Microscope for quantitative measurement of a wavefront, module and kit for a microscope, method and computer program for computational reconstruction of a wavefront - Patents.com

Abstract

The present invention relates to a microscope for quantitative measurement of wavefronts, comprising: - means for illuminating a sample (T), objective lens (2), and spacing p greater than 500 μm. μ The present invention relates to a microscope comprising a regular two-dimensional array of lenses (3) having an aperture ratio of less than 10, an image sensor (4) positioned in a capture space (Ec) for receiving light scattered by a sample (T) and acquiring spatial and angular information relating thereto to the object wavefront, and a computational entity that performs computational reconstruction of the object wavefront from the spatial and angular information. Other aspects of the present invention relate to methods adapted for carrying out the functions of the computational entity of the microscope, computer programs and products incorporating the same, and modules and kits for microscopes.

Description

[Technical field]

[0001] In a first aspect, the present invention relates to a microscope for quantitative measurement of a wavefront, the microscope comprising an ordered two-dimensional array of lenses designed to enable measurement of subtle and non-trivial variations in the wavefront from a biological sample, or obtaining high-resolution 3D images, including from a microscopic sample.

[0002] A second aspect of the invention relates to a method for the computational reconstruction of a wavefront, in which a computational entity of a microscope is adapted to perform the functions for which it is configured.

[0003] In a third aspect, the invention relates to a computer program for the computational reconstruction of a wavefront, comprising program instructions which, when executed on a processor, implements the method of the second aspect.

[0004] In a fourth aspect, the invention relates to a computer program product comprising a tangible medium on which is stored a computer program of the third aspect.

[0005] A fifth aspect of the invention relates to a module for a microscope, connected to a camera port of the microscope, for constructing the microscope of the first aspect of the invention.

[0006] A sixth aspect of the invention relates to a kit for a microscope comprising the module of the fifth aspect and an illumination module to be connected to an illumination port of the microscope. [Background technology]

[0007] Computational reconstruction of the wavefront is a problem of particular interest in optical microscopy, since it provides information about the bright field emitted by a 3D sample, i.e. a quantitative measurement of the wavefront. Currently, this kind of reconstruction is achieved by two types of microscopes: 1) Microscopy based on interferometric detection of wavefronts by holographic processes. This type of system presents the disadvantages of its low stability and the fact that only monocular information is obtained. 2) A microscope having a structure similar to that of a wavefront sensor based on the Shack-Hartmann (HS) principle, i.e. including the features defined in the preamble of claim 1 of the present invention, including those disclosed in the following patents: US9726875B2, US9658443B2, US9679360B2 and US9976911B2.

[0008] In said patents different techniques are proposed aiming at obtaining a spatial resolution which in US9726875B2 is at least acceptable in order to obtain a good approximation of the actual bright field, in US9658443B2 is increased but limited by the diffraction limit, in US9679360B2 is improved by obtaining a composite image combining a spatial intensity image with a bright field image, or in US9976911B2 is good but does not adversely affect the angular resolution, i.e. does not satisfy a compromise between spatial and angular resolution.

[0009] Each of the proposals made in said patents achieves a certain improvement in the spatial resolution of the microscope proposed therein, but said improvements clearly leave room for further improvement, since the achieved spatial resolution improvements have the limitations of systems based on Shack-Hartmann sensors, whose resolution is limited by the number of microlenses (independent of the spatial resolution of the pixelated sensor). Therefore, to optimize the HS sensor, the maximum possible number of microlenses must be used and its size must therefore be reduced to a minimum. However, two main limitations exist in said process: 1) Size of the diffraction spot: By illuminating a microlens with a localized plane wave, the light is focused onto a sensor (located at the image focal plane of the microlens) and forms a diffraction spot. The position of this spot relative to the center of the microlens can be related to the tilt angle of the localized plane wave. The diameter of the spot φ for a particular illumination wavelength λ dif is the following formula:

number

number

[0010] On the other hand, to be able to effectively sample a diffraction spot in order to calculate its centroid and therefore its relative displacement, said spot must occupy at least 4 pixels according to the Nyquist criterion, in this case a size of 4 pixels is the optimum value, since said value allows effective sampling of the spot by the sensor and thus optimizes the number of wavefront angles determined by the displacement of the spot.

number

[0011] The limitations associated with this may be seen by entering a representative value in Ec.(3). If the sensor has a pixel size of Δx=6 μm, a wavelength of λ=0.5 μm, and we take a representative value for the size of the microlens d=100 μm, the focal length for optimizing the sensor is F=2.5 mm according to Ec.(3). In this case the microlens must therefore be placed 2.5 mm from the sensor. If the size of the microlens is reduced, said value is reduced proportionately, which is possible from a practical point of view since said focal length values ​​are very close to the practical limits of both manufacturing and alignment.

[0012] From these calculations it is easy to understand why a value of d=150 μm for the diameter of the microlenses and a focal length of approximately f=6 mm are commonly used in commercial models. Said diameter directly determines the spatial resolution of the system. Moreover, said size limits the number of pixels at which the wavefront is sampled for a sensor of a certain size. Typically, in commercial HSs, the number of microlenses and therefore the number of pixels of the reconstruction varies between 20×20 and 100×100 (corresponding to pixelated sensors whose total size is found in the range between 3.0×3.0 mm and in extreme cases 15.0×15.0 mm).

[0013] 2) Angular resolution. The maximum angle that may be sampled for each local plane wave is related to the maximum displacement of the spot within that region: considering that the microlenses form an optical barrier, said displacement corresponds to half the size of the microlenses.

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[0014] Therefore, if a microlens with d=150 μm and f=6 mm is used, the maximum measurable angle of the incident wavefront is θ max = 0.7°. This is the reason why HS sensors are typically used to measure small variations in the wavefront, certainly limiting their use in measuring scattered light in biological samples.

[0015] The above reasoning is that, in the best case, the commercially available HS has a max = 1.0°.

[0016] On the other hand, the mechanism proposed in the aforementioned patent, which is based on the Shack-Hartmann principle, presents a certain degree of complexity, and it would therefore be advantageous to also propose an alternative, simpler mechanism. Summary of the Invention [Problem to be solved by the invention]

[0017] It is therefore necessary to present an alternative to those based on the state of the art, as in the aforementioned patent, which compensates for the shortcomings found in the state of the art by providing a microscope that is structurally similar to those based on the Shack-Hartmann principle, but which offers results in terms of spatial resolution much better than those offered by microscopes based on the state of the art principle, as well as its simplification, and moreover offers greater stability and robustness than those based on interferometric procedures, and does not give only monocular information. [Means for solving the problem]

[0018] For this purpose, the invention relates in a first aspect to a microscope for the quantitative measurement of a wavefront, which microscope, in an already known manner, comprises: - illumination means for illuminating the sample, - a microscope objective constructed and arranged to receive and focus light scattered by a sample when illuminated by said illumination means; - a regular two-dimensional array of lenses arranged at the aperture stop of said microscope objective or at the location of an intermediate image thereof; - an image sensor formed by a plurality of light receiving elements arranged in a capture space in a focal plane of a regular two-dimensional array of lenses, receiving the light scattered by a sample after passing through the microscope objective lens and the regular two-dimensional array of lenses, and acquiring spatial and angular information of an object wavefront related to the light from the sample, some of the light receiving elements facing respective lenses; and at least one computational entity operatively coupled to said image sensor and configured and arranged to perform a computational reconstruction of said object wavefront based on said spatial and angular information; The present invention relates to a microscope including:

[0019] Unlike the microscopes known in the state of the art, the one proposed according to the first aspect of the invention has a center-to-center spacing p of every two consecutive lenses of the regular two-dimensional array of lenses.μ is larger than 500 μm, and the aperture ratio is less than 10.

[0020] According to an embodiment, the center-to-center spacing of every two consecutive lenses of the regular two-dimensional array of lenses is between 900 μm and 1100 μm, and the aperture ratio is between 5 and 7.

[0021] In a preferred embodiment, the spacing between the centers of every two consecutive lenses of the regular two-dimensional array of lenses is between 990 μm and 1010 μm, preferably 1000 μm, and the aperture ratio is between 5.8 and 6.2, preferably 6.

[0022] The microscope proposed by the first aspect of the invention is extremely compact due to the few components and arrangements of components it contains, allowing the measurement of wavefronts in microscopic 3D samples. Its implementation requires minimal modifications to the configuration of conventional microscopes. It may therefore be easily implemented in conventional microscopes. Similar to the Shack-Hartmann technique, this novel microscope is based on the use of an array or regular two-dimensional arrangement of lenses. However, the characteristics of the lens array are notably different. Shack-Hartmann devices feature narrow spacing (approximately 100 μm) and a large aperture ratio (or f-number) (approximately f # ≈25), whereas, as mentioned above, the microscope proposed in the first aspect of the invention uses microlenses with a wide spacing (preferably at or around 1000 μm) and a small aperture ratio (preferably f # ≒6 or approximately f # A lens array with a focal length of approximately 6 is used.

[0023] The microscope of the present invention, due to the particular conditions of the spacing between the centers of each two consecutive lenses of a regular two-dimensional array of lenses, and their aperture ratio, which differ significantly from the usual conditions used in the state of the art, makes it possible to obtain results that are not achievable with the Shack-Hartmann technique, such as the measurement of slight and not slight variations in the wavefront from a biological sample, or obtaining 3D images with a higher resolution than that provided by the host microscope in which the microscope proposed by the first aspect of the present invention with respect to an embodiment may be implemented.

[0024] Various kinds of shapes for the regular two-dimensional array of lenses are valid and are encompassed by the microscope of the first aspect of the invention, but this should preferably follow a hexagonal lattice shape or a square matrix shape.

[0025] Advantageously, the illumination means is configured to illuminate the sample with partially or fully coherent light, and in a preferred embodiment with a light beam of such width that in the absence of a sample or for a transparent sample the object wavefront is completely flat and the capture in the capture space is only the bright field provided by the central lens of said regular two-dimensional array of lenses.

[0026] A regular two-dimensional array of particular lenses, and in particular their spacing p μ According to an embodiment benefiting from the aperture ratio, the computational entity (or entities) performs a double sampling in two reciprocal spaces of: - a first sampling or angular sampling for obtaining angular information of an object wavefront in a space in which the ordered two-dimensional array of lenses is arranged, the first sampling or angular sampling being performed by a computation entity to determine the angular information and, together with it, determining its spatial frequency content as a function of one or more positions in the capture space in which the image sensor receives and captures the light emitted by the sample after passing through the microscope objective lens and the ordered two-dimensional array of lenses; and - a second sampling or spatial sampling in the capture space to obtain spatial information from the intensities received by each light receiving element or pixel of the image sensor. The method is configured to:

[0027] Generally, each light-receiving element or pixel of an image sensor is subject to the following constraints:

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[0028] According to an embodiment, the computational entity is configured to perform a transposition of one of the two reciprocal spaces and to look up both spatial and angular information in a reconstruction space that is virtually located in the same space or in the object space, said reconstruction space being made up of an L / N area (where L is the number of light receiving elements or pixels of the image sensor and N is the number of lenses in the regular two-dimensional array of lenses), so that Δx / M (M=-f μ / f ob and f ob is the focal length of the microscope objective) and μ / f ob A single local sampling of the plane wave of the object wavefront is performed, which includes the aforementioned angular sampling at a period of .

[0029] In the implementation of said embodiment, the computational entity is configured to interpret said reconstruction space as a synthetic capture system in which a regular two-dimensional array of lenses is arranged with a spacing Δx / M between the centers of every two successive lenses, such that for each spatial sampling position l, the object wavefront o(x) is locally sampled, so that the light intensity I received at each light receiving element or pixel is lm represents a measure of the angular composition of the object wavefront.

[0030] According to an embodiment, in order to perform the aforementioned computational reconstruction, the computational entity is configured to consider that in a transposed space, i.e. in each sub-region of the reconstruction space, a local sampling is performed on a plane wave of the object wavefront, each pixel of a sub-region of the transposed space corresponding to a propagation direction of the plane wave forming the object wavefront in said area.

[0031] Furthermore, according to an implementation of said embodiment, the computation entity calculates for the sub-region given by the superscript l the following formula:

number

number

[0032] According to an embodiment, the computational entity is configured to transfer the information contained in each pixel to a plane wave database, in which each pixel position represents a propagation direction of the object wavefront, and to perform said computational reconstruction, advantageously for each sub-region of the transposed space, by summing up the contributions of the different angular components represented in the plane wave database, resulting in a greyscale image in which the resulting shades of grey represent a quantitative measurement of the object wavefront.

[0033] In an embodiment, the computational entity is operatively coupled to and controls both the extensive incoherent light source and the image sensor, and is configured to perform a pre-calibration process (i.e., before proceeding with performing quantitative measurements of the sample) for characterization and parameterization of the capture space according to the following sequence: - controlling a broad incoherent source to illuminate an object space such that all lenses of a regular two-dimensional array of lenses are illuminated; - controlling an image sensor to capture an image of the sample under said widespread incoherent illumination; - applying an image processing circle detection algorithm, given the following parameters: the relative positions and sizes of the lenses in the ordered two-dimensional array of lenses, and the number of pixels contained in the sub-region bounded by each lens in the ordered two-dimensional array of lenses.

[0034] According to an embodiment, the computational entity is configured to perform the aforementioned parameterization of the capture space and to determine and impose angular dimensions relative to the capture space using the parameters obtained during the calibration process and depending on the size of the pixels of the image sensor, which are known by the computational entity.

[0035] In a second aspect, the present invention also relates to a method for computational reconstruction of a wavefront comprising performing the functions for which a computational entity of a microscope of the first aspect of the invention with respect to any of its embodiments is configured, i.e. all functional features mentioned above following the expression "the computational entity is configured" describe the method steps of the second aspect of the invention with respect to the corresponding embodiment.

[0036] In a third aspect, the invention relates to a computer program for the computational reconstruction of a wavefront, comprising program instructions which, when executed on a processor, implements the method of the second aspect.

[0037] In a fourth aspect, the invention relates to a computer program product comprising a tangible medium on which is stored a computer program of the third aspect.

[0038] The microscope of the invention, the nature and effects of which will be described below, makes it possible to obtain results which, due to its conditions, are not achievable with the aforementioned Shack-Hartmann technique.

[0039] On the other hand, the number of pixels of the wavefront reconstruction of the inventive microscope can be significantly higher than that of the HS, since it does not depend on the size of the lenses (in this case the size of the lenses of the regular two-dimensional array is preferably in the mm range, for this reason they are called "milli-lenses"). In the inventive microscope, the number of pixels of the wavefront reconstruction is obtained by dividing the number of pixels of the sensor by the number of milli-lenses. For example, if in an embodiment the inventive microscope has 5 milli-lenses in the transverse direction (for example horizontal or direction x) and a sensor with 2,500 pixels in said direction, the final reconstructed image has 500 pixels in said direction. If it is desired to have an HS sensor with the same number of pixels, it is necessary to have micro-lenses with d=12 μm for a typical sensor size of approximately 6.0×6.0 mm. As explained in the previous section, this size is far from the practical limit.

[0040] It should be noted that in the microscope of the present invention, the spatial resolution is determined by the displacement ratio. mic =10 (Scientific microscopes have a maximum M mic For the example of a microscope with a Δx = 100 magnification, the spatial resolution of the system is 0.6 μm when a pixel size of Δx = 6 μm is taken into account, i.e. the system improves the spatial resolution by three orders of magnitude compared to a typical HS sensor (note that its resolution is determined by the size of the microlenses and is therefore in the range of 100-150 μm).

[0041] On the other hand, the maximum angle of a plane wave that may be sampled by the microscope of the present invention is

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[0042] The present invention presents a simple and low-cost solution for the measurement of the wavefront scattered by a microscopic sample. It presents a much simpler system that is much more stable than those based on interferometric detection, while at the same time providing a significant improvement in spatial resolution with respect to systems based on Shack-Hartmann detectors.

[0043] Due to the fact that it requires the integration of fewer optical elements relative to conventional microscopes, its development in the form of a module compatible with commercially available microscopes is relatively simple.

[0044] Said module is therefore proposed as a fifth aspect incorporating at least the ordered two-dimensional array of microscope lenses and an image sensor of the first aspect of the invention, as well as a support for supporting same and an optical-mechanical connecting tube adapted to be connected (optically and mechanically) to the camera port of the microscope.

[0045] A sixth aspect of the present invention relates to a kit for a microscope comprising a module for a microscope of the fifth aspect and an illumination module comprising an illumination means of the microscope of the first aspect of the present invention adapted to be connected to an illumination port of the microscope.

[0046] The invention has potential applications in various fields of science and technology. On the one hand, it has direct applications in any field that requires quantitative information on a microscopic sample in a non-invasive manner, i.e. without the need for dyes to observe the different structures that form the sample. For this reason, its use in histology is of particular interest. Furthermore, it is possible to apply the invention to metrology and microelectromechanical systems (MEMS), in particular in the study of the behavior of said systems with respect to temperature, taking into account the stability of the proposed measurement system with respect to temperature.

[0047] The above and other advantages and features will be better understood upon consideration of the following detailed description of embodiments, which is to be interpreted as illustrative and not exclusive, and which is made with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0048] [Figure 1] FIG. 1 is a diagram of a microscope proposed according to a first aspect of the invention with respect to an embodiment. [Diagram 2] FIG. 2 shows a matrix arrangement, or a regular two-dimensional array, of microscope lenses as proposed by a first aspect of the invention superimposed on the pupil of a microscope objective lens. [Diagram 3] FIG. 2 is a schematic diagram of the transposition ratio between the capture space and the reconstruction space implemented by the computational entity of the microscope of the first aspect of the invention and of the method of the second aspect of the invention in relation to an embodiment of the invention. [Figure 4] FIG. 2 shows a series of images as an example of a proof-of-concept experiment of the use of the microscope of the first aspect of the invention for the computational reconstruction of a sample consisting of a large number of cotton fibres. [Diagram 5] FIG. 2 is a schematic diagram of the capture, transposition and reconstruction performed by the microscope of the first aspect of the invention and according to the method of the second aspect of the invention, with reference to an embodiment. [Figure 6] FIG. 2 is a flow diagram of the operational steps to be followed using the microscope and method proposed by the present invention with respect to an embodiment. [Figure 7]FIG. 1 is a schematic diagram of a kit for a microscope proposed according to the sixth embodiment of the present invention, with two connections, one for a camera port for the module of the fifth embodiment of the present invention and the other for an illumination port for the illumination means of the illumination module of a conventional microscope kit. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] As illustrated diagrammatically in FIG. 1 with respect to its most basic embodiment, the microscope proposed according to a first aspect of the invention comprises: - illumination means (schematically illustrated in FIG. 5 ) comprising a partially or fully coherent light source 1 for illuminating a sample T, - a microscope objective 2 constructed and arranged to receive and focus light scattered by a sample T when illuminated by said illumination means 1; - a regular two-dimensional array 3 of lenses arranged at the location of the aperture stop of said microscope objective 2 or its intermediate image, - an image sensor 4 or pixelated sensor formed by a number of light receiving elements or pixels, some of which face each lens, arranged in a capture space in the focal plane of a regular two-dimensional array of lenses or lens matrix 3, which receives light scattered by a sample T after passing through the microscope objective lens 2 and the regular two-dimensional array of lenses 3, and which obtains spatial and angular information of the object wavefront related to the light from the sample T; at least one computational entity (not shown) operatively coupled to the image sensor 4 and configured and arranged to perform a computational reconstruction of the object wavefront based on the spatial and angular information; Includes.

[0050] As shown in the previous section, the spacing p between the centers of every two consecutive lenses of the regular two-dimensional array 3 of lenses is μ is preferably 1000 μm or around 1000 μm, and the aperture ratio has a value of 6 or around 6.

[0051] Figure 2 shows a matrix arrangement of microscope lenses 3 proposed according to a first aspect of the invention, or a regular two-dimensional array 3, superimposed on the pupil of a microscope lens. Each millilens is characterised by the position of its centre with respect to the origin of the coordinates.

[0052] The ideal configuration of the microscope would satisfy the following conditions (other configurations may be used, provided that the obvious changes in the complex amplitude of the field of view as it passes through the microscope components are kept in mind): 1) The position of the milli-lens matrix 3 corresponds to the position of the aperture stop of the microscope objective 2 or the position of any of its intermediate images. 2) The shape in which the lenses of matrix 3 are arranged determines the fill factor at the aperture stop. The two most common shapes in which millilenses are arranged are a square matrix (where the centers of the millilenses are placed at the nodes of a two-dimensional square lattice) or a hexagonal lattice. However, any other shape is valid, provided that the node positions of the lattice are known. 3) The light source 1 provides a uniform collimated beam (or at least with low divergence) onto the microscope sample T. The width of the beam B (see FIG. 5) is such that, in the absence of a sample, the image recorded by the sensor presents a uniform field of view in the area corresponding to the image plane of the central millilens, and otherwise in the complete absence of light, there is no overlap between the areas corresponding to the different millilenses. 4) A pixelated sensor 4 is placed at the focal image plane of the milli-lens matrix 3 .

[0053] Under these conditions, the information captured by the microscope proposed according to the first aspect of the invention represents a double sampling process in two reciprocal spaces and therefore contains spatial and angular information simultaneously. The existence of a Fourier transform ratio between spatial and angular information gives rise to the following constraints on the pixel size:

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[0054] The distribution of intensity in the field of view detected by the sensor is:

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[0055] In this formula, l represents the lth pixel of the sensor, the function h(·) represents the 2D impulse response of the microscope objective (typically the Airy disk), and h μ (·) denotes that of the millilens matrix (generally the matrix of the Airy disk for millilenses with circular apertures). Their impulse responses are determined by the diffraction of the waves and are functionally proportional to the Fourier transform of the corresponding aperture. In addition, the function o(·) denotes the distribution of the amplitude of the measured wavefront, M=-f μ / f ob yf ob is the lateral magnification of the microscope, and f ob is the focal length of the microscope objective lens, L is the number of pixels on the sensor, and finally, the function rect(·) is a binary function that has a value of 1 inside the rectangle and a value of 0 outside it, and δ(·) is the Dirac delta function).

[0056] This equation emphasizes the presence, in the microscope and method of the present invention, of a double sampling of the complex amplitude distribution o(·) of the object, which is resolution-limited by diffraction, as in an aberration-free optical system.

[0057] In combination with and based on a particular configuration and arrangement of the components of the microscope of the first aspect of the invention, a novel reconstruction software is proposed, implemented by a computational entity and a method of the second aspect of the invention, whose first task consists in detecting the positions of the image provided by the matrix 3 of milli-lenses on the plane of the image sensor 4. These positions define different regions of frequency content that form the spectrum of the object. The spacing between the milli-lenses defines the periodicity of the frequency sampling.

[0058] On the other hand, the pixelated sensor performs a second sampling, but this time on the space content. Considering that this sampling and the previous sampling are performed in reciprocal space, it is possible to perform any legitimate scaled transposition and locate both pieces of information in the same space. Said transposition may be understood as follows: The sensor samples the spatial information with a periodicity of Δx. However, the sampling is done in the reciprocal space with a periodicity of p μ This is performed on a field of view previously sampled by a matrix of milli-lenses 3 with a periodicity of N p. The spatial resolution times bandwidth product (also known in the scientific literature as the "space-bandwidth product" (SBP)) determines the amount of information captured by the optical system. In the present invention, the matrix of milli-lenses 3 is the limiting factor. Thus, the SBP is N p μ where N is the total number of millilenses that can fit in the objective pupil and therefore provide an image to the image sensor 4. - Transposing the spatial-angular information captured by the proposed microscope gives a new expression for this information, where the periodicity of the sampling is exchanged such that a new pixelation matrix represents a plane wave local sampling of the wavefront scattered by the object, where the period of the spatial sampling is Δx / M and the period of the angular sampling is p μ / f ob It is.

[0059] This defines a space known as the "reconstruction space Er" (whose transposition space is the c-space), which is virtually located in the object space or the capture space Ec (see Figures 3 and 5). The latter consists of L / N regions, whose position is determined by the periodicity of the new space sampling. Within each region, a total of N angles are sampled with a precision that depends on the periodicity of the angle sampling in the reconstruction space Er, as shown in Figure 3, where the transposition ratio between the capture space Ec and the reconstruction space Er is illustrated.

[0060] The space may be interpreted as a synthetic capture system in which several microlenses with a spacing of Δx / M are placed directly on the plane on which the sample is placed. Thus, for each space sampling position l, the intensity I lm The object wavefront o(x) is locally sampled such that represents a measurement of the angular composition of the object wavefront. If the object wavefront is interpreted as a superposition of plane waves, then the sum of the local measurements for each spatial sampling region l, duly scaled by the intensity and the corresponding angular components, represents a sampled version of the wavefront:

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[0061] For a given microscope objective, the accuracy of the measurement of the angular components of the wavefront depends on the size and number of millilenses in the capture space.

[0062] A more detailed description of the capture, transposition and reconstruction process implemented by the microscope of the first aspect of the invention and the method of the second aspect of the invention for an embodiment for a light beam B illuminating a sample T in a capture space Ec behind which is arranged a matrix of lenses 3 and an image sensor 4 is given below with reference to FIG. 5, which diagrammatically illustrates a diagram of the reproduction space Er.

[0063] Each subregion Sr of the transposed or reconstructed space Er can be considered to perform a local sampling of the plane wave of the object wavefront. Each pixel of a subregion Sr of the transposed space Er corresponds to the propagation direction of the plane wave comprising the object wavefront in said area. For a given subregion Sr, indicated by the superscript l, the complex amplitude of the object wavefront is:

number

number

[0064] For example, when illuminating a perfectly transparent sample T as shown in the figure, the wavefront is perfectly flat and the only recording in the capture space Ec is the field of view given by the central millimeter lens. 00 The element corresponds to a plane wave traveling in the direction of the optical axis. In this way, a perfectly flat wavefront is formed from all sub-regions Sr, which corresponds to the sample T. The element is physically related to the plane wave by the system parameters as follows:

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[0065] Figure 4 shows preliminary results as an example of the operation of the microscope and method of the invention. The results were obtained with a non-optimized low-resolution microscope and a cotton fiber sample, but they show the potential of the concept presented in the present invention. The left panel of the figure shows a captured image obtained by a microscope such as the one proposed by the present invention. After performing a transposition, a matrix representing the reconstruction space Er is obtained (center panel of Figure 4). Finally, the object wavefront is obtained by processing the local contributions to the wavefront given by the pixels of the subregions (Figure 4, right panel).

[0066] 6 shows a flow diagram illustrating the steps of the method proposed by the second aspect of the invention with respect to an embodiment, or in other words the functions for which the computational entities of the microscope of the first aspect of the invention are configured, which are explained below corresponding to the legends included in each block.

[0067] Capture: A captured image obtained by the pixelated sensor 4.

[0068] Calibration: Calibration is a necessary process for characterizing and parameterizing the capture space Ec. For the same device, this process only needs to be performed once. To do this, the object space is illuminated with a broad incoherent source such that all lenses of matrix 3 are illuminated. The resulting image is then saved and an image processing circle detection algorithm is applied. Said algorithm provides all the necessary parameters: the relative positions and sizes of the lenses, as well as the number of pixels contained in the sub-region bounded by each lens.

[0069] Parameterization of the capture space: The parameters obtained during calibration and the known pixel size of the sensor 4 give the capture space Ec a valid angular dimension.

[0070] Transposition to reconstruction space: Using the parameterization data, a transposition is applied to the capture space Ec), resulting in a reconstruction space Er formed by a set of sub-regions Sr, each pixel of which represents the angular propagation direction of the wavefront.

[0071] Measurement of the angular components for each sub-region: The information contained by each pixel is transferred to a database of plane waves where each pixel position represents the propagation direction of the wavefront.

[0072] Wavefront reconstruction: For each subregion, the contributions of the different angular components, represented in a database of plane waves, are summed. The resulting grey shade represents a quantitative measurement of the wavefront.

[0073] Finally, Figure 7 is a schematic diagram of a kit for a microscope proposed according to the sixth aspect of the invention, comprising a module of the fifth aspect connected to the camera port of a commercially available microscope, each comprising an eyepiece Oc, an objective lens Ob, a tube lens Z and a folding mirror R, and an illumination module further comprising illumination means connected to the illumination port of the microscope.

[0074] The kit, which can be adapted to a commercially available microscope, consists of two parts, marked with dashed lines in the diagram of FIG.

[0075] P1) Illumination Module: An illumination means, typically a laser that produces the illumination as described in this document, and a series of lenses must be fitted to the illumination port.

[0076] P2) Module or collection module of the fifth aspect of the invention: A matrix of lenses 3, a sensor 4 and an auxiliary lens 2 (or a series of auxiliary lenses) are fitted to the camera port of the microscope so that the field of view collected by the sensor 4 has the characteristics defined in the present invention.

[0077] The main advantage of the microscope proposed by the invention is that, due to the fact that the physical capture is performed in transposition space, the resolution of the composite microlenses in the reconstruction space Er is not limited by diffraction but by the inter-pixel spacing of the camera or image sensor 4. This fact makes it possible to produce quantitative measurements of the phase with unprecedented lateral resolution.

[0078] Those skilled in the art can make changes and modifications to the embodiments described herein without departing from the scope of the invention, which is defined in the appended claims.

Claims

1. 1. A microscope for quantitative measurement of a wavefront, comprising: Illumination means (1) for illuminating the sample (T), a microscope objective (2) constructed and arranged to receive and focus light scattered by the sample (T) when illuminated by said illumination means (1); a regular two-dimensional array (3) of lenses arranged at the aperture stop of the microscope objective (2) or at the location of its intermediate image; an image sensor (4) formed by a plurality of light receiving elements arranged in a capture space (Ec) in the focal plane of the regular two-dimensional array of lenses (3), for receiving the light scattered by the sample (T) after passing through the microscope objective lens (2) and the regular two-dimensional array of lenses (3) and for acquiring an image containing spatial and angular information of an object wavefront related to the light from the sample (T), some of the light receiving elements facing each lens; and at least one computer operatively coupled to said image sensor (4) and configured and arranged to perform a computational reconstruction of said object wavefront based on said spatial and angular information; Including, The spacing p between the centers of every two consecutive lenses of the regular two-dimensional array (3) of lenses μ greater than 500 μm and an aperture ratio of less than 10.

2. 2. The microscope according to claim 1, wherein the spacing between the centres of each two consecutive lenses of the regular two-dimensional array of lenses (3) is between 900 μm and 1100 μm and the aperture ratio is between 5 and 7.

3. 3. The microscope according to claim 2, wherein the spacing between the centers of each two consecutive lenses of the regular two-dimensional array of lenses (3) is between 990 μm and 1010 μm and the aperture ratio is between 5.8 and 6.

2.

4. 4. A microscope according to any one of claims 1 to 3, wherein the illumination means is configured to illuminate the sample (T) with partially or fully coherent light.

5. 5. The microscope of claim 4, wherein the illumination means is configured to illuminate the sample (T) with a light beam (B) of such width that in the absence of a sample or for a transparent sample (T), the object wavefront is completely flat and the capture in the capture space (Ec) is only the bright field provided by the central lens of the regular two-dimensional array of lenses (3).

6. The at least one computer performs a double sampling in two reciprocal spaces of: a first sampling or angular sampling for obtaining angular information of the object wavefront in a space in which the regular two-dimensional array of lenses (3) is arranged, the computer determining the angular information and therewith determining its spatial frequency content as a function of one or more positions in the capture space (Ec) where the image sensor (4) receives and captures the light emitted by the sample (T) after passing through the microscope objective lens (2) and the regular two-dimensional array of lenses (3); a second sampling or spatial sampling in said capture space (Ec) to obtain spatial information from the intensity received by each light receiving element or pixel of said image sensor (4).

6. The microscope according to claim 1 , configured to perform the steps:

7. The size of each light receiving element or pixel of the image sensor (4) is within the following constraints: [0010] (where Δx is the size of a pixel, λ is the wavelength of the light beam with which the illumination means (1) illuminates the sample (T), and f μ is the focal length of the lenses of the regular two-dimensional array of lenses (3), and p μ is the distance between the centers of each two consecutive lenses of said lens. The microscope according to claim 6 ,

8. The at least one computer is configured to perform a transposition of one of the two reciprocal spaces and look up both spatial and angular information in a reconstruction space (Er) that is virtually located in the same space or object space, the reconstruction space (Er) being made up of an L / N area (L being the number of light receiving elements or pixels of the image sensor (4) and N being the number of lenses of the ordered two-dimensional array of lenses (3)), so that Δx / M (M=-f μ / f ｏｂ and f ｏｂ is the focal length of the microscope objective lens (2)) and μ / f ｏｂ 8. The microscope of claim 7, wherein a single local sampling of a plane wave of the object wavefront is performed, including said angular sampling at a period of

9. To perform the computational reconstruction, the at least one computer is configured to interpret the reconstruction space (Er) as a synthetic capture system in which a regular two-dimensional array of lenses is arranged with a center-to-center spacing Δx / M between every two consecutive lenses such that for each space sampling position l, the object wavefront o(x) is locally sampled, so that the light intensity I received at each light receiving element or pixel is ｌｍ 9. The microscope of claim 8, wherein represents a measure of the angular composition of the object wavefront.

10. The microscope of claim 9, wherein, to perform the computational reconstruction, the at least one computer is configured to consider that in each subregion of the reconstruction space (Er) a local sampling is performed on a plane wave of the object wavefront, and each pixel of a subregion of the reconstruction space (Er) corresponds to a propagation direction of a plane wave forming the object wavefront of the subregion.

11. The at least one computer calculates, for a subregion given by superscript l, the following formula: [0025] (In the formula, [0030] is the intensity of the pixel corresponding to position m, n in subregion I, and k ｍｎ is the direction vector of the plane wave corresponding to the pixel located at position m, n) 11. The microscope of claim 10, configured to determine the complex amplitude of the object wavefront according to:

12. 12. A microscope according to claim 10 or 11, wherein the at least one computer is configured to transfer the information contained in each pixel to a database of plane waves in which each position of a pixel represents a propagation direction of the object wavefront.

13. 13. The microscope of claim 11 or 12, wherein the at least one computer is configured to perform the computational reconstruction by summing, for each sub-region of the reconstruction space (Er), the contributions of the different angular components represented in the database of plane waves, resulting in a grayscale image in which the resulting shades of gray represent a quantitative measurement of the object wavefront.

14. 14. The microscope of claim 10, wherein the at least one computer is operatively coupled to and controls both an extensive incoherent light source and the image sensor (4) and is configured to perform a pre-calibration process for characterizing and parameterizing the capture space (Ec), controls the extensive incoherent light source to illuminate an object space such that all lenses of the regular two-dimensional array of lenses (3) are illuminated, controls the image sensor (4) to obtain an image of a sample under the extensive incoherent illumination, applies an image processing circle detection algorithm, and provides all of the following parameters: the relative positions and sizes of the lenses of the regular two-dimensional array of lenses (3) and the number of pixels contained in a sub-area bounded by each lens of the regular two-dimensional array of lenses (3).

15. The microscope according to claim 14, wherein the at least one computer is configured to perform the parameterization of the capture space (Ec) to determine and impart angular dimensions to the capture space according to parameters obtained during a calibration process and depending on the size of the pixels of the image sensor (4).

16. 16. The microscope according to any one of claims 1 to 15, wherein the regular two-dimensional array of lenses (3) follows a hexagonal lattice shape.

17. 16. The microscope according to any one of claims 1 to 15, wherein the regular two-dimensional array of lenses (3) follows a square matrix shape.

18. A method for computational reconstruction of a wavefront comprising the implementation of functions for which a computer of a microscope according to any one of claims 1 to 17 is configured.

19. A computer program for the computational reconstruction of a wavefront comprising program instructions which, when executed on a processor, implements the method according to claim 18.

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