Method and system for optical characterization of volume scattering media

Abstract

The invention relates to a method for optical characterization of a sample, the method comprising: positioning the sample in a field of view of a first microscope objective located in an object arm of an interferometer; generating a first plurality of Nin incident light waves by means of the illumination device; acquiring, for each incident light wave of the given wavefront, a second plurality of N [omega] interference signals, each interference signal resulting from interference between the sample backscattered wave and the reference wave, the interference signals acquired for N [omega] different frequencies, according to one example; determining a polychromatic reflection matrix comprising all interference signals collected for Nin incident light waves and N [omega] frequencies; numerically determining a focus reflection matrix based on the polychromatic reflection matrix; based on the first focus reflection matrix, at least one map having physical parameters of the sample is determined.

Description

Technical Field

[0001] The present invention relates to methods and systems for optical characterization of bulk scattering media. The present invention is particularly applicable, but not limited to, biomedical imaging for characterizing biological tissue. Background Art

[0002] The optical characterization of bulk scattering media such as biological tissue is a research discipline at the intersection of physics and biology. The goal is to image the structures that make up biological tissue with a diffraction-limited micrometer resolution (i.e., on the order of the wavelength of light). The general principle is as follows: a light wave illuminates the sample and the light scattered by the structures in the medium is collected by a set of lenses to form a magnified image of the tissue, for example in a camera. Microscopes known to the general public are usually transmission microscopes due to their simplicity of design and use. However, in vivo imaging is limited by the necessity to place the source and the camera on both sides of the sample. Therefore, reflection methods are particularly advantageous for applications including microscopic in vivo imaging.

[0003] For thin samples (L < 50 μm), the waves backscattered by the sample obey the reflectivity of the object; only the numerical aperture of the optical system determines the axial and lateral resolution of the microscopic image. However, when it comes to imaging deep tissues, the phenomenon of multiple scattering becomes a limitation. Specifically, biological samples are heterogeneous media whose optical indices fluctuate on different spatial scales. These index inhomogeneities not only have a negative impact on the propagation of light waves (aberrations), thereby reducing the contrast and resolution of the image, but also limit the number of useful photons that can be collected (multiple scattering). Since the contribution of single scattering decreases exponentially with depth, it cannot be combined with the contribution of the sample at a depth corresponding to the scattering mean free path l. s The contributions of aberrations and multiply scattered photons can be distinguished outside the characteristic distance (typically 50-100 μm in tissue).

[0004] To overcome this problem, two types of microscopes were developed: confocal microscopy in the early 1960s and interferometric microscopy in the early 1990s. Confocal microscopy improves the resolution and penetration depth (typically 100-300 μm) of conventional microscopy by spatially filtering the scattered photons. Interferometric microscopy, especially OCT (optical coherence tomography), adds temporal filtering to the spatial filtering of confocal microscopy. This can further improve the achievable performance to several 100 μm. s (ie, ~100-500 μm.) However, even though photons have spatiotemporal windows, the performance of interferometric microscopy in terms of resolution and penetration depth is still limited by aberrations and multiple scattering.

[0005] Therefore, the general problem of 3D imaging of biological samples still exists: sBeyond this, the resolution is 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 index of the atmosphere cause wavefront distortions and negatively affect the quality of images of the sky taken from the Earth. Adaptive optics methods based on wavefront measurement combined with wavefront control devices (usually deformable mirror arrays) are able to compensate for wavefront distortions and thus improve resolution. For this purpose, guide stars or bright spots projected into the sky are used to optimize the correction. With the advent of small deformable mirrors and SLMs (spatial light modulators), these adaptive optics techniques were subsequently transplanted to optical microscopy. However, these methods have a number of disadvantages. First, the decorrelation time of biological tissues requires fast compensation of aberrations and limits the number of angular and / or spatial degrees of freedom of the wavefront measurement and control devices. Therefore, adaptive optics in biological environments is limited to compensating relatively low-order aberrations. A further limiting factor is the restricted field of view in which the aberration compensation is effective. It corresponds to the so-called isochromatic zone, i.e., the region in which the incident and reflected wavefronts experience the same distortion. However, in the transmission mean free path l t At depths of the order of 1000 micrometers (typically 1 mm in biological tissue), the size of these isocyanine zones becomes less than a dozen micrometers. Obtaining correction for the entire field of view requires repeating the adaptive optics process for each isocyanine zone, which makes high-resolution imaging over large fields of view and depths impractical. To cope with the limited size of isocyanine zones, so-called multi-conjugate adaptive optics devices have been developed, but their experimental implementation is particularly complex. Ultimately, deep multiple scattering (i.e., the number of isocyanine zones within the transmission mean free path l) is a very important factor affecting the accuracy of the imaging system. t ) is still a problem that has not been solved by adaptive optics.

[0006] A third approach has been developed that is neither based on the generation of artificial stars nor on wavefront optimization according to imaging quality criteria. This is a matrix approach to optical imaging and aberration correction. Matrix methods for the propagation of light waves in heterogeneous media were first developed for transmission, in particular for communication through highly scattering media, see S.M. Popoff et al. [Ref. 1].

[0007] In recent years, matrix methods have been developed in reflection for imaging through highly scattering media, see the article by A. Badon et al. [Ref. 2], which involves the experimental determination of the "focal plane" or "focusing" reflection matrix for a time window defined between the source plane and the image plane and conjugate to the object focal plane of the microscope objective.

[0008] In practice, in the main experimental setup described in [Ref 2], the laser beam from a femtosecond laser source is spatially shaped by a spatial light modulator (SLM) acting as a dynamic diffraction grating. The SLM then emits a set of plane waves that are focused to different focal points, which are determined by the vector r in the object focal plane of the microscope objective. in Mark each focus. For each focus r in , reflected field E r (u out , r in , t) are collected by the same microscope objective and compared with the reference wave E0 (u) in a two-dimensional acquisition device (e.g., a CCD camera). out ,0,t) interference occurs, the two-dimensional acquisition device is arranged in a conjugate plane with the pupil plane of the microscope objective and is represented by the vector u out The interference pattern between the two waves is integrated over time t to obtain the reflection matrix R of the time window ur The coefficient R(u out , r in ):

[0009] [Formula 1]

[0010]

[0011] The symbol * represents a conjugate matrix. In practice, each coefficient R(u out , r in ) amplitude and phase. The time of flight T is controlled by the length of the reference arm of the interferometer with the aid of a mirror, the position of which is adjusted by a piezoelectric actuator (PZT). For most applications, the time of flight is adjusted to the ballistic time in order to eliminate the majority of multiply scattered photons and retain only the photons that are singly scattered by the reflectors contained in the focal plane of the microscope objective. For each incident focus r in the focal plane in , reflection coefficient R(u out , r in ) is recorded in the plane corresponding to the exit microscope objective pupil (denoted by the vector u out The coordinate u out By performing a two-dimensional Fourier transform, we can determine the focal plane with respect to the exit focal plane (given by the vector u out The reflection coefficient R(u) in the conjugate plane out , r in ). For each incident focus r in the focal plane in , reflection coefficient R(u out , r in ) are recorded and stored along the column vectors. Finally, this set of column vectors forms the reflection matrix R in the focal plane rrThis yields a focused reflection matrix for the time window, whose diagonal elements (r in =r out ) forms a "frontal" slice of the sample image, as obtained in full-field OCT (for a description of full-field OCT imaging, see, for example, published patent application US20040061867 [reference 3]). In addition, the off-diagonal elements provide information on aberrations and multiple scattering levels.

[0012] Based on the focal plane reflection matrix in the focal plane, a new matrix method for optical imaging is described, which allows the correction of aberrations simultaneously over multiple isoval domains of the field of view. See, for example, US20210310787 [Ref. 4]; in [Ref. 4], a new matrix called the "distortion matrix" is introduced for characterizing heterogeneous media. The matrix contains only the set of focal points r in The matrix is ​​the distorted part of the wavefront backscattered by the sample. This matrix reveals the spatial correlation of the light field associated with isovains. Analysis of the correlations between these different wavefronts allows the extraction of local aberration laws for the entire field of view. This gives the transfer matrix between the object focal plane inside the sample and the source and image planes located outside it. Once phase conjugated or phase inverted, these matrices provide all the focusing laws needed to optimally and locally correct the aberrations at every point in the field of view.

[0013] However, the characterization method described in [Ref 4] is limited to imaging a single transverse plane in the sample (called the coherence plane). In order to obtain a three-dimensional image of the sample, it must be scanned axially. Axial scanning limits the speed of acquisition and thus the volume that can be obtained, especially for in vivo applications or dynamic imaging of cell tissues.

[0014] Most importantly, the Applicant has shown that the characterization method described in [Reference 4] allows for lateral aberration correction only for the coherence plane whose position is determined by the position of the reference mirror in the interferometric setup. However, inhomogeneities in the optical index in the medium lead to axial displacements and deformations of the coherence volume relative to its position and flatness, compared to what would be expected if the optical index of the medium were uniform. In other words, in the case of inhomogeneities in the optical index in the medium, the object focal plane of the microscope objective and the coherence plane no longer coincide. This limits the signal-to-noise ratio, generating large lateral aberrations and axial distortions in the image.

[0015] This specification proposes a method and system for optical characterization of volume scattering media, which is also based on a matrix approach. The method and system allow ultrafast volumetric characterization of samples (typically less than one second) and also provide a reflectivity image whose axial dimension is no longer determined by the flight time of the scattered photons, but by the actual scattering depth in the sample. Summary of the invention

[0016] According to a first aspect, the present description relates to a method for optical characterization of a sample formed of a bulk scattering medium, the method comprising:

[0017] - a step of positioning the sample in the field of view of a first microscope objective, the microscope objective being located in the object arm of a first interferometer, the first interferometer also comprising a reference arm;

[0018] - generating a first plurality N having different wavefronts by means of an illumination device comprising a broad spectral band light source in The steps of the incident light wave;

[0019] -For each incident light wave of a given wavefront, by including N out The step of collecting a second plurality of interference signals by a detector of an elementary detector, each interference signal being generated by interference in a detection plane of the detector between a wave backscattered by the sample illuminated by the incident light wave and a reference wave from a reference arm, the interference signal being a signal for N interference signals between the backscattered wave and the reference wave. ω different frequencies or N ω Different path differences are collected;

[0020] - Determine the size to be N in xN out xN ω The three-dimensional multicolor reflection matrix The three-dimensional multi-color reflection matrix includes in The incident light wave and N ω All interference signals collected at each frequency;

[0021] - based on the polychromatic reflectance matrix, by applying a first propagator, numerically determining at least a first focal volume reflectance matrix, the first focal volume reflectance matrix comprising a set of responses for the sample between a source point and a receiving point, the source point and the receiving point being respectively associated with a position in the sample at a depth z in and depth z out The voxel of the sample at r in (x' in , y' in , z in ) and r out (x' out , y' out , z out ) conjugation;

[0022] - Based on the first focal volume reflection matrix, determining at least one map of a physical parameter of the sample.

[0023] In this specification, the field of view of a microscope objective includes the object focal plane of the objective. The size of the field of view is limited by the characteristics of the microscope objective, the size of the detection surface, and the magnification of the imaging device between the object focal plane and the detection surface. The size of the field of view can also be adjusted according to the lateral extent of the sample that the operator wishes to characterize.

[0024] In this specification, a wide spectral band source is one whose spectral bandwidth Δω is greater than or equal to the value Δω min The source of this value allows to obtain the desired axial resolution δz to determine the map of the physical parameter: in, is the average optical index of the sample, and c0 is the speed of light. According to an exemplary embodiment, the spectral bandwidth of the source is about 10 nm to about 400 nm. The source is, for example, but not limited to, a spectrally scanned source or a wide spectral band source, such as a light emitting diode (LED), a halogen source, a femtosecond laser, or a superluminescent diode.

[0025] The Applicant has shown that the determination of the focal volume reflection matrix obtained based on a polychromatic reflection matrix makes it possible to avoid the axial and lateral distortions inherent in the techniques described in the prior art [Ref. 4].

[0026] In practice, it is possible to form, for example, a confocal image with a resolution only limited by diffraction, such that the coherence volume coincides with the geometrical focal plane.

[0027] Unless stated otherwise, in this specification a map of a physical parameter of a sample is to be understood as a planar or volumetric map of said parameter.

[0028] Therefore, maps of physical parameters of a sample include front reflectance or volume reflectance confocal images, maps of the reflection point spread function (or RPSF) around a sample voxel, planar or volumetric maps of single scattering rates, planar or volumetric maps of multiple scattering rates, and planar or volumetric maps of the optical index of the sample.

[0029] A front reflectance confocal image is an estimate of the reflectance of a cross section of the sample. A volume reflectance confocal image is an estimate of the reflectance of the volume of the sample. In the remainder of this specification, a reflectance confocal image may be referred to simply as a "confocal image".

[0030] A voxel is defined as a volume resolution unit. In this specification, a voxel can also be referred to by its center of mass, called a "focus". The size of the volume resolution unit is determined by the lateral and axial resolutions δz of the optical system, which is defined by a set of optical elements located between the sample and the illumination and detection planes.

[0031] In the rest of this specification, the reflection point spread function around a focus will be referred to simply as "spread function around a focus" or "RPSF at the focus". A "graph of spread function" refers to a graph of reflection point spread functions (RPSF) around multiple foci of a sample.

[0032] According to one or more embodiments, the optical characterization method according to the first aspect further includes:

[0033] - determining a first reflectance confocal image based on the focal volume reflectance matrix;

[0034] - based on the focal volume reflectance matrix, determining a map of spread functions and determining a map of positions of intensity maxima of each spread function;

[0035] - determining a second reflected confocal image corrected for alignment and / or focus defects of the first interferometer based on the first reflected confocal image and the map of positions of said intensity maxima. Thus, the Applicant has demonstrated a first advantage of determining the focal volume reflection matrix in order to determine a confocal image corrected for alignment and / or focus defects of the first interferometer.

[0036] According to one or more exemplary embodiments, the optical characterization method according to the first aspect further includes:

[0037] - determining a plurality of maps of the spread function for a plurality of values ​​of the integrated optical index of the sample based on said first focal volume reflectance matrix;

[0038] - determining a plurality of maps of positions of intensity maxima of the spread function based on said plurality of maps of the spread function, each map being obtained for a value of the integrated optical index;

[0039] - a map determining the optimal value of the integrated optical index for which, at each focus, the intensity maximum of the diffusion function is at a maximum;

[0040] - numerically determining a second propagation operator based on a map of optimal values ​​of the integrated optical index; and

[0041] A second focal volume reflection matrix is ​​determined using the second propagation operator.

[0042] In this specification, the integrated optical index of a sample refers to the optical index of the sample integrated between the proximal surface of the sample (ie, on the microscope objective side) and the focus in question.

[0043] According to one or more exemplary embodiments, the method further comprises, based on the graph of optimal values ​​of the integrated optical index, determining a graph of the optical index of the medium at each focus. The second propagation operator can then be determined based on the optical index graph. Therefore, according to one or more exemplary embodiments, the optical characterization method according to the first aspect further comprises:

[0044] - determining a plurality of maps of the spread function for a plurality of values ​​of the integrated optical index of the sample based on said first focal volume reflectance matrix;

[0045] - determining a plurality of maps of positions of intensity maxima of the spread function based on said plurality of maps of the spread function, each map being obtained for a value of the integrated optical index;

[0046] - a map determining the optimal value of the integrated optical index for which, at each focus, the intensity maximum of the diffusion function is at a maximum;

[0047] - determining a map of the optical index of the medium at each focus, based on said map of optimal values ​​of the integrated optical index;

[0048] - numerically determining a second propagation operator based on a map of the optical index of said medium; and

[0049] A second focal volume reflection matrix is ​​determined using the second propagation operator.

[0050] Regardless of whether the optimization is based on a direct integrated index or a sample index derived from the integrated index to maximize the intensity of the RPSF at the focus, the second propagation operator corresponds to a propagation operator that allows the coherence volume and the geometric focal plane to substantially coincide by taking into account the index inhomogeneity in the bulk scattering medium.

[0051] The resulting second focal volume reflection matrix can be used to create a confocal image of the reflectivity of the medium, wherein the axial focus and distortion defects of the initial confocal image are corrected. The map of the RPSF associated with the second focal volume reflection matrix can also be used to locally quantify the multiple scattering rate for each voxel of the image. This parameter gives a local reliability index of the image and locally characterizes the scattering properties of the medium. The map of the RPSF also allows quantification of the single scattering rate, whose depth gradient gives a local estimate of the scattering mean free path.

[0052] According to one or more exemplary embodiments, the method further comprises determining a distortion matrix defined between a correction basis and a focusing basis based on the second focal volume reflection matrix, the determining of the distortion matrix comprising:

[0053] - at the entrance or exit, projecting the second focal volume reflection matrix onto the correction basis to obtain a projected reflection matrix at the entrance or exit, respectively;

[0054] - in the correction basis, a distortion matrix is ​​determined by the term-by-term product between the reflection matrix of the projection and a reference reflection matrix defined for a reference medium;

[0055] - locally determining the invariants of the distortion matrix in order to identify the aberration laws in a sub-domain of the field of view in a correction basis;

[0056] - Based on the aberration law, a transmission matrix between the voxels of the field of view and the correction basis is estimated.

[0057] The focusing basis at incidence or exit, respectively, is a set of source points or a set of receiving points conjugated to the voxels forming the sample.

[0058] Local correction of aberrations performed in this way produces confocal images with optimized contrast and resolution over the entire volume of the specimen.

[0059] According to one or more exemplary embodiments, the distortion matrix is ​​determined in the same way, but from a first focal volume reflection matrix, which is obtained with the aid of a first propagation operator when there is no propagation operator optimization step to maximize the spread function at each focus.

[0060] The term-by-term product between matrices is also called the Hadamard product.

[0061] According to one or more exemplary embodiments, the reference medium is a homogeneous medium having an optical index equal to the average index of the propagation medium. Using a plane mirror in the focal plane of the microscope objective, a reference reflection matrix can be determined theoretically for the reference medium. Depending on the degree of a priori knowledge of the propagation medium, the reference medium may take a more complex form (e.g., a multilayer medium, etc.). In this case, the reference matrix may be calculated numerically. To construct the distortion matrix, the phase of the corresponding element of the projected reflection matrix is ​​subtracted from the phase of each element of the reference reflection matrix.

[0062] According to one or more exemplary embodiments, the correction basis defining the distortion matrix is ​​a basis that maximizes the size of the isovist zone contained in the field of view, for example, a set of points of a plane conjugate to the plane of the aberroscope (if the plane of the aberroscope is two-dimensional), or, for example, a plane conjugate to the pupil plane of a microscope objective.

[0063] According to one or more exemplary embodiments, as described in [Reference 4], locally determining the invariant of the distortion matrix includes: performing singular value decomposition on the distortion matrix, performing singular value decomposition on a normalized distortion matrix (i.e., a matrix in which the modulus of each element has been normalized but its phase is preserved), and performing singular value decomposition on a normalized correlation matrix of the distortion matrix (i.e., a correlation matrix of the first distortion matrix in which the modulus of each element has been normalized). Other methods are also feasible for locally determining the invariant, for example, an iterative phase inversion process applied to the distortion matrix.

[0064] According to one or more exemplary embodiments, the light source is a variable wavelength source, and for each incident light wave of a predetermined wavefront, the second plurality N ω The interference signal in the interference signal is N for the incident light wave ω The frequency (or wavelength) of the incident wave is collected by a fixed path difference between the object arm and the reference arm of the first interferometer. In this specification, the frequency (or wavelength) of the incident wave refers to the center frequency (or center wavelength) of the spectrum of the incident wave.

[0065] According to one or more exemplary embodiments, for each incident light wave of a predetermined wavefront, an interference signal in the second plurality of interference signals is for N interference signals between the object arm and the reference arm of the first interferometer. ω path differences. A polychromatic reflection matrix can then be obtained from the interference signals collected for different path differences or time of flight differences using a simple temporal Fourier transform operation. In one or more exemplary embodiments, the polychromatic reflection matrix is ​​generated between an emission measurement basis, which is a plane wave basis (incident pupil plane) and a receiving measurement basis (detection plane). The propagation operator for determining the first focal volume reflection matrix based on the polychromatic reflection matrix includes, for example, a first incident basis change that enables it to go from the incident pupil plane to the emission focal plane, and a second exit basis change that enables it to go from the detection plane to the receiving focal plane (also referred to as the exit focal plane). Such propagation operators are known to those skilled in the art. These propagation operators can, for example, include Fresnel transform operations.

[0066] According to one or more exemplary embodiments, N in The incident light waves are spatially coherent and have wavefronts controlled by the means for spatially shaping the wavefront.

[0067] For example, the means for spatially shaping the wavefront comprises a scanning device, and N in The incident light waves are plane waves, each of which has a wave vector with a different direction.

[0068] However, the predetermined wavefront is not necessarily a plane wave with wave vectors of different directions.For example, the illumination device may include a spatial wavefront modulator such as a variable mirror or a liquid crystal modulator to generate different predetermined wavefronts.

[0069] When the mutual coherence function of the electromagnetic fields is uniform in the spatial plane, the incident light waves are spatially coherent.

[0070] In contrast, light waves are spatially incoherent when the mutual interference function of the electromagnetic fields has a support that is limited only by diffraction (ie, of the order of half a wavelength).

[0071] In this specification, a partially spatially coherent wave is a spatially coherent wave whose mutual coherence function has a finite support with a width greater than half the wavelength.

[0072] According to one or more exemplary embodiments, the light source for emitting spatially coherent incident light waves is a spectrally scanned single mode laser, for example, a laser emitting wavelengths in a spectral range of about 800 nm to about 875 nm.

[0073] According to one or more exemplary embodiments, the light source is a low spatial coherence source, and the method further comprises:

[0074] - generating, based on each spatially incoherent or partially spatially coherent light wave from the light source, by means of a second interferometer, two polarized illumination waves with orthogonal polarizations and with a spatial offset in a plane conjugate to the focal plane of the first microscope objective;

[0075] - Change the spatial offset to produce different wavefronts N in incident wave;

[0076] - sending said polarized waves with orthogonal polarizations respectively to the object arm and the reference arm of said first interferometer for each spatial offset by means of a polarization beam splitter element;

[0077] - collecting the second plurality of interference signals by means of a detector, each interference signal being generated by interference in a detection plane of the detector between a wave backscattered by the sample illuminated by one of the polarized waves having orthogonal polarization and a reference wave generated by reflection of another of the polarized waves having orthogonal polarization by a reference mirror of the reference arm, the interference signal being N interference signals between the backscattered wave and the reference wave ω Different path differences are collected;

[0078] -Based on N in The spatial offset and N ω A set of interference signals collected by the path differences are used to determine the polychromatic reflection matrix.

[0079] The low spatial coherence source is, for example, a light emitting diode or a halogen lamp.

[0080] Therefore, the characterization method has the advantages of being faster in acquisition time and optimized in signal-to-noise ratio for obtaining confocal images of one or more cross sections of a sample.

[0081] According to a second aspect, the present description relates to an optical characterization system for implementing the method for optical sample characterization according to the first aspect.

[0082] Thus, the present description relates to a system for optical characterization of a sample formed of a bulk scattering medium, the system comprising:

[0083] - a first interferometer having an object arm and a reference arm, the object arm comprising a first microscope objective having a given field of view, in operation the sample being positioned in the given field of view;

[0084] - an illumination device comprising a broad spectral band light source, the illumination device being configured to generate a first plurality of incident light waves having different wavefronts;

[0085] -Including N out The detector is configured to collect, for each incident light wave of a given wavefront, a second plurality of interference signals, each interference signal being generated by interference in a detection plane of the detector between a wave backscattered by a sample illuminated by the incident light wave and a reference wave from a reference arm, the interference signal being a plurality of interference signals for each incident light wave of a given wavefront. ω different frequencies or N ω Different path differences are collected;

[0086] - a processing unit configured to:

[0087] Determine the size N in xN out xN ω The three-dimensional multicolor reflection matrix The three-dimensional multi-color reflection matrix includes in The incident light wave and N ω All interference signals collected at each frequency;

[0088] - based on the polychromatic reflectance matrix, numerically determining at least a first focal volume reflectance matrix by applying a first propagation operator, the first focal volume reflectance matrix comprising a set of responses for the sample between a source point and a receiving point, the source point and the receiving point being respectively associated with a region located in the sample at a depth z in and depth z out The voxel of the sample at r in (x' in , y' in , zin ) and r out (x' out , y' out , z out ) conjugation;

[0089] - Based on the first focal volume reflection matrix, determining at least one map of a physical parameter of the sample.

[0090] According to one or more exemplary embodiments, the first interferometer is a Linnik interferometer, and the reference arm includes a reference mirror and a second microscope objective.

[0091] However, other arrangements of the first interferometer are also possible, which a person skilled in the art will be able to implement according to his common knowledge. In particular, the first interferometer may not include a reference mirror on the reference arm and / or a microscope objective.

[0092] According to one or more exemplary embodiments, the detector comprises a two-dimensional acquisition device, such as a CCD or CMOS camera.

[0093] In other exemplary embodiments, the detector includes a spectrometer. ω The interference signal is collected at a frequency.

[0094] According to one or more exemplary embodiments, the light source is a low spatial coherence source; and the illumination device comprises:

[0095] - a second interferometer configured to generate, based on each spatially incoherent or partially spatially coherent light wave from the light source, two polarized illumination waves with orthogonal polarizations and with a spatial offset in a plane conjugate to the focal plane of the first microscope objective; and

[0096] - means for varying the spatial offset; and wherein

[0097] - the first interferometer is a Linnik interferometer and comprises

[0098] - a polarization beam splitter element configured to send each of said polarized waves having orthogonal polarizations and having said spatial offset to the object arm and the reference arm, respectively;

[0099] - means for varying the path difference between the reference arms; and wherein

[0100] - Each interference signal of the second plurality of interference signals is generated by interference in the detection plane of the detector between a wave backscattered by the sample illuminated by one of the polarized waves having orthogonal polarization and a reference wave generated by reflection of another of the polarized waves having orthogonal polarization by the reference mirror of the reference arm, the interference signal being for N between the backscattered wave and the reference wave ω Different path differences are collected;

[0101] -Based on N in The spatial offset and N ω A set of interference signals collected by the path differences are used to determine the polychromatic reflection matrix. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Other advantages and features of the present invention set forth above will become apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0103] - Figure 1A A first example of a system for optical characterization of a volume scattering medium according to the present specification is shown;

[0104] - Figure 1B shows a diagram illustrating different symbols used to describe a basis of a matrix in a characterization method according to the present specification;

[0105] - Figure 2A Shows the description Figure 1A In one example of the characterization system shown, the entrance pupil N of the microscope objective lens arranged on both the reference arm and the object arm of the first interferometer is in Illustration of scanning with light pulses;

[0106] - Figure 2B Shows the description Figure 1A A diagram showing the intensity distribution of interference signals on a detection surface of a characterization system;

[0107] - Figure 2C An example representation of a polychromatic reflection matrix is ​​shown, which includes for N in The incident light wave and N ω All interference signals obtained at wavelengths, the matrix being obtained by the characterization method according to the present specification;

[0108] - Figure 3A A diagram illustrating the response of a medium between a source point of an incident focusing basis and a receiving point of an exit focusing basis is shown to illustrate elements of an example focal volume reflection matrix according to the present specification;

[0109] - Figure 3Bshows an image illustrating a physical parameter map obtained based on a focal volume reflection matrix, which is the object of the method according to the present description;

[0110] - Figure 3C shows a diagram illustrating that the focal plane and the coherence plane do not coincide, which results in a degradation of the quality of the transmit and receive focusing operations when constructing the focal volume matrix;

[0111] - Figure 4A shows a diagram illustrating a spread function (RPSF) obtained using a first Fresnel propagation operator at focal points located at different focal planes;

[0112] - Figure 4B shows a diagram illustrating the variation of the intensity maximum of the RPSF with the depth z of the focal plane;

[0113] - Figure 5 An image illustrating the effect of numerical bifocusing on a confocal image of a sample formed from a test pattern is shown;

[0114] - Figure 6 shows a quantitative study of RPSF measured in a sample formed from cornea in a characterization method according to the present description;

[0115] - Fig. 7A shows a diagram illustrating subdivision of a field of view for extracting a local RPSF;

[0116] - Figure 7B An image illustrating a multiple scattering rate map of a sample formed by the cornea at a depth of z = 250 μm is shown;

[0117] - Figure 7C An image illustrating a local RPSF map of a sample formed by the cornea at a depth of z = 250 μm is shown;

[0118] - Fig.7D shows images of two longitudinal sections (B-scans) illustrating the optical index n(x, y, z) estimated in a sample formed by a cornea according to the characterization method of the present description;

[0119] - Figure 8 shows an image illustrating the results of a method for numerically correcting lateral aberrations at a depth of z=250 μm in a sample formed by the cornea by means of a characterization method according to the present description;

[0120] - Fig. 9 shows the phase of the transmission matrix by means of an iterative aberration correction process in an exemplary method according to the present description and a confocal image of a B-scan of a sample formed by the cornea at a depth of z=250 μm before and after numerical aberration correction;

[0121] - Fig. 10A Another example of a system for optical characterization of a volume scattering medium according to the present specification is shown, wherein the first interferometer is an interferometer other than a Linnik interferometer;

[0122] - Fig. 10B Another example of a system for optical characterization of a volume scattering medium according to the present specification is shown, Fig. 10A The system is similar to that of , but uses a spectrometer detector;

[0123] - Fig.11 Another example of a system for optical characterization of volume scattering media according to the present description is shown for implementing an exemplary method according to the present description, wherein a polychromatic reflectance matrix in the time domain and in a focus basis is recorded.

[0124] In the various embodiments that will be described with reference to the accompanying drawings, similar or identical elements are provided with the same reference numerals. DETAILED DESCRIPTION

[0125] In the following detailed description, in order to ensure clarity of the disclosure, only certain embodiments are described in detail, but these examples are not intended to limit the general scope of the principles upon which this description is based.

[0126] The various embodiments and aspects described in this specification can be combined or simplified in a variety of ways. In particular, unless otherwise specified to the contrary, the steps of various methods can be repeated, interchanged or run in parallel. In this specification, whenever it is mentioned that a calculation or processing step is particularly used to implement a method step, it should be understood that each calculation or processing step can be implemented by software, hardware, firmware, microcode or any suitable combination of these technologies. When using software, each calculation or processing step can be implemented by computer program instructions or software code. These instructions can be stored in or transferred to a computer-readable storage medium (or computing unit) and / or executed by a computer (or computing unit) to implement these calculations or processing steps. Figure 1A A first example of a system 101 for optical characterization of a sample 10 consisting of a bulk scattering medium is shown, the system 101 being configured to implement an optical characterization method according to the present description. Figure 1B A simplified diagram illustrating a basis of matrices used for description in a characterization method according to the present specification is shown.

[0127] System 101 includes an illumination device 130 having a wide spectral band light source 132 (e.g., a spectrally scanned source) configured to emit a light beam having a frequency ω (or wavelength λ) capable of emitting light in a spectral band [ω min ,ω max ](or wavelength range [λ min ,λmax ) around the center frequency ω n (or center wavelength λ n ) varies, for example as schematically shown in spectrum 131. Light source 132 emits, for example, near infrared (eg, about λ min = 800nm ​​to about λ max = 875) and can be launched at a given speed (e.g., about 10 2 nm / s to about 10 5 The light source is, for example, an optical fiber based light source, and the illumination device can include an output optical fiber 133 connected to a collimator 134 configured to emit a substantially collimated spatially coherent light beam.

[0128] The system 101 further comprises an interferometer 120 having an object arm and a reference arm separated by a cube beam splitter 121. The system 101 comprises a first microscope objective 110 arranged in the object arm of the interferometer, the sample being positioned in the field of view of the microscope objective in operation.

[0129] exist Figure 1A In the example shown, the interferometer 120 is a Linnik interferometer which, in addition to the first objective 110 located on the object arm, comprises a second microscope objective 122 located in the reference arm. In this example, the reference arm also comprises a reference mirror 123.

[0130] like Figure 1A As shown, in this example, the light emitting device further comprises a device 135 for scanning the collimated light beam and a set of lenses 136, 137, 138. Lenses 137 and 138, respectively referred to as a scanning lens and a tubular lens, form a 4f component of a given magnification, and lens 138 is configured to focus the light beam exiting from the 4f component into a plane InP corresponding to the plane of the entrance pupil of the microscope objective 110 (“entrance pupil plane”). The scanning device 135 comprises two scanning mirrors, for example rotated by a galvanometer motor, to scan the incident light beam in the entrance pupil plane InP of the microscope objective 110 arranged on the object arm of the interferometer 120 in two spatial directions (received by the scanning mirrors). Figure 1A It should be noted that Figure 1A In the example of FIG. 4 , since a Linnik interferometer is used, the incident light beam also scans the entrance pupil plane of the second microscope objective 122 arranged on the reference arm of the interferometer.

[0131] The system 101 also includes a detector 140 having a detection plane ImP and a processing unit 150 configured to process data generated by the detector, as will be described below. The detector includes, for example, a camera configured to acquire a two-dimensional image at a given acquisition frequency. For example, the camera allows the acquisition of an image of 256 x 256 pixels at a given frequency (e.g., about 75 kHz).

[0132] Of course, other configurations of the optical characterization system 101 are possible, and those skilled in the art will be able to implement these configurations using general knowledge in the art. For example, the system 101 can be deployed in a polarization configuration. In this case, the cube beam splitter is a polarization cube beam splitter. Two quarter wave plates can be placed between the microscope objective and the cube beam splitter. A polarization analyzer can be arranged upstream of the detector 140 to recombine the two polarizations.

[0133] Furthermore, broadband sources do not necessarily have to be frequency-variable. For example, a camera could be replaced by a spectrometer that performs frequency selection during detection, such as Fig. 10B as described.

[0134] Furthermore, instead of changing the frequency of the source, the path difference (or “flight time”) between the object arm and the reference arm of the interferometer can also be changed, for example by moving the block consisting of the reference mirror 123 and the second microscope objective 122 .

[0135] Instead of the Linnik interferometer, other interferometer arrangements are also possible. For example, as will be seen in reference Fig. 10A As described, the reference wave in the interferometer can come directly from the source.

[0136] exist Figure 1B The symbols used in this specification to identify the various bases described, in particular the reflection matrices used for the characterization of the sample, are given in FIG. Figure 1B Only some of the elements that characterize the system are shown. These symbols apply to Figure 1A but more generally also applies to all characterization systems of this specification.

[0137] The focal plane of the microscope objective 110 is denoted as FP and is intended to receive the sample 10. InP is used to denote the entrance pupil plane of the microscope objective, or any plane conjugate to the entrance pupil plane of the microscope objective, and is referred to as the incident plane wave basis. The entrance pupil is intended to receive incident light waves for illuminating the field of view of the sample to be characterized. The symbol u in represents a point in the entrance pupil plane InP, represented by its Cartesian coordinates (V in , W in). OutP is used to represent the exit pupil plane of the microscope objective, or any plane conjugate to the exit pupil plane of the microscope objective, and is called the exit plane wave basis. The exit pupil is intended to receive light waves reflected by the field of view of the sample to be characterized. The symbol u out represents a point in the exit pupil plane OutP, represented by its Cartesian coordinates (V out , W out ) indicates. Figure 1B As shown, the incident and exit paths, which include the entrance pupil and the exit pupil, respectively, are separated by a beam splitter element 121. In the incident path, SP represents the source plane of the optical system, which is conjugate to the focal plane of the microscope objective, represented in this example by the lens 11 forming a 4f assembly with the microscope objective 110. The symbol p in represents a point in the source plane SP, represented by its Cartesian coordinates (x in ,y in ). In the exit path, ImP represents the imaging plane of the optical system, which is conjugate to the focal plane of the microscope objective, represented in this example by the lens 12 forming a 4f assembly with the microscope objective 110. Symbol p out Denotes a point in the imaging plane ImP, represented by its Cartesian coordinates (X out ,y out )express.

[0138] The voxel r of the sample in With coordinates (ρ′ in , z in ), where point r in The horizontal coordinate ρ′ in (x' in , y' in ) and point ρ in the source plane SP in Conjugate. Voxel r in The (volume) basis of conjugate source points of is called the incident focal basis.

[0139] The voxel r of the sample out With coordinates (ρ′ out , z out ), where point r out The horizontal coordinate ρ′ out (x′ out , y′ out ) and point ρ in the imaging plane ImP out Conjugate. Voxel r out The (volume) basis of conjugate receiving points of is called the incident focal basis. In operation, the illumination device 130 allows the implementation of the characterization method according to the examples of the present specification, as described below. For a given frequency (or wavelength), the illumination device generates a first plurality N of different predetermined wavefronts. in For example, Figure 1A As shown, N in The incident light waves are controlled by the scanning device 135 and have N wave vectors with different directions. in plane waves. For example, the lens (exit lens) 124 ensures the optical conjugation between the object focal plane FP of the microscope objective 110 and the detection plane ImP of the detector 140. Therefore, according to one example, in operation, the scanning device 135 cooperates with the 4f components 136, 137 and the focusing lens 138 to scan the pupil planes InP of the microscope objectives 110 and 122 arranged on the object arm and the reference arm of the Linnik interferometer 120, respectively, in two spatial directions. The resulting N in The incident light waves each have a wave vector k in , the direction and norm of the wave vector depend in particular on the scanning angle specified by the scanning device 135 and the frequency ω (or wavelength λ) of the incident wave.

[0140] In addition, for each incident light wave of the predetermined wavefront, the detector 140 collects a second plurality of N ω interference signals, each interference signal being generated by the interference between the wave backscattered by the sample 10 illuminated by the incident light wave in the detection plane ImP of the detector and the reference wave from the reference arm, N ω The interference signal is for N ω For example, Figure 1A In the example, the irradiation device 130 includes a spectral scanning light source, which collects N by changing the wavelength of the light source. ω An interference signal.

[0141] The fields backscattered by the two arms of the interferometer interfere at the detection plane ImP of the detector 140. For the frequency ω of the incident wave, the interference term between the two arms corresponds to the field E reflected by the sample s The phase conjugate E of the reflected field from the reference arm r * The product of, according to the following equation:

[0142] [Formula 2]

[0143]

[0144] where ρ′ out (x' out , y' out ) is the spatial coordinate (x) in the detection plane ImP (receiving measurement plane) out ,y out ) and u in (v in , w in) is the spatial coordinate (v in , w in ) vector.

[0145] In operation, lens 138 allows the incident light beam to be focused in the pupil plane of the microscope objective. The associated focal spot of size δu is limited only by diffraction (numerical aperture of the system upstream of the interferometer). The microscope objective transforms this focal spot into a quasi-plane wave that illuminates the sample in a wide field of view. The quasi-plane wave will be partially imaged on the detection plane ImP.

[0146] By way of explanation, Figure 2A Shown by N in The incident light beam scans the entrance pupil InP of the microscope objective 110, 122. The scanning mirror 135 allows the physical pupil of the microscope objective to be scanned by Figure 2A The black circle 201 in the figure represents the circumference of the physical pupil. The black circle indicates the position of each of the incident light beams focused on the entrance pupil InP of the microscope objective 110, 122. The spatial step length between each light beam is recorded as δu in Taking into account the characteristics of the microscope objective used, the characteristic size of each focal spot is, for example, δu=8 μm. This corresponds to a maximum illumination of the field of view Δr=λf / δu=2 mm, where f is the focal length of the microscope objective 110. The spatial step length δu in corresponds to a sampling of the electromagnetic field in the plane of the emission pupil and indicates the accuracy with which the incident wavefront is resolved when it is numerically resynthesized using the measured reflection matrix. The spatial step size can therefore be advantageously adjusted depending on the level of aberrations introduced by the medium; a sufficiently fine spatial step size can be chosen to capture the fastest spatial fluctuations in the distortion experienced by the light wave when passing through the medium. Depending on the intended application, the entire entrance pupil 201 can be scanned or only a subsection thereof. For example, in order to be able to obtain a high-resolution confocal image, the entire entrance pupil can be scanned, such as Figure 2A shown.

[0147] For each illumination of the sample, the interference term is measured by the detector in the detection plane ImP

[0148] Figure 2B The field of view Δρ and spatial sampling δρ of the camera in the detection plane are shown out , each interference signal (or interference pattern) is recorded under this spatial sampling. Symbol N out represents the number of pixels (or unit detectors) of the camera through which the interference signal is collected. The field of view corresponds to the magnified image of the focal plane of the microscope objective, with a magnification G, where G is the magnification of the imaging system consisting of the microscope objective and the exit lens 124. Figure 1AIn the example system shown, G = -f s / f om , where f s and f om is the focal length of lens 124 and the focal lengths of microscope objectives 110 and 122.

[0149] Each irradiation u in All interference patterns recorded for each frequency ω are stored as a 3D polychromatic reflection matrix, Size is N out ×N in ×N ω .

[0150] According to an example, Figure 2C This 3D polychromatic reflection matrix is ​​shown to show its three-dimensional structure. The first dimension represents the detection point p in the detection plane of the camera. out , whose coordinates (x out ,y out ) by index Given. Among them, as well as The second dimension represents the coordinates u of each incident beam focused in the entrance pupil plane InP in .u in The coordinates (v in , w in ) by index Given, where as well as

[0151] The third dimension, marked by the index k, corresponds to the set of frequencies ω at which the interference signal is recorded.

[0152] Therefore, in this example, the estimation matrix can be used The theoretical total acquisition time is: acq =N in t s , and t s =N ω / f c corresponds to the acquisition time of an image of the volume of interest for a single shot, and f c is the acquisition frequency of the camera. The number of frequencies (or wavelengths) N ω is adjusted according to the thickness L of the medium to be imaged. In particular, N ω =Δω / δω, where Δω=ω max -ω min For measurement is the bandwidth, and δω is the frequency sampling determined by the thickness L of the medium to be imaged, so that in, is the average optical index of the sample, and c0 is the speed of light. For example, for N in =11 2 An incident plane wave, L = 500 μm and f c = 100 kHz, the acquisition time of the image of the volume of interest is t s =1ms, matrix The total acquisition time is t acq =121ms.

[0153] The second step of the method according to the present description consists in applying a propagation operator and based on the polychromatic reflection matrix Numerically determining at least a first focal volume reflection matrix The focal volume reflection matrix includes a set of responses of the sample between the receiving point and the source point conjugate to the voxel of the bulk medium 10. The response of the sample at a given frequency between the source point and the detection point is the reflection coefficient of the sample measured at the detection point when a light wave of that frequency is emitted from the source point. The position of the voxel is given by the vector r used for emission in =(x′ in , y′ in , z in ) and the vector r for receiving out =(x′ out , y′ out , z out ) identifier. A voxel is defined as a volume unit with a given resolution. For example, in Figure 1A In the example of the system shown, the size of such a volume resolution unit is determined by the lateral resolution of the device along (x, y) and the depth of field or spectral band of the source along z. Unlike the prior art [Ref. 4], voxels can be located at different depths in the medium, as described below. The depth z in the volume medium 10 is the depth along an axis (z) parallel to the optical axis of the microscope objective, for example relative to the origin z ref = 0, which corresponds to zero path difference between the reference arm and the sample arm.

[0154] In this example, based on the multi-color reflection matrix Reflection matrix for the first focal volume The numerical determination of includes a first incident basis change that enables it to go from the entrance pupil plane to an emission focus basis (incident focus basis) and a second exit basis change that enables it to go from the detection plane to a reception focus basis (exit focus basis). In the remainder of this specification, these basis changes are referred to as emission and reception numerical focusing operations, or dual focusing operations.

[0155] Figure 3A The different depths (z in and zout ) in and r out Numerical focusing process of two voxels at .

[0156] In order to perform the multicolor reflection matrix To the Focus Volume Matrix , using, for example, the Fresnel propagation operator to perform a double focusing operation for both transmit and receive. To this end, the polychromatic reflection matrix First, we can use a simple spatial Fourier transform to obtain the output coordinate ρ out Projected onto the virtual Fourier plane (u′ out ) on the exit:

[0157] [Formula 3]

[0158]

[0159] Then it is recorded as F(u, z in / out -z f ,ω) are applied to the resulting matrix The incident and outgoing f The transmitting and receiving focal planes at θ are numerically translated to the depth z in the sample. in or depth z out , where z f is the focal length of the microscope objective 110:

[0160] [Formula 4]

[0161]

[0162] exist Figure 4A For different focal depths z=z in =z out An example of a Fresnel mask F, 411, 421 is shown. It is represented as follows:

[0163] [Formula 5]

[0164]

[0165] in, is the assumed optical index of the medium under study (considered to be homogeneous at the moment), is the axial wave number. The coordinate u′ out and u in Apply an inverse spatial Fourier transform to project the incident and reflected fields onto the focusing basis r out and r in In order to obtain the focal volume reflection matrix

[0166] Then, applied to the multi-color focusing reflective matrix The numerical Fourier transform of the frequency ω is used to distinguish the scattered photons according to their flight time in the sample. The resulting matrix is ​​denoted as R rr =[R(r out , r in , t)]. Ideally, that is, under the single scattering approximation and for a uniform index The medium, each flight time is related to the depth in the sample The photons scattered at

[0167] To express the value of the focal volume reflection matrix Rrr, the applicant uses the system described in 1A, with a thickness of L = 400 μm and an average index of Multicolor matrix measured on opaque human cornea The numerical aperture of the microscope objective is ON = 0.3. The acquisition parameters are as follows: N in =441,N ω =80,λ min =800nm,λ max =875nm, the field of view in the object focal plane of the microscope objective is 716x716μm.

[0168] Figure 3B According to an example, The matrix R rr =[R(r out , r in , t)], where z=z in =z out =250 and ballistic time And the columns 302 of the sub-matrix rearranged in two dimensions are shown.

[0169] Matrix R rr Each column 302 of the subsection 302 gives the out For the virtual source point r in The reflected field measured by the emitted light wave. In the ideal case without aberration or multiple scattering, the reflected field around the source point r in An Airy disk is observed. However, here the reflected field is in This takes the form of a diffuse halo, which indicates that the assumptions about the optical index are not necessarily optimal, as will be described in more detail below.

[0170] However, the broadband focusing matrix R rr A set of relevant observations is provided, which will now be used to quantitatively characterize the samples under investigation.

[0171] First, by considering the matrix R rr The following elements can construct a confocal image of a time window

[0172] [Formula 6]

[0173]

[0174] therefore, Figure 3B A confocal image 303 of the cornea is shown, where z = 250 μm. This image corresponds to the diagonal elements of the sub-matrix 302 rearranged in two dimensions. Confocal image is an estimate of the reflectivity of the cornea in the coherence plane associated with a single scattered photon whose flight time is given by Given.

[0175] The quality of this estimate depends on the quality of the focus in the medium. The expression h in / out (r,r in / out ) represents the relationship between each point r in the sample and each point r in / out The impulse response between Figure 1B ). To quantify the focus quality, the relevant observable is the reflection point spread function (RPSF) around the reflection focus, which can be measured along the submatrix R rr The submatrix assumes the same incident and exit focal planes (z in =z out ):

[0176] [Formula 7]

[0177] RPSF(r p , Δx, Δy, t) = <|R({x p -Δx / 2,y p -Δy / 2,z p}, {x p +Δx / 2,y p +Δy / 2,z p}, t)| 2 >

[0178] where <...> is the same point r p A pair of points r as the center in and r out The average value of r p =(r in +r out ) / 2. Δr=r out -r inis the relative position (coordinates Δx, Δy) between these two points. This intensity distribution is relevant because it allows the detection of aberrations independently of the local reflectivity of the medium. Specifically, by considering the incident and exit PSFs to be locally isoplanatic, it is possible to calculate the intensity distribution around each point r by p The translation of redefines the invariant local PSF Make Under this assumption, for a medium of random reflectivity, the RPSF gives the convolution product between the incoherent incident and outgoing spread functions of the imaging device:

[0179] [Formula 8]

[0180]

[0181] For specular objects, the RPSF gives the convolution product between the coherent incident and outgoing spread functions:

[0182] [Formula 9]

[0183]

[0184] In these two asymptotic states, the physical quantities detected by RPSF are not exactly the same, but their spatial distributions show that the point r p The local aberration level at . Figure 3B shows that at depth z p = 250 μm, in an opaque cornea. This has the following manifestations: excess intensity associated with photons scattered by the coherent plane, and incoherent background associated with multiple scattering events occurring upstream of this same plane. The spatial extension of the excess intensity observed on the RPSF and the position of the maximum of the RPSF give information on the quality of the focus.

[0185] Figure 3B It is shown that the maximum of the RPSF (305) is not centered at Δr=0. This is due, among other things, to alignment problems of the first interferometer. Therefore, the confocal intensity does not correspond to the relative position Δr=0, but to the position Δr of the RPSF intensity maximum. max Therefore, new confocal images corrected for these alignment issues can be generated

[0186] [Formula 10]

[0187]

[0188] Image 304 shows the newly acquired confocal image. The comparison of 303 shows that the first use of RPSF is to enable it to overcome alignment problems in the device.

[0189] Furthermore, if the numerical focusing process is ideal apart from these alignment issues, RPSF support will extend to a single resolution unit. Figure 3B It is shown that this is not the case for the opaque cornea studied.

[0190] Figure 3C The main cause of poor focus quality is shown: the focal plane and the coherence plane do not coincide. The finite depth of field of the microscope objective can lead to both transmit and receive focus defects.

[0191] Figure 4A It is shown how the RPSF can be used to adjust the position of the coherence plane and the focal plane. Figure 4A is based on the defocus distance Z at the focal plane of the microscope objective (ON=0.3) of the sample arm m Proof-of-concept experiment with a test pattern of φ = 100 μm. Figure 4A The results for different focal planes z=z are shown. in =z out and the RPSF obtained from the coherence plane coinciding with the position of the test pattern, where t = 2z m / c0.

[0192] When z <z m (or similarly, z>z m ), the spread of the RPSF (411, 412) is characteristic of a focus defect. Then an estimate is defined to determine z m This estimate corresponds to the position z that maximizes the confocal intensity. opt , that is, the maximum value of RPSF, denoted as RPSF max For z = z opt , Figure 4A (421, 422) clearly show that the lateral extension of the RPSF is minimal, i.e., only limited by diffraction.

[0193] Figure 4B shows the RPSF max With the change of depth z. It is indeed found that z opt =z m , and the confocal intensity is outside the depth of field (|zz m |>20μm) is 3.5 times. Figure 3C The numerical focusing optimization process is summarized. By maximizing the confocal intensity (RPSF max ), enabling the coincidence of the coherence plane corresponding to the measured interference property and the geometric focus associated with the position of the sample relative to the microscope objective. Figure 5The effect of numerical double focusing on a confocal image of a test pattern (510) is shown. Although the initial image 511 is degraded due to focusing defects, the numerical focusing process at the entrance and exit produces an image of the test pattern 512 with optimal contrast and a confocal resolution that is only limited by diffraction (δρ ~ λ / (4NA)). After correction, all details of the test pattern are clear and distinct. Therefore, the technical effect of the above-mentioned method is observed for generating confocal images from a full-field spectral OCT device. Therefore, the reflectivity map of the sample can be determined based on the first focal volume reflectance matrix.

[0194] A similar focus optimization method is applied to the reflection matrix measured in the cornea. However, in this experiment, the cornea is immersed in water with a known index nwater. Most importantly, the cornea has a spatially inhomogeneous optical index n(r). First, it is assumed that the mean index of the medium is invariant with respect to lateral translations and that its optical index n depends only on the depth z: n(r) = n(z). The first step of the method is to calculate the reflection matrix n(r) = n(z) for an index n = nwater. Apply the propagation operator F(., z0-z f ,ω), will initially be at depth z in the absence of samples f The focal plane at z0 is brought to the sample surface at depth z0:

[0195] [Formula 11]

[0196]

[0197] An estimate of the distribution of the optical index n(z) is then obtained as follows For each flight time t, the average optical index is modified in the propagation operator F ([Eq. 5]) by Thus modifying the coherence plane The position of the RPSF is scanned to maximize the confocal intensity (RPSF max The value of n that maximizes this quantity gives the depth z = c0t / (2n int ) at the integrated optical index n int (z) is an estimate of the integral exponential distribution n int (z) is defined as the integral of the optical index n(z) of the medium from the surface (z = z0) of the medium to the depth z:

[0198] [Formula 12]

[0199]

[0200] By discretizing this equation with respect to the flight time t, it is then possible to obtain the integral exponential distribution n by inverting the linear system of equations. int The estimated value of (z) Derive an estimate of the local optical index n(z)

[0201] In order to obtain the correlation of n with respect to the horizontal coordinate (x, y), we can first calculate the reflection matrix Perform field segmentation (see Fig. 7A ), and repeat the above process for each sub-region of the field of view. Thus, by considering a sub-region of the field of view centered at each abscissa (x', y'), it is possible to obtain an estimate of the spatial distribution of the optical index n(x', y', z) in the medium exist Fig.7D (Images 71, 72) show the estimated optical index in the cornea Two longitudinal sections (B scans) of is the deviation from the mean optical index of the cornea, here the estimated value

[0202] It should be noted that the estimated value n of the spatial distribution of the optical index n (referred to in this specification as the "optical index map") can be used to construct a new propagation operator F in order to optimally describe the propagation of light from the incident and exit pupil planes of the device to all voxels (i.e., the focal basis) of the medium under investigation. This propagation operator can then be used to construct a new focus reflection matrix and provide a confocal image that is closer to the actual reflectivity of the medium.

[0203] Figure 6 A confocal image 610 obtained after the process of numerically focusing the data is shown. It should be noted that the image can therefore be constructed based on depth z rather than time of flight t, which is a limitation of conventional OCT images where the axial dimension is determined by the path difference between the interferometer arms.

[0204] Compared with previous confocal images (304, Figure 3B ) shows that the image quality has been improved in terms of contrast and resolution.

[0205] However, due to aberration phenomena (excluding focusing defects) and multiple scattering, the quality of the image can still be improved.

[0206] Figure 6 The last claim is illustrated by showing a quantitative study of the RPSF measured in the cornea after focusing. In particular, Figure 6 shows the area around the confocal point Δr maxVariation of the radial distribution of the RPSF with depth 620. The extent of the confocal peak increases with depth, quantifying the loss of resolution caused by the aberrations experienced by the light waves when propagating through the sample. For example, at a depth z = 250 μm, the extension of the RPSF at half maximum 630 is 15 μm, which is far beyond the diffraction limit (δρ ~ 1.4 μm). Finally, the variation of the RPSF with depth shows that an increasingly incoherent background appears beyond z = 200 μm. This is a drag relative to the average value of the integration time of the camera; this component is determined and is therefore related to the occurrence of multiple scattering events upstream of the focal plane.

[0207] In addition to the variation of aberrations and multiscattering with depth, it is also possible to study the depth distribution of the image around a set of points r in the field of view. p The RPSF is measured locally to laterally probe the level of aberration and multiple scattering.

[0208] Fig. 7A It is shown that in order to extract Figure 7C For example, the spatial extent of the local RPSF shows that the focus quality is significantly better in the lower left of the field of view. Figure 6 The confocal image shown is a significantly better estimate of the reflectivity of the medium in this portion of the field of view. The lateral variation of the RPSF also shows the non-isovigny character of the aberrations caused by the inhomogeneity of the optical index distributed in the specimen. As will be seen later, local compensation of the aberrations is advantageous and the matrix method is a suitable tool for this.

[0209] In addition to aberrations, the confocal intensity RPSF can be used max and incoherent background RPSF bg Measure the ratio of multiple scattering relative to single scattering:

[0210] [Formula 13]

[0211]

[0212] The factor of 2 in the denominator takes into account the effect of coherent backscattering, which results in the confocal amplification of the multiple scattering intensity by a factor of 2. Figure 7B The parameter ρ at depth z = 250 μm is shown MS The figure reveals that Figure 6 The presence of streaks that are already more or less visible in the confocal images of the cornea. Such streaks in the corneal stroma are characteristic of diseases such as keratoconus. In addition to this qualitative analysis, the ratio ρ MS It is the first step in the quantitative local imaging of light scattering properties in biological samples. Depending on the information sought, other parameters can be extracted, such as the single scattering ratio (or confocal scattering ratio):

[0213] [Formula 14]

[0214]

[0215] This parameter decreases with z and enables local quantification of the scattering mean free path in the medium. More generally, quantification of single and multiple scattering phenomena allows local measurement of transport parameters such as the scattering mean free path, the transport mean free path or the scattering coefficient. The reflection matrix method also allows the growth of diffuse halos to be studied at relatively short flight times, providing better spatial resolution than standard techniques such as diffuse optical tomography.

[0216] Besides the quantification of aberrations and multiple scattering phenomena in the medium, it will now be shown how aberrations induced by the sample can be locally compensated using a numerically refocusing reflection matrix in order to obtain confocal images with optimal contrast and resolution close to the diffraction limit.

[0217] To this end, the incident / exit transmission matrix T between the incident / exit correction basis and the voxels of the medium can be estimated in / out To this end, a local analysis is performed here to estimate the applicable focusing law for each voxel of the image.

[0218] By way of illustration, a plane wave basis is chosen as the correction basis. The first step in the matrix correction process for correcting the aberrations is to transform the focus reflection matrix R of the time window by a simple spatial Fourier transform. rr (t = 2nz / c0) is projected into the correction basis at the exit (or incident):

[0219] [Formula 15]

[0220]

[0221] According to the dual matrix Construct the distortion matrix D kr , the distortion matrix is ​​defined as follows:

[0222] [Formula 16]

[0223]

[0224] Where T0 is the reference matrix for simulating the propagation of a plane wave in a homogeneous medium (i.e. without aberration): In reference [4], it is shown how, under the isoplanatic assumption, the singular value decomposition of the matrix D can be used to estimate the aberrometer transmittance A(k out ). Here, since the aberrations are not spatially invariant, subdividing the field of view facilitates local analysis of the distortion matrix and local estimation of a suitable focusing law for each voxel of the image. The field of view is divided into overlapping regions, the overlapping regions being defined by the central midpoint r pand spatial extension Δr. With the focus r located in each region in All distortion components of the associated field can be extracted and stored in a local distortion matrix:

[0225] [Formula 17]

[0226] D′(k out , r in , r p )=D(k out , r in )W Δr (r in -r p )

[0227] where, for |x′|<Δx, |y|<Δy, and |z′|<Δz, W Δr (r) = 1, for other cases, W Δr is zero. Ideally, each local distortion matrix should contain a set of foci r belonging to the same isohalo in In practice, the isochromatic condition cannot be fully achieved. Therefore, the spatial window W must be Δr The size of Δr is a delicate compromise: it must be small enough to approximate the isovain condition, but large enough to contain a sufficient number of disordered independent realizations. Each local distortion matrix D′(r p ) gives the first outgoing eigenvector u1(r p )=[U1(k out , r p )]. Therefore, the transmission matrix T is constructed out =[T out (k out , r p )]

[0228] [Formula 18]

[0229]

[0230] The transmission matrix T can be obtained by an iterative phase inversion process out Another estimate of . Based on the following recurrence relation:

[0231] [Formula 20]

[0232]

[0233] The index "T" indicates the matrix transpose operation, W0(r p ) corresponds to the initial transmittance arbitrarily chosen by the operator. This can be considered uniform, w0(r p )=[1 … 1]T Or it can be determined based on a priori knowledge of the aberrations in the medium to accelerate the convergence of the iterative process. Depending on the choice made by the operator, the phase reversal process can be iterated m times, and the resulting vector W m =[W m (k out , r p )] is the aberrometer at point r p Therefore, the transmission matrix T is constructed. out =[T out (k out , r p )]

[0234] [Formula 21]

[0235]

[0236] Regardless of how the transmission matrix T is estimated out , and then apply the estimate The phase conjugation of is used to correct the aberration at the medium exit, and a new focusing reflection matrix is ​​constructed by the following matrix product

[0237] [Formula 22]

[0238]

[0239] Among them, the symbol Corresponding to the matrix The same process can be repeated at the incident position to estimate the transmission matrix T in , and iterates by gradually reducing the extension Δr of the spatial window to correct for higher and higher aberrations associated with smaller and smaller isoplanatic patches. At the end of the matrix aberration correction process, The diagonal elements of the corrected confocal image are obtained:

[0240] [Formula 23]

[0241]

[0242] Figure 8 The results of the method for numerical correction of lateral aberrations in the cornea at a depth z=250 μm are shown. Here an iterative phase inversion method is considered to estimate the transfer matrix T out and T in .

[0243] Figure 8 For 105x105μm 2The RPSF changes 801, 802, 803 for a sub-area of ​​the field of view at each iteration. As the incidence and exit iterations proceed, the RPSF becomes more refined because more and more local aberrations are corrected. The aberration laws 804, 805 obtained at the incidence and exit at the end of the process are Figure 8 In the ideal case, due to spatial reciprocity, they should be the same (T in =T out ). Their differences are caused in particular by imperfections in the imaging system (e.g. alignment imperfections). Therefore, the matrix aberration correction process not only corrects the aberrations caused by the inhomogeneity of the optical index in the medium, but also corrects the imperfections in the imaging system. Comparison of sub-area confocal images before and after correction and The advantages of performing lateral aberration correction after axial aberration correction are explained.

[0244] Fig. 9 shows the transfer matrix T obtained at the end of the iterative aberration correction process in This contains all the aberration laws for each sub-region of the field of view ( Fig. 7A ). Images 903 and 904 show frontal cross-sections of the volume confocal images of the cornea before and after numerical aberration correction at a depth of z = 250 μm. Images 905 and 906 show longitudinal cross-sections (B-scans) of the volume confocal images of the cornea before and after numerical aberration correction. After matrix aberration correction, the contrast of the confocal image is significantly improved: the confocal intensity in the field of view is increased by an average of 7 times. The increase in resolution is also significant: studies of RPSF have shown a 3-fold increase in resolution.

[0245] The experimental validation test was performed using Figure 1A Of course, other characterization systems according to the present specification can also be used to implement the method according to the present specification.

[0246] therefore, Fig. 10A , Fig. 10B and Fig.11 Systems for optical characterization of samples formed from bulk scattering media according to other exemplary embodiments are shown.

[0247] exist Fig. 10A In the example of the system 102 shown, Figure 1A One difference in the system shown is the field from the reference arm. The reference wave in the first interferometer comes directly from the source via a beam splitter 1032. For a fiber-based light source 132 ( Fig. 10AIn the case of the sample arm, the reference arm comprises an optical fiber 1033 and a collimator 1034, allowing the reference wave to be projected as a plane wave at the detector 140. In free space, the reference arm will consist of a lens system ideally chosen to minimize the path difference between the reference wave and the wave single scattered by a reflector located in the plane of interest in the sample arm. In this case, the interference between the wave reflected by the sample illuminated by a predetermined set of different incident wavefronts and a reference plane wave that varies only in frequency is measured. This directly gives the backscattered field E backscattered by the sample s (ρ out ,u in , ω). By comparing this field with the reference field E s (ρ out ,u in , ω) numerically multiply to obtain the matrix

[0248] exist Fig. 10B In the example of the system 103 shown, the source considered is a broadband source. By using a detector 160 comprising a spectrometer, the spectral dependence can be measured. The spectrometer comprises, for example, a dispersive element 161 (e.g., a grating) and a detection device 162. Each element of the detection device has a coordinate p in the plane ImP out The device measures the field at each point and at a frequency of ω. Figure 1A The matrix of system measurements is equivalent to the matrix

[0249] Fig.11 Another example of a system for optically characterizing a volume scattering medium according to the present specification is shown in FIG. Figure 1A Compared to the system shown, Fig.11 The illustrated system 104 has the advantage of being faster in acquisition time and optimized in signal-to-noise ratio, particularly when it is necessary to obtain confocal images of a sample over a small number of cross sections of the sample.

[0250] The system 104 comprises an illumination device 1130 having a spatially incoherent light source 1132 arranged at the focus of a lens 1133, the illumination device being configured to emit a plurality of incident light waves for illuminating a given field of view of the sample 10 through the microscope objective 110. The system 104 further comprises a detector 1140 arranged at the focus of the lens 1124 and a processing unit (not shown) which in particular receives the photoelectric signal from the detector 1140. The characterization system 104 comprises two interferometers arranged in series. A first interferometer 1120 and a second interferometer forming part of the illumination device 1130. The second interferometer is, for example, a Michelson interferometer in an air wedge configuration, which enables the generation of two orthogonal polarized illumination beams tilted relative to each other at the exit. The first interferometer 1120 is, for example, a Linnik interferometer having a polarization beam splitter 1121 and a quarter wave plate 1126, 1128 on each arm. The afocal systems 1111, 1112 enable 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.

[0251] The spatially and temporally incoherent incident field is linearly polarized at 45 degrees to the parallel direction (e||) and the normal direction (e⊥) of the plane of the device by the polarizer 1134. The components of the wave polarized in the directions e|| and e⊥ are transmitted and reflected by the polarization beam splitter 1131, respectively. Each arm of the second interferometer includes a quarter wave plate (1135, 1137) and a mirror (1136, 1138). On one arm, the mirror (1136) is tilted relative to the optical axis. When the two waves return, the two waves from each arm leave the interferometer as two tilted orthogonal polarization beams. However, the two beams are coherent with each other because they come from the same incident wave.

[0252] In the first interferometer 1120, the two beams are separated again by a polarization beam splitter 1121. The e|| polarized beam is transmitted in the reference arm. The e⊥ polarized beam is reflected in the object arm. The presence of quarter wave plates 1126 and 1128 on each of the two arms allows optimal transmission of the two beams after they are reflected by the sample in the object arm and the mirror on the reference arm. The two beams are recombined at the exit of the interferometer using an analyzer 1125 that is polarized at 45 degrees to e|| and e⊥. They are therefore able to interfere in the focal plane of the lens 1124. A detector (e.g. a CCD or CMOS camera) records the corresponding interference signal. The tilting of the mirror 1136 causes the reference beam and the object beam to be offset relative to each other in the camera. In this way, different points ρ can be measured. in and ρ out Therefore, with the help of Fig.11 The system described is able to measure the polychromatic reflectance matrix R in the time domain and on a basis conjugate to the focal plane of the microscope objective.ρρ :

[0253] [Formula 24]

[0254]

[0255] The flight time t of the photon is controlled by the path difference between the reference arm and the sample arm of the first interferometer 1120. The flight time can therefore be scanned by simultaneously moving elements 1122, 1123 and 1126 of the reference arm of the first interferometer. in and point ρ out The relative position of is controlled by the inclination of the reference mirror 1136 in the second interferometer.

[0256] In some exemplary embodiments, by ρρ The coefficient of the coordinate ρ out Performing a spatial Fourier transform and a temporal Fourier transform on the variable t yields the same result as that obtained by Figure 1A The device record matrix is ​​equivalent to the matrix Only the incident and outgoing coordinates are swapped. This can be done simply by swapping the matrices The same numerical processing as described above is applied to the exit and incident coordinates of .

[0257] In other embodiments, it is possible to ρρ , by applying a time shift to the measured interferometric signal, the focusing reflection matrix R is obtained rr :

[0258] [Formula 25]

[0259]

[0260] Among them, A in / out is the spatial apodization term of the field (related to the geometric decay of the Green's function in integral diffraction theory), and Δt in / out is the same as the ballistic photon from point r in / out propagates to the emission in the sample (ρ in ) / Detection(ρ out ) plane. This time shift operation is equivalent to applying the Fresnel propagation operator in the Fourier domain. If the data is collected in time, then the back propagation of the measured field in the time domain is more advantageous in terms of time and storage than shifting to the frequency domain.

[0261] Although described through a number of detailed exemplary embodiments, the optical characterization method and system include various variations, modifications and improvements, which are obvious to those skilled in the art and should be understood to form part of the scope of the present invention as defined by the appended claims.

[0262] References

[0263] Reference 1: S.M. Popoff et al. “Measuring the Transmission Matrix in Optics”, Phys. Rev. Lett. 104, 100601, 2010.

[0264] Reference 2: A. Badon et al. “Smart optical coherence tomography for ultra-deep imaging through highly scattering media”, Sci. Adv. 2016;2:e1600370.

[0265] Reference 3: US20040061867.

[0266] Reference 4: US20210310787.

Claims

1. A method for optical characterization of a sample (10) formed of a bulk scattering medium, the method comprising: - a step of positioning the sample (10) in the field of view of a first microscope objective (110), the microscope objective being located in the object arm of a first interferometer (120), the first interferometer also comprising a reference arm; - generating a first plurality N of different wavefronts by means of an illumination device (130) comprising a broad spectral band light source (132) in The steps of the incident light wave; -For each incident light wave of a given wavefront, by including N out The detector (140) of the unit detectors collects the second plurality (N ω ) step of detecting interference signals, each interference signal being generated by interference in the detection plane of the detector between a wave backscattered by the sample illuminated by the incident light wave and a reference wave from the reference arm, the interference signal being a signal for N ω different frequencies or N ω Different path differences are collected; - Determine the size to be N in x N out x N ω The three-dimensional multicolor reflection matrix The three-dimensional multi-color reflection matrix includes in The incident light wave and N ω All interference signals collected at different frequencies (N in x N ω ): - Based on the polychromatic reflection matrix, numerically determining at least a first focal volume reflection matrix (R rr ), the first focal volume reflection matrix comprises a set of responses for the sample between a source point and a receiving point, the source point and the receiving point being respectively associated with a sample (10) at a depth z in and depth z out The voxel of the sample at r in (x' in , y' in , z in ) and r out (x' out , y' out , z out ) conjugation; - Based on the first focal volume reflection matrix, determining at least one map of a physical parameter of the sample.

2. The optical characterization method according to claim 1, wherein: The at least one graph of a physical parameter of the heterogeneous medium comprises: a confocal image, a graph of a point spread function (RPSF) around a plurality of reflection foci (rp), a graph of a single scattering rate, a graph of a multiple scattering rate, a graph of an optical index of a sample.

3. The optical characterization method according to any one of the preceding claims, further comprising: - Based on the focal volume reflectance matrix, determining a first reflectance confocal image - based on the focal volume reflectance matrix, determining a map of spread functions and determining a map of positions of intensity maxima of each spread function; - determining a second reflection confocal image corrected for alignment and / or focus defects of the first interferometer based on the first reflection confocal image and the map of the positions of the intensity maxima 4. The optical characterization method according to any one of the preceding claims, further comprising: - determining a plurality of maps of a spread function for a plurality of values ​​of an integrated optical index of the sample based on the first focal volume reflectance matrix; - determining a plurality of maps of positions of intensity maxima of the spread function based on said plurality of maps of the spread function, each map being obtained for a value of the integrated optical index; - a map determining the optimal value of the integrated optical index for which, at each focus, the intensity maximum of the diffusion function is at a maximum; - numerically determining a second propagation operator based on the map of optimal values ​​of the integrated optical index; as well as A second focal volume reflection matrix is ​​determined using the second propagation operator.

5. The method according to claim 4, further comprising: Based on the map of optimal values ​​of the integrated optical index, a map of the optical index of the medium at each focus is determined.

6. The optical characterization method according to claim 5, wherein: Based on the map of the optical indices, the second propagation operator is determined.

7. The method according to any one of claims 4 to 6, further comprising: - based on the second focal volume reflection matrix, determining a distortion matrix (D) defined between a correction basis and a focus basis, comprising: - At the entrance or exit, projecting the second focal volume reflection matrix onto the correction basis to obtain a projected reflection matrix at the entrance or exit, respectively - in the correction basis, a distortion matrix is ​​determined by the term-by-term product between the reflection matrix of the projection and a reference reflection matrix defined for a reference medium; - locally determining the invariants of the distortion matrix in order to identify the aberration laws in a sub-domain of the field of view in a correction basis; - Based on the aberration law, estimate the transfer matrix (T out ).

8. The optical characterization method according to any one of the preceding claims, wherein: - the light source is a variable wavelength source and for each incident light wave of a predetermined wavefront, the second plurality (N ω ) The interference signal in the interference signal is N for the incident light wave ω The central wavelength and the fixed path difference between the object arm and the reference arm of the first interferometer are collected.

9. The optical characterization method according to any one of claims 1 to 7, wherein: - for each incident light wave of a given wavefront, the second plurality (N ω The interference signal in the interference signal is the N between the object arm and the reference arm of the first interferometer. ω The path difference is collected.

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

11. The optical characterization method according to claim 10, wherein: The N of the first plurality of light waves in The incident light waves are plane waves, each of which has a wave vector with a different direction.

12. The optical characterization method according to any one of claims 1 to 9, wherein: The light source is a low spatial coherence source, and the method further comprises: - generating, by a second interferometer, based on each spatially incoherent or partially spatially coherent light wave from the light source, two polarized illumination waves with orthogonal polarizations and with a spatial offset in a plane conjugated to the focal plane (FP) of the first microscope objective; - Change the spatial offset to produce different wavefronts N in incident wave; - sending said polarized waves with orthogonal polarizations respectively to the object arm and the reference arm of said first interferometer for each spatial offset by means of a polarization beam splitter element (1121); - collecting the second plurality (N) by the detector (1140) ω ) interference signals, each interference signal is generated by interference in the detection plane of the detector between a wave backscattered by the sample illuminated by one of the polarized waves with orthogonal polarization and a reference wave generated by reflection of another of the polarized waves with orthogonal polarization by the reference mirror of the reference arm, the interference signal being N interference signals between the backscattered wave and the reference wave ω Different path differences are collected; -Based on N in The spatial offset and N ω A set of interference signals (N in x N ω ), determine the polychromatic reflection matrix.

13. An optical characterization system for a sample (10) formed of a bulk scattering medium, the system comprising: a first interferometer (120) having an object arm and a reference arm, the object arm comprising a first microscope objective (110) having a given field of view, in which, in operation, the sample (10) is positioned; - an illumination device (130) comprising a broad spectral band light source (132) configured to generate a first plurality (N) of light having different wavefronts in ) incident light wave; -Including N out A detector (140) of unit detectors, the detector being configured to collect, for each incident light wave of a given wavefront, a second plurality (N ω ) interference signals, each interference signal is generated by the interference between the wave backscattered by the sample illuminated by the incident light wave and the reference wave from the reference arm in the detection plane of the detector, and the interference signal is for N ω different frequencies or N ω Different path differences are collected; - a processing unit (150) configured to: - Determine the size to be N in x N out x N ω The three-dimensional multicolor reflection matrix The three-dimensional multi-color reflection matrix includes in The incident light wave and N ω All interference signals collected at different frequencies (N in x N ω ); - Based on the polychromatic reflection matrix, numerically determining at least a first focal volume reflection matrix (R rr ), the first focal volume reflection matrix comprises a set of responses for the sample between a source point and a receiving point, the source point and the receiving point being respectively associated with a sample (10) at a depth z in and depth z out The voxel of the sample at r in (x' in , y' in , z in ) and r out (x' out , y' out , z out ) conjugation; - Based on the first focal volume reflection matrix, determining at least one map of a physical parameter of the sample.

14. The optical characterization system of claim 13, wherein: The first interferometer is a Linnik interferometer, and the reference arm includes a reference mirror and a second microscope objective.

15. A system according to any one of claims 13 and 14, wherein: - the light source is a low spatial coherence source; and the illumination device (130) comprises - a second interferometer configured to generate, based on each spatially incoherent or partially spatially coherent light wave from the light source, two polarized illumination waves with orthogonal polarizations and with a spatial offset in a plane conjugated to the focal plane (FP) of the first microscope objective (110); as well as - means for varying said spatial offset; and in - the first interferometer (1120) is a Linnik interferometer and comprises - a polarization beam splitter element (1121) configured to send each of said polarized waves having orthogonal polarizations and having said spatial offset to an object arm and a reference arm, respectively; - means for varying the path difference between the reference arms; and in - the second plurality (N ω ) Each of the interference signals is generated by interference in the detection plane of the detector between a wave backscattered by the sample illuminated by one of the polarized waves with orthogonal polarizations and a reference wave generated by the other of the polarized waves with orthogonal polarizations reflected by the reference mirror of the reference arm, and the interference signal is for N between the backscattered wave and the reference wave ω Different path differences are collected; -Based on N in The spatial offset and N ω A set of interference signals (N in x N ω ), determine the polychromatic reflection matrix.

Images