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    • 专利摘要:
    The present invention concerns a microscope for quantitative wavefront measurements, comprising: - means for illuminating a sample (T); - a target (2); - an ordered two-dimensional arrangement of lenses (3) with a spacing p{mi } greater than 500 μm and a relative aperture less than 10; - an image sensor (4) located in a capture space (Ec) to receive the light scattered by the sample (T), and acquire spatial information and angular information of the object wavefront associated therewith; and - a computational entity to perform a computational reconstruction of the object wavefront from the spatial and angular information. Other aspects of the invention concern a method, a computer program and a product that includes it, adapted to perform the functions of the computational entity of the microscope, as well as a module and a kit for a microscope. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2779500A1
    申请号:ES201930116
    申请日:2019-02-15
    公开日:2020-08-17
    发明作者:Corral Manuel Martinez；Tortosa Genaro Saavedra；Ortiga Emilio Sanchez；Peter Török
    申请人:Universitat de Valencia；
    IPC主号:
    专利说明:

    [0002] MICROSCOPE FOR QUANTITATIVE WAVE FRONT MEASUREMENTS, MODULE AND KIT FOR MICROSCOPE, METHOD AND COMPUTER PROGRAM FOR COMPUTATIONAL WAVE FRONT RECONSTRUCTION
    [0004] Technical sector
    [0006] The present invention concerns, in a first aspect, a microscope for quantitative wavefront measurements, comprising an ordered two-dimensional arrangement of lenses designed in such a way as to allow the measurement of smooth and non-smooth variations of the wavefront from samples biological, or obtaining high-resolution 3D images, including microscopic samples.
    [0008] A second aspect of the present invention concerns a method for the computational reconstruction of the wavefront, adapted to perform the functions for which the computational entity of the microscope is configured.
    [0010] In a third aspect, the present invention concerns a computer program for the computational reconstruction of the wavefront, which includes program instructions that, when executed in a processor, implement the method of the second aspect.
    [0012] In a fourth aspect, the present invention concerns a computer program product that comprises a tangible medium on which the computer program of the third aspect is stored.
    [0014] A fifth aspect of the present invention concerns a microscope module, to be coupled to a camera port of a microscope in order to construct the microscope of the first aspect of the present invention.
    [0016] A sixth aspect of the present invention concerns a kit for a microscope, which comprises the module of the fifth aspect and an illumination module that can be coupled to an illumination port of a microscope.
    [0018] Prior state of the art
    [0020] The computational reconstruction of the wavefront is a problem of special interest in optical microscopy since it provides information on the light field emitted by 3D samples, that is, quantitative measurements of the wavefront. Currently this type of reconstruction is provided by two types of microscopes:
    [0021] 1) Microscopes based on interference detection! wavefront using a holographic process. This type of system has the disadvantage of its low stability and of obtaining only monocular information.
    [0023] 2) Microscopes with a structure similar to wavefront sensors based on the Hartmann-Shack (H-S) principle. That is, those that include the characteristics defined in the preamble of claim 1 of the present invention. Such is the case of those described in the following patents: US9726875B2, US9658443B2, US9679360B2 and US9976911B2.
    [0025] In such patents different techniques are proposed focused on obtaining a spatial resolution that is, in US9726875B2, at least acceptable to obtain a good approximation of the real light fields, that is, in US9658443B2, increased but limited by the diffraction limit, which that is, in US9679360B2, improved by obtaining a composite image that combines a spatial intensity image with a light field image, or that is, in US9976911B2, good but without negatively affecting the angular resolution, that is, a compromise between spatial and angular resolution.
    [0027] Although each of the proposals made in such patents manages to improve the spatial resolution of the microscopes proposed in them to a certain degree, such improvement is clearly improvable, since the improvement in spatial resolution achieved has the limitations of sensor-based systems. Hartmann-Shack, which are limited in resolution by the number of microlenses (regardless of the spatial resolution of the pixelated sensor). Because of this, to optimize an H-S sensor, the largest possible number of microlenses should be used, the size of which, consequently, should be reduced to a minimum. However, there are two main limitations to this process:
    [0029] 1) The size of the diffraction spot : When illuminating a microlens with a locally flat wave, the light is focused on the sensor (located in the image focal plane of the microlens) in the form of a diffraction spot. The position of this spot with respect to the center of the microlens can be related to the angle of inclination of the locally flat wave. The diameter of the spot, 0 dî, for a given lighting wavelength, l, is related to the diameter, d, and the focal point of each microlens, /, by the following formula:
    [0030]
    [0031] where
    [0033]
    [0034] On the other hand, in order to effectively sample the diffraction spot in order to calculate its centroid and, with it, 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 optimal value since this value allows the spot to be effectively sampled with the sensor and, in turn, allows optimizing the number of wavefront angles determined by the movement of the stop,
    [0036] 4Ax = 0 d¿ / = 2Ad (3)
    [0037] You can see the limitation that this supposes when introducing typical values in Eq. (3). For the case of a sensor with a pixel size of Ax = 6 ^ m, a wavelength of A = 0.5 ^ m, and taking a typical value of microlens size, d = 100 ^ m, the focal length that would optimize the sensor, according to Eq. (3), is f = 2.5 mm. Therefore, the microlenses should be positioned 2.5 mm from the sensor in this case. If the size of the microlenses were reduced, this value would be reduced in the same proportion, but this is possible from a practical point of view, since said focal length value is very close to the practical limit of both manufacturing and alignment.
    [0039] From these calculations it is easy to understand why a value of d = 150 ^ m is typically used as the diameter of the microlenses and focal length of approximately f = 6 mm in commercial models. This diameter directly determines the spatial resolution of the system. Furthermore, this size limits the number of pixels in which the wavefront is sampled for a given sensor size. Typically, in commercial HS the number of microlenses, and therefore the number of pixels of the reconstruction, varies between 20x20 and 100x100 (corresponding to pixelated sensors whose total size is in the range between 3.0 x 3.0 mm and, in extreme cases , 15.0 x 15.0 mm).
    [0041] 2) Angular resolution.
    [0043] For each locally flat wave, the maximum angle that can be sampled is related to the maximum displacement of the spot within its region. Since the microlenses create optical barriers, said displacement corresponds to half the size of microlenses,
    [0045]
    [0046] Therefore, if microlenses of d = 150 and f = 6 mm were used, the maximum measurable angle of the incident wavefront would be dmax = 0.7 ° . This is why HS sensors are normally used to measure smooth wavefront variations and their use to measure scattered light in biological samples is certainly limited.
    [0048] The above reasoning demonstrates why at best commercial HSs do not measure locally flat wave slopes greater than dmax = 1.0 or
    [0049] On the other hand, the mechanisms proposed in the aforementioned patents based on the Hartmann-Shack principle have a certain complexity, so it would also be interesting to propose simpler alternative mechanisms.
    [0051] It appears, therefore, necessary to offer an alternative to the state of the art that covers the gaps found in it, by providing a microscope that, like the patents mentioned above, is structurally similar to those based on the Hartmann-Shack principle but that offers results in terms of spatial resolution much better than those provided by microscopes of the state of the art based on this principle, as well as a simplification thereof, and that, likewise, offers greater stability and robustness than those based on interferential procedures, and information not only monocular.
    [0053] Explanation of the invention
    [0055] To this end, the present invention concerns, in a first aspect, a microscope for quantitative wavefront measurements, comprising, in a manner known per se:
    [0056] - lighting means for illuminating a sample;
    [0058] - a microscope objective configured and arranged to receive and focus light scattered by the sample when illuminated with said illumination means;
    [0060] - an ordered two-dimensional arrangement of lenses located at the aperture diaphragm of said microscope objective or at the location of an intermediate image thereof;
    [0061] - an image sensor formed by a plurality of photodetector elements, which is located in a capture space on the focal plane of the two-dimensional arrangement array of lenses to receive said light scattered by the sample after passing through said microscope objective and said ordered two-dimensional arrangement of lenses, and acquire spatial information and angular information of the object wavefront associated with said light from said sample, remaining facing each various lens of said photodetector elements; and
    [0063] - at least one computational entity operatively connected to said image sensor and which is configured and arranged to perform a computational reconstruction of said object wavefront from said spatial and angular information.
    [0065] Unlike the microscopes known in the state of the art, in the one proposed by the first aspect of the present invention the spacing p # between the centers of each two contiguous lenses of the ordered two-dimensional arrangement of lenses is greater than 500 pm and its relative aperture is less than 10.
    [0067] According to an exemplary embodiment, the spacing between the centers of each two contiguous lenses of the ordered two-dimensional array of lenses has a value that is between 900 pm and 1100 pm, and their relative aperture has a value that is between 5 and 7 .
    [0069] For a preferred embodiment, the spacing between the centers of each two contiguous lenses of said ordered two-dimensional array of lenses has a value between 990 pm and 1010 pm, preferably 1000 pm, and their relative aperture has a value that is between 5, 8 and 6.2, preferably 6.
    [0071] The microscope proposed by the first aspect of the invention, due to the few components it includes and their arrangement, is very compact and allows the measurement of the wavefront in 3D microscopic samples. Its implementation requires minimal variation on the configuration of a conventional microscope. Therefore, it is easy to implement on conventional microscopes. Like Hartmann-Shack technology, this new microscope is based on the use of a matrix or ordered two-dimensional arrangement of lenses. However, the characteristics of lens arrays are very different. Whereas Hartmann-Shack devices use microlenses with small spacing (around 100 pm) and large relative aperture (or "f-number" in English) (around f # »25), as indicated Above, the microscope proposed by the first aspect of the present invention uses lens arrays of large spacing (preferably at or around 1000 pm) and small relative aperture (preferably at or around f # »6).
    [0072] The microscope of the present invention allows obtaining results that are unattainable with the Hartmann-Shack technology, such as the measurement of smooth and non-smooth variations of the wavefront from biological samples, or obtaining 3D images with higher resolution than that provided by the microscope itself. host in which the one proposed by the first aspect of the present invention can be implemented, for an exemplary embodiment thanks to the specific spacing conditions between the centers of each two contiguous lenses of the ordered two-dimensional arrangement of lenses, and their relative aperture, which differ significantly from the usual conditions used in the state of the art.
    [0074] Different types of geometry for the ordered two-dimensional arrangement of lenses are valid and covered by the microscope of the first aspect of the present invention, although it preferably follows a hexagonal lattice geometry or a square matrix geometry.
    [0076] Advantageously, the light illumination means are configured to illuminate the sample with partially or totally coherent light, and, for a preferred embodiment, with a beam of light with a width such that, in the absence of a sample or for a transparent sample, the object wavefront will be completely flat and the acquisition in the capture space will be exclusively the light field given by a central lens of said ordered two-dimensional arrangement of lenses.
    [0078] According to an exemplary embodiment, which benefits from the particular ordered two-dimensional arrangement of lenses, especially their spacing p # and their relative aperture, the computational entity (or computational entities) is configured to perform the following double sampling in two reciprocal spaces:
    [0080] - a first sampling, or angular sampling, in the space in which the ordered two-dimensional arrangement of lenses is found to obtain the angular information of the object wavefront, determining it, the computational entity, and with it its content of spatial frequencies, in a function of the position or positions in the capture space in which the image sensor receives and acquires said light emitted by the sample after passing through the microscope objective and the ordered two-dimensional arrangement of lenses; and
    [0081] - a second sampling, or spatial sampling, in the capture space to obtain the spatial information from the intensity received by each photodetector element, or pixel, of the image sensor.
    [0083] In general, each photodetector element, or pixel, of the image sensor meets the following constraint:
    [0086] where Dx is the size of the pixel, Á the wavelength of the illumination beam with which the illumination means illuminate said sample, f # the focal length of the lenses of said ordered two-dimensional array of lenses, and p # the spacing between the centers of every two contiguous lenses of said lenses.
    [0088] According to an example of embodiment, the computational entity is configured to carry out a transposition of one of the two reciprocal spaces to place both information, the spatial and the angular, in the same space, or reconstruction space, located virtually on the object space , where said reconstruction space consists of L / N regions, where L is the number of photodetector elements, or pixels, of the image sensor and N is the number of lenses of the ordered two-dimensional arrangement of lenses, so that a unique local sampling of plane waves of the object wavefront that includes the aforementioned spatial sampling with a period of Dx / M where M = - f / fb and f is the focal length of the microscope objective, and the aforementioned angular sampling with a period of p / f b.
    [0090] For an implementation of said embodiment, the computational entity is configured to, in order to perform the aforementioned computational reconstruction, interpret the aforementioned reconstruction space as a synthetic capture system in which an ordered two-dimensional arrangement of lenses is placed. spacing between the centers of every two contiguous lenses Dx / M , so that for each spatial sampling position l, the object wavefront o ( x) is sampled locally, so that the intensity of the light received at each photodetector element or pixel Iim, represents a measure of the angular composition of the object wavefront.
    [0092] The computational entity is configured to, according to an embodiment, in order to carry out the aforementioned computational reconstruction, consider that in each subregion of the transposed space, that is, the reconstruction, a local sampling is carried out in plane waves of the front of object wave, where each pixel of a subregion of the transposed space corresponds to a direction of propagation of the plane waves that make up the object wavefront in said area.
    [0094] Likewise, the computational entity is also configured, according to an implementation of said embodiment, to determine, for a subregion given by the superscript l , the complex amplitude of the object wavefront according to the following expression:
    [0095] O = m = Z - N / 2 n = Z - N / 2 eXPIÍkmn ]
    [0097] where I 1mn is the intensity of the pixel corresponding to the position m, n within the subregion I and kmn the direction vector of the plane wave that corresponds to the pixel located in the position m, n.
    [0099] According to an exemplary embodiment, the computational entity is configured to transfer the information contained by each pixel to a plane wave base in which each position of the pixel represents a propagation direction of the object wavefront, and, advantageously, to carry out the aforementioned computational reconstruction by adding, for each subregion of the transposed space, the contribution of the different angular components expressed in the plane wave base, to provide a grayscale image, where the resulting gray level represents a quantitative measure of the object wavefront.
    [0101] For an exemplary embodiment, the computational entity is operatively connected to an incoherent extensive light source and to the image sensor to control both, and is configured to perform a pre-calibration process (that is, before proceeding with measurements quantitative data of a sample), for the characterization and parameterization of the capture space, according to the following sequence:
    [0103] - controlling the incoherent wide source to illuminate the object space so that all lenses in the ordered two-dimensional array of lenses are illuminated,
    [0105] - controlling the image sensor to acquire, under said extensive incoherent illumination, an image of the sample, and
    [0107] - applying a circle detection image processing algorithm to provide the following parameters: relative position and size of the lenses of the ordered two-dimensional array of lenses and number of pixels contained within the sub-regions delimited by each lens of the ordered two-dimensional array Of lenses.
    [0109] According to an exemplary embodiment, the computational entity is configured to perform the aforementioned parameterization of the capture space, determining and granting the angular dimensions to the capture space by means of the parameters obtained during the calibration process, and depending on the pixel size of the sensor image, which is known to the computational entity.
    [0110] The present invention also concerns, in a second aspect, a method for the computational reconstruction of the wavefront, which comprises performing the functions for which the computational entity of the microscope of the first aspect of the invention is configured, for any of its examples of realization.
    [0112] In a third aspect, the present invention concerns a computer program for the computational reconstruction of the wavefront, which includes program instructions that, when executed in a processor, implement the method of the second aspect.
    [0114] In a fourth aspect, the present invention concerns a computer program product that comprises a tangible medium on which the computer program of the third aspect is stored.
    [0116] The microscope of the present invention, due to the conditions thereof, the nature and effects of which will be described below, allows obtaining the results unattainable with the Hartmann-Shack technology indicated above.
    [0118] On the one hand, the number of pixels of the reconstruction of the wavefront of the microscope of the present invention can be significantly higher than that of an HS since it is not linked to the size of the lenses (in this case the size of the lenses of the ordered two-dimensional arrangement is preferably of the order of mm, which is why they will be called mililents). In the microscope of the present invention, the number of pixels of the wavefront reconstruction is obtained by dividing the number of sensor pixels by the number of millilents. If, for example, for an exemplary embodiment, the microscope of the present invention consisted of 5 millilents in a transverse direction (for example the horizontal or x direction) and a 2,500 pixel sensor in said direction, the final reconstruction would have 500 pixels in that direction. If you wanted to have an HS sensor with the same number of pixels, for a typical sensor size of about 6.0 x 6.0 mm, it would be necessary to have microlenses of d = 12 ^ m. As explained in the previous section, this size is far from the practical limit.
    [0119] It should be taken into account that in the microscope of the present invention the spatial resolution is given by the transposition ratio. For the case of a moderate magnification microscope, for example Mmic = 10 (scientific microscopes operate with magnifications of up to Mmic = 100), the spatial resolution of the system if a pixel size of Ax = 6 ^ m is considered would be 0 , 6 ^ m. That is, this system improves spatial resolution by 3 orders of magnitude compared to a typical HS sensor (whose resolution, remember it is determined by the size of the microlenses, and therefore it is of the order of 100 to 150 pm).
    [0121] On the other hand, the maximum angle of the plane waves sampled by the microscope of the present invention is given by
    [0123] sin 6 max = N "0
    [0124] 2fob
    [0126] where N is the number of millilents in the corresponding sampling direction, "0 is the size of the millilents, and f ob (mm) = 200 µmmic. Considering typical values, for example p0 = 1 mm, f ob = 12mm and N = 5, the maximum angle that could be sampled would be 6max = 15 °. Therefore, the microscope of the present invention makes it possible to measure, for the locally flat waves that make up the wavefront, inclinations between 15 and 30 times greater than a HS.
    [0128] The present invention represents a compact and low cost solution for the measurement of the scattered wavefront by microscopic samples. It represents a much more stable and simple system than those based on interferential detection while significantly improving spatial resolution compared to systems based on Hartmann-Shack detectors.
    [0130] Because it requires the incorporation of few optical elements with respect to a conventional microscope, its development in the form of a module adaptable to a commercial microscope is relatively simple.
    [0132] Therefore, in a fifth aspect, such a module is proposed, which includes at least the ordered two-dimensional arrangement of lenses and the image sensor of the microscope of the first aspect of the present invention, as well as a support thereof and a tube. optical-mechanical coupling device adapted to be coupled (optically and mechanically) to a camera port of a microscope.
    [0134] A sixth aspect of the present invention concerns a microscope kit comprising the microscope module of the fifth aspect and an illumination module comprising the illumination means of the microscope of the first aspect of the present invention adapted to be coupled to a port of illumination of a microscope.
    [0136] The present invention has potential application in various fields of science and technology. On the one hand, it represents a direct application in any field that requires quantitative information from microscopic samples in a non-invasive way, it is that is, without requiring a dye to observe the different structures that make up the sample. Therefore, its use in histology is of special interest. In the same way, it is possible to apply the present invention in metrology and in the study of MEMS systems (microelectromechanical systems), especially, the behavior of said systems as a function of the temperature given to the stability of the proposed measurement system with respect to it. .
    [0138] Brief description of the drawings
    [0140] The foregoing and other advantages and characteristics will be more fully understood from the following detailed description of some embodiments with reference to the attached drawings, which should be taken by way of illustration and not limitation, in which:
    [0141] Figure 1 shows a diagram of the microscope proposed by the first aspect of the present invention, for an embodiment.
    [0143] Figure 2 shows the arrangement of the array or ordered two-dimensional arrangement of lenses of the microscope proposed by the first aspect of the invention, superimposed on the pupil of the objective thereof.
    [0145] Figure 3 is a schematic illustration of the transposition relationship between the capture space and the reconstruction space, carried out by the computational entity of the microscope of the first aspect of the present invention, for an embodiment of the same and of the method of the second aspect of the invention.
    [0147] Figure 4 shows a series of exemplary images of a proof-of-concept experiment using the microscope of the first aspect of the invention for the computational reconstruction of a sample made up of cotton fibers.
    [0148] Figure 5 is a schematic representation of the capture, transposition and reconstruction carried out with the microscope of the first aspect of the present invention and according to the method of the second aspect of the invention, for an embodiment.
    [0150] Figure 6 shows a flow chart with the operational steps to be followed by means of the microscope and method proposed by the present invention, for an embodiment.
    [0151] Figure 7 schematically illustrates the microscope kit proposed by the sixth aspect of the present invention with two couplings, one to the camera port, for the module of the fifth aspect of the invention, and the other to the illumination port, for the media. lighting of a kit lighting module, of a conventional microscope.
    [0153] Detailed description of some examples of realization
    [0155] As illustrated in Figure 1 schematically, for its most basic embodiment, the microscope proposed by the first aspect of the present invention comprises:
    [0156] - lighting means formed by a light source 1, partially or totally coherent, to illuminate a sample T (illustrated schematically in Figure 5);
    [0158] - a microscope objective 2 configured and arranged to receive and focus light scattered by the sample T when illuminated with the illumination means 1;
    [0160] - an ordered two-dimensional arrangement of lenses 3 located at the aperture diaphragm of the microscope objective 2 or at the location of an intermediate image thereof;
    [0161] - an image sensor 4, or pixelated sensor, formed by a plurality of photodetector elements or pixels, which is located in a capture space on the focal plane of the ordered two-dimensional arrangement of lenses or lens array 3 to receive the scattered light through the T sample after passing through the microscope objective 2 and the ordered two-dimensional arrangement of lenses 3, and acquiring spatial information and angular information of the object wavefront associated with the light coming from the T sample, with several of the lenses facing each lens. photodetector elements; and
    [0163] - at least one computational entity (not illustrated) operatively connected to the image sensor 4 and which is configured and arranged to perform a computational reconstruction of the object wavefront from the spatial and angular information.
    [0164] As indicated in a previous section, the spacing p # between the centers of each two contiguous lenses of the ordered two-dimensional array of lenses 3 is preferably at or around 1000 pm, and their relative aperture has a value of o around 6.
    [0166] Figure 2 illustrates the arrangement of the array or ordered two-dimensional arrangement of lenses 3 of the microscope proposed by the first aspect of the invention, superimposed on the objective pupil thereof. Each millilent is characterized by the position of its center with respect to the origin of coordinates.
    [0167] The ideal configuration of the microscope fulfills the following conditions (other configurations can be used as long as the due alterations produced in the complex amplitude of the field when passing through the components of the microscope are taken into account):
    [0169] 1) The position of the matrix of millilents 3 corresponds to the position of the aperture diaphragm of the microscope objective 2 or to that of any intermediate image thereof.
    [0171] 2) The geometry in which the lenses are arranged in the matrix 3 determines the fill factor at the aperture diaphragm. The two most common geometries in which the millilents are arranged are in a square matrix (the centers of the millilents are located at the nodes of a two-dimensional square network) or in a hexagonal network. However, any other geometry is also valid as long as the positions of the nodes of the network are known.
    [0173] 3) Light source 1 provides a uniform and collimated beam (or at least low divergence) on the microscopic sample T. The width of beam B (see Figure 5) is such that, in the absence of sample, the image recorded by the sensor presents a uniform field in the area corresponding to the image plane of the central millilent and total absence of light in the rest, without there being any overlap between the areas corresponding to different millilents.
    [0175] 4) The pixelated sensor 4 is located on the image focal plane of the millilent matrix 3.
    [0176] Under these conditions, the information captured by the microscope proposed by the first aspect of the present invention represents a double sampling process in two reciprocal spaces, thus containing spatial and angular information simultaneously. The existence of a Fourier transformation relationship between spatial and angular information, gives rise to the following restriction on the size of the pixel:
    [0181] where Dx is the size of the pixel, A the wavelength of the illumination beam with which the illumination means 1 illuminate the sample T, f # the focal length of the millilents and "# the spacing between the centers or nodes of each two contiguous millilents of the matrix 3.
    [0182] The intensity distribution of the field detected by the sensor can be expressed mathematically as:
    [0187] In this equation, l represents the l- th pixel of the sensor, the function h () represents the 2D impulse response of the microscope objective (typically an Airy disk) and hu (-) that of the millilent matrix (typically a matrix of Airy discs, in the case of mililents with a circular opening). These impulse responses are determined by wave diffraction and are functionally proportional to the Fourier transform of the corresponding aperture. Furthermore, the function o () represents the amplitude distribution of the wavefront to be measured, M = - f / f b and fb the lateral magnification of the microscope, fb the focal length of the microscope objective, and L the number of pixels of the sensor. Finally the recf (-) function is a binary function that is worth 1 inside a rectangle and 0 outside it, and d) the Dirac delta function.
    [0189] This expression shows the existence in the microscope and method of the present invention, of a double sampling of the complex distribution of amplitudes of the object, or (), limited in resolution by diffraction like any optical system free of aberrations.
    [0191] In combination with the particular configuration and arrangement of the components of the microscope of the first aspect of the present invention, and based on them, a new reconstruction software is proposed, to be implemented by the computational entity and by the method of the second aspect of the invention, the first task of which is to detect the positions of the images provided by the matrix of millilents 3 in the plane of the image sensor 4. These positions define different regions of the frequency content that make up the spectrum of the object. The mililent spacing defines the periodicity of the frequency sampling.
    [0193] On the other hand, the pixelated sensor performs a second sampling but now on the spatial content. Since this sampling and the previous one are carried out in reciprocal spaces, it is possible to carry out a transposition, duly scaled, of any of them to place both information in the same space. Said transposition can be understood as follows:
    [0194] - The sensor samples the spatial information periodically Ax. However, said sampling is carried out on a field previously sampled by the matrix of millilents 3 in its reciprocal space, with a periodicity. The product spatial resolution x bandwidth (also known in the scientific literature as space-bandwidth product (SBP)) determines the amount of information captured by an optical system. In the present invention, the matrix of millilents 3 is the limiting element, therefore the SBP is given by N • , where N is the total number of millilents that fit inside the pupil of the objective and therefore provide images on the sensor of Picture 4.
    [0196] - The transposition of the space-angular information captured with the proposed microscope gives rise to a new expression of this information in which an exchange of periodicity of the samplings occurs, so that the new pixelated matrix represents a local sampling of plane waves of the wavefront scattered by the object. Now the period of spatial sampling is Ax / M and that of angular sampling is p / fb.
    [0197] In this way, we define what will be called the reconstruction space Er (whose transposed space is the capture space) which is located virtually on the object or capture space Ec (see Figures 3 and 5). This consists of L / N regions, whose position is given by the periodicity of the new spatial sampling. Within each region, a total of N angles are sampled with a precision that depends on the periodicity of the angular sampling in the reconstruction space Er, as illustrated in Figure 3, where the transposition relationship between the capture space is illustrated. Ec and the reconstruction space Er.
    [0199] Said space can be interpreted as a synthetic capture system in which microlenses with Ax / M spacing are placed directly on the plane in which the sample is located. With this, for each spatial sampling position, l, the object wavefront o ( x) is sampled locally, so that the intensity of each pixel Im represents a measure of the angular composition of the object wavefront. If the object wavefront is interpreted as a superposition of plane waves, the sum of the local measurements for each spatial sampling region l, duly scaled with the intensity and the corresponding angular component, represents a sampled version of said wavefront:
    [0200] For a given microscope objective, the precision in the measurement of the angular components of the wavefront depends on the size and number of millilents in the capture space.
    [0202] The following explains in more detail, with reference to Figure 5, the process of capture, rearrangement and reconstruction carried out with the microscope of the first aspect of the present invention and according to the method of the second aspect of the invention, for an example of embodiment, for an illumination beam B with which a sample T is illuminated behind which are arranged, in the capture space Ec, the lens matrix 3 and the image sensor 4, schematically illustrating a representation of the reproduction space Er.
    [0204] It is possible to consider that each Sr subregion of the transposed or reconstruction space Er performs local sampling in plane waves of the object wavefront. Each pixel of a subregion Sr of the transposed space Er corresponds to a direction of propagation of the plane waves that make up the object wavefront in said area. It can be considered that for a given subregion Sr, denoted by the superscript l, the complex amplitude of the object wavefront is given by:
    [0208] O = XX ¡lmn e XP [ik mn ]
    [0209] m = -N / 2 n = -N / 2
    [0211] where i lmn is the intensity of the pixel corresponding to position m, n within the Sr subregion and kmn is the direction vector of the plane wave that corresponds to the pixel located at position m, n.
    [0213] For example, when illuminating a completely transparent sample T, as shown in the figure, the wavefront will be completely flat and the recording in the capture space Ec will be exclusively the field given by the central milli-lens. When transposing, each Sr subregion will have only one component, given by element k00. Said element corresponds to a plane wave traveling in the direction of the optical axis. In this way, from all Sr subregions, a totally flat wavefront would be composed, which is the one corresponding to sample T. These elements are physically related to plane waves through the parameters of the system as follows:
    [0214] f í í A
    [0215] k „= exp i 2p m Pm + n Pm
    [0216] VV f or b ) V f oob y)
    [0217] A preliminary result is shown in Figure 4 as an example of the operation of the microscope and method of the present invention. Said result was obtained with a low resolution and not optimized microscope and with a sample of cotton fibers, however, it allows to show the potential of the concept presented in this invention. The left panel of said figure shows the capture obtained by means of a microscope such as the one proposed by the invention. After performing the transposition, a matrix is obtained that represents the reconstruction space Er (central panel of Figure 4). Finally, from the processing of the local contributions to the wavefront given by the pixels of the subregions, the object wavefront is obtained (Figure 4 right panel).
    [0219] Figure 6 shows a flow chart showing the steps of the method proposed by the second aspect of the present invention, or in other words, the functions for which the computational entity of the microscope of the first aspect of the invention is configured , for an example of embodiment, which are described below in correspondence with the legends included in each block.
    [0221] CAPTURE: Capture obtained by the pixelated sensor 4.
    [0222] CALIBRATION: Calibration is a necessary process in the characterization and parameterization of the capture space Eq. For the same assembly, this process will need to be carried out only once. To do this, the object space is illuminated with an incoherent large source, so that all the lenses in matrix 3 are illuminated. Once this is done, the image obtained is saved and a circle detection image processing algorithm is applied. This algorithm provides all the necessary parameters: relative position and size of the lenses, as well as the number of pixels contained within the subregions delimited by each lens.
    [0224] PARAMETERIZATION OF THE CAPTURE SPACE: By means of the parameters obtained during calibration, and knowing the pixel size of sensor 4, the correct angular dimensions are given to the capture space Eq.
    [0226] TRANSPOSITION TO THE RECONSTRUCTION SPACE: A transposition is applied to the capture space E), using the data of the parameterization. With them, an Er reconstruction space is obtained formed by a series of Sr subregions in which each pixel represents an angular direction of propagation of the wavefront.
    [0228] MEASUREMENT OF THE ANGULAR COMPONENTS FOR EACH SUBREGION: The information contained by each pixel is transferred to a plane wave base in which each position of the pixel represents a propagation direction of the wave front.
    [0229] WAVE FRONT RECONSTRUCTION: For each subregion, the contribution of the different angular components expressed in a plane wave base is added. The resulting gray level represents a quantitative measure of the wavefront.
    [0230] Finally, Figure 7 schematically illustrates the microscope kit proposed by the sixth aspect of the present invention, which includes the module of the fifth aspect coupled to the camera port of a commercial microscope that includes a respective eyepiece Oc, objective Ob , tube lens T and flip-up mirror R, as well as an illumination module including illumination means coupled to the illumination port of the microscope.
    [0232] The kit adaptable to a commercial microscope consists of two parts indicated in the scheme of Figure 7 by a broken line:
    [0234] P1) Lighting module: It is required to adapt lighting means, generally a laser and a set of lenses that produce the lighting described in this document to the lighting port.
    [0236] P2) Module of the fifth aspect of the invention, or collection module: The lens matrix 3, the sensor 4 and an auxiliary lens 2 (or a set of them) are adapted to the port of the microscope camera in such a way that the field collected by sensor 4 has the characteristics defined in the invention.
    [0238] The main advantage of the microscope proposed by the present invention is that because the physical capture is carried out in the transposed space, the resolution of the synthetic microlenses of the Er reconstruction space is not limited by diffraction but by the spacing between the camera pixels or image sensor 4. This fact allows to provide a quantitative measurement of phases with an unprecedented lateral resolution.
    [0240] A person skilled in the art could introduce changes and modifications in the described embodiments without departing from the scope of the invention as defined in the attached claims.
    权利要求:
    Claims (21)
    [1]
    1. - Microscope for quantitative wavefront measurements, comprising:
    - lighting means (1) for illuminating a sample (T);
    - a microscope objective (2) configured and arranged to receive and focus light scattered by the sample (T) when illuminated with said illumination means (1);
    - an ordered two-dimensional arrangement of lenses (3) located at the aperture diaphragm of said microscope objective (2) or at the location of an intermediate image thereof;
    - an image sensor (4) formed by a plurality of photodetector elements, which is located in a capture space (Ec) on the focal plane of the ordered two-dimensional arrangement of lenses (3) to receive said light scattered by the sample ( T) after passing through said microscope objective (2) and said ordered two-dimensional arrangement of lenses (3), and acquiring spatial information and angular information of the object wavefront associated with said light coming from said sample (T), remaining facing each various lens of said photodetector elements; and
    - at least one computational entity operatively connected to said image sensor (4) and which is configured and arranged to perform a computational reconstruction of said object wavefront from said spatial and angular information;
    the microscope being characterized in that the spacing p ^ between the centers of each two contiguous lenses of said ordered two-dimensional arrangement of lenses (3) is greater than 500 pm and that their relative aperture is less than 10.
    [2]
    2. - Microscope according to claim 1, wherein the spacing between the centers of each two contiguous lenses of said ordered two-dimensional array of lenses (3) has a value that is between 900 pm and 1100 pm, and their relative aperture has a value that is between 5 and 7.
    [3]
    3. - Microscope according to claim 2, wherein the spacing between the centers of each two contiguous lenses of said ordered two-dimensional arrangement of lenses (3) has a value between 990 pm and 1010 pm, preferably 1000 pm, and its Relative aperture has a value that is between 5.8 and 6.2, preferably 6.
    [4]
    4. - Microscope according to any one of the preceding claims, wherein said light illumination means are configured to illuminate said sample (T) with partially or totally coherent light.
    [5]
    5. - Microscope according to claim 4, wherein said light illumination means are configured to illuminate said sample (T) with a beam of light (B) with a width such that, in the absence of a sample or for a sample (T) transparent, the object wavefront will be completely flat and the acquisition in the capture space (Ec) will be exclusively the light field given by a central lens of said ordered two-dimensional arrangement of lenses (3).
    [6]
    6. - Microscope according to any one of the preceding claims, in which said computational entity, which is at least one, is configured to perform the following double sampling in two reciprocal spaces:
    - a first sampling, or angular sampling, in the space in which the ordered two-dimensional arrangement of lenses (3) is found to obtain the angular information of the object wavefront, determining it, the computational entity, and with it its frequency content space, depending on the position or positions in the capture space (Ec) in which the image sensor (4) receives and acquires said light emitted by the sample (T) after passing through the microscope objective (2) and the arrangement ordered two-dimensional lens (3); and
    - a second sampling, or spatial sampling, in the capture space (Ec) to obtain the spatial information from the intensity received by each photodetector element, or pixel, of the image sensor (4).
    [7]
    7.- Microscope according to claim 6, wherein the size of each photodetector element, or pixel, of said image sensor (4) meets the following restriction:
    D A x J <- -U
    Pv
    where Dx is the size of the pixel, - the wavelength of the illumination beam with which the illumination means (1) illuminate said sample (T), f the focal length of the lenses of said ordered two-dimensional arrangement of lenses (3 ) and p ^ the spacing between the centers of each two contiguous lenses of said lenses.
    [8]
    8. Microscope according to claim 7, wherein said computational entity, which is at least one, is configured to carry out a transposition of one of said two reciprocal spaces to locate both information, spatial and the angular, in the same space, or reconstruction space (Er), located virtually on the object space, where said reconstruction space (Er) consists of L / N regions, where L is the number of photodetector elements, or pixels, of the image sensor (4) and N is the number of lenses of the ordered two-dimensional arrangement of lenses (3), so that a single local sampling of plane waves of the object wavefront is performed that includes said spatial sampling with a period of Dx / M where M = - f / fb and f0b is the focal length of the microscope objective (2), already said angular sampling with a period of p / fb.
    [9]
    9. - Microscope according to claim 8, in which the computational entity, which is at least one, is configured to, in order to perform said computational reconstruction, interpret said reconstruction space (Er) as a capture system synthetic in which an ordered two-dimensional arrangement of spaced lenses is placed between the centers of every two contiguous lenses Dx / M, so that for each spatial sampling position l, the object wavefront o ( x) is sampled locally, so that the intensity of the light received in each photodetector element or pixel Iim represents a measure of the angular composition of the object wavefront.
    [10]
    10. - Microscope according to claim 9, in which the computational entity, which is at least one, is configured to, in order to perform said computational reconstruction, consider that in each sub-region of the reconstruction space (Er) is performs a local sampling in plane waves of the object wavefront, where each pixel of a subregion of the reconstruction space (Er) corresponds to a direction of propagation of the plane waves that make up the object wavefront in said area.
    [11]
    11. - Microscope according to claim 10, in which the computational entity, which is at least one, is configured to determine, for a subregion given by the superscript l, the complex amplitude of the object wavefront according to the following expression :
    N / 2 N / 2
    O = mn
    m = Z - N / 2 n = Z - N / 2 ¡1 exp [ikmn ]
    where I lmn is the intensity of the pixel corresponding to the position m, n within the subregion I and kmn the direction vector of the plane wave that corresponds to the pixel located at the position m, n.
    [12]
    12. - Microscope according to claim 10 or 11, in which the computational entity, which is at least one, is configured to transfer the information contained by each pixel to a plane wave base in which each position of the pixel represents a propagation direction of the object wavefront.
    [13]
    13. - Microscope according to claim 11 or 12, in which the computational entity, which is at least one, is configured to carry out said computational reconstruction by adding, for each sub-region of the reconstruction space (Er), the contribution of the different angular components expressed in said plane wave base, to provide a gray scale image, where the resulting gray level represents a quantitative measure of the object wavefront.
    [14]
    14. - Microscope according to any one of claims 10 to 13, in which the computational entity, which is at least one, is operatively connected with an incoherent extensive light source and with said image sensor (4) to control them to both, and is configured to perform a previous calibration process, for the characterization and parameterization of the capture space (Ec), controlling said incoherent extensive source to illuminate the object space so that all the lenses of the ordered two-dimensional arrangement of lenses (3) are illuminated, controlling said image sensor (4) to acquire, under said incoherent extensive illumination, an image of the sample, and applying a circle detection image processing algorithm to provide all of the following parameters: relative position and size of the lenses of the ordered two-dimensional arrangement of lenses (3) and number of pixels contained within the delimited subregions as for each lens in the ordered two-dimensional array of lenses (3).
    [15]
    15. - Microscope according to claim 14, in which the computational entity, which is at least one, is configured to perform said parameterization of the capture space (Ec), determining and granting the angular dimensions to the capture space (Ec ) using the parameters obtained during the calibration process, and depending on the pixel size of the image sensor (4).
    [16]
    16. - Microscope according to any one of the preceding claims, wherein said ordered two-dimensional arrangement of lenses (3) follows a hexagonal lattice geometry.
    [17]
    17. - Microscope according to any one of claims 1 to 15, wherein said ordered two-dimensional arrangement of lenses (3) follows a square matrix geometry.
    [18]
    18. - Method for the computational reconstruction of the wavefront, which comprises performing the functions for which the computational entity of the microscope according to any one of claims 1 to 17 is configured.
    [19]
    19.
    [20]
    20. - Module for microscope, comprising at least the ordered two-dimensional arrangement of lenses (3) and the image sensor (4) of the microscope according to any one of claims 1 to 17, as well as a support thereof and an optical-mechanical coupling tube adapted to be attached to a camera port of a microscope.
    [21]
    21. - Kit for microscope, comprising:
    - the microscope module of claim 20; and
    - an illumination module comprising the illumination means (1) of the microscope according to any one of claims 1 to 17 adapted to be coupled to an illumination port of a microscope.
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    同族专利:
    公开号 | 公开日
    ES2779500B2|2021-08-05|
    EP3926312A1|2021-12-22|
    WO2020165481A1|2020-08-20|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2014018584A1|2012-07-24|2014-01-30|Trustees Of Boston University|Partitioned aperture wavefront imaging method and system|
    US20140263963A1|2013-03-15|2014-09-18|The Board Of Trustees Of The Leland Stanford Junior University|Volume imaging with aliased views|
    US20140334745A1|2013-05-10|2014-11-13|Trustees Of Princeton University|Resolution light-field imaging|
    US20160062100A1|2014-08-26|2016-03-03|The Board Of Trustees Of The Leland Stanford Junior University|Light-field microscopy with phase masking|
    US9976911B1|2015-06-30|2018-05-22|Beam Engineering For Advanced Measurements Co.|Full characterization wavefront sensor|
    US20180203217A1|2015-07-17|2018-07-19|Leica Microsystems Cms Gmbh|Light sheet microscope for simultaneously imaging a plurality of object planes|
    US9726875B2|2014-09-30|2017-08-08|Agilent Technologies, Inc.|Synthesizing light fields in microscopy|
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