• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
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    • 专利摘要:
    The invention relates to a method for observing a sample, for example a biological sample. It comprises a step of illuminating the sample with a light source and forming a plurality of images, by an imager, the images representing the light transmitted by the sample in different spectral bands. From each image, a complex amplitude representative of the light wave transmitted by the sample is determined in a determined spectral band. The method, which can be iterative, then comprises: - the backpropagation of each complex amplitude in a plane passing through the sample, - the determination of a weighting function from the retro-propagated complex amplitudes, - the propagation of said function of weighting in a plane according to which the matrix photodetector extends, - the updating of each complex amplitude, in the plane of the sample, as a function of the weighting function thus propagated.
    公开号:FR3036800A1
    申请号:FR1554811
    申请日:2015-05-28
    公开日:2016-12-02
    发明作者:Cedric Allier；Thomas Bordy；Olivier Cioni；Lionel Herve；Sophie Morel
    申请人:Commissariat a lEnergie Atomique CEA；Commissariat a lEnergie Atomique et aux Energies Alternatives CEA；
    IPC主号:
    专利说明:

    [0001] TECHNICAL FIELD The technical field of the invention is related to the observation of a sample, in particular a biological sample, by imaging without a lens, by implementing a holographic reconstruction algorithm. improved performance. PRIOR ART The observation of samples, and in particular biological samples, by imaging without a lens has been developing significantly over the past ten years. This technique makes it possible to observe a sample by placing it between a light source and a matrix photodetector, without having an optical magnification lens between the sample and the photodetector. Thus, the photodetector collects an image of the light wave transmitted by the sample. This image is formed of interference figures between the light wave emitted by the source and transmitted by the sample, and diffraction waves, resulting from the diffraction by the sample of the light wave emitted by the source. These interference patterns are sometimes called diffraction patterns, or designated by the term "diffraction pattern".
    [0002] WO2008090330 discloses a device for the observation of biological samples, in this case cells, by imaging without a lens. The device makes it possible to associate, with each cell, an interference pattern whose morphology makes it possible to identify the type of cell. Imaging without a lens appears as a simple and inexpensive alternative to a conventional microscope. Moreover, its field of observation is much larger than can be that of a microscope. It is understandable that the application prospects related to this technology are important. This document also mentions the possibility of illuminating the sample using light sources of different wavelengths. US2012 / 0218379, subsequent to the previous document, incorporates the main teachings of WO2008090330, while mentioning the possibility of using a color matrix photodetector, but the color information is then processed to form a monochrome image. In general, the image formed on the matrix photodetector, having interference patterns, can be processed by a digital propagation algorithm, so as to estimate optical properties of the sample. Such algorithms are well known in the field of holographic reconstruction. For this, the distance between the sample and the photodetector being known, we apply a propagation algorithm, taking into account this distance, as well as the wavelength. It is then possible to reconstruct an image of an optical property of the sample. A digital reconstruction algorithm is for example described in US2012 / 0218379. It is also known that such algorithms can generate an image affected by a significant background noise, termed by the English term "Twin Image", meaning "twin image". Such noise is due to the fact that the starting image formed by the photodetector contains only partial information as to the light wave collected by the photodetector. Indeed, an image gives only information relating to the real part of the light wave, obtained by the measured intensity. But such an image does not include information relating to the imaginary part of the light wave to which the detector is exposed, in particular its phase. The reconstruction algorithm therefore uses incomplete information, which results in the appearance of noise on the reconstructed image.
    [0003] Such background noise can complicate the interpretation of the reconstructed images by digital propagation; it is important to reduce the influence by appropriate algorithms. To do this, the publication "Lensless phase contrast microscopy based on multiwavelenght Fresnel diffraction", Optics Letters Vol. 39, No. 2, January 15, 2014, describes an algorithm for improving the quality of reconstructed images. This publication describes a lensless imaging device based on the use of three light sources of different wavelengths (685 nm, 785 nm, 940 nm, respectively). The sample is illuminated successively by these three light sources. The photodetector then acquires as many images as sources of light, these images being formed in a plane, said plane of the detector, according to which the sensor extends. Each image has a wavelength.
    [0004] A first image, of a first wavelength, is retro-propagated, according to said first wavelength, in a plane according to which the object extends, said plane of the object, so as to obtain in this object plane, a first complex field. The phase of this first complex field, in the object plane, is multiplied by a ratio between the first wavelength and a second wavelength. This complex field is then propagated, according to said second wavelength, from the object plane to the detector plane, whereupon its module is replaced by the module of the image acquired at said second wavelength. It is then retro-propagated 5 in the plane of the object, for a second iteration. The iterative process continues until a convergence criterion is reached. The document WO2014035238, which some inventors are the authors of the publication mentioned above, includes the same teachings. The inventors propose an alternative method to that proposed in the previous publication. SUMMARY OF THE INVENTION A first object of the invention is a method of observation of a sample, comprising the following steps: i) illumination of said sample with the aid of a light source, capable of producing a wave light propagating along an axis of propagation. ii) acquiring, using a photodetector, a plurality of images of the sample, formed in a detection plane, the sample being disposed between the light source and the photodetector, each image being representative a light wave, transmitted by the sample, under the effect of said illumination, each image being acquired in a spectral band different from one another, the method being characterized in that it also comprises the following steps: iii) determining, from each image acquired according to each spectral band, an initial complex amplitude of the light wave transmitted, according to said spectral band, in said detection plane, from each complex amplitude established in said detection plane, according to a spectral band, determination of a complex amplitude of the transmitted wave, according to each spectral band, in a plane according to which the sample extends, v) combination of a urality of complex amplitudes determined in step iv), at different spectral bands, for calculating a weighting function in the plane of the sample, 3036800 4 vi) projection of said weighting function in the detection plane, so as to obtain, for each spectral band, a weighting function in said detection plane, vii) updating of each complex amplitude of the transmitted light wave, according to each spectral band, in the detection plane, at using said determined weighting function, in said spectral band, during step vi), viii) repeating steps iv to vii until a stop criterion is reached. Thus, in an alternative manner to the method explained in the prior art, each iteration comprises a propagation, called retro-propagation, of the plane of the detector towards the plane of the sample, of a plurality of complex amplitudes, in different spectral bands. These different complex amplitudes are combined, in the plane of the sample, to form a weighting function. This combination, in the plane of the sample, of a plurality of complex amplitudes, corresponding to different spectral ranges, has the effect of smoothing the noise affecting each of them, this noise being the consequence of the back propagation .
    [0005] The weighting function is then propagated from the plane of the sample to the detection plane, where it is used to form a new estimate of the complex amplitude of the light wave to which the sample is exposed, and this in each spectral band considered. According to one embodiment, the weighting function, in said sample plane, is calculated by calculating a weighted sum of different complex amplitudes, or their logarithm, in the plane of the sample, of the transmitted light wave, said complex amplitudes being respectively associated with different spectral bands. According to one embodiment, the weighting function in said sample plane is determined by calculating a weighted sum of the module and / or the argument of different complex amplitudes in the sample plane of the sample. transmitted light wave, said complex amplitudes being respectively associated with different spectral bands. The method may comprise any of the following features, taken alone or in technically feasible combinations: in step iii), the modulus of the complex amplitude of the transmitted wave in a spectral band is determined by normalizing the intensity of the image measured by the photodetector 16, in said spectral band, by a reference intensity measured by said photodetector in the absence of sample. In step iv), the determination of the complex amplitude in a plane of the sample, in a spectral band, is obtained by applying a propagation operator, along an axis of propagation, to said complex amplitude, defined in the same spectral band, in the detection plane. during step vi), said weighting function, in the detection plane, is obtained by applying a propagation operator, along the axis of propagation, to the weighting function, determined, in the plane of the sample, during step v). during step vii), the modulus of the complex amplitude of the transmitted light wave, in a spectral band, in the detection plane, is calculated as a function of the modulus of said initial complex amplitude, in said spectral band. During step vii), the argument of the complex amplitude of the light wave transmitted, according to a spectral band, in the detection plane, is calculated according to the argument of the determined weighting function, in said detection plane and at said spectral band, during step vi). During step v), said weighting function may be common to all of the spectral bands. Alternatively, this step v) may comprise the determination of a plurality of weighting functions, each weighting function being associated with a spectral band. The method may comprise, after step viii), step ix): forming an image representative of the module or the argument of the complex amplitude of the wave, transmitted by the sample, in the plane of the sample or in the detection plane, according to at least one spectral band (X,). Another object of the invention is a device for observing a sample comprising: a light source capable of illuminating said sample, a photodetector, the sample being placed between the light source and the photodetector, the photodetector being adapted to form a plurality of images, in a detection plane, of a light wave transmitted by the sample under the effect of illumination by said light source, each image being obtained according to a spectral band different from each other, a processor, able to process said plurality of images by executing instructions, programmed in a memory, implementing a method as previously described.
    [0006] FIGURES FIG. 1 represents a first example of a device for implementing the invention, the sample analyzed being an anatomopathology slide. FIG. 2 represents a first example of a device for implementing the invention, the sample analyzed being a body fluid comprising particles.
    [0007] Figure 3 shows the detection plane, on which an image is formed, as well as the plane of the sample. This figure also illustrates the links between the main quantities implemented in the various embodiments presented. FIG. 4 represents a logic diagram describing the sequence of the main steps of an iterative reconstruction process.
    [0008] FIG. 5 represents a second example of a device for implementing the invention, the sample analyzed being an anatomopathology slide. FIGS. 6A, 6B and 6C represent reconstructed images, in a plane of the sample, in a first spectral band, according to an iterative reconstruction algorithm, these images being respectively obtained following a number of iterations respectively equal to 1, 3 and 10.
    [0009] FIGS. 7A, 7B and 7C represent reconstructed images, in a plane of the sample, in a second spectral band, according to an iterative reconstruction algorithm, these images being respectively obtained following a number of iterations respectively equal to 1 , 3 and 10.
    [0010] FIGS. 8A, 8B and 8C represent reconstructed images, in a plane of the sample, in a third spectral band, according to an iterative reconstruction algorithm, these images being respectively obtained following a number of iterations respectively equal to 1, 3 and 10. FIGS. 9A, 9B and 9C show composite images obtained by combining the reconstructed images in the first, second and third spectral bands, these images respectively corresponding to 1, 3 and 10 iterations. These images are color images, represented here in black and white. DESCRIPTION OF PARTICULAR EMBODIMENTS FIG. 1 represents an exemplary device that is the subject of the invention. A light source 11 is able to produce a light wave 12, called the incident light wave, in the direction of a sample 10, along a propagation axis Z. The sample 10 may be a biological sample that it is desired to characterize. It may for example be a tissue slide, or anatomopathology blade, having a thin layer of tissue deposited on a transparent plate 15. By thin thickness is meant a thickness preferably of less than 100 μm. and preferably less than 10 μm, typically a few micrometers. Such a sample is shown in FIG. 1. It is observed that the sample extends along a plane Po, referred to as the plane of the sample, perpendicular to the propagation axis Z. The sample 10 can also comprise a medium 14, solid or liquid, comprising particles 1, 2, 3, 4, 5 to characterize, such a case being shown in Figure 2. It may for example be biological particles in a culture medium, or in a body fluid. By biological particle is meant a cell, a bacterium or other microorganism, a fungus, a spore ... The term particles may also mean microbeads, for example metal microbeads, glass microbeads or organic microbeads, commonly used implemented in biological protocols. It may also be insoluble droplets bathed in a liquid medium, for example lipid droplets in an oil-in-water emulsion. Thus, the term particle refers to both endogenous particles, initially present in the examined sample, and exogenous particles, added to this sample prior to analysis.
    [0011] In general, a particle has a size advantageously less than 1 mm, or even less than 500 μm, and preferably a size of between 0.5 μm and 500 μm. The distance between the light source and the sample is preferably greater than 1 cm.
    [0012] It is preferably between 2 and 30 cm. Preferably, the light source, seen by the sample, is considered as point. This means that its diameter (or diagonal) is preferably less than one-tenth, better one-hundredth of the distance between the sample and the light source. Thus, preferably, the light arrives at the sample in the form of plane waves, or can be considered as such.
    [0013] The light source 11 is capable of producing a plurality of incident light waves 121 12, each said light wave 12, extending along a Me spectral band X. The spectral bands 121 ... 12 are different from each other. others, and preferably do not overlap. In the exemplary device shown in FIGS. 1 and 2, the light source comprises three sources of light-emitting diode (LED) lights, 111, 112 and 113 emitting respectively in the light emitting diodes. spectral bands Xi = 450 nm -465 nm; X2 = 520 nm - 535 nm; X3 = 620 nm -630 nm. Preferably, there is no overlap between the different spectral bands; a negligible recovery, for example concerning less than 25%, better less than 10% of the emitted luminous intensity, is however possible. In these examples, the light source 11 comprises a multiple LED diode brand Cree (registered trademark) and reference XLamp (registered trademark) MC-E. This diode comprises four individually addressable elementary electroluminescent diodes, of which only three are used in the context of this invention, the fourth being a white LED.
    [0014] Other configurations of light sources 11 are possible and described later. These sources of elemental lights can be temporally coherent sources, such as laser diodes. The light source 11 is preferably punctual. It may in particular comprise a diaphragm 18, or spatial filter. The aperture of the diaphragm is typically between 30 μm and 1 μm, preferably between 50 μm and 500 μm, for example 150 μm.
    [0015] The diaphragm may be replaced by an optical fiber having a first end facing an elementary light source 111, 112 or 113, and having a second end facing the sample. The light source 11 preferably comprises a diffuser 17 disposed between each source 5 of elementary light 111, 112 and 113 and the diaphragm 18. The inventors have found that the use of such a diffuser makes it possible to overcome constraints centering each elemental source with respect to the opening of the diaphragm. In other words, the use of such a diffuser makes it possible to use an elementary light source 11 ,, with 1 i 3, slightly off-center with respect to the opening of the diaphragm 18. In this example, the diaphragm is supplied by Thorlabs under the reference P150S. Preferably, each elementary light source 11 is of small spectral width, for example less than 100 nm, or even 20 nm. The term spectral width refers to the width at half height of the emission band of the light source considered. In this example, the diffuser used is a 40 ° diffuser (Light Shaping Reference Diffuser 40 °, manufactured by Luminit). The function of such a diffuser is to distribute the light beam produced by a source of elemental light 11 ,, according to a cone of angle a, a being equal to 40 ° in this case. Preferably, the diffusion angle α varies between 10 ° and 60 °. The sample 10 is disposed between the light source 11 and a matrix photodetector 16. The latter preferably extends parallel to, or substantially parallel to, the transparent plate 15 supporting the sample. The term substantially parallel means that the two elements may not be strictly parallel, an angular tolerance of a few degrees, less than 10 ° being allowed. The photodetector 16 is an imager capable of forming an image according to a detection plane P.
    [0016] In the example shown, it is a matrix photodetector comprising a matrix of pixels, of the CCD type or a CMOS. CMOS are the preferred photodetectors because the size of the pixels is smaller, which makes it possible to acquire images whose spatial resolution is more favorable. In this example, the detector is a CMOS sensor marketed by Omnivision under the reference 0V5647. This is a CMOS RGB sensor, comprising 2592 x 1944 pixels, with an inter-pixel pitch of 1.4 μm. The effective area of the photodetector is 3.6 x 2.7 mm2.
    [0017] The detection plane P preferably extends perpendicularly to the propagation axis Z of the incident light wave 12. Preferably, the photodetector comprises a matrix of pixels, above which a protection window is arranged. transparent. The distance between the pixel matrix and the protection window is generally from a few tens of μm to 150 or 200 μm. The photodetectors whose inter pixel pitch is less than 3 μm are preferred in order to improve the spatial resolution of the image. The photodetector may include a mirror image return system to a pixel array, in which case the detection plane corresponds to the plane along which the image return system extends. In general, the detection plane P corresponds to the plane in which an image is formed. The distance d between the sample 10 and the pixel matrix of the photodetector 16 is, in this example, equal to 300 μm.
    [0018] In general, and irrespective of the embodiment, the distance d between the sample and the pixels of the photodetector is preferably between 50 μm and 2 cm, preferably between 100 μm and 2 mm. Note the absence of magnification optics between the photodetector 16 and the sample 10. This does not prevent the possible presence of microlenses focusing at each pixel of the photodetector 16, the latter having no function of magnification of the image. FIG. 3 represents a sample 10, comprising diffracting objects 32 arranged around non-diffracting or little diffracting zones 31, described hereinafter as poor zones. The sample may be solid, for example in the case of tissue deposited on an anatomopathology slide. It can also be liquid, for example in the case of a body fluid or a cell culture medium. The photodetector 16 is capable of producing an image I of a light wave 22, transmitted by the sample 10 when the latter is illuminated by an incident wave 12, in the i th spectral band X. The spectral band of the transmitted light wave 22, comprises all or part of the spectral band of the incident wave 12,. The light wave 22, transmitted by the sample in the spectral band X, results from the interaction of the sample 10 with the incident light wave 12 ,, produced by the elementary light source H 1. Under the effect of the incident light wave 12 ,, the sample 10 can generate a diffracted wave 5, capable of producing, at the level of the detection plane P, interference, in particular with a part of the incident light wave 12, transmitted by the sample. These interferences give rise, on the image acquired by the photodetector, to a plurality of elementary diffraction patterns, each elementary diffraction figure comprising a central zone and several concentric diffraction rings. Each elementary diffraction pattern is due to a diffractive object 32 in the sample. Moreover, the sample can absorb a portion of the incident light wave 12. Thus, the light wave 22, in a spectral band X, transmitted by the sample, and to which the matrix photodetector 16 is exposed, can comprise: a component resulting from the diffraction, previously described, this diffraction component 15 being able especially to result in the presence of elementary diffraction patterns on the photodetector 16, each elementary diffraction pattern may be associated with a diffractive element 32 of the sample. Such a diffractive element may be a cell, or a particle or other diffractive object 32 present in the sample 10, a component resulting from the absorption of the incident light wave 12 in the sample. A processor 20, for example a microprocessor, is able to process each image generated by the matrix photodetector 16. In particular, the processor is a microprocessor connected to a programmable memory 22 in which is stored a sequence of instructions for performing the operations. image processing and calculations described in this description. The following are the steps of an iterative method for obtaining an image of the sample 10, in connection with FIGS. 3 and 4. Pre-step: initialization 30 In a first step 100 of acquisition of 1, each elementary light source 11, of the light source 11 is successively activated, each light source producing an incident light wave (121, ..
    [0019] 12N), in a spectral band (X1, ... XN), along a propagation axis Z, in the direction of the sample 10. At each acquisition, the matrix photodetector collects an image 1, corresponding to an X spectral band, the index i, relating to the spectral band, being between 1 and N, N being the number of spectral bands considered. In the example shown in FIGS. 1 and 2, the light source 11 comprises three elementary light sources 111, 112 and 113, the photodetector collects three images li, 12, 13, respectively corresponding to the spectral bands Xi, X2 and X3. . The sample is placed at an axial coordinate z = 0, along the axis of propagation Z. We denote by r a radial coordinate, that is to say a coordinate in a plane perpendicular to the axis of propagation Z The plane z = d corresponds to the plane of detection, while the plane z = 0 corresponds to a plane passing through the sample, referred to as the plane of the sample, and denoted by Po. If Id (r) = I (r) designate the value of the intensity collected, in the spectral band X, by the pixel of the radial coordinate detector r in the detection plane, it is possible to establish, with the aid of the image 1, a complex amplitude ard (r) = a "I (r) of the wave 22, at said pixel of coordinate r, whose module can be expressed according to the expression: the >> 1 = ii>) The exponent d expresses the fact that the complex amplitude is determined according to the plane P of the sample, of equation z = d The complex amplitude a "I (r) comprises a module and an argument, such that: r) = (r) e (r) where: (r) denotes the modulus of the complex amplitude of the light wave detected by the photodetector, in the Me spectral band X ,, at a radial coordinate r in the detection plane; d (pi (r) denotes the phase of the complex amplitude of the light wave detected by the photodetector, in the Me spectral band X, and said radial coordinate r in the detection plane.
    [0020] However, the matrix photodetector does not provide any information relating to the phase of the light wave. Also, during step 100, it is considered that e1cil (r) is equal to an arbitrary initial value, for example equal to 1. The complex amplitude a (r) can be expressed in a standardized manner, according to the expression: (R) Act (r) = 'line where: Irne an designates the average intensity of the light emitted by the light source 11 in the Me band spectale X; this average intensity can be determined experimentally, by arranging the photodetector 16 facing the light source 11, 10 without a sample interposed between one and the other, and by calculating the average of the pixels of the image acquired by the photodetector 16 - Act (r) designates the normalized complex amplitude of the light wave 22, detected by the matrix photodetector 16 in the Me spectral band X. The normalization can also be performed by dividing the complex amplitude a "I (r) by 15 irne an) this term represents the luminous intensity, at the radial coordinate r, measured in the absence of a sample.The normalized complex amplitude Act (r) comprises a module and an argument, such that: (r) = (r) e (r) where: m (r) denotes the modulus of the normalized complex amplitude Aq (r) - 1 1 d (f) (r) denotes the phase of the standardized complex amplitude which is also the phase of the complex amplitude a "I (r). The first step 100 makes it possible to assign to each complex amplitude a I (r) or to each normalized complex amplitude Act (r) an initial value from the image I, detected by the photodetector in the Me spectral band X , so that: d = (r) = il>) 3036800 d I (r) A = 1 (r) = mi (r) = imean The index p is the iteration range of the iterative process described Step 100 being an initialization step, the value 1 is assigned to this index.
    [0021] By addressing all or part of the pixels r of the photodetector 16, a complex image, or complex field, of the light wave 22 at the plane of the detector is obtained, this image bringing together the complex amplitudes afi (r) or the amplitudes Act (r) standardized complexes. In the remainder of the description, only the normalized complex amplitude Act I (r) is considered, since the reasoning also applies to the complex amplitude a (r).
    [0022] This first step is repeated for each spectral band (Xi ... XN) detected by the photodetector. Step 2: Retropropagation in the Po plane of the sample During a second step 200, the normalized complex amplitude 11 , P (r) of the wave 22, to which the detector is exposed, is estimated in FIG. sample plane Po. This estimation is carried out by backpropagation of the normalized complex amplitude Ac p (r), determined in the detection plane P, from the detection plane P to the plane of the sample Po. index p designates the rank of the iteration. During the first iteration (p = 1), the normalized complex amplitude Acilp = i (r) = Act I (r) obtained after the first step 100 is used. During the following 20 iterations (p> 1), uses the complex amplitude resulting from the previous iteration, as will be detailed later. According to the well-known principles of digital holographic reconstruction, by carrying out a product of convolution between the complex amplitude of the light wave 22 ,, relating to the spectral band X, determined in the detection plane z = d, and an operator propagation time 25 h (r, z), it is possible to reconstruct a complex amplitude of this same light wave at any point of coordinates (r, z) of the space, and in particular in the plane Po of the sample. In other words, the normalized complex amplitude A 1 p (r) of the light wave 22 can be obtained at a coordinate point (r, z) from A 1p = d (r), depending on the operation : 14 OR 3036800 15 A (r) = (r) * hAi (r, z-d), where ha, denotes the propagation operator in the spectral band X. When the reconstruction is carried out according to the direction the propagation of the light, for example from the sample to the photodetector, is called propagation. When the reconstruction 5 is carried out in the opposite direction of the propagation of light, for example from the photodetector to the sample, it is called back propagation. The propagation operator can in particular be based on the Fresnel diffraction model. In this example, the propagation operator is the Fresnel-Helmholtz function, such that: zh (r, z) = 7.1z eJ`77, exp (jn- Az where X denotes the wavelength. , p = (r) = 119.3) (r) = Ad (r) * hAi (r, -d) 1 if d (r - r ') 2 = - id - e Ai, p (r) where r 'denotes the radial coordinates in the plane of the photodetector (z = d), r denotes the radial coordinates in the reconstruction plane (z = 0), - X, denotes the length of the central wave of the spectral band considered. A (r) is thus obtained by backpropagation of Acil, p (r) according to the distance d separating the detection plane P from the plane of the sample Po.
    [0023] This second step is repeated for each spectral band (Xi ... XN) emitted by the light source 11 or, more generally, for each spectral band (Xi ... XN) respectively associated with each image (11. ..IN) detected by the photodetector 16. It is possible, at this stage, to establish an image of the modulus or phase of the complex amplitude A (r) of each light wave 22, in the plane of the sample Po, the complex amplitude being normalized or not, by calculating the value of A (r) at the various coordinates r in the plane of the sample. Each image of the complex amplitude module A (r) is representative of the intensity of the light wave at the sample, while each image of the 3036800 argument 16 the complex amplitude A (r) is representative of the phase of the intensity of the light wave at the sample level. When, as in the present case, three spectral bands centered respectively on wavelengths in blue, green and red are used, the information contained in the three images makes it possible to obtain a color image. of the sample. Note that the normalized complex amplitude 11 P (r) is equivalent to a transmission function of the incident wave 12, by the sample 10 at the radial coordinate r. Step: Determination of the Weighting Function In step 300, in the sample plane, a weighting function, Fpc (r), of the complex amplitude of the wave is formed. light transmitted by the sample in the different spectral bands X, considered. According to this example, the weighting function Fpc) (r), in the plane of the sample, can be common to each spectral band. It is obtained by combining the normalized complex amplitudes 11, p (r) of the light wave transmitted by the sample, in the plane Po of the sample and in the different spectral bands X. According to one example, the function of weighting is obtained by a weighted sum of each complex amplitude determined during step 200, in the Po plane of the sample, according to the expression 1 Fp ° (r) = 20 where 1 <denotes a weighting factor associated with the Me spectral band X. The weighting factors may be equal to each other, for example equal to 1/3. Other ways to determine the weighting function in the sample plan are detailed below. Step 4: Propagation of the weighting function in the plane of the detector Step 400 aims to propagate, from the plane of the sample to the plane of the detector P, the weighting function Fpc) (r), determined, during the previous step, in the plane of the sample Po.
    [0024] The propagation operator being dependent on the wavelength, this propagation is carried out for each spectral band X, considered. Thus, for each spectral band X ,, Fid, p (r) = F ° (r) * z = d). When the propagation operator is a Fresnel-Helmholtz operator as previously defined, (r - r ') 2 1 j2n-e ± F (r) = j jide Ai, if (r') exp (jn-Àtd The wavelength dependent propagation operator determines as many weighting functions in the detection plane as spectral bands considered. r 'designates the radial coordinates in the plane of the sample (z = 0), r denotes the radial coordinates in the reconstruction plane, that is to say the detector plane (z = d), X, denotes the central wavelength of the spectral band considered. Step 1: Updating the Complex Amplitude in the Detector Plane In step 500, the value of the weighting function is used in the detection plane z = d to update the detector. estimation of the normalized complex amplitude A (r) of the light wave 22 ,, to which the photodetector 16 is exposed, in the spectral band X. The update formula is: ice -d (r) = m F (r) c- (r) x 'P = M (r) x e-19I, P (r) t, p Fice, p (r) e where: IFici, p (r) 1 denotes the modulus of Fid, p (r); m (r) denotes the modulus of the initial normalized complex amplitude 4 = 1 determined, from the image h, during the first step 100. This term has a function of attachment to measured data; 25 - designates an estimate of the phase of the complex amplitude of the wave 22, in the Me spectral band X, 3036800 18 A (r) designates the complex amplitude of the light wave 22i transmitted by the sample, in the plane of the photodetector 16, this complex amplitude constituents t the basis of the next iteration.
    [0025] Following this step, a new iteration can begin, the input data of this new iteration p + 1 being it + i (r) = it (r), this new iteration starting with the back propagation of each standardized complex amplitude il + i (r), for the different spectral bands considered, in the plane Po of the sample, according to step 200.
    [0026] The steps 200 to 500 are carried out iteratively, either according to a predetermined number of iterations pmax, or until a convergence criterion is reached, the latter being able, for example, to be expressed in the form of a difference between the estimation of two same quantities between two successive iterations. When this difference is below a given threshold s, the convergence criterion is reached. For example, the process is stopped when one of these conditions is reached F, d, p (r) Fi (r) <E; F (r) F, c1, p + (r) Fi P (r) -F P + 1 (r) <E; Aci "p + 1 (r) 1 <E, Ar g (Ap (r) - Ap + 1 (r)) <E; 20 This list is not limiting. At the end of the process, we have an estimate of the complex amplitude of the light wave 22, transmitted by the sample, and to which the photodetector is exposed, in the plane of the detector P, of equation z = d and / or in the plane of the sample Po, of equation z = 0, and this for each spectral band considered.Using the different reconstructed complex amplitudes A (r) in the plane of the sample, we obtain a precise representation of the latter, in each spectral bands considered, in particular by forming images from the module or the phase of said complex amplitudes.
    [0027] As previously mentioned, when the spectral bands are distributed in the visible spectrum, the images of the module or of the phase can be combined, for example superimposed so as to obtain color representations.
    [0028] It is recalled that this algorithm, although described in relation to a normalized complex amplitude Ai, also applies to the non-normalized complex amplitude ai. Contribution of the weighting function One of the important points of this iterative algorithm is the constitution of the weighting function P (r) in the plane of the sample. Indeed, in general, the determination of the complex amplitude of a light wave from an image acquired by a photodetector is insufficient because the information as to the phase of the wave is not recorded. by the photodetector, the latter being sensitive only to the intensity, corresponding to the modulus of the complex amplitude of the wave.
    [0029] Thus, as indicated in the description of step 100, the complex amplitude a "1 (r) or the normalized complex amplitude Act I (r) determined during this step do not include information as to the phase of the light wave they represent This lack of information is reflected, during the retro-propagation of the plane of the detector P to the plane of the sample Po, which is the subject of step 200, by the formation of artifacts designated by the term "twin image." The inventors have found that these artifacts mainly affect the poor areas 31 arranged in the vicinity of diffracting elements 32, that is to say the areas between two Adjacent diffracting elements 32. In addition, they found that these artifacts are susceptible to fluctuation with wavelength, thus combining the complex amplitudes in different wavelengths, backpropagated in the sample plane. , artifacts, at level of poor areas 31, are statistically This statistical smoothing then increases the signal-to-noise ratio of the retrospectively complex image in the plane of the sample. In general, the method amounts to: obtaining an initial estimate A1 (r) of the complex amplitude of the wave 22, transmitted by the sample, in the plane of the detector, and this in several spectral bands ( step 100); To retransmit each of these complex amplitudes in the plane of the sample, to obtain, at each spectral band, a complex amplitude in the plane of the sample 11 (r) (step 200) constitute a weighting function of each complex amplitude F ° (r) in the plane of the sample (step 300), so as to reduce the influence of twin image interfacies; propagating said weighting function in the plane of the detector, for at least one spectral band, (step 400); update the estimation of the complex amplitude 11` p (r) of the wave 22, transmitted by the sample, in the plane of the detector, and this in several spectral bands, using the function weighting Fidp (r) propagated in the plane of the detector (step 500). The updating formula of step 500 shows that, at each iteration, the modulus m (r) (respectively Ki (r)) of the normalized complex amplitude A (r) (respectively of the complex amplitude afip (r)), in the detection plane, corresponds to the one determined, in step 100, by each image I, formed by the photodetector 16 in the spectral band X. In other words, during the different iterations, the in the detection plane, the complex amplitude a "I (r) or the normalized complex amplitude A (r) does not change and corresponds to that derived from the intensity measured by the photodetector.
    [0030] On the other hand, the algorithm tends to change, at each update, the argument of the expressions dd - d complex A (r) or a - (r) and in particular the estimation of the phase (f) ( r) the latter t, pt, p being considered equal to the phase of the weighting function Fidp (r) propagated in the plane of the detector, at each wavelength X.
    [0031] Also, according to this algorithm, each iteration comprises: updating the complex amplitude A (r) of each light wave in the plane of the sample Po (step 200); an update of the argument of each complex amplitude, A (r) and in particular of its phase, in the detection plane (step 500).
    [0032] 3036800 21 Elaboration of the weighting function. A first way of constituting the weighting function is to perform a fair weighting between the different spectral bands X, considered. For example, the weighting function may take the form F ° (r) = k, 11 (r), where k, E, 5 denotes the weighting factor, or weight, assigned to the Me spectral band X, as previously described. in connection with step 300. Each weighting factor k can have the same value, for example 1/3. According to one variant, and this applies in particular in the case where the sample analyzed is colored, according to a spectral range Xo, the modules of the complex amplitudes of first light waves 22i whose X spectral bands are close to the spectral range Xo have a higher value than the modules of the complex amplitudes of second light waves whose spectral bands are farther from the wavelength Xo. In such a case, it is preferable to underweight the complex amplitudes of the first light waves, and to overweight the complex amplitudes of the second light waves.
    [0033] For example, if the sample is stained with a blue dye, which in our example corresponds to the first spectral band Xi, the weighting factor 1 (1 is lower than the weighting factors k2 and k3 respectively associated with the spectral bands X2 (green) and X3 (red).
    [0034] According to another variant, the module and the argument of each complex amplitude are weighted by independent weighting factors, such that 1 1 Fpc) (r) 1 = ki 111, p (r) 1 L K1 i Arg (Fpc) (r)) = k ', Arg (11 (r)) k, and k', being weighting factors respectively associated with the modulus and the argument 25 of the complex amplitude of the light wave 22 , in the sample plane, in the spectral band X. According to another variant, the combination of the complex amplitudes A (r) takes the form of a sum of logarithms, according to the expression: ln (Fpc) ( r)) = E, k, ln [A (,), p (r)].
    [0035] According to another variant, in the plane of the sample, a weighting function Fpc) (r) is not determined, but a plurality of weighting functions Fi ° p (r), each function being associated with a spectral band X. Each weighting function Fi ° p (r), associated with a Me wavelength, is obtained by combining several complex amplitudes 11 (r), respectively associated with different spectral bands. According to a first example, considering three spectral bands: k1,2 0 I k2,2 k1,3 1 [Al, p k3,2 k2,3A (r) k3,3 (r) 10 Thus, according to this mode of realization, the weighting function takes the form of a vector F ° (r), of dimension N, N being the number of spectral bands considered, each term of which (r) is a weighting function associated with an X spectral band. This weighting function can be obtained by the following matrix product: Fp ° (r) = K Aci; Where K is a weighting matrix, each term k ,,, of the weighting matrix representing the weight associated with the complex amplitude Alf3p (r) associated with the spectral band X, for calculating the weighting function associated with the spectral band X. The matrix K is a square matrix of dimension N x N, N being the number of spectral bands considered.
    [0036] The weighting function is preferably normalized, so that each term Fi% is expressed as: Fi 1 p (r) = 119 (r) E c I ed, the term -i --- eta nt a normalization term. According to a second example of this embodiment, again considering three spectral bands, 3036,800 IFT, p (r) 1 -k1,1 k1,2 23 k1,4 k2,4 k1,5 k2,5 k1, 6 k2.6 (r) 1 (r) 1 k2.1 k2.2 k 1, 3 k2.3 k 3, 3 k4.3 k 5, 3 k6.3 k 3, 4 k 3, 5 k 3, 6 (r) 1 (r) 1 arg (n, p (r)) arg (n, p (r)) arg (Ft (r)) k3.1 k3.2 k4.4 k 5.5 k4, 6 (r) 1 arg (IIi), p (r)) arg (11, p (r)), arg (np (r)) k4.1 k4.2 k 5.5 k6.5 k 5, 6 k5,1 k5,2 k6,4 k6,6- _k6,1 k6,2 Thus, according to this embodiment, the weighting function takes the form of a vector Fpc) (r), dimension 2N, N being the number of spectral bands considered, each term representing either the module or the argument of a weighting function F (r) associated with a spectral band X. This weighting function can be obtained by the following matrix product:))> Fp (r) = K A3> p Where K is a weighting matrix, of dimension 2N x2N, each term k ,,, of the weighting matrix representing the weight associated with either the argument or the phase, of the complex amplitude A ij3, p (r) associated with the spectral band 4. According to this embodiment, each coordinate of the vector Ai °, represents either the module or the argument, of a complex amplitude, A (r) in a spectral band j.
    [0037] As in the example, the weighting function is preferably normalized, so that each term ep is expressed as: 3 1 (r) 1 = 3 ki'i 1AJ ° 'p (r) 1 j = 1 and 6 Arg (F 10, p (r)) = z, = 14 arg (Ac 1, P (r)) Whatever may be the case, the coefficients of a weighting matrix can be determined beforehand either arbitrarily or based on experimental tests. For example, it is possible to establish a linear regression coefficient at between two components i and j of the vector A (r), considering a plurality of axial positions (r) in the plane of the sample, to obtain sufficient statistics. The coefficient ku of the weighting matrix is then to be determined as a function of this linear regression coefficient at, possibly affected by a term taking into account the dispersion around the linear regression model. In such a case, the diagonal of the weighting matrix can consist of coefficients k 'equal to 1. This makes it possible to establish a weighting function ep, associated with the wavelength λ, taking into account the correlation between the different terms of the vector A (r).
    [0038] 10 Variants concerning the light source or the photodetector. In the examples given with reference to FIGS. 1 and 2, the light source 11, able to emit a light wave 12 in different spectral bands, comprises three sources of elementary lights 111, 112, 113, taking the form of light-emitting diodes, emitting respectively in a first spectral band Xi, a second spectral band X2 and a third spectral band X3, the spectral bands being different from each other, and preferably do not overlap. The light source 11 may also include a white light source 11 placed upstream of a filtering device 19, for example a filter wheel, capable of interposing a bandwidth filter X between the white light source. and the sample, as shown in FIG. 5, so that the image I, formed by the photodetector 16 is representative of said bandwidth X. Several filters, having different passbands from each other, are then successively interposed between the light source 11 ,, and the sample 10. Alternatively, the filter device 19 may also be a tri-band filter, defining a plurality of spectral bands. An example of a filter suitable for the application is the Tri-Band filter 458, 530 & 628 nm - Edmund Optics, defining spectral bands respectively centered on the wavelengths 457 nm, 530 nm and 628 nm. This allows illumination of the sample simultaneously using 3 wavelengths. The use of a diffuser 17, as previously described, between the light source and the diaphgrame 18 is preferable, and this whatever the embodiment.
    [0039] The photodetector 16 may, as previously described, be an RGB matrix photodetector, which makes it possible to acquire successively or simultaneously different images in different spectral bands. In this case, the light source may be a white light source 11 ''. in which case the different images can be acquired simultaneously.
    [0040] It can also be a monochromatic photodetector 16, in which case the light source 11 is able to generate, successively, a light wave in different spectral bands. In such a configuration, the light source comprises either several sources of elemental lights. 111, 112, 113, or a filter device 19, as previously described. In such a case, the sample is successively exposed to incident light waves 121.12, ... 12N, where N is the number of spectral bands considered. An image I, fflN), representative of the light wave 22, transmitted by the sample is then acquired at each exposure.
    [0041] 15 Tests performed. Tests were performed according to the configuration shown in Figure 1 and described above. The sample is an anatomo-pathology slide, with a section of colon stained by Hematoxylin Eosine Safran. The light source is disposed at a distance equal to 5 cm from the sample, this distance 20 separating the diaphragm 18 from the sample 10. FIGS. 6A, 6B and 6C represent an image of the module lep (r) 1 of the complex amplitude A ° 1 (r) of the wave 221 transmitted by the sample, in the plane Po of the sample, in the first spectral band X extending between 450 and 465 nm, these images being obtained after a number of iterations p respectively equal to 1, 3 and 10.
    [0042] FIGS. 7A, 7B and 7C show an image of the module 111, p (r) 1 of the complex amplitude of the wave 222 transmitted by the sample, in the plane Po of the sample, in the first band spectral X2 extending between 520 and 535 nm, these images being obtained after a number of iterations p respectively equal to 1, 3 and 10.
    [0043] FIGS. 8A, 8B and 8C show an image of the module Inp (r) 1 of the complex amplitude np (r) of the wave 223 transmitted by the sample, in the plane Po of the sample, in the first X3 spectral band extending between 620 and 630 nm, these images being obtained after 3036800 26 a number of iterations p respectively equal to 1, 3 and 10. Note that the average gray level of these images is greater than the gray level of the images of FIGS. 6A, 6B, 6C, 7A, 7B and 7C. This is due to the fact that the sample has the red-violet coloring of the sample.
    [0044] Figs. 9A, 9B and 9C show the combination of images 6A-7A-8A, 6B-7B-8B, respectively; 6C-7C-8C. These figures make it possible to have a color representation of the sample, while simultaneously taking into account the three spectral bands Xi, X2 and X3. On each series of images, an increase in contrast is observed as a function of the number of iterations. . One can also see the formation of images whose spatial resolution is satisfactory when the number of iterations is less than or equal to 10, which limits the calculation time to a few seconds. The method is therefore suitable for the observation of samples in a wide field and at high rates.
    [0045] It makes it possible to obtain images in one or more spectral bands, making it compatible with the dyeing methods commonly practiced in the field of anatomocytopathology. 20
    权利要求:
    Claims (12)
    [0001]
    REVENDICATIONS1. Method for observing a sample (10), comprising the following steps: i) illuminating said sample with the aid of a light source (11), capable of producing a light wave (12) propagating along an axis for propagating (Z), acquiring, using a photodetector (16), a plurality of images (11 ... IN) of the sample, formed in a detection plane (P), sample being disposed between the light source (11) and the photodetector (16), each image being representative of a light wave (22i) transmitted by the sample under the effect of said illumination, each image (I,) being acquired according to a spectral band (X,) different from one another, the method being characterized in that it also comprises the following steps: iii) determining, from each acquired image (I,) according to a spectral band (XI of an initial complex amplitude (111 (r)) of the transmitted light wave (22,), according to said spectral band, da ns said detection plane (P), iv) from each complex amplitude (Agp = i (r), 4 (r)) established in said detection plane (P), according to a spectral band (XI determination of a complex amplitude (11 p (r)) of the transmitted wave (22i), according to each spectral band, in a plane Po according to which the sample extends, v) combination of a plurality of complex amplitudes (11) p (r)) determined in step iv), at different spectral bands, to calculate a weighting function (Fp ° (r), Fp ° (r)) in the sample plane (Po) , vi) projecting said weighting function (Fp ° (r), F ° (r)) into the detection plane (P) so as to obtain, for each spectral band (XI, a weighting function (F1 ( r)) in said detection plane (P), vii) updating each complex amplitude (11 (r)) of the transmitted light wave (22,), according to each spectral band (XI in the detection plane ( P), using said weighting function (Fidp (r)) obtained, in said spectral band (XI during step vi), viii) repeating steps iv to vii until a stop criterion is reached. 3036800 28
    [0002]
    The method according to claim 1, wherein the weighting function, in said sample plane (Fpc) (r), Fpc) (r)) is calculated by calculating a weighted sum of different complex amplitudes (11 , P ( r)), or their logarithm, in the plane of the sample (P0), of the transmitted light wave (22i), said complex amplitudes A (r) being respectively associated with different spectral bands (X,) .
    [0003]
    The method according to claim 1, wherein the weighting function in said sample plane (Fpc) (r), Fip (r)) is determined by calculating a weighted sum of the modulus (111 , P (r 1) and / or the argument (arg (11 , p (r)) of different complex amplitudes (11 , p (r)), in the plane of the sample (P0), of the transmitted light wave (22i), said complex amplitudes 11 , p (r) being respectively associated with different spectral bands (X,).
    [0004]
    A method according to any one of the preceding claims, wherein, in step iii), the modulus (rnp (r)) of the complex amplitude (4 (r)) of the transmitted wave (22, ) in a spectral band (X,) is determined by normalizing the intensity (I, (r)) of the image (I,) measured by the photodetector 16, in said spectral band, by a reference intensity (I0 (r)) measured by said photodetector 16 in the absence of sample.
    [0005]
    5. Method according to any one of the preceding claims, wherein, in step (iv), the determination of the complex amplitude (A P (r)) in a plane of the sample (P0), in a spectral band (XI is obtained by applying a propagation operator (h), along an axis of propagation (Z), to said complex amplitude (11 (r)), defined in the same spectral band (XI in the plane detection (P).
    [0006]
    6. Method according to any one of the preceding claims, in which, during step 25 vi), said weighting function (Fpdi (r)), in the detection plane (P), is obtained by applying a propagation operator (h), along the axis of propagation Z, to the weighting function (Fpc) (r), Fpc) (r)) determined in the plane of the sample (P0) during step v).
    [0007]
    7. Method according to any one of the preceding claims, in which, during step (vii), the modulus (111gp (r) 1) of the complex amplitude (11 (r)) of the transmitted light wave 3036800 29 (22), according to a spectral band (XI in the detection plane (P), is calculated as a function of the modulus (1111 (r) 1) of said initial complex amplitude (il'ilp = i (r)) in said spectral band (X,).
    [0008]
    The method according to any one of the preceding claims, wherein, in step (vii), the argument of the complex amplitude (11 (r)) of the transmitted light wave (22,), according to a spectral band (XI in the detection plane (P) is calculated according to the argument of the weighting function (Fidp (r)) determined in said detection plane and to said spectral band (XI during step vi).
    [0009]
    9. Method according to any one of the preceding claims, wherein, in step v), said weighting function (Fpc) (r)) is common to all of the spectral bands.
    [0010]
    The method of any one of claims 1 to 8, wherein step v) comprises determining a plurality of weighting functions (Fpdi (r)), each weighting function being associated with a spectral band. (X,).
    [0011]
    11. The method as claimed in any one of the preceding claims, comprising, following step viii), step ix): forming an image representative of the module or the argument of the complex amplitude (11 ( r), 111 , p (r) 1) of the wave (22), transmitted by the sample (10), in the plane of the sample (Po) or in the detection plane (P), in at least one spectral band (X,). 25
    [0012]
    12. A device for observing a sample (10) comprising: a light source (11), able to illuminate said sample, a photodetector (16), the sample being arranged between the light source (11) and the photodetector (16), the photodetector being adapted to form a plurality of images (11..1, ... IN), in a detection plane (P), of a light wave (22,) transmitted by the sample under the effect of 3036800 illumination by said light source, each image being obtained in a spectral band (X,) different from one another, a processor (20) capable of processing said plurality of images by executing instructions, programmed in a memory (22), implementing a method according to one of claims 1 to 11. 10
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1554811|2015-05-28|
    FR1554811A|FR3036800B1|2015-05-28|2015-05-28|METHOD OF OBSERVING A SAMPLE|FR1554811A| FR3036800B1|2015-05-28|2015-05-28|METHOD OF OBSERVING A SAMPLE|
    PCT/FR2016/051250| WO2016189257A1|2015-05-28|2016-05-26|Method for observing a sample|
    US15/577,129| US10564602B2|2015-05-28|2016-05-26|Method for observing a sample|
    EP16733151.1A| EP3304214A1|2015-05-28|2016-05-26|Method for observing a sample|
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