond this, there remains the general problem of three-dimensionally imaging biological samples with a resolution limited only by diffraction. In the early 2000s, inspired by pioneering research in astronomy, aberration correction methods for optical microscopes were developed. In astronomy, fluctuations in the refractive index of the atmosphere create distortions in the wavefront, which negatively affect the quality of the images of the sky formed from Earth. Compensatory optical methods based on wavefront measurement combined with wavefront control devices (typically arrays of deformable mirrors) can correct for wavefront distortions and improve resolution. For this purpose, guide stars or bright spots projected into the sky are used to optimize the correction. These compensatory optical techniques have been applied to optical microscopes with the advent of small deformable mirrors and SLMs (spatial light modulators). However, these methods have several drawbacks. First, the uncorrelated time of biological tissue requires rapid compensation for aberrations, which limits the number of angular and / or spatial degrees of freedom of the wavefront measurement and control devices. Therefore, compensatory optics in a biological environment is limited to compensating for relatively low-order aberrations. A further limiting factor is that the range of the field of view for which aberration compensation is effective is limited. This corresponds to what is generally called an isotropic region, i.e., the region up to the region where the incident and reflected wavefronts undergo the same distortion. However, the size of these isotropic regions is on the order of tens of micrometers or less at a depth of the transport mean free path l t (typically 1 mm in biological tissue). To access correction across the entire field of view, it is necessary to repeat the compensatory optical process for each isotropic region, and high-resolution imaging over a wide field of view and at depth is unrealistic. To address the limitation of the size of the isotropic regions, so-called multi-conjugate compensation optical devices have been developed, but their experimental implementation is particularly complex. Finally, deep multiple scattering (i.e., that beyond the transport mean free path l t ) remains an unaddressed problem in compensatory optics.

[0006] A third approach has been developed that is neither based on artificial star generation nor on wavefront optimization based on image quality criteria. This is the matrix approach to optical imaging and aberration correction. For the matrix approach to light wave propagation in inhomogeneous media, see the non-patent document 1 (Reference Document 1), the paper by S.M. Popoff et al., which was first developed for transmission, particularly for communication through highly scattering media.

[0007] Recently, the matrix approach has been developed for imaging in reflection through highly scattering media. See the non-patent document 2, the paper by A. Badon et al. This approach involves experimentally determining a "focal plane" or "focusing" reflection matrix with a time window defined between the source plane and the image plane conjugate to the object focal plane of the microscope objective.

[0008] In practice, in the main experimental setup described in the non-patent document 2 (Reference Document 2), the laser beam from a femtosecond laser source is spatially shaped by a spatial light modulator (SLM) that functions as a dynamic diffraction grating. A set of plane waves is emitted from the SLM, and the plane waves are focused to different foci, each marked by the vector r in of the object focal plane of the microscope objective. For each focus r im , the reflected field E r (u out ,r in ,t) is collected through the same microscope objective and is placed on a plane conjugate to the pupil plane of the microscope objective, where it interferes with a reference wave E0(u out ,0,t) in a two-dimensional light-receiving element, such as a CCD camera, identified by the vector u out . By integrating the interference pattern of these two waves over time t, the coefficients R ur of the columns of the time-windowed reflection matrix R out ,r in ) can be accessed.

[0009]

Equation

[0010] A new matrix approach for optical imaging is described that enables simultaneous correction of aberrations across multiple isotropic regions of the field of view based on this focal plane reflection matrix. See, for example, Patent Document 2 (Reference Document 4). In Patent Document 2 (Reference Document 4), a new matrix called the "strain matrix" for characterizing inhomogeneous media was introduced. This matrix contains only the distorted portion of the wavefront backscattered from the sample for a series of foci r in This matrix can reveal the spatial correlation of the light irradiation field related to isotropy. By analyzing the correlation between these different wavefronts, the local aberration law of the entire field of view can be extracted. This allows access to the transmission matrix between the objective focal plane within the sample and the light source plane and image plane outside it. Once phase conjugated or inverted, these matrices provide all the focusing laws necessary to optimally and locally correct aberrations at every point in the field of view.

[0011] However, the characterization method described in Patent Document 2 (Reference Document 4) is limited to imaging a single cross-section called the coherence plane in the sample. To obtain a 3D image of the sample, it is necessary to scan the sample axially. Axial scanning limits the acquisition speed and thus the accessible volume, especially in in-vivo applications or dynamic imaging of cell tissues.

[0012] Above all, the present applicants have shown that the characterization method described in Patent Document 2 (Reference Document 4) allows only lateral aberration correction of the coherence plane determined by the position of the reference mirror in the interferometer setup. However, the inhomogeneity of the optical refractive index in the medium causes axial displacement and deformation of the coherence volume in comparison to the position and flatness that would be expected if the optical refractive index of the medium were uniform. In other words, when the optical refractive index in the medium is inhomogeneous, the objective focal plane of the microscope objective lens and the coherence plane no longer coincide. This limits the signal-to-noise ratio and causes large lateral aberrations and axial distortions in the image.

[0013] This specification proposes a method and a system for evaluating the optical properties of a scattering bulk medium. This is also based on a matrix approach, enabling ultra-fast volumetric characterization of a sample (typically in less than 1 second), and also providing access to a reflectivity image where the axial dimension is determined by the actual depth of the scatterers in the sample rather than by the flight time of scattered photons.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0016] According to a first aspect, this specification relates to a method for evaluating the optical properties of a sample formed from a scattering bulk medium, the method comprising: - A step of placing the sample within the field of view of a first microscope objective lens, wherein the microscope objective lens is disposed on an objective arm of a first interferometer, and the first interferometer further comprises a reference arm; - Generating a first plurality of N incident light waves having different wavefronts by an illumination device including a broad spectral band light source; in - For each incident light wave of a specific wavefront, obtaining a second plurality of interference signals by a detector including N element detectors, wherein each interference signal is due to interference between a wave backscattered by the sample illuminated by the incident light wave and a reference wave from the reference arm on the detection surface of the detector, and the interference signals are obtained for N different frequencies or N different path differences between the backscattered wave and the reference wave; - Determining a three-dimensional multicolor reflection matrix of size N×N×N, wherein the three-dimensional multicolor reflection matrix includes all interference signals obtained for N incident light waves and N frequencies; out - Numerically determining at least a first focused volume reflection matrix by applying a first propagator based on the multicolor reflection matrix, wherein the first focused volume reflection matrix is for voxels r(x´,y´,z) and r(x´,y´,z) of the sample located at depths z and z in the sample, respectively; ω - ω - - in × out × ω three-dimensional multicolor reflection matrix

[0017]

Number

[0018] In this specification, the field of view of a microscope objective lens includes the objective focal plane of the objective lens. The size of the field of view is limited by the characteristics of the microscope objective lens, the size of the detection surface, and the magnification of the imaging device between the objective focal plane and the detection surface. Also, the size of the field of view can be adjusted according to the lateral range of the sample that the operator wants to evaluate the characteristics of.

[0019] In this specification, a wide spectral band light source is a light source whose spectral band Δω is a value Δω that enables obtaining a desired axial resolution δz for determining a map of physical parameters min of the above light source,

[0020]

Equation

[0021]

Equation

[0022] The present applicants have shown that by determining the focused volume reflection matrix obtained based on the multi-color reflection matrix, the axial and lateral distortions inherent in the technique described in Patent Document 2 (Reference Document 4) can be avoided.

[0023] In fact, for example, it becomes possible to form a confocal image with a resolution limited only by diffraction and to match the coherence volume with the geometric focal plane.

[0024] In this specification, a map of physical parameters of a sample is understood as a planar map or a volume map of the parameters, unless otherwise specified.

[0025] Accordingly, a map of physical parameters of a sample includes a front or volume reflection confocal image, a map of a reflection point spread function (or RPSF) around a voxel of the sample, a planar or volume map of a single scattering rate, a planar or volume map of a multiple scattering rate, and a planar or volume map of an optical refractive index of the sample.

[0026] A front reflection confocal image is an estimated value of the reflectance of a cross-section of the sample. A volume reflection confocal image is an estimated value of the reflectance of a volume of the sample. In the remainder of this specification, a reflection confocal image may simply be referred to as a "confocal image".

[0027] A voxel is defined as a volume resolution cell. In this specification, a voxel may also be referred to by its center of gravity, which is also called a "focus". The size of the volume resolution cell is determined by the lateral resolution of an optical system defined by an assembly of optical elements disposed between the sample and the illumination surface and the detection surface, and the axial resolution δz.

[0028] In the remainder of this specification, the reflection point spread function around a focus is simply referred to as the "spread function around the focus" or the "RPSF at the focus". A "map of spread functions" means a map of reflection point spread functions (RPSFs) around a plurality of foci of the sample.

[0029] According to one or more embodiments, the optical property evaluation method according to the first aspect further includes - determining a first reflection confocal image based on the focused volume reflection matrix; - Based on the focused volume reflection matrix, determining a map of the spreading function and determining a map of the positions of the intensity maximum values of each spreading function; - Based on the first reflection confocal image and the map of the positions of the intensity maximum values, determining a second reflection confocal image corrected for the alignment and / or focusing defects of the first interferometer; It includes. Therefore, the present applicants have demonstrated the first advantage of determining a focused volume reflection matrix in order to determine a confocal image corrected for the alignment and / or focusing defects of the first interferometer.

[0030] According to one or more exemplary embodiments, the optical property evaluation method according to the first aspect further - Based on the first focused volume reflection matrix, determining a plurality of maps of the spreading function for a plurality of values of the integrated optical refractive index of the sample; - Based on the plurality of maps of the spreading function, determining a plurality of maps of the positions of the intensity maximum values of the spreading function, each map being obtained for a value of the integrated optical refractive index; - At each focus, determining a map of the optimal value of the integrated optical refractive index at which the maximum intensity value of the spreading function is maximized; - Based on the map of the optimal value of the integrated optical refractive index, numerically determining a second propagator; and - Using the second propagator to determine a second focused volume reflection matrix; It includes.

[0031] In this specification, the integrated optical refractive index of a sample refers to the optical refractive index of the sample integrated between the proximal surface of the sample (i.e., the microscope objective side) and the focus in question.

[0032] According to one or more exemplary embodiments, the method further includes determining a map of the optical refractive index of the medium at each focus based on a map of the optimal values of the integrated optical refractive index. Then, the second propagator can be determined based on the optical refractive index map. Thus, according to one or more exemplary embodiments, the method for evaluating optical properties according to the first aspect further includes - determining a plurality of maps of the spreading function for a plurality of values of the integrated optical refractive index of the sample based on the first volume reflection matrix of the aggregate; - determining a plurality of maps of the positions of the maximum intensity values of the spreading function based on the plurality of maps of the spreading function, each map being obtained for a value of the integrated optical refractive index; - determining a map of the optimal value of the integrated optical refractive index at which the maximum intensity value of the spreading function is maximized at each focus; - determining a map of the optical refractive index of the medium at each focus based on the map of the optimal value of the integrated optical refractive index; - numerically determining a second propagator based on the map of the optical refractive index of the medium; and determining a second volume reflection matrix of the aggregate using the second propagator. including.

[0033] The second propagator corresponds to a propagator that enables substantially matching the coherence volume and the geometric focal plane by taking into account the non-uniformity of the refractive index in the scattering bulk medium, whether optimized based on the directly integrated refractive index or optimized based on the sample refractive index derived from the integrated refractive index to maximize the intensity of the RPSF at the focus.

[0034] The obtained second focused volume reflection matrix can be used to establish a confocal image of the reflectivity of the medium, and this confocal image has the axial focusing and distortion defects of the first confocal image corrected. The map of the RPSF associated with this second focused volume reflection matrix can also be used to quantify the local multiple scattering rate for each voxel of the image. This parameter gives a local reliability index of the image and locally characterizes the scattering properties of the medium. The map of the RPSF also makes it possible to quantify the depth gradient of the single scattering rate that gives a local estimate of the scattering mean free path.

[0035] According to one or more exemplary embodiments, the method further includes determining a distortion matrix defined between a correction basis and a focusing basis based on the second focused volume reflection matrix, and the determination of the distortion matrix includes - at the entrance or exit, projecting the second focused volume reflection matrix onto the correction basis to obtain a projected reflection matrix at the entrance or exit respectively; - determining the distortion matrix by a term-by-term product between the projected reflection matrix in the correction basis and a reference reflection matrix defined for a reference medium; - locally determining an invariant of the distortion matrix to identify an aberration rule in a subdomain of the field of view in the correction basis; - estimating a transmission matrix between a voxel of the field of view and the correction basis based on the aberration rule. and includes.

[0036] The focusing basis is, at the entrance or exit respectively, a set of light source points or light receiving points conjugate to the voxels forming the sample.

[0037] The local correction of the aberration performed in this way generates a confocal image with optimized contrast and resolution over the entire volume of the sample.

[0038] According to one or more exemplary embodiments, the distortion matrix is determined in a similar manner, but is determined from the first focused volume reflection matrix obtained by the first propagator if the optimization step of the propagator for maximizing the intensity of the spreading function is not performed at each focus.

[0039] The element-by-element product between matrices is also called the Hadamard product.

[0040] According to one or more exemplary embodiments, the reference medium is a homogeneous medium having an optical refractive index equal to the average refractive index of the propagation medium. The reference reflection matrix for this reference medium can be theoretically established by placing a plane mirror on the focal plane of the microscope objective lens. Depending on the degree of prior knowledge of the propagation medium, the reference medium may take a more sophisticated form (e.g., a multilayer medium, etc.). In this case, the reference matrix can be calculated numerically. To construct the distortion matrix, the phase of each element of the projection reflection matrix is subtracted from the phase of the corresponding element of the reference reflection matrix.

[0041] According to one or more exemplary embodiments, the correction basis in which the distortion matrix is defined is a basis that maximizes the size of the isotropic region included in the field of view, for example, when the aberration element is two-dimensional, the set of points on the plane conjugate to the surface of the aberration element, or, for example, the plane conjugate to the pupil plane of the microscope objective lens.

[0042] According to one or more exemplary embodiments, the local determination of the invariant of the distortion matrix includes, as described in Patent Document 2 (Reference Document 4), the singular value decomposition of the distortion matrix, the singular value decomposition of the normalized distortion matrix (i.e., the one in which the absolute value of each element is normalized and the phase is retained), and the singular value decomposition of the normalized correlation matrix of the distortion matrix (i.e., the correlation matrix of the first distortion matrix in which the absolute value of each element is normalized). Other methods for local determination of the invariant, such as the iterative phase inversion process applied to the distortion matrix, are also possible.

[0043] According to one or more exemplary embodiments, the light source is a variable wavelength light source, and for each incident light wave of a specific wavefront, a second plurality of N ω interference signals are obtained for N ω frequencies (or wavelengths) of the incident light wave and a fixed path difference between the objective arm and the reference arm of the first interferometer. As used herein, the frequency (or wavelength) of an incident wave means the central frequency (or central wavelength) of the spectrum of the incident wave.

[0044] According to one or more exemplary embodiments, for each incident light wave of a predetermined wavefront, the interference signals of the second plurality of interference signals are obtained for N ω path differences between the objective arm and the reference arm of the first interferometer. Then, a multi-color reflection matrix can be obtained from the interference signals obtained for different paths or time-of-flight differences using a simple time Fourier transform operation. In one or more exemplary embodiments, the multi-color reflection matrix is generated between a radiometric basis that is a plane wave basis (entrance pupil plane) and a receiving measurement basis (detection plane). A propagator for determining a first focused volume reflection matrix based on the multi-color reflection matrix includes, for example, a first entrance basis transformation that can be shifted from the entrance pupil plane to the radiation focal plane, and a second exit basis transformation that can be shifted from the detection plane to the receiving focal plane (also called the exit focal plane). Such propagators are known to those skilled in the art. These propagators can include, for example, a Fresnel transform operation.

[0045] According to one or more exemplary embodiments, the N in incident light waves are spatially coherent and have a wavefront controlled by wavefront spatial shaping means.

[0046] For example, the wavefront spatial shaping means includes scanning means, and the N in incident light waves are plane waves having wave number vectors in different directions.

[0047] However, a predetermined wavefront does not necessarily have to be a plane wave having wave number vectors in different directions. For example, the illumination device may include a spatial wavefront modulator such as a deformable mirror or a liquid crystal modulator to generate different predetermined wavefronts.

[0048] The incident light wave is spatially coherent when the mutual coherence function of the electromagnetic field is uniform within a plane of space.

[0049] Conversely, when the mutual coherence function of the electromagnetic field is limited only by diffraction, that is, has a support of about half the wavelength, the light wave is spatially incoherent.

[0050] As used herein, a partially spatially coherent wave refers to a wave whose mutual coherence function has a finite support and whose width is greater than half the wavelength.

[0051] According to one or more exemplary embodiments, the light source for emitting a spatially coherent incident light wave is a spectral scanning single mode laser, for example, a laser that emits in a spectral range equal to wavelengths between about 800 nm and about 875 nm.

[0052] According to one or more exemplary embodiments, the light source is a low spatial coherence light source, and the method further includes - Based on each spatially incoherent or partially coherent light wave from the light source, generating, by a second interferometer, two orthogonally polarized illumination waves having a spatial shift in a plane conjugate to the focal plane of the first microscope objective lens; - Changing the spatial shift to generate N in incident waves of different wavefronts; - For each spatial shift, sending the orthogonally polarized polarization waves to the object arm and the reference arm of the first interferometer, respectively, by a polarization splitting element. - A step of acquiring the second plurality of interference signals by a detector, wherein each interference signal is, at a detection surface of the detector, a wave backscattered by a sample illuminated by one of the polarized light waves of the orthogonal polarization, and a reference wave generated by reflection of the other of the polarized light waves of the orthogonal polarization by a reference mirror of a reference arm. The interference signal is caused by interference between the backscattered wave and the reference wave, and the interference signal is acquired for N ω different optical path differences; - The multi-color reflection matrix is determined based on a set of interference signals acquired for N in spatial shifts and N ω optical path differences.

[0053] The low spatial coherence light source is, for example, a light emitting diode or a halogen lamp.

[0054] The characteristic evaluation method described in this way has the advantages of being fast in terms of acquisition time for obtaining a confocal image of one or more cross-sections of a sample and being optimal in terms of signal-to-noise ratio.

[0055] According to a second aspect, this document relates to an optical characteristic evaluation system for implementing a method for evaluating characteristics of an optical sample according to the first aspect.

[0056] Therefore, this document relates to a system for optical characteristic evaluation of a sample formed from a scattering bulk medium, the system comprising - A first interferometer having an objective arm and a reference arm, the objective arm including a first microscope objective lens having a predetermined field of view where a sample is placed during operation; - An illumination device including a wide spectral band light source and configured to generate a first plurality of incident light waves having different wavefronts; - N outA detector including a plurality of element detectors and configured to acquire a second plurality of interference signals for each incident light wave of a specific wavefront, each interference signal being caused by interference between a wave backscattered by a sample illuminated by the incident light wave and a reference wave from a reference arm on a detection surface of the detector, the interference signals being acquired for N ω different frequencies or N ω different path differences, a detector, and - A processing unit, - N in incident light waves and all interference signals acquired for N ω frequencies, a three-dimensional multi-color reflection matrix of size N in ×N out ×N ω to determine,

Number

[0057] According to one or more exemplary embodiments, the first interferometer is a Linnik interferometer, and the reference arm includes a reference mirror and a second microscope objective lens.

[0058] However, other arrangements are possible for the first interferometer that can be implemented by a person skilled in the art using general knowledge. In particular, the first interferometer may not include a reference mirror and / or a microscope objective lens on the reference arm.

[0059] According to one or more exemplary embodiments, the detector includes a two-dimensional light receiving element such as a CCD camera or a CMOS camera.

[0060] In other exemplary embodiments, the detector includes a spectrometer. ω The acquisition of the interference signal for N frequencies can be performed at the time of detection.

[0061] According to one or more exemplary embodiments, the light source is a low spatial coherence light source, and the illumination device - a second interferometer configured to generate two orthogonally polarized illumination waves having a spatial shift in a plane conjugate to the focal plane of the first microscope objective lens based on each spatially incoherent or partially coherent light wave from the light source, and - means for changing the spatial shift, where - the first interferometer is a Linnik interferometer, - a polarization splitting element configured to send each of the orthogonally polarized light waves having the spatial shift to the objective arm and the reference arm, respectively, and - means for changing the optical path difference between the reference arms, where - each interference signal of the plurality of second interference signals is due to the interference between the wave backscattered by the sample illuminated by one of the orthogonally polarized light waves on the detection surface of the detector and the reference wave generated by the reflection of the other of the orthogonally polarized light waves by the reference mirror of the reference arm, and the interference signal is obtained for N different optical path differences between the backscattered wave and the reference wave ω and - the multi-color reflection matrix in has N spatial shifts and N ωIt is determined based on the set of interference signals obtained for each path difference.

Brief Description of the Drawings

[0062] Further advantages and features of the above invention will become apparent from the detailed description provided with reference to the following drawings.

[0063]

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[0064] In various embodiments in the drawings referred to hereinafter, similar or identical elements are labeled with the same reference numerals.

DETAILED DESCRIPTION OF THE INVENTION

[0065] In the following detailed description, to ensure clarity of the disclosure, only specific embodiments are described in detail, but these examples are not intended to limit the general scope of the principles underlying this specification.

[0066] The various embodiments and aspects described herein can be combined or simplified in many ways. In particular, the various method steps can be repeated, interchanged, or performed in parallel, unless otherwise specified. Whenever reference is made herein to a computational or processing step, particularly for the implementation of a method step, it is understood that each computational or processing step can be implemented by software, hardware, firmware, microcode, or any suitable combination of these techniques. When software is used, each computational or processing step can be implemented by computer program instructions or software code. These instructions can be stored in or transmitted to a computer-readable storage medium (or computing unit) and / or executed by a computer (or computing unit) to perform these computational or processing steps. FIG. 1A shows a first example of a system 101 for optical characterization of a sample 10 consisting of a scattering bulk medium, the system 101 being configured to implement the optical characterization method according to the present invention. FIG. 1B is a simplified diagram showing the basis in which matrices are described in the characterization method according to the present invention.

[0067] The system 101 includes an illumination device 130 having a broad spectral bandwidth light source 132. For example, a spectrally scanned light source [ω min ,ω max ] spectral band (or [λ min ,λ max ]) with a center frequency of ω n (or wavelength λ n ), which is shown diagrammatically in spectrum 131. The light source 132 is configured to emit a light beam having a frequency ω (or wavelength λ), which may vary around λ min = 800 nm and approximately λ max = 875 nm, and the spectrum between these two wavelengths is, for example, about 10 2 nm / s and about 10 5It can be scanned at a predetermined speed between nm / s. The light source is, for example, a fiber-based light source, and the illumination device can include an output fiber 133 connected to a collimator 134 configured to emit a substantially parallel spatially coherent beam.

[0068] System 101 further includes an interferometer 120 having an object arm and a reference arm separated by a cube splitter 121. System 101 includes a first microscope objective lens 110 disposed within the object arm of the interferometer, and the sample is disposed within the field of view of the microscope objective lens during operation.

[0069] In the example shown in FIG. 1A, the interferometer 120 is a Linnik interferometer and includes, in addition to the first objective lens 110 on the object arm, a second microscope objective lens 122 disposed within the reference arm. In this example, the reference arm further includes a reference mirror 123.

[0070] As shown in FIG. 1A, the light emitting device of this example also includes means 135 for scanning the parallel beam and a set of lenses 136, 137, 138. The lenses 137 and 138, which are respectively called the scanning lens and the tube lens, form a 4f assembly with a predetermined magnification, and the lens 138 is configured to focus the beam emerging from the 4f assembly onto a plane InP corresponding to the plane of the entrance pupil of the microscope objective lens 110 (the "entrance pupil plane"). The scanning means 135 includes, for example, two scanning mirrors rotated by a galvanometer motor, and scans the incident light beam (represented by a single-headed arrow in FIG. 1A) in both spatial directions at the entrance pupil plane InP of the microscope objective lens 110 disposed on the object arm of the interferometer 120. It should be noted that in the example of FIG. 1A, since a Linnik interferometer is used, the incident beam also scans the entrance pupil plane of the second microscope objective lens 122 disposed on the reference arm of the interferometer.

[0071] System 101 further includes a detector 140 having a detection surface ImP and a processing unit 150 configured to process data generated by the detector, which will be described later. The detector includes, for example, a camera configured to acquire a two-dimensional image at a predetermined acquisition frequency. For example, the camera enables acquisition of an image of 256×256 pixels at a predetermined frequency, such as about 75 kHz.

[0072] Of course, other configurations of the optical property evaluation system 101 that those skilled in the art can implement using general knowledge in the field are also possible. For example, the system 101 can be deployed in a polarization configuration. In this case, the cube splitter is a polarization cube splitter. Two quarter-wave plates can be disposed between the microscope objective lens and the cube splitter. A polarization analyzer for recombining the two polarizations can be disposed upstream of the detector 140.

[0073] Also, the broadband light source does not necessarily need to have a variable frequency. For example, as described with reference to FIG. 10B, it is possible to replace the camera with a spectrometer, in which case the frequency is selected at the time of detection.

[0074] Furthermore, instead of changing the frequency of the light source, it is equivalently possible to change the path difference (or "time of flight") between the object arm and the reference arm of the interferometer. For example, it can be changed by moving a block including the reference mirror 123 and the second microscope objective lens 122.

[0075] Also, instead of the Linnik interferometer, other interferometer arrangements are possible. For example, as described with reference to FIG. 10A, the reference wave in the interferometer can come directly from the light source.

[0076] The notation used herein for identifying the various bases in which reflection matrices, particularly for the characterization of samples, are described is shown in FIG. 1B. For simplicity, FIG. 1B shows only some of the elements of the characterization system. This notation is applicable to the arrangement of FIG. 1A, but more generally, it is applicable to all characterization systems in this specification.

[0077] The focal plane of the microscope objective lens 110 is denoted as FP and is intended to receive the sample 10. InP is used to represent the plane of the entrance pupil of the microscope objective lens, or any plane conjugate to the plane of the entrance pupil of the microscope objective lens, and is called the incident plane wave basis. The entrance pupil is intended to receive the incident light wave for illuminating the field of view of the sample to be characterized. The notation u in represents a point within the entrance pupil plane InP, and that point is defined by the orthogonal coordinates (v in , w in ). OutP is used to represent the plane of the exit pupil of the microscope objective lens, or any plane conjugate to the plane of the exit pupil of the microscope objective lens, and is called the exit plane wave basis. The exit pupil is intended to receive the light wave reflected by the field of view of the sample to be characterized. The notation u out represents a point within the exit pupil plane OutP, and that point is defined by the orthogonal coordinates (v out , w out ). As shown in FIG. 1B, the incident path and the exit path each including the entrance pupil and the exit pupil, respectively, are separated by the beam splitter element 121. In the incident path, SP represents the light source plane of the optical system, which is conjugate to the focal plane of the microscope objective lens, and in this example, is represented by the lens 11, which forms a 4f assembly with the microscope objective lens 110. The notation ρ in represents a point within the light source plane SP, and its orthogonal coordinates (x in , y in ) define it. In the exit path, ImP represents the image plane of the optical system, which is conjugate to the focal plane of the microscope objective lens, and in this example, is represented by the lens 12, which forms a 4f assembly with the microscope objective lens 110. The notation ρ out represents a point within the image plane ImP, and its orthogonal coordinates (x out , yout is defined by

[0078] The voxel r of the sample in has coordinates (ρ´ in , z in ), where the point r in has an abscissa ρ´ in (x´ in , y´ in ) that is conjugate to the point ρ in the light source plane SP in . The (volumetric) basis of the conjugate light source point of the voxel r in is called the incident focal basis.

[0079] The voxel r of the sample out has coordinates (ρ´ out , z out ), where the point r out has an abscissa ρ´ out (x´ out , y´ out ) that is conjugate to the point ρ in the image plane ImP out . The (volumetric) basis of the conjugate light-receiving point of the voxel r out is called the incident focal basis. During operation, the illumination device 130 enables the implementation of a characteristic evaluation method according to an example herein as follows. For a given frequency (or length), the illumination device generates a first plurality of N in incident light waves consisting of different given wavefronts. For example, as shown in FIG. 1A, the N in incident light waves are N in plane waves having wave number vectors in different directions controlled by the scanning means 135. For example, the lens 124 (exit lens) ensures the optical conjugation between the objective focal plane FP of the microscope objective 110 and the detection plane ImP of the detector 140. Thus, according to an example, during operation, the scanning means 135, in cooperation with the 4f assemblies 136, 137 and the condenser lens 138, scans the pupil planes InP of the microscope objectives 110 and 122 respectively arranged on the objective arm and the reference arm of the Linnik interferometer 120 in both spatial directions. The N in incident light waves generated in this way each have a wave number vector k inIt has a direction and a norm that depend, in particular, on the scanning angle determined by the scanning means 135 and the frequency ω (or wavelength λ) of the incident wave.

[0080] Furthermore, for each incident light wave of a given wavefront, the detector 140 acquires a second plurality of N ω interference signals. Each interference signal is due to the interference between the wave backscattered by the sample 10 illuminated by the incident light wave and the reference wave from the reference arm at the detection surface ImP of the detector, and the N ω interference signals are acquired for N ω different frequencies. For example, in the example of FIG. 1A, the illumination device 130 comprises a spectroscopic scanning light source, and the N ω interference signals are acquired by varying the wavelength of the light source.

[0081] The fields backscattered by the two arms of the interferometer interfere at the detection surface ImP of the detector 140. The interference term between the two arms for the frequency ω of the incident wave is, according to the following formula, the product of the field E s reflected by the sample and the phase conjugate E r * of the reference field from the reference arm.

[0082]

Equation

[0083] Here, ρ out (x out , y out ) is a vector of the spatial coordinates (x out , y out ) in the detection surface ImP (light-receiving measurement surface), and u in (v in , w in ) is a vector of the spatial coordinates (v in , w in ) in the pupil plane InP (radiation measurement surface) of the microscope objective 110.

[0084] During operation, the lens 138 enables the incident beam to be focused on the pupil plane of the microscope objective lens. The size δu of the associated focal spot is limited only by diffraction (the numerical aperture of the optical system upstream of the interferometer). This focal spot is converted by the microscope objective lens into a pseudo-plane wave that illuminates the sample over a wide field of view. The latter is partially imaged onto the detection plane ImP.

[0085] As an example, FIG. 2A shows the scanning of the entrance pupils InP of the microscope objective lenses 110, 122 by N in incident light beams. The scanning mirror 135 enables the scanning of the physical pupil of the microscope objective lens. In FIG. 2A, the periphery of the pupil is indicated by the black circle 201. The black disks indicate the positions of the individual incident beams focused on the entrance pupil InP of the microscope objective lenses 110, 122. The spatial pitch between each beam is δu in and is expressed in. Considering the characteristics of the microscope objective lens used, the characteristic size of each focal spot is, for example, δu = 8 μm. This corresponds to the maximum illumination of a field of view of Δr = λf / δu = 2 mm. Here, f is the focal length of the microscope objective lens 110. The spatial pitch δu in corresponds to the sampling of the electromagnetic field at the exit pupil plane and indicates the accuracy with which the incident wavefront is decomposed when numerically recombining using the measured reflection matrix. Therefore, this spatial pitch can be advantageously adjusted according to the level of aberration generated by the medium and can be chosen fine enough to capture the fastest spatial variations of the distortion experienced by the light wave as it passes through the medium. Depending on the intended application, it is possible to scan the entire entrance pupil 201 or only a part thereof. For example, to obtain a high-resolution confocal image, as shown in FIG. 2A, the entire entrance pupil can be scanned.

[0086] For each illumination of the sample, the interference term

[0087]

Number

[0088] Figure 2B shows the field of view Δp on the detection surface of the camera and the spatial sampling δρ at which each interference signal (or interference pattern) is recorded. out The notation N out represents the number of pixels (or element detectors) of the camera at which the interference signal is acquired. The field of view corresponds to an image obtained by magnifying the focal plane of the microscope objective lens by a factor G, where G is the magnification of the imaging system consisting of the microscope objective lens and the exit lens 124. In the exemplary system shown in FIG. 1A, G = -f s / f om where f s and f om are the focal lengths of the lens 124 and the microscope objective lenses 110 and 122.

[0089] All interference patterns recorded for each illumination u in and each frequency ω are stored as a three-dimensional multi-color reflection matrix of the following equation.

[0090]

Equation

[0091] FIG. 2C shows such a three-dimensional multi-color reflection matrix, as an example, to show its three-dimensional structure. The first dimension represents the detection point ρ on the detection surface of the camera out whose coordinates (x out , y out ) are given by the following indices.

[0092]

Equation

[0093]

Equation

[0094]

Equation

[0095]

Number

[0096]

Number

[0097] The third dimension indicated by the exponent k corresponds to a series of frequencies ω at which the interference signal is recorded.

[0098] Therefore, in this example, the matrix

[0099]

Number

[0100]

Number

[0101]

Number

[0102]

Number

[0103]

Number

[0104]

Number

[0105]

Number

[0106]

Number

[0107]

Number

[0108] The second step of the method according to this specification is to apply a propagator and, based on the multi-color reflection matrix

[0109]

Number

[0110]

Number

[0111] Multicolor reflection matrix

[0112]

Number

[0113]

Number

[0114] Figure 3A shows the numerical focusing procedure in two voxels located at different depths (z in and z out ) at points r in and r out in the medium under study.

[0115] Multicolor reflection matrix

[0116]

Number

[0117]

Number

[0118]

Number

[0119]

Number

[0120] Next, the Fresnel mask (denoted as F(u,z in / out -z f ,ω)) is applied to the entrance and exit of the obtained matrix

[0121]

Number

[0122]

Number

[0123] Examples (411, 421) of the Fresnel mask F are shown in FIG. 4A for different focal depths z = z in = z out This is represented by the following equation.

[0124]

Number

[0125]

Number

[0126]

Number

[0127]

Number

[0128] Next, the polychromatic focusing reflection matrix

[0129]

Number

[0130]

Number

[0131]

Number

[0132]

Number

[0133] To show the value of the focused volume reflection matrix R rr the present applicants used the system described in FIG. 1A to measure the multi-color matrix

[0134]

Number

[0135]

Number

[0136] FIG. 3B shows, as an example, z = z in = z out = 250 μm and the ballistic time

[0137]

Number

[0138]

Number

[0139]

Number

[0140] Matrix R rr Each column 302 of subsection 302 of gives the reflected field measured by each virtual receiving point r in for light waves radiated from the virtual light source point r out . In the ideal case without aberration or multiple scattering, an Airy disk would be observed around the light source point r in . However, here, the reflected field takes the form of a diffused halo around the point r in , indicating that the assumptions made about the optical refractive index are not necessarily optimal. This will be explained in more detail later.

[0141] However, the broadband focusing matrix R rr provides access to a series of related observable quantities that are utilized to quantitatively characterize the sample under study.

[0142] First, the confocal image with a time window

[0143]

Number

[0144] [Number]

[0145] Therefore, FIG. 3B shows a confocal image 303 of the cornea at z = 250 μm. The latter corresponds to the diagonal elements of the submatrix 302 after rearrangement into a two-dimensional form. The confocal image

[0146] [Number] is an estimated value of the corneal reflectance at the coherence plane associated with single-scattered photons given by the time-of-flight

[0147] [Number] where the medium is. The quality of this estimated value depends on the quality of the focusing in the medium. Equation h

[0148] (r, r in / out )(r, r in / out ) represents the impulse response between each point r in / out and each point r in the sample (see FIG. 1B). To quantify the quality of the focusing, the relevant observable quantity is the reflectance point spread function (RPSF) around the reflection focus, which is the submatrix R in = z out ) is assumed to be the same (z rr (z, t) is accessible by measuring the distribution of the backscattered energy along the anti-diagonal of.

[0149] [Number] Here, <...> is the average for the pair of points r p = (r in + r out ) / 2, centered on the same point r p . Δr = r in and r out = r out - r inis the relative position (coordinates Δx, Δy) between these two points. This intensity profile is relevant because it can examine the aberration regardless of the local reflectivity of the medium. Specifically, by considering the incident and outgoing PSFs to be locally isotropic, by translating around each point r p it is possible to redefine the invariant local PSF

[0150]

Number

[0151]

Number

[0152]

Number

[0153] For a specular surface, the RPSF gives access to the convolution product between the coherent incident spread function and the outgoing spread function.

[0154]

Number

[0155] In these two asymptotic regions, the RPSF does not exactly examine the same physical quantity, but its spatial distribution indicates the level of local aberration at the point r p The figure 3B shows the depth z pShows the RPSF305 obtained at = 250 μm. It has the following appearance. It is the excess intensity associated with photons scattered by the coherence surface and the incoherent background associated with multiple scattering events occurring upstream of this same surface. The position of the maximum value of the RPSF and the spatial spread of the excess intensity observed in the RPSF provide information about the quality of the focusing.

[0156] Figure 3B shows that the maximum value (305) of the RPSF is not centered at Δr = 0. This is due in particular to the alignment problem of the first interferometer. Thus, the confocal intensity corresponds not to the relative position Δr = 0, but to the position Δr of the intensity maximum of the RPSF. max Therefore, a new confocal image corrected for these alignment problems

[0157]

Number

[0158]

Number

[0159] Image 304 shows the new confocal image obtained. From the comparison with the raw confocal image

[0160]

Number

[0161] Furthermore, except for these alignment problems, if the numerical focusing process is ideal, the support of the RPSF should extend to a single resolution cell. As described above, Figure 3B shows that this is not the case for the opaque cornea under study.

[0162] Figure 3C shows the main reason for the low quality of this focusing, i.e., the mismatch between the focal plane and the coherence plane. The finite depth of field of the microscope objective lens can cause focusing defects for both emission and reception.

[0163] Figure 4A shows a method for adjusting the positions of the coherence plane and the focal plane using the RPSF. This figure is based on a proof-of-concept experiment regarding a test pattern with the focal point shifted by a distance of z = 100 μm below the focal plane (ON = 0.3) of the microscope objective lens of the sample arm. Figure 4A shows the RPSF obtained for different focal planes z = z m = z in = z out and for the coherence plane t = 2z m / c0 that coincides with the position of the test pattern.

[0164] z < z m (or equivalently z > z m ), the spread (411, 412) of the RPSF is a characteristic of the focusing defect. Then, an estimate for determining the value of z m is defined. This estimate corresponds to the position z max at which the confocal intensity, i.e., the maximum value of the RPSF (denoted as RPSF opt ), is maximized. For z = z opt , Figure 4A (421, 422) clearly shows that the lateral spread of the RPSF is minimized, i.e., restricted only by diffraction.

[0165] Figure 4B shows the variation of the RPSF max with depth z. In fact, z opt = z m , and it was found that the confocal intensity is 3.5 times stronger outside the depth of field (|z - z m | > 20 μm). Figure 3C summarizes the numerical focusing optimization process. The confocal intensity (RPSF maxBy maximizing , it is possible to align the coherence surface corresponding to the interference characteristics of the measurement with the geometric focus related to the position of the sample with respect to the microscope objective lens. FIG. 5 shows the effect of numerical double focusing on the confocal image of the test pattern (510). While the initial image 511 is degraded by focusing defects, the numerical focusing processes at incidence and emission produce an image of the test pattern 512 with optimal contrast and confocal resolution limited only by diffraction (δp approximately λ / (4NA)). After correction, all details of the test pattern are sharp and highly contrasted. Thus, the technical effect of the above-described method for generating a confocal image from a full-field spectroscopic OCT apparatus is observed. In this way, it is possible to determine the reflectivity map of the sample based on the first focusing volume reflection matrix.

[0166] A similar focusing optimization method is applied to the reflection matrix measured in the cornea. However, in the experiment in question, the cornea is immersed in water with a known refractive index n water (n eau ) Most importantly, the cornea has a spatially non-uniform optical refractive index n(r). First, assuming that the average refractive index of the medium is invariant with respect to lateral movement and its optical refractive index n depends only on the depth z, let n(r)=n(z). The first step of this method is to bring the focal plane, which is initially located at the depth z f to the surface of the sample located at the depth z0 by applying to the following matrix water (refractive index n=n eau ).

[0167]

Number

[0168]

Number

[0169] [Number]

[0170] Next, the estimated value of the profile of the optical refractive index n(z)

[0171] [Number] is obtained as follows. For each flight time t, the RPSF corrects the average optical refractive index

[0172] [Number] and thus corrects the position of the coherence plane in the propagator F ([Equation 5])

[0173] [Number] so that it is scanned to maximize the confocal intensity (RPSF max ). The value of n that maximizes this quantity gives the estimated value of the integrated optical refractive index n

[0174] [Number] at the depth z. The integrated refractive index profile n int (z) is defined as the integral of the optical refractive index n(z) of the medium from the surface of the medium (z = z0) to the depth z. int (z)

[0175] [Number]

[0176] By discretizing this equation with respect to the flight time t, the estimated value of the integrated refractive index profile n int (z)

[0177] [Number] From this, by reversing the linear equation, an estimated value of the local optical refractive index n(z)

[0178]

Number

[0179] To obtain the dependence of n on the abscissa (x, y), first, for the reflection matrix

[0180]

Number

[0181]

Number

[0182]

Number

[0183]

Number

[0184]

Number

[0185] The estimated value n of the spatial distribution of the optical refractive index n (simply referred to as the "optical refractive index map" in this specification) can be used to construct a new propagator F in order to optimally describe the propagation of light from the entrance and exit pupil planes of the device to all voxels of the medium under study (i.e., the focusing substrate). Next, this propagator can be used to construct a new focusing reflection matrix, which can provide a confocal image closer to the actual reflectivity of the medium.

[0186] Figure 6 shows a confocal image 610 obtained following this numerical focusing process. It should be noted that in this way, instead of the flight time t, which is a limitation of conventional OCT images where the axial dimension is determined by the path difference between the arms of the interferometer, an image can be constructed according to the depth z.

[0187] Previous confocal image

[0188] [Number] (304, Figure 3B) shows an improvement in the image quality in terms of both contrast and resolution.

[0189] However, the quality of this image can be further improved due to aberration phenomena and multiple scattering (excluding focusing defects).

[0190] Figure 6 illustrates this last claim by showing a quantitative study of the RPSF measured in the cornea after focusing. In particular, confocal Δr maxShows the variation of the radial distribution of the surrounding RPSF at a depth of 620. The spread of the confocal peak increases with depth, quantifying the loss of resolution caused by the aberration experienced by the light wave as it propagates through the sample. For example, at a depth z = 250 μm, the full width at half maximum 630 of the RPSF is 15 μm. This far exceeds the diffraction limit (δp ≈ 1.4 μm). Finally, the variation of the RPSF with depth shows that as z > 200 μm, a more and more incoherent background appears. This is resistant to the average over the integration time of the camera. This element is deterministic and is thus related to the occurrence of multiple scattering events occurring upstream of the focal plane.

[0191] Beyond the variation with depth, the levels of aberration and multiple scattering can be investigated laterally by studying the RPSF locally measured around a series of points r p in the field of view.

[0192] Figure 7A illustrates the subdivision of the field of view performed to extract the local RPSF shown in Figure 7C. The spatial spread of the local RPSF shows that, for example, the focusing quality is much better in the lower left of the field of view. Thus, the confocal image shown in Figure 6 is a much better estimate of the reflectivity of the medium in this part of the field of view. The lateral variation of the RPSF also shows the anisotropic nature of the aberration caused by the optical refractive index inhomogeneities distributed through the sample. As will be shown later, local compensation of the aberration is advantageous, and the matrix approach is a suitable tool for this.

[0193] Beyond aberration, the ratio of multiple scattering to single scattering can be measured using the confocal intensity RPSF max and the incoherent background RPSF bg .

[0194]

Number

[0195] The factor 2 in the denominator accounts for the effect of coherent backscattering, by which the multiple scattering intensity is confocal amplified by the same factor 2. Figure 7B shows the map of the parameter ρ MS at a depth z = 250 μm. This map reveals the presence of streak-like marks that were already somewhat visible in the confocal image of Figure 6. Such streak-like marks in the stroma of the cornea are characteristic of diseases such as keratoconus. Beyond this qualitative analysis, the ratio ρ MS represents a first step towards a quantitative and local image of the light scattering properties in a biological sample. Depending on the information sought, other parameters such as the single scattering rate (or confocal scattering rate) can be extracted.

[0196]

Number

[0197] The decrease with z of this parameter enables the local quantification of the scattering mean free path in the medium. More generally, the quantification of single and multiple scattering phenomena enables the local measurement of transport parameters such as the scattering mean free path, the transport mean free path, or the scattering coefficient. The reflection matrix approach also makes it possible to study the growth of the diffuse light halo with a relatively short flight time and provides a much better spatial resolution than standard techniques such as diffuse light tomography.

[0198] Beyond the quantification of aberrations and multiple scattering phenomena in the medium, it is shown here how it is possible to locally correct the aberrations induced by the sample in order to obtain a confocal image with an optimal contrast and resolution close to the diffraction limit using a numerically refocused reflection matrix.

[0199] For this purpose, the incident / outgoing radiation matrix T in / out between the incident / outgoing correction basis and the voxels of the medium can be estimated. For this, a local analysis is performed here to estimate the focusing rule suitable for the voxels of each image.

[0200] As an example, a plane wave basis is selected as the correction basis. The first step of the matrix correction process for correcting aberrations is to project the time-windowed focused reflection matrix

[0201] [Number] onto this correction basis at the exit (or entrance).

[0202] [Number]

[0203] From this double matrix

[0204] [Number] a distortion matrix D kr is constructed as follows.

[0205] [Number] Here, T0 is the reference matrix that models the propagation of plane waves if the medium is uniform (i.e., aberration-free) as follows.

[0206] [Number] In Patent Document 2 (Reference Document 4), it was shown that under the assumption of isotropy, the transmittance A(k out ) of the aberration sub can be estimated from the phase of the first eigenvector U1 by singular value decomposition of the matrix D. Here, since the aberration is not spatially invariant, it is advantageous to subdivide the field of view, locally analyze the distortion matrix, and locally estimate the focusing rule suitable for each voxel of the image. The field of view is divided into overlapping regions defined by its central midpoint r p and its spatial extent Δr. The focal point r in located within each regionAll distorted components of the associated field can be extracted and stored in a local strain matrix.

[0207]

Number

[0208]

Number

[0209]

Number

[0210] Another estimate of the transmission matrix T out can be obtained by an iterative phase-retrieval process. This is based on the following recurrence relation.

[0211]

Number

[0212]

Number

[0213]

Number

[0214] Regardless of the method for estimating the transmittance matrix T out , next, the phase conjugation of the estimated value

[0215]

Number

[0216]

Number

[0217]

Number

[0218]

Number

[0219]

Number

[0220]

Number

[0221]

Number

[0222] Figure 8 shows the results of a numerical correction method for lateral aberration in a cornea with a depth z = 250 μm. Here, the iterative phase-shifting method is considered to estimate the transmission matrices T out and T in .

[0223] Figure 8 shows the changes 801, 802, 803 of the RPSF in each iteration for a subzone of a field of view of 105×105 μm 2 . As the incident and outgoing iterations progress, the RPSF becomes more refined and local aberrations are increasingly corrected. The aberration laws 804, 805 at the incident and outgoing obtained at the end of the process are also shown in Figure 8. Ideally, due to spatial reciprocity, they should be identical (T in = T out)。They are different especially due to the imperfections of the imaging system such as alignment defects. Therefore, the matrix aberration correction process corrects not only the aberration caused by the non-uniformity of the optical refractive index in the medium but also the imperfections of the imaging system. Comparison of the confocal images of the subzones before and after correction

[0224]

Number

[0225] Figure 9 shows the phase of the transmission matrix T in obtained at the end of the iterative aberration correction process (901, 902). This includes all the aberration rules for each subzone of the field of view (Figure 7A). Images 903, 904 show the front cross-section of the volume confocal image of the cornea at a depth z = 250 μm before and after numerical aberration correction. Images 905, 906 show the longitudinal cross-section (B-scan) of the volume confocal image of the cornea before and after numerical aberration correction. After matrix aberration correction, the confocal image has higher contrast. The confocal intensity increases by an average of 7 times over the entire field of view. The improvement in resolution is also significant. From the study of the RPSF, it can be seen that the resolution has been improved by 3 times.

[0226] The experimental verification test was carried out using the characteristic evaluation system of the type shown in Figure 1A. Naturally, other characteristic evaluation systems according to the present specification for implementing the method according to the present specification are also possible.

[0227] Therefore, Figures 10A, 10B and 11 show a system for the optical characteristic evaluation of a sample formed from a scattering bulk medium according to other exemplary embodiments.

[0228] In the example of system 102 shown in FIG. 10A, one difference from the system shown in FIG. 1A lies in the field from the reference arm. The reference wave in the first interferometer comes directly from the light source via the beam splitter 1032. In the case of the fiber-based light source 132 (as in FIG. 10A), the reference arm includes an optical fiber 1033 and a collimator 1034 that enable projecting the reference wave in the form of a plane wave at the detector 140. In free space, the reference arm is composed of a lens system ideally selected to minimize the path difference between the reference wave and the wave singly scattered by a reflector located in the plane of interest in the sample arm. In this case, the interference between the wave reflected by the sample illuminated by a given set of different incident wavefronts and the reference plane wave that only varies in frequency is measured. Thereby, direct access to the field

[0229]

Number

[0230]

Number

[0231]

Number

[0232] In the example of system 103 shown in FIG. 10B, the light source in question is a broadband light source. The use of the detector 160 including a spectrometer enables measurement of the spectral dependence. The spectrometer includes, for example, a dispersive element 161 (such as a diffraction grating) and a detection device 162. Each element of the detection device measures the field at the coordinates ρ out of the plane ImP and at the frequency ω. This device provides a matrix equivalent to that measured by the system of FIG. 1A

[0233]

Number

[0234] A further example of a system for evaluating the optical properties of a scattering bulk medium according to this specification is schematically shown in FIG. 11. The system 104 shown in FIG. 11 has the advantage of being faster in terms of acquisition time and improved in terms of signal-to-noise ratio compared to the system shown in FIG. 1A, particularly when it is desired to obtain confocal images for a small number of cross-sections of the sample.

[0235] The system 104 includes an illumination device 1130 having a spatially incoherent light source 1132 disposed at the focal point of a lens 1133, and is configured to emit a plurality of incident light waves for illuminating a given field of view of the sample 10 through a microscope objective lens 110. The system 104 also includes a detector 1140 disposed at the focal point of a lens 1124, and a processing unit (not shown) that particularly receives the optoelectronic signal from the detector 1140. The property evaluation system 104 includes two interferometers arranged in series. A first interferometer 1120 and a second interferometer that forms part of the illumination device 1130. The second interferometer is, for example, a Michelson interferometer in an air wedge configuration and is capable of generating two illumination beams of orthogonal polarization that are inclined with respect to each other at the exit. The first interferometer 1120 is, for example, a Linnik interferometer having a polarization beam splitter 1121 and quarter-wave plates 1126, 1128 on each arm. Afocal systems 1111, 1112 make it possible to conjugate the surfaces of the mirrors 1136 and 1138 of the second interferometer to the pupil planes of the microscope objective lenses 1122 and 110 of the first interferometer.

[0236] Spatially and temporally incoherent incident fields are linearly polarized at 45 degrees with respect to the directions parallel (e||) and perpendicular (e⊥) to the plane of the device by the polarizer 1134. The components of this wave polarized in the directions e|| and e⊥ are transmitted and reflected, respectively, by the polarization beam splitter 1131. Each arm of the second interferometer contains a quarter-wave plate (1135, 1137) and a mirror (1136, 1138). In one arm, the mirror (1136) is tilted with respect to the optical axis. When coming back, the two waves from each arm emerge from the interferometer as two tilted orthogonally polarized beams. However, since these two beams are derived from the same incident wave, they are coherent with each other.

[0237] In the first interferometer 1120, the two beams are again separated by the polarization beam splitter 1121. The e|| polarized beam is transmitted in the reference arm. The e⊥ polarized beam is reflected in the object arm. The presence of quarter-wave plates 1126, 1128 in each of the two arms enables optimal transmission when the two beams are reflected by the sample in the object arm and the mirror in the reference arm. These two beams are recombined using an analyzer 1125 polarized at 45 degrees with respect to e|| and e⊥ at the exit of the interferometer. Thus, they can interfere at the focal plane of the lens 1124. A detector, such as a CCD or CMOS camera, records the corresponding interference signal. Due to the tilt of the mirror 1136, the reference beam and the object beam are shifted relative to each other in the camera. In this way, it is possible to measure the impulse response between different points ρ in and ρ out . Therefore, the system described with reference to FIG. 11 enables the measurement of the multicolor reflection matrix R pp in the time domain and in a basis conjugate to the focal plane of the microscope objective lens.

[0238]

Number

[0239] The flight time t of the photons is controlled by the path difference between the reference arm and the sample arm of the first interferometer 1120. Therefore, the flight time can be scanned by simultaneously moving the elements 1122, 1123, and 1126 of the reference arm of the first interferometer. Point ρ in and ρ out The relative positions of are controlled by the tilt of the reference mirror 1136 of the second interferometer.

[0240] In some exemplary embodiments, for the coefficients of the matrix R pp both a temporal Fourier transform with respect to the variable t and a spatial Fourier transform with respect to the coordinate ρ out are performed to obtain a matrix equivalent to that recorded by the apparatus of FIG. 1A

[0241]

Number

[0242]

Number

[0243] In other embodiments, based on the matrix R pp a focused reflection matrix R rr is obtained by applying a time shift to the interference signal measured as follows.

[0244]

Number

[0245] Although described by numerous detailed exemplary embodiments, the optical property evaluation method and system include various variations, modifications, and improvements that will be apparent to those skilled in the art, and it is understood that these various variations, modifications, and improvements form part of the scope of the invention defined by the following claims.

[0246] [References] Reference 1: S.M. Popoff et al. “Measuring the Transmission Matrix in Optics”, Phys. Rev. Lett. 104, 100601, 2010. Reference 2: A. Badon et al. “Smart optical coherence tomography for ultra - deep imaging through highly scattering media”, Sci. Adv. 2016; 2: el600370. Reference 3: US20040061867 Reference 4: US20210310787

Claims

1. An optical property evaluation method for a sample (10) formed from a scattering bulk medium, the optical property evaluation method comprising: - placing the sample (10) within the field of view of a first microscope objective lens (110), the microscope objective lens being disposed on the objective arm of a first interferometer (120), the first interferometer further comprising a reference arm; - Generating a first plurality of N incident light waves having different wavefronts by an illumination device (130) including a wide spectral band light source (132) ｉｎ and - For each incident light wave of a specific wavefront, a step of obtaining a second plurality (N ｏｕｔ pieces) of interference signals by a detector (140) including N ω element detectors, wherein each interference signal is due to the interference between the wave backscattered by the sample illuminated by the incident light wave and the reference wave from the reference arm on the detection surface of the detector, and the interference signal is obtained for N ω different frequencies or N ω different path differences; - with a size of N ｉｎ × N ｏｕｔ × N ω three-dimensional multicolor reflection matrix 【Number 1】 A step of determining, wherein the three-dimensional multicolor reflection matrix is the N ｉｎ incident light waves and all of the interference signals obtained for N ω frequencies (including N ｉｎ × N ω） ); - Based on the multi-color reflection matrix, by applying a first propagator, at least a first focused volume reflection matrix (R ｒｒ ) is numerically determined, wherein the first focused volume reflection matrix is the voxel r ｉｎ and z ｏｕｔ of the sample (10) located at each of them, respectively, and the set of responses of the sample between the light source point and the light receiving point conjugate to the voxel r ｉｎ (x′ ｉｎ , y′ ｉｎ , z ｉｎ ) and r ｏｕｔ (x′ ｏｕｔ , y′ ｏｕｔ , z ｏｕｔ ) is included, - determining at least one map of physical parameters of the sample based on the first focused volume reflection matrix; An optical property evaluation method comprising the steps of:

2. The at least one map of the physical parameters of the inhomogeneous medium is a confocal image, a map of the point spread function (RPSF) around a plurality of reflection foci (r ｐ ), a map of the single scattering rate, a map of the multiple scattering rate, and a map of the optical refractive index of the sample. The optical property evaluation method according to claim 1.

3. - determining a first reflection confocal image based on the focused volume reflection matrix; 【Number 2】 - determining a map of the spread function based on the focused volume reflection matrix and determining a map of the positions of the intensity maxima of each spread function; - determining a second reflection confocal image corrected for alignment and / or focusing defects of the first interferometer based on the first reflection confocal image and the map of the positions of the intensity maxima; - determining a second reflection confocal image corrected for alignment and / or focusing defects of the first interferometer based on the first reflection confocal image and the map of the positions of the intensity maxima; 【Number 3】 - determining a second reflection confocal image corrected for alignment and / or focusing defects of the first interferometer based on the first reflection confocal image and the map of the positions of the intensity maxima; The optical property evaluation method according to claim 1 or claim 2, further comprising the steps of:

4. - determining a plurality of maps of the spread function for a plurality of values of the integrated optical refractive index of the sample based on the first focused volume reflection matrix; - determining a plurality of maps of the positions of the intensity maxima of the spread function based on the plurality of maps of the spread function, each map being obtained for a value of the integrated optical refractive index; - determining a map of the optimum value of the integrated optical refractive index at which the maximum intensity value of the spread function is maximized at each focus; - numerically determining a second propagator based on the map of the optimum value of the integrated optical refractive index; and - determining a second focused volume reflection matrix using the second propagator; The optical property evaluation method according to any one of claims 1 to 3, further comprising the steps of:

5. The optical property evaluation method according to claim 4, further comprising the step of determining a map of the optical refractive index of the medium at each focus based on the map of the optimum value of the integrated optical refractive index.

6. The optical property evaluation method according to claim 5, wherein the second propagator is determined based on the map of the optical refractive index.

7. An optical property evaluation method further comprising a step of determining a distortion matrix (D) defined between a correction basis and a focusing basis based on the second focused volume reflection matrix, the optical property evaluation method comprising: - At the entrance or the exit, projecting the second focused volume reflection matrix onto the correction basis to obtain a projected reflection matrix at the entrance or the exit, respectively [Number 4] and obtaining; - Determining the distortion matrix by an element-by-element product between the projected reflection matrix in the correction basis and a reference reflection matrix defined for a reference medium; - Locally determining the invariant of the distortion matrix to specify an aberration law in a subdomain of the field of view in the correction basis. - Based on the aberration rule, estimating a transmission matrix (T ｏｕｔ ) between the voxels of the field of view and the correction basis; The optical property evaluation method according to any one of claims 4 to 6, comprising:

8. The light source is a variable wavelength light source, and for each incident light wave of a predetermined wavefront, the plurality (N ω pieces) of the interference signals are obtained for N ω central wavelengths of the incident light waves and a fixed path difference between the objective arm and the reference arm of the first interferometer. The optical property evaluation method according to any one of claims 1 to 7.

9. For each incident light wave of a specific wavefront, the interference signals of the second plurality (N ω ), are obtained for N ω path differences between the objective arm and the reference arm of the first interferometer, the optical property evaluation method according to any one of claims 1 to 7.

10. said N ｉｎ The method for evaluating optical characteristics according to any one of claims 1 to 9, wherein the N incident light waves are spatially coherent and have a wavefront controlled by the wavefront shaping means.

11. The N of the first plurality of light waves ｉｎ The method of claim 10 , wherein the incident light waves are plane waves having wave vectors in different directions.

12. The light source is a low spatial coherence light source, and the optical property evaluation method further comprises: - Based on each spatially incoherent or partially coherent light wave from the light source, generating, by a second interferometer, two orthogonally polarized illumination waves having a spatial shift in a plane conjugate to the focal plane (FP) of the first microscope objective lens; - N of different wavefronts ｉｎ changing the spatial shift to generate N incident waves of different wavefronts; - For each spatial shift, sending the orthogonally polarized light waves to the object arm and the reference arm of the first interferometer, respectively, by a polarization beam splitter (1121). - A step of obtaining the second plurality (N ω pieces) of interference signals, wherein each interference signal is, at the detection surface of the detector, a wave backscattered by the sample illuminated by one of the polarized light waves of orthogonal polarization and a reference wave generated by the reflection of the other of the polarized light waves of orthogonal polarization by the reference mirror of the reference arm. The interference signal is obtained for N ω different optical path differences between the backscattered wave and the reference wave; - The multicolor reflection matrix is the N ｉｎ space shifts and N ω interference signals obtained for the path differences (N ｉｎ × N ω ) based on the set of steps determined by the The optical property evaluation method according to any one of claims 1 to 9, comprising:

13. An optical property evaluation system for evaluating the optical properties of a sample (10) formed from a scattering bulk medium, the system comprising: - A first interferometer (120) having an object arm and a reference arm, the object arm including a first microscope objective lens (110) having a predetermined field of view in which the sample (10) is disposed during operation; - An illumination device (130) including a wide spectral band light source (132) and configured to generate a first plurality (N ｉｎ pieces) of incident light waves having different wavefronts, -N ｏｕｔ A detector (140) including N element detectors and configured to obtain a second plurality (N ω pieces) of interference signals for each incident light wave of a specific wavefront, each interference signal being due to the interference between the wave backscattered by the sample illuminated by the incident light wave and the reference wave from the reference arm at the detection surface of the detector, the interference signal being obtained for N ω different frequencies or N ω different path differences between the backscattered wave and the reference wave, and a detector - A processing unit (150), - the N ｉｎ incident light waves and N ω all of the interference signals (N ｉｎ × N ω ) obtained for the ｉｎ × N ｏｕｔ × N ω 3D multi-color reflection matrix of size N 【Number 5】 determining - Based on the multi-color reflection matrix, by applying a first propagator, the depth z in the sample (10) ｉｎ and z ｏｕｔ for the voxels r ｉｎ located at (x' ｉｎ , y' ｉｎ , z ｉｎ ) and r ｏｕｔ (x' ｏｕｔ , y' ｏｕｔ , z ｏｕｔ ), numerically determine at least a first focused volume reflection matrix (R ｒｒ ) that includes the set of responses of the sample between the conjugate light source points and light receiving points - Based on the first focused volume reflection matrix, determining at least one map of the physical parameters of the sample, and a processing unit (150) configured to perform the same, and a processing unit (150) configured to perform the same. An optical property evaluation system including the above.

14. The first interferometer is a Linnik interferometer, and the reference arm includes a reference mirror and a second microscope objective lens. The optical property evaluation system according to claim 13.

15. The light source is a low spatial coherence light source, and the illumination device (1130) - Based on each spatially incoherent or partially coherent light wave from the light source, a second interferometer configured to generate two polarized illumination waves of orthogonal polarization having a spatial shift in a plane conjugate to the focal plane (FP) of the first microscope objective lens (110), and - Means for changing the spatial shift, including Here, - The first interferometer (1120) is a Linnik interferometer, - A polarization splitting element (1121) configured to send each of the polarized light waves of orthogonal polarization having the spatial shift to the objective arm and the reference arm respectively, and - Means for changing the optical path difference between the reference arms, including Here, - For each of the second plurality (N ω pieces) of interference signals, each interference signal is due to the interference between the wave backscattered by the sample illuminated by one of the polarization waves of orthogonal polarization on the detection surface of the detector and the reference wave generated by the reflection of the other of the polarization waves of orthogonal polarization by the reference mirror of the reference arm. The interference signal is obtained for N ω different path differences between the backscattered wave and the reference wave, - The multi-color reflection matrix is the N ｉｎ space shifts and N ω The interference signal (N ｉｎ ×N ω ) based on the set of claims 13 or 14, the optical property evaluation system according to claim 13 or claim 14.

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