• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention describes a microscope and method for obtaining 3d images of several transparent or semi-transparent samples basically comprising: causing, while maintaining a constant acquisition angle, a relative displacement according to the direction of detection between the illumination sheet and the sample; obtaining, for said acquisition angle, a single 2d projection image formed by a representative parameter for each pixel; modify the acquisition angle by means of a relative rotation between the illumination sheet and the sample combined with a relative vertical translation between the illumination sheet and the sample, and repeat the previous steps; and generate, from the set of 2d projection images obtained, a 3d image of each of the samples. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2567379A1
    申请号:ES201431546
    申请日:2014-10-21
    公开日:2016-04-21
    发明作者:Jorge Ripoll Lorenzo；Alicia ARRANZ DE MIGUEL
    申请人:Universidad Carlos III de Madrid；
    IPC主号:
    专利说明:

    Microscope and procedure for the generation of 3D images of a sample collection
    OBJECT OF THE INVENTION
    The present invention belongs to the field of flat beam lighting techniques used in optical microscopes to obtain images of several transparent or semi-transparent samples such as embryos, tissues and other biological samples, as well as other materials.
    A first object of the present invention is a new method capable of obtaining 3D images of transparent or semi-transparent samples with a higher quality than that of current optical microscopes.
    A second object of the present invention is a microscope specially designed to carry out the above procedure.
    BACKGROUND OF THE INVENTION
    Embryo studies and similar biological samples through an optical microscope present, unlike what happens with individual cells, particular problems related to light absorption and loss of resolution due to light scattering. To solve these problems, significant improvements have been made in recent years on flat laser beam microscopes, whose invention dates back to 1903. See, for example, the document "Ultramicroscopy ~ by Siedentopf and Zsigmondy (Analen der Physik 10 (1) , 1903) After minor improvements proposed by Voie et al, in J. Micros. 170, 1993; (technique called OPFOS by the authors), or by Fuchs et al in Opto Exp. 2002 called uThin laser light sheet microscope "or TSLlM In 2004, Stelzer's group presented a flat laser beam microscope that he called SPIM (Selective Plane Lighting Microscope, Se / ective Plane flumination Microscope according to its acronym in English), which has applications in both in vivo and tissue imaging fixed and transparent or semi-transparent samples in general.
    A flat laser beam microscope is mainly formed by a camera coupled to a high numerical aperture lens and arranged according to a direction called "detection direction", and a lighting medium capable of emitting a thin sheet of light according to a direction called " lighting direction "which is perpendicular to the detection direction, following the original configuration of Siedentopf and Zsigmondy coupled to a detection chamber. With this configuration, the camera can obtain a fluorescence image 20 of the part of the sample illuminated by the illumination sheet or plane. If the sample is also moved in the direction of the detection axis and several images 20 are taken in different positions, a set or stack of images 20 is generated where each of the images 20 corresponds to a position of the illumination plane with respect to the sample. This stack of 2D images contains information on the z-position (depth of the sample according to the detection direction) obtained by moving the sample, and on the x and y positions, present in each image 20. The image stack 20 can then be merged to generate an image 30 of the sample, as described in US 7,554,725 of Stelzer et al.
    A drawback of the flat laser beam microscopy technique is that it has a worse resolution in the detection axis than the image plane. That is, in image 30 the resolution on the x and y axes is more accurate than the resolution on the z axis. To solve this anisotropy, the SPIM or mSPIM multi-view technique has been developed (see Huisken document US 2011/115895). This technique consists essentially of including an additional lighting arm to obtain at least two opposing lighting measurements 180 degrees. By a slight pivot of less than 10 degrees from the plane of light on the plane of illumination the resolution of the image can be improved. If an additional camera is additionally included, four simultaneous measurements can be made corresponding to all possible combinations between camera / lighting arm. These images 20 are subsequently merged to generate a single, higher quality 3D image of the sample in question.
    Another of the proposed ways to improve anisotropy and image quality is to combine several angular measures into a single 3D measure. That is, the sample is rotated around its own axis, usually a vertical axis, so that several stacks of 2D images (commonly called "angular measurements") are captured, each of which corresponds to a rotation angle of The different sample. This has been the proposal published by S. Preibisch et al, Nature Methods 7 (2010), who propose the use of reference markers to properly align these angular measures.
    For a clearer understanding of this technique, Figs. 1 a and 1 b showing two examples of flat laser beam microscopes (100). In Fig. 1a the sample (107) is arranged on a support (101) inside a bath (102). A beam (103) of Gaussian, Bessel or Airy linear lighting strikes a cylindrical lens (104) that focuses on it thanks to a lighting objective (105) to generate the vertical flat illumination sheet (106). This sheet (106) of vertical flat lighting strikes the sample (107) according to the lighting direction (DI), and the fluorescent light (108) is collected by a detection lens (109) oriented according to the detection direction (OD) ), which is perpendicular to the lighting direction (DI). A similar microscope (100) is shown in Fig. 1b, although in this case the formation of the flat illumination sheet (106) takes place by vertical scanning of the linear lighting beam (103) by means of a galvanometric mirror (104 ' ) or similar. In both cases, the support (101) can rotate around its vertical axis to allow several angular measurements to be taken in accordance with the technique proposed by Preibisch.
    Fig. 2 shows a detail of the formation of a stack of images 20 of the sample with a flat laser beam microscope (100) according to Fig. 1a or 1b. It shows how the sheet
    (106) moves according to the direction of detection, taking a picture 20 for each of said positions. The final result is to obtain a stack of images 20. To carry out the method proposed by Preibisch et al, this process is repeated several times for different angles of rotation of the sample around the vertical axis, which allows to obtain an image 3D of the sample with greater isotropy.
    However, the introduction of these angular measures means increasing the exposure time and the duration of the experiment proportionally to the number of angular measures. Indeed, since the exact position of the center of rotation is unknown, merging all angular images obtained in a flat laser beam microscope requires the use of markers (fiducials) to generate the final 3D image (see S. Preibisch, et al , Nature Methods 7, 2010), which requires high computing power, storage capacity and complicates the experimental measurement.
    DESCRIPTION OF THE INVENTION
    The present invention solves the above problem thanks to a new microscope and 3D image generation procedure that requires much less computing power and storage capacity than current flat laser beam techniques. This is not only advantageous in terms of the requirements of a microscope designed to carry out this procedure, but also allows 3D images to be generated with a higher resolution and lower anisotropy. In the context of the acquisition of images of samples in vivo, the rapidity in obtaining the images is crucial, since it depends on this that useful information can be obtained for the understanding of certain biological processes. In addition, the new microscope and procedure allows to obtain images of several different samples located vertically one on top of the other, which is not possible with the current equipment.
    In order to ensure the clarity of the description, a series of terms that will be used in this document are described below.
    Lighting direction: Direction along which the illumination sheet or plane through the sample is projected.
    Illumination sheet: Thin sheet of light emitted towards the sample according to the direction of illumination. The plane containing the lighting sheet, called the lighting plane, is normally vertical.
    Detection address: Address according to which the camera lens is arranged to obtain 2D images of the sample. It is perpendicular to the illumination sheet, and therefore is normally horizontal. The objective of the camera can be high or low magnification, and its numerical aperture high or low.
    2D Image: Each of the individual images obtained by the camera. Each such individual image corresponds to a position of the illumination sheet in relation to the sample.
    Stack of 2D images: Set of 2D images obtained by the camera and corresponding parallel positions of the illumination sheet as a result of either the displacement of the sample according to the direction of detection or the displacement of the illumination sheet in the same direction.
    3D Image: Image generated from a set of stacks of 2D images corresponding to illuminate the sample from different orientations.
    Projection image: 2D image that can be generated by illuminating the sample and obtaining, on the side of the sample opposite to the one from which it has been illuminated, a projection image that can be assimilated to the "shadow" that has been projected. It can also be obtained from a stack of 2D images by applying a parameter to each pixel of the image, for example a statistical parameter such as variance, maximum value, minimum value, average value, correlation between pixels, etc.
    Acquisition angle: The angle is the horizontal plane between the direction of detection and a vertical plane of the sample, for example the plane of symmetry or similar in the case of certain organisms. As mentioned, this remains constant during the acquisition of each stack of 2D images.
    The inventors of the present invention have developed a new microscope and method for the generation of the 3D image that combines the techniques commonly used in flat laser beam microscopy with those commonly used in microscopy
    OPT.
    The OPT technique (Optical Projection Tomography, Oplical Projection Tomography), described in US20060122498 A1, is relatively similar to X-ray tomography. It is essentially based on optically illuminating the sample homogeneously and obtaining, on the side of the sample opposite to the one from which it is illuminated, an image that can be assimilated to the "shadow" that the sample casts on a plane, or in the case of measuring the fluorescence, the total emission of the illuminated volume. This "shadow" or fluorescence emission, normally called projection image, has different shades of gray depending on the absorption of light and / or fluorescence emission that occurs in different parts of the sample. If the sample is illuminated from several angles, it is possible to implement a reconstruction algorithm on all the images obtained to generate a 3D image of said sample. This reconstruction algorithm is usually based on solving the Radon transform, originally developed for the X-ray 3D image.
    OPT (Optical Projection Tomography, Sharpe Science 2002) microscopes, unlike flat laser beam microscopes, illuminate the entire sample simultaneously, and base their 3D reconstruction on angular measurements, analogously to a Computerized Tomograph of X-ray (CT) but with optical measures, both absorption and fluorescence. A disadvantage of OPT microscopes is the need to use low numerical apertures to keep all or at least half of the sample within the focal plane, thus reducing both the sensitivity and resolution of these devices. On the other hand, OPT measurements are simple to implement, since each angular measure consists of a single projection of the entire volume, being able to use existing algorithms such as Filtered Back Projection, or even modeling the dispersion of light present in the sample and obtain images of samples with a high dispersion coefficient.
    Fig. 3 shows schematically the operation of an OPT microscope (200). The assembly consists essentially of a detection lens (201) coupled to a camera and directed towards the sample (202) to collect the fluorescence or trans-illumination
    (203) caused by the homogeneous illumination emitted by an element that is not shown in the image. The sample (202), which is contained in a bath (204) fixed to a support (205), revolves around its own axis, usually a vertical axis, so that several images corresponding to different angles are taken. Subsequently, the Radon reverse transform is implemented (using filtered backprojection, Filtered Backprojection, for example, or another reconstruction algorithm) to construct a single 3D image of the sample.
    The microscope and procedure proposed by the inventors of the present application combines characteristics of both techniques in a way that improves the resolution and quality of the final 3D image, combining both devices into one. The microscope and method of the invention employ, like the flat laser beam technique, a lighting sheet to obtain clear information from inside the sample. This allows to increase the numerical aperture of the camera, since the distance between the lens and the illumination plane is always known, thus improving the resolution of the images obtained. On the other hand, as in the OPT technique, the microscope and method of the invention do not store a 2D image for each position of the illumination sheet, but for each acquisition angle it stores only one representative parameter of each pixel. That is, for each acquisition angle a single 2D projection image (similar to the OPT technique) is stored, instead of a whole stack of 2D images (as in the flat laser beam technique). This allows not only to decrease the system requirements, but also to increase the acquisition speed. Furthermore, by means of a vertical translation and the use of a container in which several samples can be placed, this new invention allows to obtain images of several samples arranged one above the other, as can be seen in Fig. Four.
    Accordingly, the present invention describes a method for generating 3D images of a collection of samples by a microscope comprising an image acquisition means directed towards the collection of samples according to a detection direction, and a lighting medium configured to emit a flat illumination sheet towards the sample collection according to a direction perpendicular to the detection direction. As discussed earlier in this document, the sample collection comprises several samples located one above the other. The procedure includes the following steps:
    1) Provoke, maintaining a constant acquisition angle, a relative displacement according to the direction of detection between the illumination sheet and the sample.
    As is known in the art, this relative displacement can be carried out in two ways: keeping the illumination sheet still and moving the sample according to the detection direction; or keeping the sample still and moving the illumination sheet. In either case, the fact is that the illumination sheet performs a "scan" of the sample along the detection direction.
    2) Obtain, for said acquisition angle, a single 2D projection image formed by a representative parameter for each pixel.
    According to a preferred embodiment of the invention, the representative parameter for each pixel is a statistical parameter, such as the maximum value, the variance, the minimum value, the standard deviation, the average value, the correlation between pixels, etc. In this case, obtaining the statistical parameter can be carried out by acquiring the images in continuous mode for each acquisition angle. That is, causing a continuous relative movement between the illumination sheet and the sample according to the direction of detection while acquiring images at high speed (more than 100 images per second). Being interested in the statistics of each pixel, the exposure times can be extremely short, with noise signal values lower than those used in a flat laser beam microscope.
    According to another alternative embodiment of the invention, the representative parameter for each pixel is the sum of the intensities of each pixel. The sum of the intensities of each pixel can be obtained by causing a continuous relative movement between the illumination sheet and the sample according to the detection direction while keeping the exposure of the image acquisition medium open. That is, in the projection image obtained each pixel has as its final value the sum of intensities that pixel has received during the entire process of moving the illumination sheet throughout the sample.
    3) Modify the acquisition angle by a relative rotation between the illumination sheet and the sample combined with a relative vertical translation between the illumination sheet and the sample, and repeat the previous steps.
    Once the projection image is obtained and stored for a given acquisition angle, the acquisition angle is modified and the process is repeated, and so on for a number of configurable acquisition angles, so that a set of images of projection at 20. This angular information would effectively correspond to an OPT measurement carried out with a high numerical aperture objective, an unrealizable measure with the low numerical aperture requirements necessary for OPT. In addition, by not illuminating the entire volume of the sample simultaneously, dramatically reduce the
    photobleaching and phototoxicity effects.
    As previously mentioned, the fact that this technique obtains
    and store only one projection image at 20 for each acquisition angle contrasts with the multi-angular flat laser beam technique proposed by Preibisch (Nat. Meth. 2010) or with the multiview SPIM proposed by Huisken, in which all a stack of images 20 for each acquisition angle, since the storage and processing of all that information considerably slows down the process and imposes greater requirements in relation to the equipment necessary to carry it out.
    On the other hand, a relative vertical translation between the illumination sheet and the sample is added to the step of modifying the acquisition angle by using a long-haul motor. That is, what is known as helical OPT (hOPT), equivalent to helical or spiral CT, is applied. As mentioned, this can be done either by moving the sample vertically, or by moving the illumination sheet vertically together with the detection system. In any case, the combination of the rotation with the translation generates a helical movement that allows obtaining information from several samples arranged one above the other or from a single very elongated sample, which is not possible with the current flat laser beam technique .
    Additionally, in a preferred embodiment of the invention it further comprises modifying the position of the sample within a plane perpendicular to the axis of rotation of said sample to always center the same portion of the sample in front of the detection direction. That is to say, the sample is recentra in the microscope's field of vision by means of a movement in the mentioned plane (which can also be defined as perpendicular to the vertical translation, or plane that contains the directions of detection and illumination). This allows to analyze very large tissues or samples, which is currently not possible.
    4) Generate, from the set of projection images obtained, a 3D image of each of the samples.
    Finally, a reconstruction algorithm is implemented to the information of the set of projection images obtained to generate a single 3D image of the sample. For example, the projection images can be introduced in a Filtered Back Projection algorithm or in a projection reconstruction algorithm such as the Radon reverse transform, generating the 3D image of the sample.
    It is important to highlight that a conventional flat laser beam microscope does not obtain its 3D images in this way. In these cases, the 3D image is constructed from the image stack 20 obtained, or in the case of the Preibsch multi-angle SPIM or the Huisken mSPIM, the fusion of several stacks of 2D images corresponding to each angle. In no invention of flat laser beam in general the use of the inverse transform of Radon to generate a 3D image is described.
    Additionally, according to another preferred embodiment of the invention, the method further comprises the step of combining several 3D images obtained using different parameters to obtain an improved final 3D image. For example, if the 3D image obtained using the maximum intensity is subtracted from the 3D image obtained using the sum of intensities, the contrast can be improved by removing the background. On the other hand, it is possible that the 3D image obtained from the variance provides additional information that does not have an image obtained from the intensity, with more anatomical detail, for example.
    A second aspect of the invention is directed to a microscope specially designed to carry out the described process comprising an acquisition means of Fig. 3 shows a schematic view of a conventional OPT type microscope according to the prior art.
    S images directed towards the sample collection according to a detection direction, and a lighting medium configured to emit a flat illumination sheet towards the sample collection according to a direction perpendicular to the detection direction, where the sample collection comprises several samples located one on top of the other. In addition, the microscope of the invention comprises:
    10 a) Means to cause, maintaining a constant acquisition angle, a relative displacement according to the direction of detection between the illumination sheet and the sample. b) Means for obtaining, for said acquisition angle, a single projection image in 20 formed by a representative parameter for each pixel.
    fifteen c) Means for modifying the acquisition angle by a relative rotation between the illumination sheet and the sample combined with a relative vertical translation between the illumination sheet and the sample.
    twenty d) Means to generate, from the set of projection images in 20 obtained, a 3D image of each of the samples. In a preferred embodiment of the invention, the means for causing a relative displacement according to the direction of detection between the illumination sheet and the sample are configured to perform a continuous displacement.
    25 In another preferred embodiment of the invention, the means for performing a relative vertical translation between the illumination sheet and the sample comprise a long-distance electric motor.
    30 BRIEF DESCRIPTION OF THE FIGURES Figs. 1a and 1b respectively show a schematic view of two examples of conventional flat laser beam microscope according to the prior art.
    35 Fig. 2 shows a detail of the acquisition of a set of 2D images of the sample.
    Fig. 4 shows a support designed to house the collection of samples arranged one above the other.
    Fig. 5 (a), 5 (b), 5 (c) and 5 (d) show respectively: Fig. 5 (a) shows a numerical scheme of an image 20 of an object; Fig. 5 (b) shows examples of projections on the image 20 of Fig. 5 (a) corresponding to different parameters for a rotation angle of 0 °; Fig. 5 (c) shows examples of projections on the image 20 of Fig. 5 (a) corresponding to different parameters for a rotation angle of 90 °; and Fig. 5 (d) shows examples of projections on the image 20 of Fig. 5 (a) corresponding to the "Sum" parameter for the angles of rotation 0 °, 90 °, 180 ° and 270 °.
    Fig. 6 shows a flow chart of the procedure that is carried out to obtain the projection image for each angle.
    PREFERRED EMBODIMENT OF THE INVENTION
    Figures 5 (a) -5 (d) represent a simplified example showing the process of obtaining angular projection images of a two-dimensional object corresponding to different parameters. The parameters used in this example are the mean, the minimum value, the variance, the sum and the maximum value.
    Fig. 5 (a) shows the object with the values obtained after the acquisition of five images corresponding to a given acquisition angle that, due to the simplification of this example, would be monodimensional. It could be said that it is a stack of 5 1 D images (five rows) representing 5 sections of the object.
    From this data, several projection parameters can be generated. Fig. 5 (b) shows an example of obtaining several projection images from the image stack corresponding to an acquisition angle of O degrees. Note that, in the present invention, it is not necessary to store all the images of the image stack obtained, since the calculation of the parameter can be performed as the images are obtained during the "scanning" of the object according to the detection direction .
    Once the corresponding projection image is generated, the sample is rotated and a new stack of images is acquired from which another projection image will be obtained. If there is a vertical displacement, this would normally take place after the rotation. Fig. 5 (c) shows an example corresponding to an acquisition angle of 90 ° for the same parameters. For simplicity, the same stack of images as in Fig. 5 (b) has been used, although as is logical in a real case,
    I would get a different stack of images from the previous one.
    This process is repeated for a specific number of angular measurements, in this specific example four measurements corresponding to 0 °, 90 °, 180 ° and 270 °. The result is shown in Fig. 5 (d), which shows the four projection images corresponding to the sum parameter. These projection images are then introduced in a code that solves the inverse Radon transform, for example, or in a more advanced tomographic projection code that takes into account the presence of scattering, generating the final 3D image.
    The complete procedure is shown in the algorithm shown in Fig. 6. First, an image 20 corresponding to a given acquisition angle is taken. Next, the illumination sheet is moved and another image 20 is taken. This process is repeated as many times as necessary until images have been taken covering the entire required volume. Next, the determined parameter for each one is calculated
    of the pixels of the obtained images 20, which results in the projection image corresponding to that first acquisition angle. The resulting projection image 20 is stored.
    Then, after printing a rotation to the second acquisition angle combined with a vertical offset to the sample, the above operations are repeated. This whole process is repeated until a complete round around the sample or samples has been completed. Finally, the set of projection images that has been stored is introduced into an algorithm to generate the final 3D image. For example, using a Filtered Back Projection type algorithm or a Radon reverse transform algorithm.
    A concrete example of the use of this procedure could be a zebrafish embryo sample that expresses a protein. This embryo can be introduced into a transparent container (an FEP tube, for example), or it can be embedded in agarose so that it can move in the measurement plane, and can be rotated around an axis of rotation. Once this sample was placed on the microscope of the invention, the light source would be turned on to create a flat beam of light. Once the parameter to be saved has been chosen (the maximum intensity of the 2D image stack, for example), we would proceed to move the light plane with respect to the sample from an initial position to an final position, saving the projection image in 2D that contains the statistical information of this displacement. This process would be repeated for each measurement angle up to a total of M angles, generating a stack M projection images.
    5 These projection images would be introduced in a Filtered Back Projection algorithm,for example, or in a predefined projection reconstruction algorithm, generating the3D image of the sample.
    Another example could consist of N tissue samples with fluorescent staining, previously
    10 cleared and fixed. These N samples can be introduced into a single transparent container, or they can be embedded in agarose so that they can be moved both in the measurement plane and vertically, and the set of samples can be rotated around an axis of rotation. The result would be similar to that shown in Fig. 4. Once these samples were placed on the microscope of the invention, the
    15 lighting source to create a flat beam of light. Once the parameter to be saved has been chosen (the maximum intensity of the image stack 20, for example), the lighting plane would be moved relative to the sample from an initial position to an final position, saving the projection image in 20 containing the statistical information of this displacement. Once the scanning of the light plane is finished,
    20 would proceed to the rotation, and, simultaneously, the vertical translation of the sample. This process would be repeated until the entire sample has been scanned, requiring several complete rotations with a total of M angles, generating a stack of M helical projection images. These projection images would be introduced in a Filtered Back Projection algorithm with helical displacement, for example, or in a
    25 projection reconstruction algorithm with predefined vertical translation, generating the 3D image of all samples simultaneously.
    权利要求:
    Claims (13)
    [1]
    one. Method for generating images 30 of a sample collection by means of a microscope, characterized in that the microscope comprises an image acquisition means directed towards the sample collection according to a detection direction, and a lighting means configured to emit a sheet of flat illumination towards the sample collection according to a direction perpendicular to the detection direction, where the sample collection comprises several samples located one above the other, the procedure comprising the following steps: -provoking, maintaining a constant acquisition angle, a relative displacement according to the direction of detection between the illumination sheet and the sample; - obtaining, for said acquisition angle, a single projection image in 20 formed by a representative parameter for each pixel; -modify the acquisition angle by a relative rotation between the illumination sheet and the sample combined with a relative vertical translation between the illumination sheet and the sample, and repeat the above steps; and -generating, from the set of projection images in 20 obtained, an image 30 of each of the samples.
    [2]
    2. Method according to claim 1, wherein the representative parameter for each pixel is a statistical parameter.
    [3]
    3. Method according to claim 2, wherein the statistical parameter is selected from the maximum value, the variance, the minimum value, the standard deviation, the average value and the correlation between pixels.
    [4]
    Four. Method according to any of claims 2-3, wherein obtaining the statistical parameter is carried out by causing a continuous relative movement between the illumination sheet and the sample according to the detection direction and acquiring images at high speed.
    [5]
    5. Method according to claim 1, wherein the representative parameter for each pixel is the sum of the intensities of each pixel.
    [6]
    6. Method according to claim 5, wherein the sum of the intensities of each pixel is obtained by causing a continuous relative movement between the sheet of
    lighting and the sample according to the detection direction while keeping the exposure of the image acquisition medium open.
    [7]
    7. Method according to any of the preceding claims, wherein the step of modifying the acquisition angle further comprises modifying the position of the sample within a plane perpendicular to the axis of rotation of said sample to always center the same portion of the sample against The detection address.
    [8]
    8. Method according to any of the preceding claims, wherein the step of generating a 3D image of the sample comprises applying a filtered back projection algorithm to the set of projection images obtained.
    [9]
    9. Method according to any of claims 1-7, wherein the step of generating a 3D image of the sample comprises applying the inverse transform of Radon to the set of projection images obtained.
    [10]
    10. Method according to any of the preceding claims, which further comprises combining several 3D images obtained using different representative parameters to obtain an improved 3D image.
    [11]
    eleven. Microscope for the generation of 3D images of a collection of samples capable of carrying out the method of any of the preceding claims, characterized in that it comprises an image acquisition means directed towards the collection of samples according to a detection direction, and a lighting medium configured to emit a flat illumination sheet towards the sample collection according to a direction perpendicular to the detection direction, where the sample collection comprises several samples located one above the other, which also comprises: means for causing, maintaining a constant acquisition angle, a relative displacement according to the direction of detection between the illumination sheet and the sample; - means for obtaining, for said acquisition angle, a single 2D projection image formed by a representative parameter for each pixel; - means for modifying the acquisition angle by means of a relative rotation between the lighting sheet and the sample combined with a relative vertical translation between the lighting sheet and the sample; Y
    - means to generate, from the set of 2D projection images obtained, a 3D image of each of the samples.
    [12]
    12. Microscope according to claim 11, wherein the means for causing a
    5 relative displacement according to the direction of detection between the illumination sheet and the sample are configured to perform a continuous displacement.
    [13]
    13. Microscope according to any of claims 11-12, wherein
    Means for performing a relative vertical translation between the illumination sheet and the sample 10 comprise an electric motor.
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