Methods and systems for optical characterisation of a bulk scattering medium

Description

Domaine technique de l'invention

[0001] This description relates to methods and systems for the optical characterization of a volumetric and scattering medium. This description applies particularly, but not exclusively, to biomedical imaging for the characterization of biological tissues. Etat de la technique

[0002] Optical characterization of volumetric and scattering media such as biological tissues is a research discipline at the intersection of physics and biology. The goal is to image the structures that compose biological tissues with a micrometer resolution limited by diffraction, i.e., on the order of the optical wavelength. The general principle is as follows: a light wave illuminates the sample, then the light scattered by the structures of the medium is collected by an array of lenses to form a magnified image of the tissue, for example, on a camera. Microscopes familiar to the general public are usually transmission microscopes, due to their simplicity of design and use. However, they are limiting for in-vivo imaging since it is necessary to position the light source and the camera on opposite sides of the sample.The reflective approach is therefore preferred for applications that include, in particular, in-vivo microscopic imaging.

[0003] For thin samples (L < 50 µm), the backscattered light accurately reflects the object's reflectivity; only the numerical aperture of the optical system determines the axial and transverse resolution of the microscopic image. However, when imaging deeper tissues, the phenomenon of multiple scattering becomes limiting. Indeed, biological samples are heterogeneous media whose refractive index fluctuates across different spatial scales. These refractive index inhomogeneities not only degrade the propagation of the light wave (aberrations), thus reducing image contrast and resolution, but also limit the number of useful photons that can be collected (multiple scattering).Since the simple scattering contribution decreases exponentially with depth, it becomes impossible to discriminate it from the contributions of aberrant and multiplely scattered photons by the sample beyond a characteristic distance corresponding to the mean free path of scattering. l s (typically 50-100 µm in tissues).

[0004] To address this problem, two types of microscopes emerged: the confocal microscope in the early 1960s and the interferometric microscope in the early 1990s. The confocal microscope, through spatial filtering of scattered photons, increases the resolution and penetration depth of the conventional microscope (typically 100-300 µm). The interferometric microscope, and particularly OCT (Optical Coherence Tomography), offers a more comprehensive approach to optical imaging. Optical Coherence Tomography " adds temporal filtering to the spatial filtering of the confocal microscope. This further increases the achievable performance by up to a few l s (i.e. ~ 100 - 500 µm). However, despite the spatio-temporal windowing of photons, the performance of the interferometric microscope in terms of resolution and penetration depth is still limited by the phenomena of aberrations and multiple scattering.

[0005] A general challenge remains, therefore, to image biological samples in three dimensions, beyond a few l s with a resolution limited only by diffraction. In the early 2000s, inspired by pioneering work in astronomy, aberration correction methods were developed for optical microscopy. In astronomy, fluctuations in the refractive index of atmospheric layers create wavefront distortions and degrade the quality of images of the sky formed from Earth. So-called adaptive optics methods, based on wavefront measurement combined with a wavefront control device (typically an array of deformable mirrors), make it possible to compensate for wavefront distortion and thus improve resolution. For this, a guide star or a bright point projected onto the sky is used to optimize the correction. These adaptive optics techniques were then transposed to optical microscopy with the advent of small deformable mirrors and SLMs (an acronym for "Small-Method Microscope"). Spatial Light Modulator These methods, however, have several drawbacks. First, the decorrelation time of biological tissues necessitates rapid aberration compensation and limits the number of angular and / or spatial degrees of freedom of wavefront measurement and control devices. Adaptive optics in biological environments is therefore restricted to compensating for relatively low orders of aberration. Furthermore, an even more limiting factor is the narrow field of view over which aberration compensation is valid. This corresponds to what is commonly called an isoplanetary zone, i.e., a zone up to and from which the incident and reflected wavefronts undergo the same distortions. However, the size of these isoplanetary zones becomes less than ten micrometers at a depth on the order of a mean free path of transport. l t (typically 1 mm in biological tissues). Achieving correction for the entire field of view requires repeating the adaptive optics process for each isoplanetary region, making high-resolution imaging over large fields of view and at great depths impractical. To overcome the limited size of isoplanetary regions, so-called multi-conjugate adaptive optics devices have been developed, but they are particularly complex to implement experimentally. Finally, deep multiple scattering (i.e., beyond a mean free path of transport) is another issue. l t ) remains a problem that is not addressed by adaptive optics.

[0006] A third approach, which relies neither on generating an artificial star nor on wavefront optimization based on image quality criteria, has been developed. This is a matrix approach to optical imaging and aberration correction. The matrix approach to light wave propagation within heterogeneous media was initially developed in transmission, particularly for communication through highly scattering media – see the article by SM Popoff. et al. [Ref. 1].

[0007] Recently, the matrix approach has been developed in consideration for imaging through highly scattering media – see article by A. Badon et al. [Ref. 2]. This approach includes the experimental determination of a reflection matrix " plan focal " Or " focalisée » temporally windowed and defined between a source plane and an image plane, conjugate with an object focal plane of a microscope objective.

[0008] In practice, in the main experimental setup described in [ref. 2], a laser beam from a femtosecond laser source is spatially shaped by a spatial light modulator (SLM) acting as a dynamic diffraction grating. A set of plane waves is then emitted by the SLM, with the plane waves focused at different focal points, each identified by a vector r in of the object focal plane of the microscope objective. For each focal point r in , the reflected field E r ( u out , r in ,t) is collected through the same microscope objective and interferes with a reference wave E 0 ( u out , 0,t) on a two-dimensional acquisition device, for example a CCD camera, arranged in a plane conjugate to the pupil plane of the microscope objective and located by the vector u out . The interference pattern between these two waves, integrated over time t gives access to the coefficients R ( u out , r in ) of a column of the reflection matrix R ur temporally windowed: R u out , r in = ∫ dt E r u out , r in , t × E 0 * u out , 0 , t + τ where the symbol * denotes the conjugate matrix. In practice, the amplitude and phase of each coefficient R ( u out , r in ) are recorded by phase-shift interferometry. The time of flight τ is controlled by the length of the interferometer's reference arm using a mirror whose position is adjusted by a piezoelectric actuator (PZT). The time of flight is adjusted to the ballistic time for most of the applications considered, so as to eliminate a large portion of the multiple-scattered photons and retain only the photons simply scattered by the reflectors contained in the focal plane of the microscope objective. For each point r in of focusing at the point of entry into the focal plane, the reflection coefficient R ( u out , r in ) is recorded in a plane conjugate to that of the pupil of the output microscope objective (located by the vector u out A two-dimensional Fourier transform on the coordinate u out allows us to determine the reflection coefficient R ( r out , r in ) in a plane conjugate to the exit focal plane located by the vector r out . For each point r in of focusing at the point of entry into the focal plane, the reflection coefficient R ( r out , r in ) is recorded and stored along a column vector. Finally, the set of column vectors forms the reflection matrix R rr in the focal plane. This yields a temporally windowed focused reflection matrix whose diagonal elements ( r in = r out ) form a "cut en face » of the sample image as it would be obtained by full-field OCT (for a description of full-field OCT imaging, see for example published patent application US20040061867 [Ref.3]). In addition, off-diagonal features provide information on the level of aberration and the level of multiple scattering.

[0009] From this focal plane reflection matrix, a new matrix approach for optical imaging allowing the simultaneous correction of aberrations over several isoplanetary domains of the field of view has been described. See, for example, US20210310787 [Ref. 4]. In [Ref. 4], a new matrix, called " matrice de distorsion This matrix was introduced for the characterization of heterogeneous media. It contains only the distorted portion of the wavefronts backscattered by the sample for a set of focal points. r in . This matrix reveals the spatial correlations of the light field related to isoplanetism. Analyzing the correlations between these different wavefronts allows us to extract the local aberration laws across the entire field of view. This makes it possible to access the transmission matrices between the object focal plane within the sample and the source and image planes located outside of it. Once phase-conjugated or inverted, these matrices provide the complete focusing laws that allow for optimal and local correction of aberrations at each point in the field of view.

[0010] The characterization method described in [Ref. 4] is, however, limited to imaging a single transverse plane, known as the plane of coherence, within the sample. To obtain a three-dimensional image of the sample, an axial scan would be required. An axial scan limits the acquisition speed and therefore the accessible volume, particularly for in-vivo applications or for dynamic imaging of cellular tissues, for example.

[0011] Most importantly, the applicants demonstrated that the characterization method described in [Ref. 4] only allows for transverse correction of aberrations for a coherence plane whose position is dictated by that of the reference mirror in the interferometric setup. However, inhomogeneities in the optical index within the medium induce axial displacement and deformation of the coherence volume relative to its position and flatness, which would be expected if the optical index of the medium were homogeneous. In other words, the object focal plane of the microscope objective and the coherence plane no longer coincide in the case of optical index inhomogeneities within the medium. This limits the signal-to-noise ratio, generates strong transverse aberrations, and causes axial distortions in the image.

[0012] The present description proposes methods and systems for optical characterization of a volumetric and diffusing medium, also based on a matrix approach, allowing ultra-fast volume characterization of the sample, typically less than one second, and also allowing access to reflectivity images whose axial dimension is no longer dictated by the time of flight of the scattered photons but by the actual depth of the scatterers in the sample. Résumé

[0013] The present description relates, in its first aspect, to a method for the optical characterization of a sample consisting of a volumetric and diffusing medium, the method comprising: a step of positioning said sample in the field of view of a first microscope objective, said microscope objective being located in an object arm of a first interferometer, said first interferometer further comprising a reference arm; a step of generating, by means of an illumination device comprising a broadband spectral light source, a first plurality of N incident light waves having different wavefronts; for each incident light wave with a given wavefront, an acquisition step by means of a detector comprising N out elementary detectors, of a second plurality of interference signals, each interference signal resulting from the interference, on a detection plane of the detector, between a wave backscattered by the sample illuminated by said incident light wave, and a reference wave from the reference arm, said interference signals being acquired for N ω different frequencies or N ω different path differences between the backscattered wave and the reference wave; the determination of a three-dimensional polychromatic reflection matrix R̃ ρu = [ R̃ ( ρ out , u in , ω )] of size N in x N out x N ω said three-dimensional polychromatic reflection matrix comprising all the interference signals acquired for the N in incident light waves and N ω frequencies; the numerical determination, by application of a first propagator, from said polychromatic reflection matrix, of at least a first focused volumetric reflection matrix, comprising a set of sample responses between source points and receiver points conjugated with voxels r in (x' in , y' in , z in ) and r out (x' out , y' out , z out ) of the sample located respectively at depths z in and z out in the sample; the determination, from said first focused volume reflection matrix, of at least one map of a physical parameter of said sample.

[0014] In this description, the field of view of the microscope objective includes an object focal plane of said objective. The dimensions of the field of view are limited by the characteristics of the microscope objective, the dimensions of the detection surface, and the magnification of an imaging device between the object focal plane and the detection surface. The dimensions of the field of view can also be adjusted according to the transverse extent of the sample that the operator wishes to characterize.

[0015] In this description, a source with a broad spectral bandwidth is defined as a source whose spectral bandwidth Δω is equal to or greater than a value Δω min allowing for axial resolution δz desired for determining the mapping of physical parameters: δz = 2πc 0 / ( nΔω min ), Or n is the average optical index of the sample and c 0 is the speed of light. According to embodiment examples, the spectral bandwidth of the source is between approximately 10 nm and approximately 400 nm. The source is, for example, but not limited to, a spectrally scanned source or a broad spectral bandwidth source, for example a light-emitting diode (LED), a halogen source, a femtosecond laser, or a superluminescent diode.

[0016] The applicants have shown that the determination of the focused volume reflection matrix, obtained from the polychromatic reflection matrix, makes it possible to overcome the axial and transverse distortions inherent in the techniques described in the state of the art [Ref. 4].

[0017] It becomes possible, for example to form a confocal image with a resolution limited only by diffraction, to make the coherence volume coincide with the geometric focal plane.

[0018] A mapping of a physical parameter of the sample is included in this description and unless otherwise specified as a planar or volumetric mapping of said parameter.

[0019] Thus, a mapping of a physical parameter of the sample includes, according to one or more implementation examples, a confocal image in reflection, en face or volumetric, a mapping of the energy spreading function in reflection (or RPSF for " réflective point spread function around voxels of the sample, a simple scattering rate map, planar or volumetric, a multiple scattering rate map, planar or volumetric, a sample optical index map, planar or volumetric.

[0020] A confocal image in reflection en face is an estimator of the reflectivity of a cross-section of the sample. A volume confocal reflection image is an estimator of the reflectivity of a volume of the sample. In the following description, a confocal reflection image may be simply called " image confocale "

[0021] A voxel is defined as a volumetric resolution cell. In this description, a voxel can also be named by its centroid, called " point de focalisation The dimensions of a volumetric resolution cell are determined by the transverse resolution of an optical system defined by the set of optical elements located between the sample and the illumination and detection planes, and the axial resolution. δz .

[0022] In the following description, we will more simply call the function of the energy spreading in reflection around a focal point, " fonction d'étalement autour d'un point de focalisation " Or " RPSF audit point de focalisation We will call it cartographie de la fonction d'étalement » a mapping of the energy spreading function in reflection (RPSF) around a plurality of focal points of the sample.

[0023] According to one or more embodiment examples, the optical characterization process according to the first aspect further includes: The determination, from said focused volume reflection matrix, of a first confocal reflection image; the determination, from said focused volume reflection matrix, of a map of the spreading function and the determination of a map of the position of the maximum intensity of each spreading function; the determination, from the first confocal reflection image and said map of the position of the maximum intensity, of a second confocal reflection image corrected for any misalignment and / or focusing of the first interferometer. Thus, the applicants have highlighted a first advantage of determining the focused volume reflection matrix for determining confocal images corrected for misalignment and / or focusing of the first interferometer.

[0024] According to one or more embodiment examples, the optical characterization process according to the first aspect further includes: from said first focused volume reflection matrix, the determination of a plurality of spreading function maps for a plurality of values ​​of an integrated optical index of the sample; from said plurality of spreading function maps, the determination of a plurality of maps of the position of the maximum intensity of the spreading function, each map being obtained for a value of the integrated optical index; the determination of a map of the optimal value of the integrated optical index for which, at each focal point, the maximum intensity value of the spreading function is the greatest; the numerical determination of a second propagator based on said map of the optimal value of the integrated optical index; and the determination of a second focused volume reflection matrix using said second propagator.

[0025] In this description, the integrated optical index of the sample is defined as the optical index of the sample integrated between a proximal surface of the latter (i.e., on the side of the microscope objective) and the focal point under consideration.

[0026] According to one or more exemplary embodiments, the method further comprises, from said mapping of the optimal value of the integrated optical index, the determination of a map of the optical index of the medium at each focal point. Said second propagator can then be determined on the basis of said optical index map. Thus, according to one or more exemplary embodiments, the optical characterization method according to the first aspect further comprises: from said first focused volume reflection matrix, the determination of a plurality of maps of the spreading function for a plurality of values ​​of an integrated optical index of the sample; from said plurality of maps of the spreading function, the determination of a plurality of maps of the position of the maximum intensity of the spreading function, each map being obtained for a value of the integrated optical index; the determination of a map of the optimal value of the integrated optical index for which, at each focal point, the maximum intensity value of the spreading function is the greatest; from said map of the optimal value of the integrated optical index, the determination of a map of the optical index of the medium at each focal point;the numerical determination of a second propagator based on said mapping of the optical index of the medium; and; the determination of a second focused volume reflection matrix using said second propagator.

[0027] The said second propagator, whether it is optimized from the integrated index directly or from the sample index derived from the integrated index to maximize the intensity of the RPSF at the focal point, corresponds to a propagator which makes it possible to make the coherence volume and geometric focal plane coincide substantially by taking into account index inhomogeneities in the volumetric and diffusing medium.

[0028] From the resulting second focused volume reflection matrix, it is possible to establish a confocal image of the medium's reflectivity, correcting the focus errors and axial distortions present in the initial confocal image. Furthermore, a map of the RPSF (Reflectivity of the Medium) associated with this second focused volume reflection matrix allows for the local quantification of a multiple scattering rate for each voxel in the image. This parameter provides a local reliability index for the image and locally characterizes the scattering properties of the medium. This RPSF map also allows for the quantification of a simple scattering rate, the depth gradient of which provides access to a local estimate of the mean free path of scattering.

[0029] According to one or more embodiments, the method further comprises determining, from the second focused volume reflection matrix, a distortion matrix defined between a correction basis and a focused basis, the determination of the distortion matrix comprising: the projection, at input or output, of the second focused volume reflection matrix, into the correction basis, to obtain a reflection matrix projected respectively at input or output; a determination of the distortion matrix by term-by-term product between said projected reflection matrix and a reference reflection matrix, defined for a reference medium, in said correction basis; a local determination of the invariants of said distortion matrix, in order to identify, in the correction basis, aberration laws in subdomains of the field of vision; the estimation from said aberration laws, of a transmission matrix between voxels of the field of vision and the correction basis.

[0030] A focused basis, respectively input or output, is the set of source points, respectively receiving points, combined with the voxels tiling the sample.

[0031] A local correction of aberrations thus performed makes it possible to obtain a confocal image whose contrast and resolution are optimized throughout the entire volume of the sample.

[0032] According to one or more embodiment examples, the distortion matrix is ​​determined in the same way but from the first focused volume reflection matrix, obtained by means of the first propagator when there has been no optimization step of the propagator to maximize the intensity of the spreading function at each focal point.

[0033] A term-by-term product between matrices is also known as the Hadamard product.

[0034] According to one or more embodiments, the reference medium is a homogeneous medium with an optical index equal to the average index of the propagation medium. The reference reflection matrix can be theoretically established for this reference medium using a plane mirror in the focal plane of the microscope objective. Depending on the level of knowledge a priori of the propagation medium, the reference medium may take more elaborate forms ( e.g multi-layered medium etc In this case, the reference matrix can be calculated numerically. Constructing the distortion matrix involves subtracting the phase of the corresponding element of the reference reflection matrix from the phase of each element of the projected reflection matrix.

[0035] According to one or more embodiment examples, the correction basis in which said distortion matrix is ​​defined is a basis maximizing the size of the isoplanetary domains contained in the field of view, for example the set of points of a plane conjugate with the plane of an aberrator if the latter is two-dimensional, or for example a plane conjugate with the pupil plane of the microscope objective.

[0036] According to one or more embodiments, a local determination of the invariants of said distortion matrix comprises, as described in [Ref. 4], a singular value decomposition of said distortion matrix, a singular value decomposition of a normalized distortion matrix (i.e., one in which the magnitude of each element has been normalized but the phase has been preserved), and a singular value decomposition of a normalized correlation matrix of said distortion matrix (i.e., a correlation matrix of said first distortion matrix in which the magnitude of each element has been normalized). Other methods are possible for the local determination of the invariants, such as an iterative phase-reversal process applied to said distortion matrix.

[0037] According to one or more exemplary embodiments, the light source is a source with a variable wavelength, and for each incident light wave with a given wavefront, the interference signals of the second plurality N ω interference signals are acquired for N ω frequencies (or wavelengths) of said incident light wave and a fixed path difference between the object arm and the reference arm of the first interferometer. By frequency of an incident wave (respectively wavelength), we understand in this description the central frequency (respectively central wavelength) of the spectrum of the incident wave.

[0038] According to one or more exemplary embodiments, for each incident light wave with a predetermined wavefront, the interference signals of said second plurality of interference signals are acquired for N ω The path differences between the object arm and the reference arm of the first interferometer are calculated. A simple time-domain Fourier transform then allows the polychromatic reflection matrix to be obtained from the interference signals acquired for different path differences, or times of flight. In one or more implementation examples, the polychromatic reflection matrix is ​​generated between a measurement basis at the emission endpoint, which is a plane wave basis (input pupil plane), and a measurement basis at the reception endpoint (detection plane).A propagator for determining a first focused volume reflection matrix from the polychromatic reflection matrix includes, for example, a first change of basis at the input that transforms the input pupil plane into a focal plane at the emission, and a second change of basis at the output that transforms the detection plane into a focal plane at the reception, also called the output focal plane. Such propagators are known to those skilled in the art. These propagators may, for example, include Fresnel transform operations.

[0039] According to one or more embodiment examples, the N in incident light waves are spatially coherent and exhibit wavefronts controlled by means of spatial wavefront shaping.

[0040] For example, the means of spatially shaping the wavefront include scanning means and N in incident light waves are plane waves each exhibiting a wave vector with a different direction.

[0041] The predetermined wavefronts are not necessarily plane waves with wave 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.

[0042] Incident light waves are spatially coherent when the mutual coherence function of the electromagnetic field is uniform over a plane of space.

[0043] Conversely, a light wave is spatially incoherent when the mutual coherence function of the electromagnetic field has support limited only by diffraction, i.e. on the order of half the wavelength.

[0044] In the present description, a spatially partially coherent wave is defined as a wave whose mutual coherence function has a finite support whose width is greater than half the wavelength.

[0045] According to one or more exemplary embodiments, said light source for the emission of spatially coherent incident light waves is a spectrally scanning single-mode laser, for example a laser emitting in a spectral range between wavelengths of about 800 and about 875 nm.

[0046] According to one or more exemplary embodiments, said light source is a source with low spatial coherence, the process further comprising: the generation, from each spatially incoherent or partially coherent light wave from the light source, and by means of a second interferometer, of two polarized illumination waves of orthogonal polarizations and exhibiting a spatial shift in a plane conjugate with a focal plane of the first microscope objective; the variation of said spatial shift to generate the N in incident waves of different wavefronts; the sending, for each spatial shift, of said polarized waves of orthogonal polarizations respectively on the object and reference arms of said first interferometer by means of a polarization splitter element; the acquisition, by means of the detector, of said second plurality of interference signals, each interference signal resulting from the interference, on the detector's detection plane, between a wave backscattered by the sample illuminated by one of said orthogonally polarized waves, and a reference wave generated by the reflection of the other of said orthogonally polarized waves by a reference mirror of the reference arm, said interference signals being acquired for N ω different path differences between the backscattered wave and the reference wave; said polychromatic reflection matrix being determined from the set of interference signals acquired for the N spatial shifts and N ω differences in walking speed.

[0047] The said source with low spatial coherence is, for example, a light-emitting diode or a halogen lamp.

[0048] The characterization process thus described has the advantage of being faster, in terms of acquisition time, and optimal, in terms of signal-to-noise ratio, for obtaining a confocal image of one or more cross-sections of the sample.

[0049] The present description relates, according to a second aspect, to an optical characterization system for the implementation of optical characterization processes of a sample conforming to the first aspect.

[0050] Thus, the present description concerns an optical characterization system for a sample made of a volumetric and diffusing medium, comprising: a first interferometer with a target arm and a reference arm, the target arm comprising a first microscope objective with a given field of view in which the sample is positioned during operation; an illumination device comprising a broadband spectral light source configured to generate a first plurality of incident light waves exhibiting different wavefronts; a detector comprising N out elementary detectors, configured for the acquisition, for each incident light wave of a given wavefront, of a second plurality of interference signals, each interference signal resulting from the interference, on a detection plane of the detector, between a wave backscattered by the sample illuminated by said incident light wave, and a reference wave from the reference arm, said interference signals being acquired for N ω different frequencies or N ω different path differences between the backscattered wave and the reference wave; a processing unit configured for: the determination of a three-dimensional polychromatic reflection matrix R̃ ρu = [ R̃ ( ρ out , u in , ω )] of size N in x N out x N ω said three-dimensional polychromatic reflection matrix comprising all the interference signals acquired for the N in incident light waves and N ω frequencies; the numerical determination, by application of a first propagator, from said polychromatic reflection matrix, of at least a first focused volumetric reflection matrix comprising a set of sample responses between source points and receiver points conjugated with voxels r in (x' in , y' in , z in ) and r out (x' out , y' out , z out ) of the sample located respectively at depths z in and z out in the sample; the determination, from said first focused volume reflection matrix, of at least one map of a physical parameter of said sample.

[0051] According to one or more exemplary embodiments, said first interferometer is a Linnik interferometer and the reference arm includes a reference mirror and a second microscope objective.

[0052] However, other arrangements are possible for the first interferometer that a person skilled in the art can implement with their general knowledge. In particular, the first interferometer may not include a reference mirror and / or a microscope objective on the reference arm.

[0053] According to one or more embodiment examples, the detector includes a two-dimensional acquisition device, for example a CCD or CMOS type camera.

[0054] In other embodiments, the detector includes a spectrometer. The acquisition of interference signals for N ω frequencies can then be used for detection.

[0055] According to one or more embodiments, the light source is a source with low spatial coherence; and the illumination device comprises: a second interferometer configured to generate, from each spatially incoherent or partially coherent light wave emanating from the light source, two polarized illumination waves of orthogonal polarizations and exhibiting a spatial shift in a plane conjugate to a focal plane of the first microscope objective; and means for varying the spatial shift; and wherein said first interferometer is a Linnik interferometer and comprises a polarization-splitting element configured to send respectively onto

[0056] the object and reference arms, each of said polarized waves with orthogonal polarizations and exhibiting said spatial shift; means for varying the path difference between the object arm and the reference arm; and wherein each interference signal of said second plurality of interference signals results from the interference, on the detector's detection plane, between a wave backscattered by the sample illuminated by one of said polarized waves of orthogonal polarizations, and a reference wave generated by the reflection of the other of said polarized waves of orthogonal polarizations by a reference mirror of the reference arm, said interference signals being acquired for N ω different path differences between the backscattered wave and the reference wave; said polychromatic reflection matrix is ​​determined from the set of interference signals acquired for the N spatial shifts and N ω differences in walking speed. Brève description des figures

[0057] Other advantages and characteristics of the technique presented above will become apparent upon reading the detailed description below, made with reference to the figures in which: There Fig. 1A , a first example of an optical characterization system for a volumetric and diffusing medium according to the present description; The Fig. 1B , a diagram illustrating the different notations used for the bases in which matrices are described in characterization processes according to this description; The Fig. 2A a diagram illustrating the scanning by N in light pulses from the entrance pupil of a microscope objective arranged on either of the reference and object arms of a first interferometer in an example of a characterization system as illustrated on the Fig. 1A , ; There Fig. 2B , a diagram illustrating the intensity distribution of an interference signal on a detection panel of a detector in a characterization system as illustrated in the Fig. 1A ; There Fig. 2C , a representation of an example of a polychromatic reflection matrix including all the interference signals acquired for N in incident light waves and N ω wavelengths, said matrix being obtained by means of a characterization process according to the present description; The Fig. 3A , a diagram illustrating the response of the medium between a source point of an input focusing basis and a receiving point of an output focused basis, to illustrate an element of an example of a focused volume reflection matrix as described herein; The Fig. 3B , images illustrating physical parameter maps obtained from the focused volumetric reflection matrix that is the subject of the process as described herein; The Fig. 3C , a diagram illustrating the non-coincidence of the focusing and coherence planes, resulting in degraded quality in the focusing operations at transmission and reception, for the construction of the focused volume matrix; The Fig. 4A Diagrams illustrating spread functions (RPSF) obtained with a first Fresnel propagator at focal points located at different focal planes; The Fig. 4B A diagram showing the evolution of the maximum intensity of the RPSF as a function of the depth z of the focal planes; The Fig. 5 Images showing the effect of digital dual focusing on a confocal image of a sample formed from a target; The Fig. 6 , a quantitative study of the RPSF measured in a sample consisting of a cornea, during a characterization process according to the present description; The Fig. 7A , a diagram illustrating the subdivision of the field of vision performed to extract local RPSFs; The Fig. 7B , an image illustrating a map of the multiple scattering rate at a depth of z=250 µm in the corneal sample; The Fig. 7C , an image illustrating a map of local RPSFs at a depth of z=250 µm in the corneal sample; The Fig. 7D , images showing two longitudinal sections (B-scans) of the optical index n ( x, y, z ) estimated in the corneal sample obtained by means of a characterization method as described herein; The Fig. 8 , images illustrating the result of a digital correction process for transverse aberrations in the corneal sample at a depth of z=250 µm, using a characterization process as described herein; The Fig. 9 illustrates the phase of a transmission matrix by means of an iterative aberration correction process in an example of a method according to this description and confocal images of the sample formed from the cornea at a depth z = 250 µm, as well as B-scans, before and after digital aberration correction; The Fig. 10A , another example of an optical characterization system for a volumetric and diffusing medium according to the present description, in which the first interferometer is an interferometer other than a Linnik interferometer; The Fig. 10B , another example of an optical characterization system for a volumetric and diffusing medium according to the present description, similar to that of the Fig. 10A but employing a spectrometer-type detector; The Fig. 11 , another example of an optical characterization system for a volumetric and diffusing medium according to the present description, for the implementation of an example of a method according to the present description in which a reflection matrix polychromatic in the temporal domain and in a focused basis, is recorded. In the different embodiments that will be described by reference to the figures, similar or identical elements bear the same references. Description détaillée

[0058] In the detailed description that follows, only certain embodiments are described in detail to ensure clarity of exposition, but these examples are not intended to limit the general scope of the principles arising from this description.

[0059] The various embodiments and aspects described herein can be combined or simplified in numerous ways. In particular, the steps of the various processes can be repeated, reversed, or executed in parallel, unless otherwise specified. Where reference is made in this description to calculation or processing steps for the implementation of process steps, it is understood that each calculation or processing step can be implemented by software, hardware, firmware, microcode, or any suitable combination of these technologies. When software is used, each calculation or processing step can be implemented by computer program instructions or software code.These instructions can be stored or transmitted to a computer-readable storage medium (or computing unit) and / or executed by a computer (or computing unit) in order to implement these calculation or processing steps.

[0060] There Fig. 1A illustrates a first example of a system 101 for the optical characterization of a sample 10 consisting of a volumetric and diffusing medium, the system 101 being configured for the implementation of optical characterization processes according to the present description. Fig. 1B represents a simplified diagram illustrating the bases in which matrices are described in characterization processes according to the present description.

[0061] The system 101 includes an illumination device 130 with a broadband spectral light source 132, for example a spectrally scanning source configured to emit a light beam having a frequency ω (or wavelength) λ ) which can vary over a spectral band [ ω min , ω max ] (or a range of wavelengths [ λ min , λ max ]), around a central frequency ω n (or wavelength) λ n ) as schematically illustrated in spectrum 131. The light source 132 emits, for example, in the near-infrared, for example between approximately λ min = 800 nm and approximately λ max = 875 nm and can sweep the spectrum between these two wavelengths at a given speed, for example between about 10⁻² nm / s and about 10⁵ nm / s. The light source is for example a fiber source and the illumination device may include an output fiber 133 connected to a collimator 134 configured for the emission of a substantially collimated, spatially coherent beam.

[0062] The system 101 also includes an interferometer 120 with an object arm and a reference arm separated by a separator cube 121. The system 101 includes a first microscope objective 110 arranged in the object arm of the interferometer, the sample being positioned, in operation, in a field of view of the microscope objective.

[0063] In the example shown on the Fig. 1A The interferometer 120 is a Linnik interferometer comprising, in addition to the first objective 110 on the object arm, a second microscope objective 122 arranged in the reference arm. The reference arm also includes, in this example, a reference mirror 123.

[0064] As illustrated on the Fig. 1A The light-emitting device also includes, in this example, means 135 for scanning the collimated beam as well as a set of lenses 136, 137, 138. Lenses 136 and 137, called respectively the scanning lens and the tube lens, form an assembly 4f of a given magnification, while lens 138 is configured to focus the beam exiting the assembly 4f in a plane InP corresponding to a plane of an entrance pupil of the microscope objective 110 ("entrance pupil plane"). The scanning means 135 include, for example, two scanning mirrors driven in rotation by galvanometric motors to scan the incident light beam (indicated with simple arrows on the Fig. 1A ) in the pupil plane, entrance InP of the microscope objective 110 arranged on the object arm of the interferometer 120, in both spatial directions. Note that in the example of the Fig. 1A Due to the use of a Linnik interferometer, the incident beam also scans the entrance pupil plane of the second microscope objective 122 arranged on the interferometer's reference arm. The system 101 also includes a detector 140 with an ImP detection plane and a processing unit 150 configured for processing the data generated by the detector, as will be explained later. The detector includes, for example, a camera configured for acquiring two-dimensional images at a given acquisition frequency. For example, the camera can acquire 256 × 256 pixel images at a given frequency, for example, around 75 kHz.

[0065] Of course, other configurations are possible for the optical characterization system 101, which a person skilled in the art can implement using their general knowledge in the field. For example, the system 101 can be deployed in a polarized configuration. In this case, the beam splitter is a polarization beam splitter. Two quarter-wave plates can be placed between the microscope objectives and the beam splitter. Upstream of the detector 140, a polarization analyzer can be arranged to recombine the two polarizations.

[0066] Furthermore, the broadband source is not necessarily variable frequency. It is possible, for example, to replace the camera with a spectrometer, as will be described using the Fig. 10B frequency selection occurs at detection.

[0067] Furthermore, rather than varying the frequency of the source, it is equivalently possible to vary the path difference (or "time of flight") between the object and reference arms of the interferometer, for example by moving the block consisting of the reference mirror 123 and the second microscope objective 122.

[0068] Furthermore, other interferometric arrangements are possible instead of the Linnik interferometer. For example, as will be described with reference to the Fig. 10A The reference wave in the interferometer can come directly from the source.

[0069] The notations used in this description to identify the different bases in which, in particular, the reflection matrices for sample characterization are described are recalled by means of the Fig. 1B . There Fig. 1B This only illustrates a few elements of the characterization system for the sake of simplicity. The notations apply to the assembly of the Fig. 1A but, more generally, to all the characterization systems that are the subject of this description.

[0070] The focal plane of the microscope objective 110 is designated FP and is intended to receive the sample 10. The basis of the incoming plane waves, denoted InP, is the plane of the entrance pupil of the microscope objective or any plane conjugate to the plane of the entrance pupil of the microscope objective. The entrance pupil is intended to receive the incident light waves for the illumination of a field of view of the sample that we seek to characterize. We denote u in A point in the plane of the entrance pupil InP, defined by its Cartesian coordinates (vin, win). The plane basis of the output plane waves, denoted OutP, is the plane of the exit pupil of the microscope objective or any plane conjugate to the plane of the exit pupil of the microscope objective. The exit pupil is designed to receive the light waves reflected by the field of view of the sample being characterized. We denote u out a point in the plane of the pupil such that OutP, defined by its Cartesian coordinates (v out , w out ). As illustrated on the Fig. 1B The input and output paths, comprising the input and output pupils respectively, are separated by the beam splitter element 121. On the input path, SP denotes the source plane of the optical system, conjugate with the focal plane of the microscope objective, in this example symbolized by an optic 11 forming a 4f assembly with the microscope objective 110. ρ in a point on the source plane SP, defined by its Cartesian coordinates (x in , y in ). On the output path, we denote ImP the image plane of the optical system, conjugate with the focal plane of the microscope objective, in this example symbolized by an optic 12 forming with the microscope objective 110 a 4f assembly. We denote ρ out a point of the image plane ImP, defined by its Cartesian coordinates (x out , y out ).

[0071] A voxel r in of the sample has the coordinates, ( ρ' in , z in ), where the transverse coordinate ρ' in (x' in , y' in ) of the point r in is conjugate of a point ρ in of the source plane SP. The input focal basis is defined as the (volumetric) basis of the conjugate source points of the voxels. r in .

[0072] A voxel r out of the sample has the coordinates, ( ρ' out , z out ), where the transverse coordinate ρ' out (x' out, y' out) of the point r out is conjugate of a point ρ out of the image plane ImP. The output focal base is defined as the (volumetric) base of the conjugate reception points of the voxels. r out . In operation, the illumination device 130 allows the characterization process to be implemented according to an example conforming to this description, as follows. The illumination device generates, for a given frequency (or wavelength), a first plurality of N in incident light waves consisting of different predetermined wavefronts. For example, as illustrated in the Fig. 1A , THE N in incident light waves are N in Plane waves with wave vectors in different directions are controlled by the scanning means 135. For example, a lens 124 (output lens) provides optical conjugation between the object focal plane FP of the microscope objective 110 and the detection plane ImP of the detector 140. Thus, in operation, the scanning means 135 cooperate with the setup. 4f 136, 137 and the focusing lens 138 to scan, in both spatial directions, the pupil plane InP of the microscope objectives 110 and 122 arranged respectively on the object arm and reference arm of the Linnik interferometer 120. N in each incident light wave thus generated has a wave vector k in whose direction and magnitude depend in particular on the scanning angle imposed by the scanning means 135 and the frequency ω (or wavelength) λ ) of the incident wave.

[0073] Furthermore, for each incident light wave with a predetermined wavefront, detector 140 acquires a second plurality of N ω interference signals, each interference signal resulting from the interference, on the detection plane ImP of the detector, between a wave backscattered by the sample 10 illuminated by said incident light wave, and a reference wave from the reference arm, the N ω interference signals being acquired for N ω different frequencies. For example, in the example of the Fig. 1A in which the illumination device 130 comprises a spectrally scanning light source, the N ω Interference signals are acquired by varying the wavelength of the light source.

[0074] The backscattered fields from the two arms of the interferometer interfere at the ImP detection plane of detector 140. The interference term between the two arms for a frequency ω the incident wave corresponds to the product between the field reflected by the sample E s and the conjugate in phase E r * of the reference field from the reference arm according to the equation: R ˜ ρ out , u in , ω = E s ρ out , u in , ω × E r * ρ out , u in , ω

[0075] Or ρ out (x out , y out ) is a vector of spatial coordinates (x out , y out ) in the ImP detection plane (measurement plane at the receiver) and u in (v in , w in ) is a vector of spatial coordinates (v in , w in ) in the pupil plane InP of the microscope objective 110 (emission measurement plane).

[0076] In operation, lens 138 focuses the incident beam into the pupil plane of the microscope objectives. The associated focal spot, of dimension δu The focal spot is limited only by diffraction (the numerical aperture of the system upstream of the interferometer). This focal spot is converted by the microscope objective into a quasi-plane wave illuminating the sample over a large field of view. The sample will be imaged, in part, on the ImP detection plane.

[0077] For example, the Fig. 2A This illustrates the scanning of the entrance pupil InP of microscope objectives 110, 122 by N in incident light beams. The scanning mirrors 135 allow scanning the physical pupil of the microscope objectives, the circumference of which is represented by a black circle 201 on the Fig.2A The black disks denote the positions of each of the incident beams focused on the entrance pupil InP of the microscope objectives 110, 122. The spatial spacing between each beam is noted δu in . Taking into account the characteristics of the microscope objectives used, the characteristic dimension of each focal spot is, for example, δu = 8 µm. This corresponds to a maximum illumination of the field of vision of Δr = λf / δu = 2 mm where f is the focal length of the microscope objective 110. The spatial pitch, δu in , corresponds to the sampling of the electromagnetic field in the emission pupil plane and indicates the precision with which the incident wavefronts will be resolved when digitally resynthesized from the measured reflection matrix. This spatial step can therefore be advantageously adjusted according to the level of aberrations generated by the medium; it can be chosen fine enough to capture the fastest spatial fluctuations of the distortions undergone by the light wave as it passes through the medium. Depending on the intended application, the entire entrance pupil 201 or only a sub-part of it can be scanned. For example, to obtain a highly resolved confocal image, the entire entrance pupil can be scanned as shown in the Fig. 2A .

[0078] For each illumination of the sample, the interference term R̃ ( ρ out , u in , ω ) is measured by a detector in the detection plane ImP.

[0079] There Fig. 2B illustrates the field of vision Δρ in the camera detection plane and spatial sampling δρ out under which each interference signal (or interferogram) is recorded. Nout denotes the number of pixels (or elementary detectors) of the camera on which the interference signal is acquired. The field of view corresponds to a magnified image of the focal plane of the microscope objective by a factor of G, where G is the magnification of an imaging system composed of the microscope objective and the output lens 124. In the example of the system of the Fig. 1A , G=-fs / f om with fs and f om the focal lengths of lenses 124 and microscope objectives 110 and 122.

[0080] The set of interferograms recorded for each illumination u in and each frequency ω are stored in the form of a 3D polychromatic reflection matrix, R̃ ρu = [ R ( ρ out , u in , ω )], of size N out × N in × N ω .

[0081] There Fig. 2C This illustrates, using an example, such a 3D polychromatic reflection matrix to show its three-dimensional structure. The first dimension designates the detection point. ρ out in the camera's detection plane, whose coordinates (x out, y out) are identified by the index i = p + N out − 1 × q . With p = N out + 1 / 2 + x out / δρ out And q = N out + 1 / 2 + y out / δρ out The second dimension refers to the coordinate. u in of each incident beam focused in the entrance pupil plane InP. The coordinates ( v in , w in ) of u in are identified by the index j = n + N in − 1 × m . With n = N in + 1 / 2 + v in / δu in And m = N in + 1 / 2 + y in / δu in The third dimension, identified by the index k , corresponds to the set of frequencies ω to which the interference signal is recorded.

[0082] A total theoretical acquisition time for the matrix R̃ ρu = [ R̃ ( ρ out , u in , ω )] can therefore be estimated, in this example, at t acq = N in t s , Or t s = N ω / f c corresponds to the acquisition time of an image of the volume of interest for a single illumination and f c is the camera's acquisition frequency. The number N ω The frequency (or wavelength) is adjusted according to the thickness L from the middle to be imaged. Indeed, N ω = Δ ω / δω , with Δ ω = ω max - ω min the bandwidth over which is measured R̃ ρu And δω frequency sampling dictated by thickness L of the environment to be visualized such as δ ω = c 0 / (2 nL ), with nthe average refractive index in the sample and c0 the speed of light. For example, for N in = 11 2< incident plane waves, L = 500 µm, and f c =100 kHz, the acquisition time of an image of the volume of interest is t s = 1 ms and the total acquisition time of the matrix R̃ ρu East t acq = 121 ms. A second step of the process according to this description comprises the numerical determination, by application of a propagator and from said polychromatic reflection matrix R̃ ρu of at least a first focused volume reflection matrix = [ R ( r out , r in , ω The focused volume reflection matrix comprises a set of sample responses between source and receiver points conjugated with voxels of the volume medium 10. A sample response at a given frequency between a source point and a detection point is the sample reflection coefficient, measured at the detection point when a light wave at said frequency is emitted from the source point. The position of the voxels is identified by the vectors r in = (x' in , y, in , z in ) at emission and r out = (x' out, y' out, z out) at reception. A voxel is defined as a volumetric cell of a given resolution. For example, the dimensions of such a volumetric cell of resolution are determined, in the example of a system such as that represented on the Fig. 1A , by the transverse resolution of the device along (x, y) and the depth of field or spectral band of the source along z. Contrary to the state of the art [Ref.4], the voxels can be located at different depths in the medium, as explained below. The depth z in the volumetric medium 10 is defined along an axis (z) parallel to the optical axis of the microscope objective, for example with respect to an origin z ref = 0 which corresponds to the zero path difference between the reference and sample arms.

[0083] Numerical determination of the focused volume reflection matrix R̃ rr from the polychromatic reflection matrix R̃ ρu This example includes a first basis change at the input, which allows the transition from the input pupil plane to the focused basis at transmission (input focused basis), and a second basis change at the output, which allows the transition from the detection plane to the focused basis at reception (output focused basis). These basis changes are referred to in the following description as digital focusing operations at transmission and reception, or double focusing operations.

[0084] There Figure 3A illustrates the digital focusing procedure on two voxels located at points r in And r out located at distinct depths (respectively z in and z out ) in the environment studied.

[0085] To perform the polychromatic reflection matrix conversion R̃ ρu to the focused volume matrix R̃ rr The dual focusing operation at the emission and reception sites is achieved through the application of, for example, a Fresnel lens. For this purpose, the polychromatic reflection matrix R̃ ρu = [ R̃ ( ρ out , u in , ω )] can first be projected as output into a virtual Fourier plane ( u out ′ ) by simple spatial Fourier transform on the output coordinate ρ out : R ˜ u out ′ , u in , ω = 1 N out ∑ ρ out R ˜ ρ out , u in , ω e − j 2 π λf u out ′ . ρ out

[0086] Fresnel masks, noted F (u, z in / out - z f , ω ), are then applied to the input and output of the resulting matrix R ˜ uu = R ˜ u out ′ , u in , ω to digitally translate the emission and reception focusing planes, initially located at depth zf, to a depth z in , respectively z out in the sample, where z f is the focal length of the microscope objective 110: R ˜ ′ u out ′ z out u in z in ω = F u out ′ − u in , z out − z f , ω R ˜ u out ′ u in ω F u in , z in − z f , ω

[0087] Examples of Fresnel masks F , 411, 421, are presented on the Fig. 4A for different depths z = z in = z out of focus. This is expressed as follows: F u z ω = e j 2 π n ¯ λ − k z z with n , the assumed optical index of the medium under study, considered for the moment to be homogeneous and k z = 2 π λ n ¯ 2 − u 2 f 2 , the axial wavenumber. An inverse spatial Fourier transform can be applied to the coordinates u out ′ And u in in order to project the incident and reflected fields into the focused bases r out And r in and thus obtain the focused volume reflection matrix R̃ rr = [ R ( r out , r in , ω )].

[0088] A digital Fourier transform on frequencies ω applied to the polychromatic focused reflection matrix R̃ rr This allows us to discriminate between scattered photons according to their time of flight in the sample. The resulting matrix is ​​denoted R rr = [ R ( r out , r in , t )]. Ideally, i.e., under the simple diffusion approximation and for a medium of index n homogeneous, each flight time is associated with photons scattered at depth z = c 0 t / (2 n ) in the sample.

[0089] To illustrate the advantages of the volumetric focused reflection matrix R rr , The depositors measured the polychromatic matrix R̃ ρu using a system such as the one described on the Fig. 1A on an opaque human cornea of ​​thickness L = 400 µm and average index n = 1.4. The numerical aperture of the microscope objectives is ON=0.3. The acquisition parameters are as follows: N in = 441, N ω = 80, λ min =800 nm, λ max =875 nm, field of view in the object focal plane of the microscope objective 716 × 716 µm.

[0090] There Fig. 3B This illustrates, by way of example, a subsection 301 (or cross-section) of the matrix R rr = [ R ( r out , r in , t )] built from R̃ ρu , For z = z in = z out = 250 µm and at ballistic time t = 2 nz / c 0 and a column 302 of this submatrix rearranged in a two-dimensional form.

[0091] Each column 302 of sub-part 301 of the matrix R rr gives the reflected field measured by each virtual reception point r out for a light wave emitted from the virtual source point r in . In an ideal case without aberrations or multiple scattering, an Airy disk would be observed around the source point. r in . Here, on the contrary, the reflected field takes the form of a diffuse halo around the point r in , This means that the assumption made about the optical index is not necessarily optimal, which will be described in more detail later.

[0092] However, the broadband focused matrix R rr This therefore provides access to a set of relevant observables that we will now use to quantitatively characterize the sample studied. First, a confocal image ( r A time-windowed matrix can be constructed by considering the following elements of the matrix. R rr : ℑ r = R r , r , t = 2 n ¯ z / c 0

[0093] There Fig. 3B This presents the confocal image 303 of the cornea for z = 250 µm. This image corresponds to the diagonal elements of the submatrix 302 after being rearranged into a two-dimensional form. The confocal image ( r) is an estimator of corneal reflectivity in a coherence plane associated with simply scattered photons whose time of flight is given by t = 2 nz / c 0 .

[0094] The quality of this estimator depends on the quality of focusing in the medium. We note h in / out ( r , r in / out ) the impulse response between each point r in / out and each point r in the sample (see Fig. 1B ). To quantify the quality of focusing, a relevant observable is the volumetric energy spreading function around the reflecting focal point (or RPSF for " reflection point spread function " which can be accessed by measuring the distribution of backscattered energy along the antidiagonals of the submatrix R rr ( z, t ) which considers identical input and output focal planes ( z in = z out ): RPSF r p , Δ x , Δ y , t = R x p − Δ x / 2 , y p − Δ y / 2 , z p x p + Δ x / 2 , y p + Δ y / 2 , z p t 2 where < ... > is an average over the even points r in And r out centered on the same point r p such as r p = ( r in + r out ) / 2. Δ r = r out - r in is the relative position between these two points (coordinates Δ x, Δ y). This intensity profile is relevant because it allows us to probe aberrations independently of the local reflectivity of the medium. Indeed, by considering the inlet and outlet PSFs as locally isoplanetary, we can redefine local PSFs h in / out l invariants under translation around each point r p , such as h in / out r r in / out = h in l r − r in / out , r p Under this assumption and for a medium of random reflectivity, the RPSF gives the convolution product between the incoherent input and output spreading functions of the imaging device: RPSF r p , Δ x , Δ y , t ∝ h in l Δ x , Δ y , 0 r p 2 ⊗ Δ x , Δ y h out Δ x , Δ y , 0 r p 2

[0095] For a specular object, the RPSF provides access to the convolution product between the coherent input and output spreading functions: RPSF r p , Δ x , Δ y , t ∝ h in l Δ x , Δ y , 0 r p ⊗ Δ x , Δ y h out l Δ x , Δ y , 0 r p 2

[0096] In these two asymptotic regimes, the RPSF does not probe exactly the same physical quantity, but its spatial distribution reveals the local level of aberration at the point r p . There Fig. 3B shows the RPSF 305 obtained in the opaque cornea at a depth z p = 250 µm. This presents the following appearance: an overintensity associated with photons scattered by the coherence plane and an incoherent background associated with multiple scattering events occurring upstream of this same plane. The position of the RPSF maximum and the spatial extent of the observed overintensity on the RPSF provide information on the focusing quality.

[0097] There Figure 3B shows that the maximum of the RPSF (305) is not centered at Δr = 0. This is related in particular to an alignment problem of the first interferometer. The confocal intensity therefore does not correspond to the relative position Δr = 0 but to the position Δ r max of the maximum intensity of the RPSF. A new confocal image (r) corrected for these alignment problems can thus be achieved: ℑ ′ r = R r , r + Δr max , t = 2 n ¯ z / c 0 .

[0098] Image 304 shows the new confocal image obtained. Comparison with the raw confocal image ( r ) 303 shows an initial interest in the RPSF to overcome the problems of alignment of the device.

[0099] Furthermore, if, apart from these alignment issues, the digital focusing process were ideal, the RPSF support would extend over a single resolution cell. As explained above, the Fig. 3B shows that this is not the case for the opaque cornea studied.

[0100] There Fig. 3C This illustrates the main reason for this poor focusing quality: the misalignment of the focal and coherence planes. This can lead to a focusing defect in both emission and reception due to the finite depth of field of microscope objectives.

[0101] There Fig 4A This demonstrates the usefulness of RPSF for adjusting the position of coherence and focusing planes. This figure was created from a proof-of-concept experiment on a defocused target at a distance of zm = 100 µm below the focal plane of the sample arm microscope objective (ON=0.3). Fig 4A shows the RPSF obtained for different focal planes z = z in = z out and for a coherence plan coinciding with the position of the target: t = 2z m / c 0.

[0102] When z < z m (or, equivalently, for z > z m ), the spread of the RPSF (411, 412) is characteristic of a defect in the fine-tuning. An estimator is then defined in order to determine the value of z m This estimator corresponds to the position z opt which maximizes the confocal intensity, i.e., the maximum of the RPSF, denoted RPSF max. For z = z opt , la Fig 4A (421, 422) clearly shows that the transverse extension of RPSF is minimal, i.e. only limited by diffraction.

[0103] There Fig. 4B shows the evolution of RPSF max as a function of depth z. We find that z opt = zm and the confocal intensity is 3.5 times more intense than outside the depth of field (| z - z m | > 20 µm). The Figure 3C This summarizes the digital focusing optimization process thus achieved. By maximizing the confocal intensity (RPSF max), the plane of coherence, corresponding to the interferometric nature of the measurement, can be made to coincide with the geometric focus linked to the position of the sample relative to the microscope objective. Fig. 5 shows the effect of dual digital focusing on a confocal image of the target (510). While the initial image 511 is degraded by the focusing error, the input and output digital focusing process produces an image of the target 512 with optimal contrast and confocal resolution limited only by diffraction (δ ρ ~ λ / (4 NA )) .After correction, all the details of the target are sharp and contrasted. This demonstrates a technical effect of the process described above for creating a confocal image from a full-field spectral OCT device. Thus, it is possible to determine, from the first focused volumetric reflection matrix, a reflectivity map of the sample.

[0104] A similar focusing optimization procedure is applied to the reflection matrix measured in the cornea. However, in the experiment considered, the cornea is immersed in water of known refractive index. n eau . Most importantly, the cornea has an optical index n ( r ) spatially inhomogeneous. We initially assume that the average refractive index of the medium is invariant under lateral translation and that its optical index n depends only on the depth z: n ( r ) = n ( zThe first step in the process is to bring the focusing plane, initially located at the depth z f in the absence of a sample, on the surface of the sample located at the depth z 0 by applying the propagator F (., z 0 - z f , ω) for an index n = n eau to the matrix R̃ uu : R ˜ ′ u out ′ z 0 u in z 0 ω = F u out ′ − u in , z 0 − z f , ω R ˜ u out ′ u in ω F u in , z 0 − z f , ω

[0105] An estimator n̂ ( z ) of the optical index profile n ( z ) is then obtained as follows: for each time of flight t, the RPSF is scanned while modifying the average optical index n , and consequently the position of the coherence plan z ( n ) = c 0 t / (2 n ), in the propagator F ([Math 5]) so as to maximize the confocal intensity (RPSF max). The value of n Maximizing this quantity gives an estimate of the integrated optical index n int (z ) to the depth z = c 0 t / (2 n int ). The profile of the integrated index n int ( z ) is defined as the integral of the surface area of ​​the medium ( z = z 0) at the depth z of the optical index n ( z ) of the middle: n int z = 1 z − z 0 ∫ z 0 z n z dz

[0106] By discretizing this equation with respect to flight time t , it is then possible to deduce from the estimator n̂ int ( z ) of the integrated index profile n int ( z ) an estimator n̂ ( z ) of the local optical index n ( z ) by means of an inversion of a system of linear equations.

[0107] In order to obtain the dependency of n with transverse coordinates ( x, y ), a segmentation of the field of vision (cf Fig. 7A ) can be performed beforehand on the reflection matrix R̃ rr = [R ( r out , r in , ω )] and the process described above can be repeated for each sub-zone of the field of vision. An estimator n̂ ( x', y', z ) of the spatial distribution of the optical index n ( x', y', z ) in the middle can thus be obtained by considering sub-zones of the field of vision centered on each transverse coordinate ( x', y' ). Two longitudinal sections (B-scans) of the optical index n̂ ( x', y', z ) estimated in the cornea are presented on the Fig. 7D (images 71, 72). The quantity represented, δn ( x', y', z ) = n̂ ( x', y', z ) - n , is the deviation from the average optical index of the cornea, here estimated at n = 1.4.

[0108] Note that the estimator n̂ of the spatial distribution of the optical index n (more simply called " cartographie de l'indice optique (in this description) can be used to build a new propagator F In order to optimally describe the propagation of light from the input and output pupil planes of the device to all the voxels of the medium under study (i.e., the focused basis), this propagator can then be used to construct a new focused reflection matrix and provide a confocal image much closer to the actual reflectivity of the medium.

[0109] There Fig. 6 shows the confocal image 610 obtained following this digital focusing process of the data. Note that the image can thus be constructed as a function of the depth z rather than as a function of the time of flight t , which is a limitation of conventional OCT images whose axial dimension is dictated by the path difference between the arms of the interferometer.

[0110] Comparison with the previous confocal image (r ) (304, Fig. 3B This illustrates the improvement in image quality, both in terms of contrast and resolution. However, the image quality could still be improved due to aberrations (excluding focus errors) and multiple scattering.

[0111] There Fig. 6 This last assertion is illustrated by showing a quantitative study of the RPSF measured in the cornea after focusing. It notably shows the evolution in depth of the radial distribution of the RPSF around the confocal point Δr. max . The extent of the confocal peak increases with depth, which quantifies the loss of resolution due to aberrations experienced by the light wave as it propagates through the sample. For example, the extent of the RPSF at half-height 630 is 15 µm at a depth z=250 µm, which is well above the diffraction limit (δ p~ 1.4 µm). Finally, the in-depth evolution of the RPSF shows the emergence of an increasingly significant incoherent background beyond z = 200 µm. This background resists averaging over the camera integration time; this component is deterministic and is therefore linked to the occurrence of multiple scattering events upstream of the focal plane.

[0112] Beyond their evolution in depth, the levels of aberration and multiple diffusion can also be probed cross-sectionally by studying the RPSF measured locally around a set of points r p of the field of vision.

[0113] There Fig. 7A illustrates the subdivision of the field of vision performed to extract the local RPSFs shown on the Fig. 7C The spatial extent of local RPSFs shows, for example, that focusing quality is much better in the lower left of the field of view. The confocal image shown on the Fig. 6 is therefore a much better estimator of the medium's reflectivity in this part of the field of view. The cross-sectional evolution of the RPSFs also shows the non-isoplanetary nature of the aberrations induced by the heterogeneities of optical index distributed within the sample. As we will see later, local compensation of the aberrations is advantageous, and the matrix approach is a suitable tool for this.

[0114] Beyond aberrations, a multiple scattering over single scattering ratio can be measured from the confocal intensity RPSF max and the incoherent background RPSF bg: ρ MS r p = RPSF bg r p RPSF max r p − 2 × RPSF bg r p

[0115] The factor of 2 in the denominator takes into account the phenomenon of coherent backscattering, which results in a confocal amplification of the intensity, multiplely scattered by this same factor of 2. Fig. 7B shows a map of the parameter ρ MS at a depth of z=250µm. This map reveals the presence of striations already more or less visible on the confocal image of the Fig. 6 Such striations in the corneal stroma are characteristic of diseases like keratoconus. Beyond this qualitative analysis, the ratio ρ MS This is a first step towards a quantitative and localized image of the light scattering properties in biological samples. Depending on the information sought, other parameters can be extracted, such as the simple scattering rate (or confocal scattering rate): ρ S r p = RPSF max r p RPSF max r p + RPSF bg r p

[0116] The z-value of this parameter allows for the local quantification of the mean free path of diffusion within the medium. More generally, the quantification of simple and multiple diffusion phenomena allows for the local measurement of transport parameters such as the mean free path of diffusion, the mean free path of transport, or the diffusion coefficient. Furthermore, the matrix reflection approach allows for the study of diffuse halo growth at relatively short flight times, resulting in a much better spatial resolution than standard techniques such as diffuse optical tomography.

[0117] Beyond quantifying aberration and multiple scattering phenomena in the medium, we will now show how, starting from the digitally refocused reflection matrix, it is possible to perform local compensation for sample-induced aberrations in order to obtain a confocal image with optimal contrast and a resolution close to the diffraction limit. For this, the input / output transmission matrices T in / out The relationship between an input / output correction basis and the voxels in the middle can be estimated. To this end, we perform a local analysis to estimate suitable focusing laws for each voxel in the image.

[0118] As an illustration, we choose the plane wave basis as the correction basis. The first step in the matrix aberration correction process consists of projecting the time-windowed focused reflection matrix, R rr ( t = 2 nz / c 0 ) , as output (or input) to this correction basis by a simple spatial Fourier transform: R ¯ k out r in = ∑ ρ out R ρ ′ out , z , ρ ′ in , z , t = 2 nz / c 0 e − i k out . ρ ′ out

[0119] From this dual matrix R kr we construct a distortion matrix D kr defined as follows: D k out r in = R ¯ k out r in × T 0 k out r in with T 0 a reference matrix that models the propagation of plane waves if the medium were homogeneous (i.e., without aberrations): T 0 ( k out , r in ) = e -i k < out< · r< in< . In Ref [4], it was shown how, under an isoplanetary assumption, a singular value decomposition of the matrix D allowed to estimate the transmittance A( k out ) of the aberrator from the phase of its first eigenvector U 1 Here, since aberrations are not spatially invariant, subdividing the field of view is advantageous in order to locally analyze the distortion matrix and estimate local focusing laws adapted for each voxel of the image. The field of view is divided into overlapping regions defined by their central median point. r p and their spatial extent Δ r All distorted components of the field associated with the focal points r in data located in each region can be extracted and stored in a local distortion matrix: D ′ k out r in r p = D k out r in W Δr r in − r p Or W Δ r ( r ) = 1 for | x' | < Δ x , | y | < Δ y and | z' | < Δ z , and zero otherwise. Ideally, each local distortion matrix should contain a set of focal points r in belonging to the same isoplanetary spot. In reality, the isoplanarity condition is never completely met. A delicate compromise must therefore be made on the size Δ r of the spatial window W Δ r : it must be small enough to approach the isoplanarity condition, but large enough to encompass a sufficient number of disorder-independent realizations. An SVD of each local distortion matrix D'( r p ) gives a first eigenvector of output U 1 (r p ) = [ U 1 ( k out , r p )]. An estimator T̂ out of the transmission matrix T out = [ T out ( k out , r p )] is thus constructed: T ^ out k out r p = T 0 k out r p × e j arg U 1 k out r p

[0120] Another estimator of the transmission matrix T out can be obtained through an iterative phase reversal process. It relies on the following recurrence relation: W n + 1 T r p = exp j arg D ′ r p D ′ † r p W n r p where the exponent T< refers to the matrix transposition operation and W 0 ( r p ) corresponds to the initial transmittance chosen arbitrarily by the operator. This can be taken as homogeneous, W 0 ( r p ) = [1 ··· 1] T< , or determined from our prior knowledge of aberrations in the medium in order to accelerate the convergence of the iterative process. Depending on the operator's choice, the iterative phase reversal process can be iterated m times and the resulting vector, W m = [ W m ( k out , r p )], constitutes an estimator of the aberrator's transmittance at the point r p . An estimator T̂ out of the transmission matrix T̂ out = [ T out (k out , r p )] is thus constructed: T ^ out k out r p = T 0 k out r p × W m r p

[0121] Regardless of how the transmission matrix is ​​estimated T out , the phase conjugate of the estimator T̂ out is then applied to correct the aberrations at the output of the medium and construct a new focused reflection matrix R rr = [ R ( ρ out , ρ in , z )] via the following matrix product: R ¯ rr z = T ^ out † × R ¯ kr z where the symbol † corresponds to a trans-conjugation of the matrix T̂ out . The same process can be repeated at the input to estimate the transmission matrix. T in and iterated by gradually decreasing the extent Δr of the spatial windows in order to correct increasingly higher-order aberrations associated with increasingly smaller isoplanetary patches. At the end of the matrix aberration correction process, a corrected confocal image is obtained from the diagonal elements of the matrices. R rr (z): ℑ c r = R ¯ r r

[0122] There Fig. 8 This shows the result of a digital correction process for transverse aberrations in the cornea at a depth of z=250 µm. An iterative phase-reversal method is considered here to estimate the transmission matrices. T̂ out And T in .

[0123] There Fig. 8 Figures 801, 802, and 803 show the evolution of the RPSF at each iteration for a sub-zone of the field of view of 105 × 105 µm². As the input and output iterations progress, the RPSF becomes more refined as increasingly local aberrations are corrected. The aberration laws 804 and 805 obtained at the input and output at the end of the process are also shown in Figure 804. Fig. 8 In an ideal case, due to spatial reciprocity, they should be identical ( T in = T out They differ, in particular, due to imperfections in the imaging system, notably its alignment defects. The matrix aberration correction process therefore corrects not only aberrations induced by heterogeneities in the optical index within the medium but also imperfections in the imaging system. Comparison between the confocal images of the subzone before and after correction, ( r ) And ( r), illustrate the benefit of a transverse correction of aberrations, after an axial correction of the latter.

[0124] There Fig. 9 shows the phase of the transmission matrix T in obtained at the end of the iterative aberration correction process (901, 902). This contains all the aberration laws for each sub-zone of the field of vision ( Fig. 7A Images 903 and 904 show a cross-section en face of the volumetric confocal image of the cornea at depth z= 250 µm before and after digital aberration correction. Images 905 and 906 show a longitudinal section (B-scan) of the volumetric confocal image of the cornea before and after digital aberration correction. The confocal image appears much more contrasted after matrix aberration correction: the confocal intensity is increased by a factor of 7 on average across the field of view. The gain in resolution is also significant: the RPSF analysis shows a resolution refinement by a factor of 3.

[0125] The experimental validation evidence was performed using a characterization system of the type illustrated on the Fig. 1A Of course, other characterization systems conforming to this description are possible for the implementation of processes according to this description.

[0126] Thus, the Fig 10A , Fig. 10B And Fig. 11 represent systems for the optical characterization of a sample formed of a volumetric and diffusing medium, according to other embodiment examples.

[0127] In the example of system 102 illustrated on the Fig. 10A , a difference with the system illustrated on the Fig. 1A resides in the field from the reference arm. The reference wave in the first interferometer comes directly from the source via the beam splitter 1032. For a fiber-optic light source 132 (case considered in the Fig. 10A The reference arm comprises an optical fiber 1033 and a collimator 1034, enabling the projection of a reference wave in the form of a plane wave onto the detector 140. In free space, the reference arm would consist of a lens system ideally chosen to minimize the path difference between the reference wave and the waves scattered simply by the reflectors located in a plane of interest in the sample arm. In this case, the interference is measured between the wave reflected by the sample illuminated by a set of different predetermined incident wavefronts and a reference plane wave that varies only in frequency. This provides direct access to the backscattered field. E s ( ρ out , u in , ω ) by the sample. By numerically multiplying this field by a reference field E r ∗ ρ out , u in , ω we access the matrix R̃ ρu = [ R̃ ( ρ out , u in , ω )].

[0128] In the example of system 103 illustrated on the Fig. 10B The source under consideration is a broadband source. The measurement of the spectral dependence is made possible by the use of a detector 160 comprising a spectrometer. The spectrometer includes, for example, a dispersive element 161 (e.g., a grating) and a detection device 162. Each element of the detection device measures the field at a coordinate ρ out of the ImP plane and at a frequency ω The device measures a matrix R̃ ρu = [ R̃ ( ρ out , u in , ω )] in every respect equivalent to that measured by the system of the Fig. 1A .

[0129] Another example of an optical characterization system for a volumetric and diffusing medium, as described herein, is schematically represented on the FIG. 11 The 104 system presented on the 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 presented on the FIG. 1A particularly when one wishes to obtain a confocal image of the sample on a small number of cross-sections of the sample.

[0130] The system 104 includes an illumination device 1130 with a spatially incoherent light source 1132 arranged at the focus of a lens 1133, and configured for the emission of a plurality of incident light waves intended to illuminate through the microscope objective 110 a given field of view of the sample 10. The system 104 also includes a detector 1140, arranged at the focus of a lens 1124 and a processing unit (not shown) receiving, in particular, the optoelectronic signals from the detector 1140. The characterization system 104 includes two interferometers placed in series. A first interferometer 1120 and a second interferometer which is part of the illumination device 1130. The second interferometer is for example a Michelson interferometer in an air wedge configuration allowing to generate at its output two illumination beams inclined relative to each other and of orthogonal polarizations.The first interferometer 1120 is, for example, a Linnik interferometer with a polarized beam splitter 1121 and quarter-wave plates 1126, 1128 on each arm. An afocal system 1111, 1112 allows the planes of the mirrors 1136 and 1138 of the second interferometer to be conjugated with the pupil planes of the microscope objectives 1122 and 110 of the first interferometer.

[0131] The incident field, spatially and temporally incoherent, is linearly polarized at 45 degrees to the parallel (e∥) and normal (e⊥) directions with respect to the plane of the device, by means of a polarizer 1134. The components of this wave, polarized along the e∥ and e⊥ directions, are transmitted and reflected respectively by a polarized beam splitter 1131. Each arm of the second interferometer contains a quarter-wave plate (1135, 1137) and a mirror (1136, 1138). On one of the arms, the mirror (1136) is inclined with respect to the optical axis. Upon their return, the two waves from each arm exit the interferometer as two inclined beams with orthogonal polarization. These two beams are, however, coherent with each other since they originate from the same incident wave.

[0132] In the first interferometer 1120, the two beams are again separated by the polarized beam splitter 1121. The beam polarized along e|| is transmitted in the reference arm. The beam polarized along e⊥ is reflected in the object arm. The presence of quarter-wave plates 1126, 1128 on each of the two arms allows the two beams to be transmitted optimally after they have been reflected by the sample in the object arm and the mirror in the reference arm. These two beams are recombined at the output of the interferometer using an analyzer 1125 polarized at 45 degrees with respect to e∥ and e⊥. They can thus interfere in the focal plane of the lens 1124. The detector, for example a CCD or CMOS camera, records the corresponding interference signal. The tilt of mirror 1136 causes the reference and object beams to be offset from each other on the camera. This allows us to measure the impulse response between points ρ in And ρ out distinct. Thus, the system described by means of the Fig. 11 , allows for the measurement of a polychromatic reflection matrix R ρρ in the temporal domain and in a basis conjugate to the focal plane of the microscope objective: R ρ out , ρ in , t = ∫ dτ E s ρ out , ρ in , τ × E r ∗ ρ out , ρ in − ρ out , τ − t

[0133] The time of flight t of the photons is controlled by the path difference between the reference and sample arms of the first interferometer 1120. The time of flight can therefore be scanned by simultaneously moving elements 1122, 1123, and 1126 of the reference arm of the first interferometer. The relative position of the points ρ in And ρ out is controlled by the tilt of the reference mirror 1136 in the second interferometer.

[0134] In implementation examples, by performing Fourier transforms both temporally on the variable t and spatially on the coordinate ρ out on the matrix coefficients R ρρ , we obtain a matrix R̃ uρ = [ R̃ ( u out , ρ in , ω )], equivalent to that recorded by the device of the Fig 1A Only the input and output coordinates are exchanged. The same numerical processing described previously can be applied simply by swapping the output and input coordinates of the matrix. R̃ ρu .

[0135] In other implementation examples, the focused reflection matrix can be obtained. R rr from the matrix R ρρ by applying time shifts to the measured interference signals: R r out , r in , t = ∑ ρ out ∑ ρ in A in ρ in , r in A out ρ out , r out R ρ out , ρ in , t − Δ t ρ in , r in − Δ t ρ out , r out Or A in / out is a spatial apodization term of the field (related to the geometric decay of Green's functions in integral diffraction theory) and Δ t in / out is the time of flight associated with the ballistic photon propagating from the point r in / out in conjunction with the emission plan ( ρ in ) / detection( ρ out ) in the sample. This time-shifting operation is equivalent to the Fresnel propagator applied in the Fourier domain. If the data is acquired over time, backpropagation of the measured field in the time domain can prove more advantageous in terms of both time and memory compared to a shift in the frequency domain.

[0136] Although described through a number of detailed embodiment examples, the optical characterization methods and systems include various variants, modifications and improvements which will be obvious to those skilled in the art, it being understood that these various variants, modifications and improvements form part of the scope of the invention, as defined by the following claims. Références

[0137] Réf 1 : S. M. Popoff et al. « Measuring the Transmission Matrix in Optics », Phys. Rev. Lett. 104, 100601, 2010. Réf 2 : A. Badon et al. « Smart optical coherence tomography for ultra-deep imaging through highly scattering media », Sci. Adv. 2016; 2:e1600370. Réf.3 : US20040061867 Réf. 4 : US20210310787

Claims

1. A method for the optical characterization of a sample (10) formed of a bulk scattering medium, the method comprising: - a step of positioning said sample (10) in a field of view of a first microscope objective (110), said microscope objective being located in an object arm of a first interferometer (120), said first interferometer further comprising a reference arm; - a step of generating, by means of an illuminating device (130) comprising a wide spectral band light source (132), a first plurality of Nin incident light waves having different wavefronts; - for each incident light wave of given wavefront, a step of acquiring, by means of a detector (140) comprising Nout elementary detectors, a second plurality (Nω) of interference signals, each interference signal resulting from the interference, in a detection plane of the detector, between a wave backscattered by the sample illuminated by said incident light wave and a reference wave from the reference arm, said interference signals being acquired for Nω different frequencies or Nω different path differences between the backscattered wave and the reference wave; - determining a three-dimensional polychromatic reflection matrix R̃ρu = [R̃(ρout, uin, ω)] of size Nin x Nout x Nω, said three-dimensional polychromatic reflection matrix comprising all of the interference signals (Nin x Nω) acquired for the Nin incident light waves and Nω frequencies; - numerically determining, by applying a first propagator, on the basis of said polychromatic reflection matrix, at least a first focused volumetric reflection matrix (Rrr), comprising a set of responses for the sample between source points and receiving points that are conjugate with voxels rin(x'in, y'in, Zin) and rout(x'out, y'out, zout) of the sample (10) which are located at depths zin and zout in the sample, respectively; - determining, on the basis of said first focused volumetric reflection matrix, at least one map of a physical parameter of said sample.

2. The optical characterization method as claimed in claim 1, wherein said at least one map of a physical parameter of said heterogeneous medium comprises: a confocal image, a map of the point spread function around a plurality of reflection focal points (rp) (RPSF), a map of a single scattering rate, a map of a multiple scattering rate, a map of the optical index of the sample.

3. The optical characterization method as claimed in any one of the preceding claims, the method further comprising: - determining, on the basis of said focused volumetric reflection matrix, a first reflection confocal image ((r)); - determining, on the basis of said focused volumetric reflection matrix, a map of the spread function and determining a map of the position of the intensity maximum of each spread function; - determining, on the basis of the first reflection confocal image and said map of the position of the intensity maximum, a second reflection confocal image ((r)) corrected for an alignment and / or focusing defect of the first interferometer.

4. The optical characterization method as claimed in any one of the preceding claims, the method further comprising: - on the basis of said first focused volumetric reflection matrix, determining a plurality of maps of the spread function for a plurality of values of an integrated optical index of the sample; - on the basis of said plurality of maps of the spread function, determining a plurality of maps of the position of the intensity maximum of the spread function, each map being obtained for a value of the integrated optical index; - determining a map of the optimum value of the integrated optical index for which, at each focal point, the maximum intensity value of the spread function is the greatest; - numerically determining a second propagator based on said map of the optimum value of the integrated optical index; and determining a second focused volumetric reflection matrix using said second propagator.

5. The method as claimed in claim 4, the method further comprising, on the basis of said map of the optimum value of the integrated optical index, determining a map of the optical index of the medium at each focal point.

6. The optical characterization method as claimed in claim 5, wherein said second propagator is determined on the basis of said map of the optical index.

7. The method as claimed in any one of claims 4 to 6, the method further comprising: - determining, on the basis of the second focused volumetric reflection matrix, a distortion matrix (D) defined between a correction base and a focused base, comprising: - projecting, at entrance or at exit, the second focused volumetric reflection matrix, in the correction base, to obtain a projected reflection matrix (Rkr) at entrance or at exit, respectively; - determining the distortion matrix via term-by-term product between said projected reflection matrix and a reference reflection matrix, defined for a reference medium, in said correction base; - locally determining the invariants of said distortion matrix, in order to identify, in the correction base, aberration laws in sub-domains of the field of view; - estimating, on the basis of said aberration laws, a transmission matrix (Tout) between voxels of the field of view and the correction base.

8. The optical characterization method as claimed in any one of the preceding claims, wherein: - said light source is a variable-wavelength source and, for each incident light wave of predetermined wavefront, the interference signals of said second plurality (Nω) of interference signals are acquired for Nω central wavelengths of said incident light wave and a fixed path difference between the object arm and the reference arm of the first interferometer.

9. The optical characterization method as claimed in any one of claims 1 to 7, wherein: - for each incident light wave of given wavefront, the interference signals of said second plurality (Nω) of interference signals are acquired for Nω path differences between the object arm and the reference arm of the first interferometer.

10. The optical characterization method as claimed in any one of the preceding claims, wherein the Nin incident light waves are spatially coherent and have wavefronts controlled by means for spatially shaping the wavefront.

11. The optical characterization method as claimed in claim 10, wherein said Nin incident light waves of the first plurality of light waves are plane waves each having a wave vector with a different direction.

12. The optical characterization method as claimed in any one of claims 1 to 9, wherein said light source is a low spatial coherence source, the method further comprising: - generating, on the basis of each spatially incoherent or partially coherent light wave from the light source, by means of a second interferometer, two polarized illumination waves with orthogonal polarizations and having a spatial shift in a plane conjugate with a focal plane (FP) of the first microscope objective; - varying said spatial shift to generate the Nin incident waves of different wavefronts; - sending, for each spatial shift, said polarized waves with orthogonal polarizations to the object and reference arms of said first interferometer, respectively, by means of a polarization splitter element (1121); - acquiring, by means of the detector (1140), said second plurality (Nω) of interference signals, each interference signal resulting from the interference, in the detection plane of the detector, between a wave backscattered by the sample illuminated by one of said polarized waves with orthogonal polarizations, and a reference wave generated by the reflection of the other of said polarized waves with orthogonal polarizations by a reference mirror of the reference arm, said interference signals being acquired for Nω different path differences between the backscattered wave and the reference wave; - said polychromatic reflection matrix being determined on the basis of the set of the interference signals (Nin x Nω) acquired for the Nin spatial shifts and Nω path differences.

13. A system for the optical characterization of a sample (10) formed of a bulk scattering medium, the system comprising: - a first interferometer (120) with an object arm and a reference arm, the object arm comprising a first microscope objective (110) with a given field of view in which, in operation, the sample (10) is positioned; - an illuminating device (130) comprising a wide spectral band light source (132), configured to generate a first plurality (Nin) of incident light waves having different wavefronts; - a detector (140) comprising Nout elementary detectors, configured to acquire, for each incident light wave of given wavefront, a second plurality (Nω) of interference signals, each interference signal resulting from the interference, in a detection plane of the detector, between a wave backscattered by the sample illuminated by said incident light wave and a reference wave from the reference arm, said interference signals being acquired for Nω different frequencies or Nω different path differences between the backscattered wave and the reference wave; - a processing unit (150) configured to: - determine a three-dimensional polychromatic reflection matrix R̃ρu = [R̃(ρout, uin. ω)] of size Nin x Nout x Nω said three-dimensional polychromatic reflection matrix comprising all of the interference signals (Nin x Nω) acquired for the Nin incident light waves and Nω frequencies; - numerically determine, by applying a first propagator, on the basis of said polychromatic reflection matrix, at least a first focused volumetric reflection matrix (Rrr), comprising a set of responses for the sample between source points and receiving points that are conjugate with voxels rin(x'in, y'in, zin) and rout(x'out, y'out, zout) of the sample (10) which are located at depths zin and zout in the sample, respectively; - determine, on the basis of said first focused volumetric reflection matrix, at least one map of a physical parameter of said sample.

14. The optical characterization system as claimed in claim 13, wherein said first interferometer is a Linnik interferometer and the reference arm comprises a reference mirror and a second microscope objective.

15. The system as claimed in any one of claims 13 and 14, wherein: - the light source is a low spatial coherence source; and the illuminating device (1130) comprises - a second interferometer configured to generate, on the basis of each spatially incoherent or partially coherent light wave from the light source, two polarized illumination waves with orthogonal polarizations and having a spatial shift in a plane conjugate with a focal plane (FP) of the first microscope objective (110); and - means for varying the spatial shift; and wherein - said first interferometer (1120) is a Linnik interferometer and comprises - a polarization splitter element (1121) configured to send, to the object and reference arms, respectively, each of said polarized waves with orthogonal polarizations and having said spatial shift; - means for varying the path difference between the reference arms; and wherein - each interference signal of said second plurality (Nω) of interference signals results from the interference, in the detection plane of the detector, between a wave backscattered by the sample illuminated by one of said polarized waves with orthogonal polarizations, and a reference wave generated by the reflection of the other of said polarized waves with orthogonal polarizations by a reference mirror of the reference arm, said interference signals being acquired for Nω different path differences between the backscattered wave and the reference wave; - said polychromatic reflection matrix is determined on the basis of the set of the interference signals (Nin x Nω) acquired for the Nin spatial shifts and Nω path differences.