Device for the polarimetric measurement, by spectral interferometry, of anisotropic values of a medium, and corresponding measurement method

Abstract

The present invention relates to a device for the polarimetric measurement (PT) of anisotropic values of a medium (M), the device comprising a spectrally structured light source (SSS) that delivers a series of pulses with reference spectral fringes, means for spectral polarisation encoding (MPG) which are configured to carry out spectral polarisation encoding on each of the reference pulses delivered by the light source with a measurement phase delay identical to the reference phase delay, a light detector (D) configured to capture the light intensity returned by the medium (M) for each of the reference pulses delivered by the light source, and a measurement unit configured to determine the anisotropic values of the medium (M) from the images captured by the detector.

Description

[0001] Polarimetric measurement device using spectral interferometry of anisotropic quantities of a medium, and corresponding measurement method

[0002] technical field

[0003] The invention lies in the field of polarimetric measurement by spectral interferometry.

[0004] More specifically, the invention relates to a new concept of spectrally encoded polarimetric measurement device, enabling the measurement of at least one anisotropic quantity of a given medium.

[0005] The invention applies particularly, but not exclusively, to full-field microscopic imaging of linear birefringent media, whose retardation and azimuth are measured by spectral coding. The invention also applies to full-field fluorescence polarization microscopic imaging, the spectral coding measuring the average orientation and angular dispersion of the absorption axis of the fluorophores.

[0006] The invention is particularly well suited to real-time full-field microscopy of media with very low linear birefringence (typically less than 10 -4 degrees) or media in which we wish to measure the inclination of fluorophores and their angular distribution (with a precision typically less than one degree).

[0007] Technological background

[0008] The remainder of this document focuses more specifically on describing the problems encountered in measuring the polarimetric birefringence properties of a medium. The invention is not limited to this particular application context, but is of interest to any technique aimed at characterizing the anisotropic properties of a medium and facing a similar problem.

[0009] Spectral interferometry involves interfering broad-spectrum electromagnetic fields, resulting in the appearance of grooves in their spectral envelope. Spectral interferometry is used, for example, in Optical Coherence Tomography (OCT) to image the reflectance of a medium in depth, and also in polarization to measure the polarimetric properties of a medium. The light intensity I(y) at the output of these devices is expressed as a function of the optical frequency ν as the product of the spectrum of the light source / ν(ν) and the spectral response of the devices based on spectral interferometry H(y):

[0010] / (v) = I0H(y (1)

[0011] The spectral response H( ) is expressed as a sum of several spectral modulations whose amplitude A n and phase 4> nprovide information on the properties of the study environment:

[0012] H(y) = A COS(T„V + < >„). (2)

[0013] In OCT imaging, spectral modulations correspond to different depths in the medium and the amplitude A n the corresponding reflectances. In polarimetry, polarimetric properties are distributed over a limited number of modulations, and amplitudes and phases are related to these properties.

[0014] In the state of the art, a polarimeter is a device used to non-destructively characterize the polarization state of light passing through a medium, or, depending on the optical configuration used, the polarization state of light reflected, refracted or diffracted by that medium.

[0015] Today there are several polarimetric approaches to characterize the anisotropic properties of a medium, based on a coding of the polarization: temporal coding (based on a sequential separation of the polarization states), spatial coding (based on a spatial separation of the polarization states), and spectral coding (based on a wavelength separation of the polarization states).

[0016] As illustrated in patent document FR3112605, a spectrally encoded polarimeter comprises, in its input arm, a multi-wavelength light source followed by a polarization state generator, and, in its output arm, a polarization state analyzer and a light detector. The medium to be characterized is disposed between the input and output arms. The polarization state generator includes a first polarizer and means for polarization spectral encoding (typically based on birefringent crystals). The polarization state analyzer includes means for polarization spectral decoding that translates the polarimetric properties of the medium into light intensity (typically based on birefringent crystals and a second polarizer).The principle involves spectrally encoding the incident light into polarization to make it interact with the medium and converting the modified incident polarization state into a variation in light intensity. The anisotropic characteristics of the medium can then be determined based on the light signal received by the light detector. The use of passive optical elements in such a device makes polarimetric measurement much more efficient than those based on temporal or spatial encoding.

[0017] From a theoretical standpoint, a linear birefringent medium is characterized by two measurable physical quantities: retardation, which depends on the linear birefringence and the thickness of the medium, and azimuth, corresponding to the orientation of the neutral axes of the birefringent medium relative to a reference axis. In microscopy, since the media under study have very low retardation due to their thinness, polarimetric configurations tending towards dark-field characterization (also called "quasi-dark-field" polarimetric techniques) have been proposed to improve the sensitivity of polarization measurements. These techniques rely on the implementation of a dark-field polarimetric bias and a specific design of the polarization state generator and analyzer to ensure maximum measurement sensitivity.

[0018] In order to determine the amplitudes A nand the phases of the spectral response, the light intensity λ(v) of the light signal received by the light detector is measured and sampled by a spectrometer in N v values, then a discrete Fourier transform (FT) is applied to the light intensity / (v). The light intensity is measured as a function of wavelength, which requires resampling the corresponding electrical signal so that it is defined at regular intervals in optical frequency v. If the light source is a wavelength-scanning source, the light intensity / (v) is then measured over time, using a single-channel photoelectric sensor or a spectrometer.

[0019] Although it allows the measurement of low-amplitude anisotropy in a medium, this spectral interferometry measurement technique is not suitable for real-time acquisition of observed phenomena. Indeed, measuring light intensity at each image point of the object and the optical frequency discretization process generate a large volume of data that is difficult to process in real time (for an image composed of N x xN y points and a spectral sampling N v , the data volume is then N x xN y xN vvalues), not counting the time required to perform the wavelength scan (approximately 10 ps per image point, or 10 s for a 1000 x 1000 point image). This processing time therefore limits the image recording speed to a certain frequency, which is not optimal since the light detector's acquisition capabilities are not fully utilized.

[0020] It therefore appears particularly advantageous to have a polarimetric measurement technique that is especially well-suited to media with low-amplitude anisotropy and that is efficient in terms of execution speed. In particular, there is a need for a technique that allows imaging the anisotropy of a medium at the detector's acquisition rate.

[0021] Description of the invention

[0022] In a particular embodiment of the invention, a polarimetric measurement device for at least one anisotropic quantity of a medium is proposed, said device comprising: a light source configured to generate a series of identical light pulses with a broad spectral band, the light pulses of said series being spectrally modulated according to the same sinusoidal modulation defined by a reference phase delay and spectrally phase-shifted from each other by a distinct modulation phase shift, so as to deliver a series of pulses with distinct spectral grooves, called reference pulses; spectral polarization coding means disposed between the light source and the medium, configured to spectrally polarize each of the reference pulses delivered by the light source with a measurement phase delay identical to the reference phase delay;a light detector configured to capture a series of images representative of the light intensity emitted by the medium and obtained respectively with the series of reference pulses delivered by the light source; a unit of measurement configured to determine said at least one anisotropic quantity of the medium from a combination of said captured images.

[0023] Thus, this new polarimetric measurement concept relies on the projection of spectrally structured light onto the medium (whose anisotropy is to be measured) and spectral demodulation of the light reflected by the medium, enabling ultrafast measurements through spectral coding of the medium's anisotropy. Indeed, such an approach improves the rate of polarimetric imaging of the medium.

[0024] According to a specific implementation, the light source includes:

[0025] - a first chromatic phase-delay component shaped to produce said reference phase delay corresponding to sinusoidal modulation:

[0026] - spectral phase-shifting means based on achromatic passive components shaped to apply a distinct modulation phase shift to each of the light pulses.

[0027] According to a particular implementation, the polarization spectral coding means include a second chromatic phase-delay component shaped to produce said measurement phase delay.

[0028] According to a particular implementation, the series of reference pulses delivered by the light source consists of first, second and third successive reference pulses and the series of images captured by the light detector consists of first, second and third successive images obtained respectively with the first, second and third reference pulses.

[0029] According to a particular characteristic, at least one anisotropic quantity of the medium is determined from the combination of images defined by the following equation, for a given position (X, Y) of image portion: r( 2 ) _ jW / nyr(3) \

[0030] , _ 'CCD 'CCD , ,• I 'CCD1I

[0031] Yx ' Y ~ 2 ) + rd) + l (2) + rd) 1 j

[0032] 'CCD' R 'CCD 'CCD ' R'CCD / with: luminous area of ​​the portion of the first image occupying said given position, luminous area of ​​the portion of the second image occupying said given position, luminous of the portion of the third image occupying said given position.

[0033] In one particular embodiment, the light source includes a first polarizer defining a first polarization axis, called the reference axis, and the encoding means include a second polarizer defining a second polarization axis inclined at a non-zero angle to the reference axis, the value of which is defined according to a saturation threshold of the light detector. This ensures the feasibility of dark-field spectral encoding measurements. In another particular embodiment, the device includes spectral polarization decoding means configured to spectrally decode the light reflected by the medium, said decoding means including a third polarizer defining a third polarization axis oriented perpendicular to the reference axis. This configuration is particularly well-suited to measuring a birefringent medium.For measuring the fluorescence characteristics of a medium, the aforementioned spectral decoding methods are not necessary.

[0034] According to a particular implementation, the light source includes means for polarizing light pulses, each defining a polarization axis aligned with the reference axis.

[0035] According to a particular implementation, the encoding means include a passive phase adapter shaped to induce an adaptation phase delay and the decoding means include a passive phase compensator shaped to apply a compensation phase delay which is a phase delay inverse to the adaptation phase delay.

[0036] In another embodiment of the invention, a microscopic imaging system is proposed comprising: a polarimetric measurement device as defined above in any of its implementations, said device comprising microscopic imaging means and means for obtaining polarimetric measurement data of said at least one anisotropic quantity of the medium; means for generating a polarimetric image of the medium taking into account said polarimetric measurement data of the medium.

[0037] The integration of the measurement device according to the invention into a microscopic imaging system allows for the acquisition of high-quality polarimetric images at video frame rates.

[0038] In another embodiment of the invention, a polarimetric characterization method is proposed for at least one anisotropic quantity of a medium, said method comprising the following steps: generation of a series of identical light pulses with a broad spectral band, the light pulses of said series being spectrally modulated according to the same sinusoidal modulation defined by a reference phase delay and spectrally phase-shifted from each other according to a distinct modulation phase shift, so as to deliver a series of pulses with distinct spectral grooves, called reference pulses;capture of a series of images representative of the luminous intensity returned by the medium and obtained respectively with the series of reference pulses delivered by the light source, a spectral coding consisting of spectrally coding in polarization each of the reference pulses with a measurement phase delay identical to the reference phase delay being previously applied to the reference pulses; determination of said less an anisotropic quantity of the medium from a combination of said captured images.;

[0039] In another embodiment of the invention, a computer program product is proposed which includes program code instructions for implementing the aforementioned process (in any of its various embodiments), when said program is executed on a computer.

[0040] In another embodiment of the invention, a computer-readable and non-transient storage medium is proposed, storing the aforementioned computer program product.

[0041] List of figures

[0042] Other features and advantages of the invention will become apparent from the following description, given by way of illustrative and non-limiting example, and the accompanying drawings, in which:

[0043] Fig. 1 presents a spectrally structured light source according to a particular embodiment of the invention;

[0044] Fig. 2 is a spectral representation illustrating an example of signals delivered by the light source shown in Figure 1;

[0045] Fig. 3 shows a transmission configuration of a measuring device according to a particular embodiment of the invention;

[0046] Fig. 4 presents a first configuration in reflection of a measuring device according to a particular embodiment of the invention;

[0047] Fig. 5 presents a second configuration in reflection of a measuring device according to a particular embodiment of the invention; Fig. 6 is a functional block illustrating the principle of spectral demodulation by spectrally structured illumination according to the invention;

[0048] Fig. 7 represents the simplified structure of a device implementing the process according to a particular embodiment of the invention.

[0049] Detailed description of the invention

[0050] In all figures in this document, identical elements and steps are designated by the same numerical reference.

[0051] The general principle of the invention is based on a new concept of spectral demodulation using spectrally structured light to measure the anisotropy of a medium by spectral coding.

[0052] Three particular configurations of the device according to the invention are proposed: a transmission configuration described in relation to Figure 3 and two reflection configurations described in relation to Figures 4 and 5.

[0053] In general, the measurement device according to the invention comprises, in its input arm, a light source and a spectrally encoded polarization state generator, and in its output arm, a polarization state analyzer (in the case of a linear birefringent medium) and a light detector. The light detector comprises a monochrome CCD sensor, insensitive to spectral band wavelengths of the light source. The medium to be characterized is placed between the input and output arms. The measurement device also includes a processing unit electrically connected to the light detector on one side and to the light source on the other, for the purpose of controlling these two elements. The light source is synchronized with the light detector such that a light pulse emitted by the light source corresponds to an associated image of the medium captured by the light detector.

[0054] By "insensitive to wavelengths contained in the spectral band of the light source" we mean the fact that the camera cannot differentiate the wavelengths from one another in this spectral band.

[0055] In relation to Figure 1, a spectrally structured light source is presented according to a particular embodiment of the invention. This light source, referenced SSS, is used to demodulate a polarimetric configuration by spectral coding, or more generally a spectral interferometry device, whose spectral modulation is identical to the spectral modulation of the light source.

[0056] In this particular embodiment, the SSS light source comprises three light components S1-S2-S3 having the same spectral characteristics: same bandwidth, same central wavelength, and same amplitude. The components S1-S2-S3 are broadband sources emitting unpolarized, multi-wavelength light. They are characterized by the same continuous spectral envelope, with bandwidth AX, central wavelength X0, and luminous intensity I o .

[0057] As an illustrative example, the S1-S2-S3 light-emitting diodes (LEDs) emit polychromatic light with a spectral envelope defined by a spectral width of 30 nm, a center wavelength of 530 nm, and an emission power of 100 mW. The S1-S2-S3 diodes operate in pulsed mode, emitting light successively in pulses according to a predefined emission sequence. Each light pulse is shorter than the acquisition time of the light detector used in the device. The S1, S2, and S3 diodes turn on and off successively and synchronously with the light detector D. In the example presented here, the S1 diode first generates a light pulse 11, then the S2 diode generates a second light pulse 12, and finally the S3 diode generates a third light pulse 13.A pulse duration of approximately 10 ms is particularly well-suited to an image acquisition rate of 1 / 100 s. The diodes are arranged relative to each other so that the axis of diode S1 coincides with the optical axis of the device, and the axes of diodes S2 and S3 are perpendicular to the optical axis. Furthermore, diodes S1-S2-S3 are coupled to an optical collimator (not shown) to limit the divergence of the light beams they emit.

[0058] The SSS light source includes a linear achromatic polarizer PO that defines the device's reference polarization axis. The PO polarizer converts incident multi-wavelength light into linearly polarized light whose orientation is parallel to that of the reference axis.

[0059] The SSS light source comprises an achromatic polarizing beam splitter cube PBS and an achromatic beam splitter plate BS1, both arranged on the optical axis of the device. The beam splitter cube PBS is positioned at the intersection of the axes of diode SI and diode S2, and the beam splitter plate BS1 is positioned at the intersection of the axes of diode SI and diode S3. The beam splitter cube PBS is configured to transmit the incident light from diode SI along the optical axis and to reflect the incident light from diode S2 towards the beam splitter plate BS1. The unpolarized, multi-wavelength light from diodes SI and S2 is converted into linearly polarized light by the beam splitter cube PBS, one of whose axes is aligned with the reference axis.The beam splitter BS1 is arranged to transmit the incident light from diodes SI and S2 along the optical axis and to reflect the incident light from diode S3 towards the beam splitter LPO. The unpolarized, multi-wavelength light from diode S3 is then converted into linearly polarized light by the achromatic linear polarizer PS3, whose polarization axis is aligned with the reference axis.

[0060] The term "achromatic" refers to the fact that the component polarizes the incident light independently of the wavelengths characterizing that light.

[0061] Before reaching the beam splitter BS1, the light from the polarizer PS3 also passes through a quarter-wave plate LQ1 oriented at 45° to the reference axis. This quarter-wave plate is an achromatic (or zero-order) plate whose phase delay is equal to K / 2, regardless of the wavelengths involved.

[0062] Finally, the SI and S2 diodes can be linearly biased beforehand upstream of the PBS separator cube using linear polarizers PSI and PS2, whose polarization axes are aligned with the reference axis, to correct any polarimetric defects of the PBS separator cube.

[0063] The SSS light source further comprises a chromatic phase-delay plate LPO (for example, based on a birefringent crystalline material), arranged between the beam splitter BS1 and the polarizer PO, defining the reference phase delay of the device. The phase-delay plate LPO has a neutral axis oriented at 45° with respect to the reference axis of the polarizer LPO. The phase-delay plate LPO constitutes modulation means according to the invention. The phase-delay plate LPO modulates the luminous intensity of the incident light according to a sinusoidal modulation defined by the reference phase delay, and thus produces spectrally structured light at the output. This results in the formation of pulses with spectral grooves Sel, Sc2, Sc3 as illustrated in the figure (appearance of spectral grooves in the original spectrum of the diodes).The applied reference phase delay depends on the thickness of the plate, the material used, and the spectral characteristics of the diodes. The greater the applied phase delay, the greater the number of grooves present in the light signal. Here, "chromatic," for a plate with a given phase delay, means that the plate is thick enough to produce a phase delay whose variation is greater than 2K between the beginning and end of the original spectral envelope of the diodes.

[0064] Furthermore, the presence of the PBS separator cube and the LQ1 quarter-wave plate induces a distinct phase shift in the sinusoidal modulation depending on the diode involved. This results in a shift in the spectral grooves from one spectrum to another at the output of the SSS light source. Specifically, the PBS separator cube allows the spectral grooves associated with diode S1 after the PO polarizer to be in opposite phase to the spectral grooves of diode S2, and the LQ1 quarter-wave plate allows the spectral grooves associated with diode S3 after the PO polarizer to be in quadrature phase to the spectral grooves associated with diodes S1 and S2. Thus, the PBS separator cube and the LQ1 quarter-wave plate constitute modulation phase-shifting means according to the invention.

[0065] Thus, the SSS light source delivers a series of three successive distinct spectral groove pulses Si,2, 3, the groove pulse Sel being associated with diode SI, the groove pulse Sc2 being associated with diode S2 and the groove pulse Sc2 being associated with diode S3. These are the reference light pulses of the device.

[0066] The spectrum of the three reference pulses delivered at the output of the SSS light source can be expressed mathematically as follows: Cv) = I o (1 + COS(T0V)) for the grooved spectrum Sel;

[0067] / 2 (v) = I o (1 + COS(T0V + 7r)) for the spectrum the grooved spectrum Sc2;

[0068] / 3 (v) = / 0(1 + COS(T0V + rr / 2)) for the grooved spectrum Sc3.

[0069] Figure 2 is a spectral representation illustrating the triplet of light pulses generated successively at the output of the SSS light source (expressed in arbitrary units) as a function of the wavelength X (expressed in nm). The three light pulses Sel, Sc2, Sc3 are shown superimposed on this figure for illustrative purposes only, to highlight the differences between the light spectra delivered by the SSS light source, but do not constitute the actual invention, where the three light pulses Sel, Sc2, Sc3 are not delivered simultaneously, but successively in time according to an emission sequence predefined by the processing unit. As explained above, the application of the same sinusoidal modulation (for example, of the form COS(T0V), with T0 the modulation coefficient and v the spectral frequency) and a spectral modulation phase shift p mdistinct (0 for 11, K / 2 for 13 and for 12) to the light signals 11, 12, 13 has the effect of producing grooved spectra out of phase with each other: the spectrum Sc2 associated with diode S2 is presented as a signal with spectral grooves out of phase with respect to the spectrum Sel associated with diode SI, while the spectrum Sc3 associated with diode S3 is presented as a signal with spectral grooves out of phase with respect to the spectra Sel and Sc2 (the set of grooves being constrained by the shape of the original spectral envelope of the diodes).

[0070] A minimum number of grooves N c The spectral width AX of diodes S1-S2-S3 centered at wavelength Xo is necessary to enable the feasibility of a polarimetric measurement according to the invention. This number of grooves corresponds to a minimum phase delay of the form T O V O = 2nN c 0 / at the central optical frequency v0 = c / 0, where c is the speed of light in a vacuum. In theory, the number of grooves can be infinite if the spectral modulation of diodes S1-S2-S3 is exactly the same as the spectral modulation of the spectral coding used to measure the polarimetric properties of the medium under study. If this is not the case, the number of grooves is practically limited to about ten (N c is then between 1 and 10).

[0071] As examples, the LPO phase-retarding plate can be a quartz (SiC₂), calcite (CaCO₃), yttrium vanadate (YVO₄), or tellium dioxide (TeC₂) crystal, though this list is not exhaustive. For a YVO₄-based crystal and a light source centered at 500 nm with a spectral width of 30 nm, the minimum thickness of the phase-retarding plate is 0.4 mm to create at least 10 grooves within the spectral width of the source.

[0072] According to one implementation variant, the phase delay is done by an interferometer instead of a phase-delay plate.

[0073] In this example, the SSS light source consists of a triplet of identical light-emitting diodes (LEDs), each with a spectral width of 30 nm and a central wavelength of 530 nm. This is a specific implementation, and other types of multi-wavelength light components, with identical or different spectral characteristics, can certainly be used without departing from the scope of the invention. Examples of light components include superluminescent diodes, femtosecond lasers, supercontinuum lasers, and wavelength-scanning lasers, among others. Similarly, the number of light components in the light source can be increased so that the number of light pulses in the series illuminating the medium M is greater, thereby increasing the accuracy of the measurements.

[0074] According to one embodiment, the light source (SSS) comprises a single light component generating a single light pulse and mechanical means for deflecting said single pulse onto three different optical paths exhibiting an optical phase delay that allows for a phase shift of the matched spectral modulation (in opposition and in quadrature), so as to generate a series of three reference light pulses. In this case, the mechanical deflection means must be synchronized with the light detector D.

[0075] Full-field transmission microscopy for linear birefringence measurement by spectral coding with spectrally structured illumination

[0076] Figure 3 shows a transmission configuration of a measuring device according to a particular embodiment of the invention. This particular configuration allows for full-field, dark-field polarization microscopy measurements of the linear birefringence of a medium M using spectrally structured light.

[0077] Medium M is characterized by an isotropic transmittance T x , y at each point (x,y) of the medium which corresponds to the ratio between the luminous intensity of the light coming from the medium (after interaction with it), denoted [l M ]x, y , and the luminous intensity directly from the spectrally structured source SSS (without interaction with the medium), denoted lo. In this case, the luminous intensity [l M ]x, y can be written mathematically as follows: [l M ]x, y = T x , yx lo. The medium M is for example a collagen fiber, characterized by linear birefringence and whose retardation R (related to the thickness and birefringence of the biological sample) and azimuth a (corresponding to the orientation of the fibers in the plane of the image) we seek to measure.

[0078] According to the invention, the PT measurement device comprises: in its input arm, a spectrally structured light source SSS (as presented above in relation to Figures 1 and 2) disposed at the input of a polarization spectral coding module (also called a polarization spectral generator), referenced MPG; in its output arm, a polarization spectral decoding module (also called a polarization spectral analyzer), referenced MPA, disposed at the input to a light detector D; the medium M, disposed between the MPG spectral generator and the MPA spectral analyzer; a processing unit (not shown) electrically connected to the SSS source on one side and to the detector D on the other, the detector D being synchronized to the SSS source.

[0079] The PT measurement device is integrated into an imaging system comprising other elements which are, in this example, a condenser C and a microscope objective O, arranged on either side of the medium M between the MPG generator and the MPA analyzer, and a projection lens L. The light detector D is arranged in the image plane of the projection lens L.

[0080] All these elements are arranged on the same optical axis to allow analysis by light transmission through the flow of matter to be analyzed. The reference axis of the device, defined by the polarizer PO, is denoted hereafter PO.

[0081] The MPG generator is configured to spectrally encode the light emitted by the SSS source into polarization. Each emitted wavelength is assigned a distinct polarization state, characterizing the light interacting with the medium M. The MPA analyzer is configured to translate the polarization states modified by the birefringent medium M into variations in light intensity. The light detector D, synchronized to the light pulses emitted by the SSS source, captures and digitizes images of the medium M at an acquisition rate of FPS (e.g., 1 / 100 s).

[0082] The MPG generator is configured to modulate the light signal passing through the medium of interest M according to a sinusoidal spectral modulation identical to that applied by the light source SSS.

[0083] The light detector D is a monochrome CCD camera, insensitive to wavelengths within the spectral band of the SSS light source. The light detector D is positioned in the image plane of the medium M defined by the lens and detects the light intensity it receives from the MPA analyzer. It then converts this optical image into a digital image for the processing unit, which applies mathematical operations to three successive images to display the image of the delay and azimuth at a frame rate of FPS / 3. The light detector D is further characterized by a light intensity saturation threshold SD, defined as the amount of light beyond which the detector's photosensitive layer is no longer able to detect a change in light intensity.

[0084] It is possible to adapt the nature of the light detector according to the intended application, as discussed later in relation to the measurement of dispersion of birefringence and spatial phase of the medium.

[0085] Device input arm

[0086] The MPG generator includes an achromatic linear polarizer PI whose polarization axis is inclined at an angle p with respect to the reference axis PO. The angle of inclination is defined in the plane perpendicular to the principal axis of the device (the axis of light propagation). This angle increases the sensitivity of the birefringence measurement by making the measurement proportional to the birefringence. The angle p is called the linearization factor. The value of the linearization factor p (expressed in degrees) is within the range [0; 90°] and is a function of the light intensity level lo emitted by the source SSS and the saturation threshold SD of the light detector D. In order to be close to the saturation threshold of detector D, the value of the linearization factor p is on the order of a few degrees.

[0087] The MPG generator also includes a chromatic phase-delay plate LP1 whose phase delay—referred to as the measurement phase delay—is identical to that provided by the reference phase-delay plate LPO equipping the SSS source. One of the neutral axes of the LP1 plate is aligned with the reference axis PO. In the specific example presented here, the phase-delay plates LPO and LP1 are each made of a birefringent yttrium vanadate (YVO4) crystal of the same thickness. Alternatively, the phase-delay plates LPO and LP1 could each be made of a material with a different birefringence, with thicknesses chosen so that the phase delay applied by the LPO plate is identical to that applied by the LP1 phase-delay plate.The presence of the phase-delay plate LP1 allows the generation of a multitude of polarization states close to linear states and almost aligned with the reference axis PO, each associated with a given wavelength in the spectral band of the SSS source. This phase-delay plate LP1 also allows the light signal passing through the medium of interest M to be spectrally modulated with the same sinusoidal modulation as that applied by the SSS light source. Since this sinusoidal modulation generated by LP1 is affected in amplitude and phase by the properties of the medium M, spectral demodulation of the light signal passing through the medium of interest M allows the desired anisotropic quantities (delay and azimuth) to be determined.

[0088] The MPG generator also includes an achromatic quarter-wave plate LQ+, whose neutral axes are oriented at 45° with respect to the reference axis PO. The role of the LQ+ plate is to probe the medium M with polarization states close to a circularly polarized state in order to independently measure the retardation and azimuth of the medium M.

[0089] Device output arm

[0090] The MPA analyzer includes an achromatic quarter-wave plate LQ- oriented to induce a phase delay of the inverse order to that of the plate LQ+. More precisely, the fast axis of the plate LQ+ is aligned with the slow axis of the plate LQ-. Thus, without a medium M, the combination of the plates LQ+ and LQ- behaves like a wave plate that is neutral with respect to phase shift.

[0091] The MPA analyzer also includes an achromatic polarizer P3 whose polarization axis is perpendicular to the PO reference axis.

[0092] If the plates LQ+ and LQ- do not perfectly compensate each other, the light intensity measured by detector D is no longer zero for p=0 without medium M. A third quarter-wave achromatic plate LQC, whose neutral axes are oriented at 0° with respect to the reference axis PO, is added after plate LQ- to compensate for any defects in plates LQ+ and LQ-. In this case, the polarization axis of P3 is rotated until the light intensity measured by detector D is minimized for p=0 without medium M, so as to guarantee a measurement against a dark background.

[0093] The principle of this transmission configuration is to allow an ultrasensitive and ultrafast measurement of the retardation R and the azimuth a of the medium M at the acquisition rate FPS, from only three successive images obtained respectively with the three grooved pulses Scl-Sc2-Sc3.

[0094] The underlying mathematical approach that led to the solution of the present invention is described below. This approach is based on spectral demodulation of channeled spectra to determine the retardation R and the azimuth α of the medium M. It relies in part on the block diagram in Figure 6, which illustrates the principle of spectral demodulation by synchronous detection according to the invention. The GMA block represents all the MPG-M-MPA elements (i.e., the MPG generator, the medium M, and the MPA analyzer) of the device. The spectrally structured source SSS emits a series of three successive multi-wavelength light pulses, linearly polarized along the reference axis PO. These light pulses are synchronized to the light detector D, and their durations are less than or equal to the acquisition time of the light detector D. The spectral intensity of the m is hereafter denoted by λ. iemelight pulse. As mentioned above, these light pulses have the same spectral characteristics I0(y) (same spectral width, same central wavelength, and same amplitude) and exhibit a single sinusoidal modulation r0 of their spectrum with a contrast C m equal to 1 or 0, and a phase specific to them in the form:

[0095] Let us now consider the spectrally structured source SSS as a light source for a device based on spectral interferometry and spectral response at each point (x,y) of the object defined by [H(v)] x >y = [X0] x , y + |A] XJ , COS( T0V + [ <p] x , y ).

[0096] The spectral response is therefore composed of a term X o independent of v (DC term) and a modulation term in v whose amplitude and the phase <p sont les éléments à mesurer. Si on considère un détecteur de lumière muni d'un capteur CCD monochrome (donc insensible à la longueur d'onde) comme détecteur du dispositif d'interférométrie spectrale, l'image captured by the CCD sensor for the m ieme Light pulses can be written as follows:

[0097] Æ2 = n^(v)]x,y / (m) (v)dv. (4)

[0098] It is shown below that three representative images of the light intensity returned by the medium M respectively for three different light pulses are sufficient to calculate the Fourier Transform of [ / (v)] x y to a specific TO modulation and go back to the values phase <p.

[0099] Let's take, for example, the following three grooved spectra:

[0100] The images obtained by the light detector for the three light pulses are then expressed as follows:

[0101] Combining the images associated with the three light pulses allows us to obtain: with z 2 =-l, which corresponds well to the Fourier Transform of [ / (v)] x y to the specific modulation T0.

[0102] Image I^ CD This corresponds to the amplitude term X o independent of the frequency v (DC term) of the Fourier Transform of [H(v)] x y .

[0103] Of course, other combinations of channeled spectra are possible. For example, consider the channeled spectra Sel, Sc2, and Sc3 emitted by the SSS light source (reference spectral representations illustrated in Figures 1 and 2):

[0104] Combining the images associated with the three bursts of light also allows us to obtain:

[0105] The combination of images corresponds to the term of amplitude X oindependent of the frequency v (DC term) of the Fourier Transform of [H(v)] x y .

[0106] At the output of the SSS light source, the channeled spectra associated with diodes SI, S2, and S3 correspond respectively to equations (12), (13), and (14) for diodes SI, S2, and S3, assuming that these diodes have exactly the same luminous intensity I0(y). If the diodes used (or other light components) exhibit differences in luminous intensity, the three images I^ D , I^D ctThe light captured by the light detector D for the three light pulses must be digitally weighted to ensure that the three diodes emit the same spectral envelope with an identical luminous intensity / 0(v). The weighting coefficients can be calculated by measuring the luminous intensity of each diode prior to the construction of the light source or by measuring the luminous intensity of each diode in real time using a photodetector DI placed after the beam splitter BS1 (such as the photodetector DI shown as a dashed line in Figure 1).

[0107] For a birefringent medium M, the spectral response [H(v)] x y at each point (x, y) of the midpoint can then be expressed in the form:

[0108] Equation (13) above is defined by considering a medium M with very low linear birefringence. The spectral response is therefore composed of a term independent of v (DC term) and a modulation term in v whose amplitude is linear with respect to the retardation R (which ensures good sensitivity) and whose phase is related to the azimuth a.

[0109] In order to access the amplitude and phase of the spectral modulation of the spectral coding (identical to the spectral coding of the S1-S2-S3 sources), but also the DC term to eliminate the T transmission x>y of the medium and light intensity I o of the light source, according to the invention, it suffices to consider the three successive images captured by the light detector D - namely 4co' Asco ct Asco " et obtained respectively with the three grooved spectra Sel, SC2, Sc3 - namely -delivered by the SSS light source.

[0110] We define the combination coefficient y x y , for a given position (X, Y) of a portion of the image (a pixel or group of pixels) common to the three captured images, by the following image combination:

[0111] ICCD ' the luminous intensity of the portion of the first image occupying said given position, the first image being associated with the first reference pulse,

[0112] ICCD ' the luminous intensity of the portion of the second image occupying said given position, the second image being associated with the second reference pulse,

[0113] ICCD> the light intensity of the portion of the second image occupying said given position, the third image being associated with the third reference pulse.

[0114] The coordinates (X, Y) of an image point are related to the coordinates (%, y) of an object point in the medium according to the transverse magnification of the microscope.

[0115] We can then deduce the following equation: that is, at each given position (X, Y) of the triplet of images:

[0116] Thus, this new concept of demodulating channeled spectra allows, by knowing the amplitude and phase of a single spectral modulation, the determination of the anisotropic properties of a birefringent medium at the acquisition rate of the light detector. In other words, this new concept allows imaging the amplitude and phase of a single spectral modulation at the video rate. This ingenious approach enables linear birefringence measurements to be performed independently of the transmission characteristics T x>y of the medium M and the light intensity I o from the SSS light source.

[0117] Multimodal full-field transmission microscopy for measuring linear birefringence and retardance dispersion R

[0118] For this application, only the light detector D included in the PT measurement device (described above in relation to Figure 3) is replaced by a hyperspectral camera (wide-field imaging) whose property is to sequentially provide images at different wavelengths over a given spectral range and spectral interval. The phase-delay plates LPO and LP1 must be sufficiently thick so that the reference and measurement phase delays are at least 2K greater than the spectral interval of the hyperspectral camera. The diodes SI, S2, and S3 must have a spectral width at least twice the spectral interval of the hyperspectral camera. The diodes SI, S2, and S3 alternately illuminate the medium M, with a duration corresponding to the scan time of the hyperspectral camera to cover the spectral range of the sources SI, S2, and S3.Thus, for each image provided by the hyperspectral camera and corresponding to a wavelength in the spectral range, the retardation R of the medium M is measured. The dispersion of the birefringence is then obtained by measuring the variations in the value of the retardation R as a function of the different images acquired by the hyperspectral camera, that is, as a function of the wavelength.

[0119] Multimodal transmission full-field microscopy for linear and

[0120] For this application, only the light detector D included in the PT measurement device (described above in relation to Figure 3) is replaced by a camera sensitive to spatial phase, i.e., to the wavefront originating from medium M. Diodes S1, S2, and S3 alternately illuminate medium M, with a duration corresponding to the time it takes the phase camera to measure the wavefront. Since the phase camera also provides the intensity image of medium M, the device is thus capable of providing both a birefringence image and a phase image of medium M.

[0121] Full-field reflection microscopy for measuring linear birefringence with structured illumination. Figure 4 shows a first reflection configuration of a measurement device according to a particular embodiment of the invention. This particular configuration, referenced PR, allows for full-field polarization microscopy measurement of the linear birefringence of a medium M using spectrally structured light.

[0122] As with the PT device, the PR measurement device is designed to perform an ultra-sensitive measurement of the linear birefringence of a study medium M by means of three successive images of the medium M.

[0123] Unlike the configuration of the PT device, the quarter-wave plates LQ+ and LQ-, which perform the roles of phase adapter and phase compensator according to the invention, are replaced by a single achromatic quarter-wave plate LQ (for example, identical to the quarter-wave plate LQ+). A beam splitter plate BS2, positioned between the phase-delay measurement plate LP1 and the quarter-wave plate LQ, is arranged to reflect the light returned by the medium M towards the light detector D via the polarizer P3 and the lens L.

[0124] As with the PT device, the SSS light source generates a series of three pulses and the light detector D synchronously captures images representative of the light intensity returned by the medium M for each of the pulses delivered by the source, as previously described.

[0125] It is possible to adapt the nature of the light detector D according to the intended application: typically, it can be equipped with a monochrome CCD camera for measuring retardance and azimuth, a hyperspectral camera for measuring retardance, azimuth and retardance dispersion, or a wavefront-sensitive camera for measuring retardance, azimuth and spatial phase.

[0126] Full-field reflection microscopy for fluorescence measurement by spectral coding with spectrally structured illumination

[0127] Figure 5 shows a second configuration in reflection of a measuring device according to a particular embodiment of the invention. This particular configuration, referenced FR, allows for the measurement of bloom in full-field polarization microscopy of a medium M' using spectrally structured light.

[0128] The FR measurement device is integrated into an imaging system comprising a microscope objective O positioned in front of the medium M' and a projection lens L, with the light detector D positioned in the image plane of the projection lens L. The medium M' is, for example, a cell whose actin microfilaments are labeled with eosin-conjugated phalloidin. Such a medium is characterized by a fluorescence intensity when illuminated by a wave polarized parallel to the absorption axis of the fluorophores, and we seek to measure the average orientation θ of the fluorophore absorption axis (amplitude measurement) and their angular dispersion θ (phase measurement). If the fluorophore absorption axes are all aligned along the θ direction, then θ = θ. Conversely, if the absorption axes are randomly distributed, then θ = 0.This configuration allows for a local measurement of 0 and p at a video frame rate of FPS, from three successive images obtained following three light pulses from the SSS light source.

[0129] Unlike the PR or PT configurations, the MPA analyzer is not needed because the translation of polarization states modified by the medium M into variations in light intensity occurs directly along the absorption axis of the fluorophores. Therefore, the polarizer P3, present in the previous configurations, is not required here. Furthermore, unlike the PR configuration, the phase-delay plate LP1 is positioned not between the polarizer P2 and the beam splitter BS2, but between the beam splitter MS2 and the quarter-wave plate LQ, in order to avoid polarization state modifications induced by potential polarimetric defects in the beam splitter BS2.

[0130] A wavelength filter FF is further disposed between the projection lens L and the light detector D and shaped to transmit the fluorescence light to be measured and absorb the light from the source SSS from the light returned by the medium M'.

[0131] The light detector D, synchronized to the light pulses emitted by the SSS source, captures images of the medium M' at an acquisition rate of FPS. More specifically, the light detector D detects the fluorescence light intensity it receives after reflection from the beam splitter BS2 and passage through the wavelength filter FF. It then converts the received fluorescence light intensity into a digital image for the processing unit (not shown), which applies the necessary mathematical operations to the three successive images to display an image of the average orientation of the absorption axes and the angular distribution at an acquisition rate of FPS / 3.

[0132] Device input arm

[0133] In this particular configuration, the MPG generator comprises: the achromatic linear polarizer P2 whose polarization axis is aligned with the reference axis PO, the beam splitter BS2 arranged to reflect fluorescence light from medium M' towards the output arm, the measurement phase delay plate LP1 whose phase delay difference is identical to the reference phase delay plate LPO, one of the neutral axes of the plate LP1 being rotated 45° with respect to the reference axis PO, the achromatic quarter-wave plate LQ, whose neutral axes are oriented at 0° with respect to the reference axis PO, the role of the plate LQ being to probe the medium M' with polarization states which allow independent measurement of the average orientation and angular dispersion of the absorption axes of the fluorophores of the medium M'.

[0134] Device output arm

[0135] The light reflected by the BS2 separating plate is spectrally filtered via the FF wavelength filter to allow only the fluorescence intensity from the medium M' to pass through.

[0136] Unlike the PT configuration, the tilt angle p applied to the achromatic linear polarizer P2 is set at 0° relative to the reference axis PO, and the plate LP1 is rotated 45° because the goal is not to measure the smallest possible p values. The quarter-wave plate LQ must therefore be oriented at 0° relative to the reference axis PO in order to independently measure the 0 direction and the angular dispersion p and a.

[0137] Based on the same mathematical considerations mentioned above in relation to Figure 3, the spectral response [H(v)] x y at each point (x,y) of the fluorescent medium M' is expressed in the form:

[0138] The spectral response is therefore composed of a term independent of v (DC term) and a modulation term in v whose amplitude is linear with respect to the angular distribution p of the absorption axes and whose phase is related to the average orientation of the absorption axes of the fluorophores.

[0139] To access the amplitude and phase of the spectral modulation, it suffices, according to the invention, to consider the three successive images captured by the light detector D, I ( ; D . sco et obtained respectively with the three light pulses 2 \ 3 ) delivered by the SSS light source.

[0140] The coordinates ( , Y) of an image point are related to the coordinates (x, y) of an object point in the medium according to the transverse magnification of the microscope.

[0141] We define the combination coefficient y x y, for a given position (X, Y) of a portion of the image (a pixel or group of pixels) common to the three captured images, by the following image combination:

[0142] We can then deduce the following equation: that is, at each given position ( , Y) of the triplet of images:

[0143] Thus, this new concept of demodulation of channeled spectra, thanks to the knowledge of the amplitude and phase of a spectral modulation, makes it possible to determine the anisotropic quantities of a fluorescent medium at the acquisition rate of the light detector.

[0144] Figure 7 represents the simplified structure of a processing unit implementing the measurement process according to a particular embodiment.

[0145] This processing unit 10 comprises, more specifically, a random access memory 30 (for example, RAM), a processor or microprocessor 10, and is controlled by a computer program stored in read-only memory 20 (for example, ROM or a hard drive). At initialization, the code instructions of the computer program are, for example, loaded into the random access memory 30 before being executed by the processor 10.

[0146] The processing unit (PU) is electrically connected to the light detector and the light source for controlling both, as well as to a human-machine interface, equipped, for example, with a screen to display an image representing the measured anisotropy of the medium. These elements are controlled by means of commands transmitted by the PU.

[0147] Upon receiving instructions to initiate anisotropy measurement, the processing unit UT initializes the procedure and executes the subsequent steps using the computer program. The processing unit UT transmits an emission command to the light source SSS to trigger the emission of a triplet of successive reference light pulses (a series of pulses with distinct spectral grooves, referenced Si, 2, 3 in Figures 3, 4, 5). Simultaneously, the processing unit UT transmits a capture command to the light detector D to trigger a series of successive images of the luminous flux reflected by the medium under study, obtained respectively for the triplet of successive reference light pulses delivered by the light source SSS. Each reference pulse has previously undergone passive spectral polarization encoding with a measurement phase delay identical to the reference phase delay.The processing unit (PU) then obtains a triplet of three full-field images, acquired at the detector's acquisition rate, representing the light intensity emitted from the medium. Each image is associated with one of the triplet's reference light pulses. The PU then determines the anisotropic properties of the medium under study by combining the captured images, according to the principle described above in relation to Figures 3 to 6. These polarimetric measurement data of the medium are a function of the electrical signals delivered by detector D. They are processed by the PU, which outputs a polarimetric image (PI) to the display screen, representing the measured anisotropy. This image provides real-time visual information illustrating the anisotropic characteristics of the medium M.

[0148] Figure 7 illustrates only one particular way, among several possible ways, of carrying out the steps described above. Indeed, the method according to the invention can be carried out interchangeably: on a reprogrammable computing machine (a PC, a DSP processor or a microcontroller) executing a program comprising a sequence of instructions; or on a dedicated computing machine (for example a programmable logic controller, a set of logic gates such as an FPGA or an ASIC, or any other hardware module).

[0149] In the case where the invention is implemented on a reprogrammable computing machine, the corresponding program (i.e. the sequence of instructions) may be stored in a removable storage medium (such as, for example, a floppy disk, a CD-ROM or a DVD-ROM) or not, this storage medium being readable partially or totally by a computer or a processor.

Claims

DEMANDS 1. Polarimetric (PT) measurement device for at least one anisotropic quantity of a medium (M), said device being characterized in that it comprises: a light source (SSS) configured to generate a series of identical light pulses with a broad spectral band, the light pulses of said series being spectrally modulated according to the same sinusoidal modulation defined by a reference phase delay and spectrally phase-shifted from each other by a distinct modulation phase shift, so as to deliver a series of pulses with distinct spectral grooves, called reference pulses; spectral polarization coding means (MPG) disposed between the light source and the medium, configured to spectrally polarize each of the reference pulses delivered by the light source with a measurement phase delay identical to the reference phase delay;a light detector (D) configured to capture a series of images representative of the light intensity returned by the medium (M) and obtained respectively with the series of reference pulses delivered by the light source; a unit of measurement configured to determine said at least one anisotropic quantity of the medium (M) from a combination of said captured images.

2. Device according to claim 1, wherein the light source (SSS) comprises a first chromatic phase-delay component (LPO) shaped to produce said reference phase delay corresponding to sinusoidal modulation, and the polarization spectral coding means (MPG) comprise a second chromatic phase-delay component (LP1) shaped to produce said measurement phase delay.

3. Device according to any one of claims 1 and 2, wherein the light source (SSS) comprises spectral phase-shifting means (PBS, LQ1) based on achromatic passive components shaped to apply a distinct modulation phase shift to each of the light pulses.

4. A device according to any one of claims 1 to 3, wherein the series of reference pulses delivered by the light source consists of successive first, second, and third reference pulses, and the series of images captured by the The light detector is composed of first, second and third successive images obtained respectively with the first, second and third reference pulses.

5. Device according to claim 4, wherein said at least one anisotropic quantity of the medium (M) is determined from the image combination defined by the following equation, for a given position ( , Y) of image portion: r( 2 ) _ jW / nyr(3) \ , _ 'CCD 'CCD , ,• I 'CCD1I Yx ' Y ~ A 2 ) + rd) + l (2) + rd) 1 j 'CCD ' R 'CCD 'CCD ' R 'CCD / with: luminous area of ​​the portion of the first image occupying said given position, luminous area of ​​the portion of the second image occupying said given position, luminous of the portion of the third image occupying said given position.

6. Device according to any one of claims 1 to 5, wherein the light source (SSS) comprises a first polarizer (PO) defining a first polarization axis, referred to as the reference axis, and the coding means (MPG) comprise a second polarizer (PI) defining a second polarization axis inclined at a non-zero angle with respect to the reference axis, the value of which is defined as a function of a saturation threshold of the light detector (D).

7. Device according to claim 6, further comprising spectral polarization decoding means (MPA) configured to spectrally decode in polarization the light restored by the medium, said decoding means comprising a third polarizer (P3) defining a third polarization axis oriented perpendicular to the reference axis.

8. Microscopic imaging system characterized in that it comprises: a polarimetric measurement device according to any one of claims 1 to 7, said device comprising microscopic imaging means (C, O) and means for obtaining polarimetric measurement data of said at least one anisotropic quantity of the medium; means for generating a polarimetric image (PI) of the medium (M) taking into account said polarimetric measurement data of the medium.

9. A method for the polarimetric characterization of at least one anisotropic quantity of a medium (M), said method being characterized in that it comprises the following steps: generation of a series of identical, broadband spectral light pulses, the light pulses of said series being spectrally modulated according to the same sinusoidal modulation defined by a reference phase delay and spectrally out of phase with each other according to a distinct modulation phase shift, so as to deliver a series of pulses with distinct spectral grooves, called reference pulses; - capture of a series of images representative of the light intensity returned by the medium and obtained respectively with the series of reference pulses delivered by the light source, a spectral coding consisting of spectrally coding in polarization each of the reference pulses with a measurement phase delay identical to the reference phase delay being previously applied to the reference pulses; - determination of said at least one anisotropic quantity of the medium (M) from a combination of said captured images.

10. A computer program product, comprising program code instructions that lead the device according to any one of claims 1 to 7 to perform the steps of the process according to claim 9, when said program is executed on a computer.

11. A computer-readable, non-transient storage medium storing a computer program product as defined in claim 10.

Images