• 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 new automatic device (1) that allows to change the objective (109) of acquisition of images of a flat laser beam microscope according to the magnification that is desired in each moment. The device (1) comprises: at least two supports (2) configured for the parallel coupling of at least two targets (109) oriented according to a detection direction (dd); a lateral translation means (3) configured to laterally displace said at least two supports (2) so that one of the objectives (109) faces a cuvette (102) of the plane laser beam microscope (100); and a longitudinal translation means (4) configured to longitudinally displace according to the detection direction (dd) at least the support (2) to which the objective (109) facing the trough (102) is coupled. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2630733A1
    申请号:ES201630071
    申请日:2016-01-21
    公开日:2017-08-23
    发明作者:Jorge Ripoll Lorenzo；Alicia ARRANZ DE MIGUEL；César NOMBELA ARRIETA
    申请人:4d-Nature Imaging Consulting S L；4d-Nature Imaging Consulting SL；
    IPC主号:
    专利说明:

    Automatic lens change device for flat laser beam microscope OBJECT OF THE INVENTION
    The invention pertains to the field of microscopy, and more particularly to flat laser beam illumination microscopy used to obtain images of several transparent or semi-transparent samples such as embryos, tissues and other biological samples.
    The object of the present invention is a new automatic device that allows the image acquisition objective of a flat laser beam microscope to be changed depending on the magnification desired at any given time. 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. In order to solve these problems, important improvements have been developed in recent years on flat laser beam microscopes, whose invention dates back to 1903.
    A flat laser beam microscope is essentially formed by a camera coupled to a high numerical aperture lens and arranged in 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 2D fluorescence image 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 2D images are taken in different positions, a set or stack of 2D images is generated where each of the 2D images 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 the x and y positions, present in each 2D image. The 2D image stack can then be merged to generate a 3D image of the sample, as described in


    US 7,554,725 by Stelzer et al. Subsequently, it was proposed to rotate the sample around its own axis, normally vertical, to capture several stacks of 2D images (commonly called “angular measures”) and merge them later, which allows to improve anisotropy and image quality (S Preibisch et al, Nature Methods 7 (2010)).
    For a clearer understanding of this technique, Fig. 1 is attached, which shows an example of a flat laser beam microscope (100). The sample (107) is arranged in a support (101) inside a cuvette (102) filled with a liquid. A beam (103) of Gaussian, Bessel, Airy or similar linear illumination strikes a cylindrical lens (104) that focuses 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) emitted by that particular plane of the sample (107) is collected by a target (109). ) of detection oriented according to the direction of detection (DD), which is perpendicular to the direction of illumination (DI). 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.
    On the other hand, the OPT (Optical Projection Tomography) technique, described in US20060122498 A1, is relatively similar to X-ray tomography. It is essentially based on optically illuminating the sample homogeneous and obtain, 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, usually 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.
    Recently, the inventors of the present application have submitted the patent application PCT / ES2015 / 070455 entitled "Microscope and procedure for the generation of 3D images of a collection of samples" which describes a new microscope that combines the flat laser beam technique of SPIM type (Selective Plane Illumination Microscope) with the technique of optical projection tomography (OPT). This


    The new microscope does not store a complete 2D image for each position of the illumination sheet, but for each acquisition angle it stores only a representative parameter of each pixel obtained by OPT-type techniques. That is, for each acquisition angle a single 2D projection image 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.
    The inventors of the present application have also filed the patent application P201531401, entitled "Multiple charge device for flat laser beam microscope" which describes a multiple charge device for feeding a flat laser beam microscope of a continuous flow and Sequential samples. This device fundamentally comprises a capillary conduit that crosses the measuring area of the microscope sample receiving cell having a diameter such that it only allows the passage of the samples one at a time, and an adjustable flow generating element connected to the capillary conduit capable of causing a continuous and controllable flow of samples immersed in a fluid medium through said capillary conduit. This allows a plurality of samples to be passed sequentially through the interior of the receiving cell, accelerating the process of data acquisition of multiple samples.
    In any of these cases, the current flat laser beam microscopes (100) as shown in Fig. 1 normally employ a lens (109) called "immersion." This type of objective (109) requires to be introduced into the fluid in which the sample (107) is immersed, so that said fluid is the only means that interposes between objective
    (109) and sample (107). Because of this, since the detection direction (DD) according to which the objective (109) is oriented is normally horizontal, the distal end of the objective
    (109) It must enter inside the cuvette (102) through a hole. This setting prevents the change of the lens (109). DESCRIPTION OF THE INVENTION
    The invention describes a new automatic lens change device that allows one lens to be automatically exchanged for another in case it is necessary to modify the magnification. This automatic device has two main configurations, a horizontal configuration and a vertical configuration. In the vertical configuration, the objective that is being used is moved in a vertical plane to introduce it above the cuvette until its end is immersed in the fluid that fills the cuvette. Therefore, in this case “immersion” objectives are used. In the horizontal configuration, the objective that


    being used, it travels in a horizontal plane on the outside of the cuvette, approaching it at most until its end is adjacent to a lateral wall of the cuvette. Therefore, in this case objectives called "air" are used. This horizontal configuration is also compatible with the presence of a horizontal “immersion” target fixed to the cuvette, so that by rotating the cuvette it is possible to choose whether the “immersion” device or any of the “air” devices is used . In any of the cases, this new device not only allows the change of objective, but also avoids the need for the change to be made manually, which allows to optimize the processes of obtaining images.
    The invention describes an automatic lens change device for a flat laser beam microscope comprising: at least two supports for targets, a lateral translation means and a longitudinal translation means. Each of these elements is described in more detail below:
    a) Supports
    These are at least two supports configured for parallel coupling of at least two objectives oriented according to a detection direction. That is, the supports are normally located next to each other according to a direction perpendicular to the detection direction and oriented in parallel according to said detection direction so that, when some objectives are coupled to said supports, all objectives are oriented in parallel. according to the direction of detection.
    The supports can in principle be configured in any way as long as they allow the rigid coupling of a lens. For example, they may be supports configured so that the lens snaps into place, or they may have threaded clamping elements or the like. As for the number of media, it will depend on the number of objectives necessary for the application. For example, it may be two, three, or more supports.
    The lateral displacement of the supports can be done individually for each support. In this case, the device would include a lateral translation means for each support. However, in a preferred embodiment of the invention the supports are preferably fixed to a single platform, the platform being laterally movable by the lateral translation means. That is, in this case the


    brackets move laterally all together.
    b) Lateral translation medium
    The lateral translation means is configured to move the at least two supports laterally so that one of the lenses is facing a flat laser beam microscope cuvette. As mentioned above, it can be a single lateral translation means that move all the supports together, or a lateral translation means for each support so that the supports move individually. In either case, the lateral translation means have the function of placing a specific support, corresponding to a target with a determined magnification, in front of the microscope cuvette.
    c) Longitudinal translation medium
    The longitudinal translation means is configured to move longitudinally according to the direction of detection at least the support to which the lens facing the cuvette is attached. The function of this means of longitudinal translation is to allow an adequate focus of the sample whose images are to be acquired by means of the lens facing the cuvette. It can be a single means of longitudinal translation that longitudinally displaces all the supports in unison, or a means of individual longitudinal translation for each support.
    Both longitudinal and lateral translation means can be simply implemented by small motors. This new device allows to automatically change the objective used at any time to modify the magnification. In addition, this device can have two main configurations.
    Horizontal configuration
    In a preferred embodiment of the invention, the detection direction is horizontal, so that both the lateral translation direction and the longitudinal translation direction are contained in a horizontal plane. This means that the objective in use at all times is not introduced into the cuvette, but acquires the images through the glass of a sidewall of the cuvette. Therefore, in this configuration the type of objective used is an “air” objective. In this horizontal configuration


    of the device of the invention, the sample is introduced into the cuvette from above and rotates around an axis of vertical rotation.
    In another more preferred embodiment of the invention, the end of stroke of the longitudinal translation means corresponds to a position in which the distal end of the target facing the cuvette is adjacent to a side wall of said cuvette. That is, the longitudinal translation means can move the support facing the cuvette at most until the end of the corresponding target is next to the side wall of the cuvette without touching it. The reason is obviously to prevent the distal end of the lens from damaging or breaking the side wall of the cuvette.
    In another even more preferred embodiment of the invention, the platform is longitudinally movable by the longitudinal translation means. That is, as the objective in use does not exceed the plane of the side wall of the cuvette, and since all the objectives are located in parallel, there is no danger that any objective will collide with said wall. Therefore, in this case it is not necessary for each support to move longitudinally individually, but rather the longitudinal translation means can move the entire platform assembly, and therefore all the supports at the same time.
    Vertical configuration
    According to another preferred embodiment of the invention, the detection direction is vertical, so that both the lateral translation direction and the longitudinal translation direction are contained in a vertical plane. This means that the target in use at any time is configured to enter the cuvette vertically from above. Since the cuvette is open superiorly, the target can descend until its distal end is immersed in the fluid that fills the cuvette. Therefore, in this configuration the type of objective used is an "immersion" objective. In addition, in the vertical configuration of the device of the invention, the sample is introduced into the cuvette from one side and rotates around a horizontal rotation axis.
    In another preferred embodiment of the invention, the end of stroke of the longitudinal translation means corresponds to a position in which the distal end of the target facing the cuvette is below the level of the fluid filling said cuvette.


    In another even more preferred embodiment of the invention, each individual support is longitudinally movable by the longitudinal translation means. That is, as the objective in use can descend until its end is introduced into the fluid that fills the cuvette, if all the targets were displaced together there would be a danger that, depending on the size of the cuvette and the separation distance between the supports, one of the objectives adjacent to the objective in use came to collide with one of the side walls of the cuvette. To avoid this possibility, in this configuration preferably the longitudinal translation means displaces each support individually. This can be done in different ways, either by using an individual longitudinal translation means for each support, or by using a single longitudinal translation means configured so that it can be coupled at any time only to the support facing the cuvette.
    The present invention is also generally directed to a flat laser beam microscope comprising an automatic target change device according to any of the preceding claims.
    In addition, a flat laser beam microscope that is equipped with an automatic device for changing the horizontal configuration lens as described in the previous lines may additionally comprise:
    d) A means of rotation of the cuvette around a vertical axis. This means of rotation can be configured in any way as long as it allows the tray to be rotated in a controlled manner until it is placed in a desired position depending on the most suitable image acquisition mode at any time. For example, it can be simply formed by a small electric stepper motor coupled to the bucket.
    e) An objective fixed to a lateral wall of the cuvette so that its distal end crosses said lateral wall and is immersed in the fluid that fills the cuvette. It is therefore a fixed “immersion” objective similar to those used in flat laser beam microscopes according to the prior art.
    In this way, selecting the angle of rotation of the cuvette can be selected if the fixed “immersion” objective or one of the “air” objectives of the automatic target change device is used. That is, if it is desired to use any of the objectives of the automatic target change device, the cuvette is placed in a position where


    the fixed target does not interfere with the use of said device. For example, the cuvette can be placed in a position where the fixed target is on the opposite side to that in which the automatic target change device is located, or on the opposite side from that from which it is carried out the lighting of the bucket. In this situation, the user can normally use any of the objectives of the automatic lens change device by connecting the objective in question to an image acquisition equipment that is supposed to be located on that side of the cuvette.
    Alternatively, if it is desired to use the fixed objective, the cuvette is placed in a position where said fixed objective is located on the face where the automatic lens change device and also the image acquisition equipment are located. The automatic device will be in a retracted position so that it does not interfere in obtaining the sample images using the fixed target. In this situation, the user can connect the fixed objective to the image acquisition equipment and use it normally. BRIEF DESCRIPTION OF THE FIGURES
    Fig. 1 shows an example of a flat laser beam microscope according to the prior art.
    Fig. 2 shows a view of a flat laser beam microscope equipped with an automatic lens change device according to the first horizontal configuration.
    Fig. 3 shows a view of a flat laser beam microscope provided with a lens changing device according to the second vertical configuration.
    Fig. 4 shows a view of a flat laser beam microscope according to the vertical configuration provided with a means of rotation of the cuvette and an additional lateral objective. PREFERRED EMBODIMENTS OF THE INVENTION
    Fig. 1 shows an example of a flat laser beam microscope (100) according to the prior art. As described above in this document, the sample (107) is arranged in a support (101) inside a cuvette (102) filled with a liquid. A beam (103) of Gaussian, Bessel, Airy or similar linear illumination strikes a cylindrical lens (104) that focuses it thanks to a lighting objective (105) to generate the sheet (106) of


    vertical flat lighting. This sheet (106) of vertical flat lighting strikes the sample (107) according to the direction of illumination (DI), and the fluorescent light (108) emitted by the sample is collected by a direction-oriented detection lens (109) of detection (DD), which is perpendicular to the lighting direction (DI). The support (101) can rotate around its vertical axis.
    Fig. 2 shows an example of automatic lens change device (1) according to the present invention in its horizontal configuration. As you can see, the device
    (1) of this example specifically presents three supports (2) located in a horizontal plane and oriented according to a detection direction (DD) also horizontal. The three supports (2) are rigidly fixed to a horizontal platform (5), in parallel and aligned with each other. A respective lens (109) is attached to each of the supports (2). In other words, the supports (2) are arranged such that the distal ends of the lenses (109) coupled thereto are aligned according to a direction perpendicular to the detection direction (DD).
    The platform (5) is connected to a lateral translation means (3), for example a small electric motor step by step or provided with a reducer, which is configured to move said platform (5) in a direction perpendicular to the direction of detection (DD) within a horizontal plane. This lateral translation allows to select which of the objectives (109) is aligned with the sample (107) housed inside the cuvette (102). The selection of the objective (109) can be done, for example, according to the magnification needed at a given time or for a particular sample (107).
    The platform (5) is in turn connected to a means (4) of longitudinal translation, which can also be a small electric motor step by step or equipped with a reducer, which is configured to move said platform (5) according to the direction of detection (DD). This longitudinal translation serves to properly focus the sample (107) on the lens (109) being used. In this horizontal configuration, objectives are used
    (109) of air pointing to the sample (107) through a side wall of the cuvette (102). For this reason, it is necessary to carefully calibrate the limit switch of the longitudinal translation means (4) so that the distal end of the lens (109) aligned with the cuvette
    (102) do not advance excessively and come to collide with said side wall of the cuvette (102). The existence of this end of race allows to fix the three supports (2) in parallel and aligned to the same platform (5) and move them all together, since with this configuration neither the objectives (109) not aligned with the sample (107) they can collide with the bucket (107). In this horizontal configuration, the sample (107) is arranged


    with the aid of a support (101) inside the cuvette (102) according to a vertical orientation.
    Fig. 3 shows another example of the automatic target change device (1) of the present invention in its vertical configuration. In this example, the device (1) also has three supports (2) located this time in a vertical plane and oriented according to a detection direction (DD) which in this case is vertical. The three supports (2) are fixed to a vertical platform (5), although in this case they are individually longitudinally movable according to the vertical detection direction (DD) relative to the platform (5), as will be seen later. Each of the supports (2) has a lens (109) attached, and, in a resting position of the device (1), the distal ends of the three lenses (109) are aligned in a direction perpendicular to the detection direction (DD).
    The platform (5) is connected to a lateral translation means (3) similar to those mentioned above which is configured to move the platform (5) according to a horizontal direction perpendicular to the direction (DD) of detection. As in the previous configuration, this lateral translation allows to select which of the objectives (109) is aligned with the sample (107) housed inside the cuvette (102).
    In addition, each of the supports (2) is connected to a respective longitudinal translation means (4) configured to individually move the corresponding support (2) vertically according to the detection direction. In this vertical configuration, immersion objectives (109) are used, so that once the objective (109) to be used by means of the lateral translation means (3) is selected, the corresponding longitudinal translation means (4) is used to lower only said objective
    (109) specific until its distal end enters the cuvette (102) through its open upper part and enters the liquid in which the sample (107) is immersed. Therefore, the limit switch of the longitudinal translation means (4) must be calibrated so that the distal end of the lens (109) is immersed in said liquid but without touching the sample. Since, unlike what happens in the horizontal configuration, the objectives (109) that are not used remain in their rest or retracted position, there is no danger that they may collide with the cuvette (102). In this configuration, the sample
    (107) located on a support (101) is arranged inside the cuvette (102) according to a horizontal orientation.
    Finally, Fig. 4 shows a case in which the device (1) is installed in a


    microscope (100) which also has a means (10) for rotating the cuvette (102), such as an electric motor, and an additional lens (11) fixedly coupled to a wall of the cuvette (102) of such so that its distal end is immersed in the liquid that fills the cuvette (102). These elements allow the user to select from any of the 5 movable air targets (109) by means of the device (1) described in the previous examples, and the additional immersion lens (11). To do this, if one of the lenses (109) is to be used, the rotation means (10) of the cuvette (102) is activated until the additional lens (11) is placed on the opposite side of the microscope (100) and the objective (109) in question in the manner described above. In case you want to use the objective
    10 (11), the lenses (109) are moved to a retracted position away from the cuvette (102), as shown in Fig. 4, and then the cuvette (102) is rotated until the target is colored (11) on the side of the microscope (100) where the image acquisition equipment is located.

    权利要求:
    Claims (9)
    [1]
    1. Automatic lens change device (1) for a flat laser beam microscope (100), characterized in that it comprises: -at least two supports (2) configured for parallel coupling of at least two lenses (109) ) oriented according to a direction (DD) of detection;
    - a lateral translation means (3) configured to laterally displace said at least two supports (2) so that one of the objectives (109) faces a cuvette (102) of the flat laser beam microscope (100); Y
    - a longitudinal translation means (4) configured to move longitudinally according to the direction (DD) of detection at least the support (2) to which the objective (109) is connected facing the cuvette (102).
    [2]
    2. Device (1) according to claim 1, wherein the at least two supports
    (2) are fixed to a platform (5), the platform (5) being laterally movable by the means (3) of lateral translation.
    [3]
    3. Device (1) according to claim 2, wherein the detection direction (DD) is horizontal, so that both the lateral translation direction and the longitudinal translation direction are contained in a horizontal plane.
    [4]
    Four. Device (1) according to claim 3, wherein the end of stroke of the medium
    (4) of longitudinal translation corresponds to a position in which a distal end of the lens (109) facing the cuvette (102) is adjacent to a side wall of said cuvette (102).
    [5]
    5. Device (1) according to claim 4, wherein the platform (5) is movable longitudinally by the means (4) of longitudinal translation.
    [6]
    6. Device (1) according to any of claims 1-2, wherein the detection direction (DD) is vertical, so that both the lateral translation direction and the longitudinal translation direction are contained in a vertical plane.
    [7]
    7. Device (1) according to claim 6, wherein each support (2) is individually longitudinally movable by means of longitudinal translation (4).

    [8]
    8. Device (1) according to claim 7, wherein the end of stroke of the medium
    (4) of longitudinal translation corresponds to a position in which the distal end of the lens (109) facing the cuvette (102) is below the level of the fluid filling said cuvette (102).
    [9]
    9. Flat laser beam microscope (100) characterized in that it comprises a device
    (1) automatic target change (109) according to any of the preceding claims.
    10 10. Flat laser beam microscope (100) characterized in that it comprises a device
    (1) automatic target change (109) according to any of the
    claims 2-4, and further comprising:
    - a means of rotation (10) of the cuvette (102) about a vertical axis; Y
    - a target (11) fixed to a side wall of the cuvette (102) such that its
    The distal end passes through said lateral wall and is immersed in the fluid that fills the cuvette (102), so that by selecting the angle of rotation of the cuvette (102) it can be selected if the fixed target (11) is used or any of the objectives (109) of the device (1) automatic lens change (109).


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    同族专利:
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JP2003029156A|2001-07-19|2003-01-29|Minolta Co Ltd|Solid immersion lens, objective lens using the same, and optical recording and reproducing device|
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    DE102009044983A1|2009-09-24|2011-03-31|Carl Zeiss Microimaging Gmbh|microscope|ES2695798B2|2017-07-04|2019-12-04|Univ Madrid Carlos Iii|Rotary lens shift device for flat laser beam microscope|
    法律状态:
    2018-07-04| FG2A| Definitive protection|Ref document number: 2630733 Country of ref document: ES Kind code of ref document: B1 Effective date: 20180704 |
    2019-03-06| FA2A| Application withdrawn|Effective date: 20190228 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201630071A|ES2630733B1|2016-01-21|2016-01-21|Automatic lens change device for flat laser beam microscope|ES201630071A| ES2630733B1|2016-01-21|2016-01-21|Automatic lens change device for flat laser beam microscope|
    PCT/ES2017/070028| WO2017125635A1|2016-01-21|2017-01-17|Automatic lens switching device for a planar laser beam microscope|
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