• 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 lens changing device (1) comprising a prismatic cuvette (2) perpendicular to a plane parallel to a detection direction (DD) and a lighting direction (DI) perpendicular to each other. The cross section of the prism is a polygon of more than four sides with pairs of faces (2a, 2a '; 2b, 2b'; 2c, 2c '; 2d, 2d') perpendicular to each other. In addition, at least several pairs of faces (2a, 2a '; 2b, 2b'; 2c, 2c '; 2d, 2d') comprise a face (2a ', 2b', 2c ', 2d') configured to receive a beam of flat light (6) according to the direction of illumination (DI) and one face (2a, 2b, 2c, 2c) with a lens or objective (9a, 9b, 9c, 9d) coupled to detect a fluorescent light (8) in the detection address (DD). The cuvette (2) is rotatable about an axis perpendicular to said plane, so that it is possible to orient a particular objective (9a, 9b, 9c, 9d) according to the direction of detection (DD). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2695798A1
    申请号:ES201730887
    申请日:2017-07-04
    公开日:2019-01-10
    发明作者:Lorenzo Jorge Ripoll；De Miguel Alicia Arranz
    申请人:Universidad Carlos III de Madrid；
    IPC主号:
    专利说明:

    [0001]
    [0002] Rotary lens shift device for flat laser beam microscope
    [0003]
    [0004] OBJECT OF THE INVENTION
    [0005]
    [0006] The present invention belongs to the field of microscopy, and more particularly to the flat laser beam illumination microscopy used for the obtaining of images of several transparent or semi-transparent samples such as embryos, tissues and other biological samples.
    [0007]
    [0008] The object of the present invention is a new device that allows to change the objective in a fast and simple way through rotations of the cuvette itself.
    [0009]
    [0010] BACKGROUND OF THE INVENTION
    [0011]
    [0012] The studies of embryos and similar biological samples through optical microscopy have, unlike what happens with individual cells, particular problems related to the absorption of light and the loss of resolution due to the scattering of light. To solve these problems, important improvements have been made in the past few years on flat laser beam microscopes, whose precursor invention on a flat light beam microscope dates from 1903.
    [0013]
    [0014] A flat laser beam microscope is essentially formed by a camera coupled to a lens of high numerical aperture and arranged in a direction called "direction of detection", and a lighting means capable of emitting a thin sheet of light according to a direction called " direction of illumination " that is perpendicular to the direction of detection, following the original configuration of Siedentopf and Zsigmondy coupled to a detection camera. With this configuration, the camera can obtain a 2D fluorescence image of the part of the sample illuminated by the sheet or plane of illumination. 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 position in z (depth of the sample according to the direction of detection) obtained when moving the sample, and of the x and y positions, present in each 2D image. The stack of 2D images it can then be merged to generate a 3D image of the sample, as described in US 7,554,725 to Stelzer et al. Subsequently, it was proposed to rotate the sample around its own axis, usually vertical, to capture several stacks of 2D images (commonly called "angular measurements") and merge them later, which allows to improve the anisotropy and the quality of the images (S Preibisch et al, Nature Methods 7 (2010)).
    [0015]
    [0016] For a clearer understanding of this technique, Figs. 1a and 1b showing an example of a microscope (100) of a flat laser beam. The sample (107) is disposed on a support (101) inside a cuvette (102) filled with a liquid. A beam (103) of Gaussian linear illumination, Bessel, Airy or similar, impinges on a cylindrical lens (104) that focuses it thanks to a lighting objective (105) to generate the vertical flat illumination sheet (106). This vertical plane lighting sheet (106) strikes the sample (107) according to the direction of illumination (DI), and the fluorescent light (108) emitted by that particular plane of the sample (107) is picked up by a lens (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 about its vertical axis to allow the taking of several angular measurements according to the technique proposed by Preibisch.
    [0017]
    [0018] On the other hand, the technique OPT ( Optical Projection Tomography , Optical Projection Tomography according to its acronym in English), described in document US20060122498 A1, is relatively similar to X-ray tomography. It is fundamentally based on optically illuminating the sample in a form homogeneous and obtain, on the side of the sample opposite to that from which it is illuminated, an image that can be assimilated to the "shadow" projected by the sample 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 the 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.
    [0019]
    [0020] Recently, the inventors of the present application have filed 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 SPIM (Selective Plane Illumination Microscope) laser beam technique with the technique of optical projection tomography (OPT). This new microscope does not store a complete 2D image for each position of the illumination sheet, but for each acquisition angle it stores only one parameter representative of each pixel obtained by means of 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 decrease the requirements of the system, but also increase the acquisition speed of the microscope.
    [0021]
    [0022] More recently, the inventors of the present application have filed the patent application PCT / ES2016 / 070714, entitled " Multi -load device for flat laser beam microscope" which describes a multiple loading device for feeding to a laser beam microscope plan of a continuous and sequential flow of samples. This device fundamentally comprises a capillary passage that passes through the measuring area of the sample receiving cuvette of the microscope having a diameter such that it only allows the passage of the samples one at a time, and an adjustable flow generation 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 sequentially passed through the interior of the receiving tray, accelerating the process of acquiring data from multiple samples.
    [0023]
    [0024] Even more recently, the inventors of the present application have filed patent application PCT / ES2017 / 070028, entitled "Automatic lens change device for flat laser beam microscope", which describes a device that allows to automatically change the objective of acquisition of images of a flat laser beam microscope according to the magnification desired at each moment. To this end, the device comprises supports for lenses coupled to a lateral translating means and a longitudinal translating means, so that the user can choose which specific target faces the face of the cuvette oriented according to the direction of detection.
    [0025]
    [0026] This last device, although it allows changing the objective of the flat laser beam microscope, presents as the main drawback the complexity of the mechanical assembly that must be carried out. In addition, the control of the different drive elements that move the platforms is complex, since any calculation error can cause the targets to impact with the bucket.
    [0027] DESCRIPTION OF THE INVENTION
    [0028]
    [0029] The inventors of the present application solve the above problems thanks to a new lens changing device based on a rotating polygonal shaped cube where some faces have a coupled objective and other faces perpendicular to those are transparent. Thanks to this configuration, the lens changes only require rotating the cuvette to place the face having the desired objective oriented toward the direction of detection and the transparent face perpendicular to that facing the direction of illumination. This avoids the possibility of collisions between targets and bucket, and also greatly simplifies the operations necessary for changes of objective.
    [0030]
    [0031] Note that, although in most cases there was talk of goal changes, it would be equally possible to arrange a lens instead of a lens on the corresponding face of the pair of faces. Therefore, in this context it should be understood that each mention to a "face endowed with an objective" can also be interpreted as a "face endowed with a lens".
    [0032]
    [0033] The present invention thus discloses a rotary lens change device for a flat laser beam microscope comprising a cuvette having the shape of an axis prism perpendicular to a plane parallel to a detection direction and a lighting direction. As usual in flat laser beam microscopes, the direction of detection and the direction of illumination are fixed and perpendicular to each other. The square cuvettes used so far in this field meet these conditions. However, the device of the present invention clearly differs from them because it also has the following additional characteristics:
    [0034]
    [0035] a) The shape of the cross section of the prism parallel to this plane is a polygon of more than four sides that has pairs of faces perpendicular to each other.
    [0036]
    [0037] b) Each pair of faces of at least several pairs of faces perpendicular to each other comprises a face configured to receive a flat beam of light to illuminate a sample according to the direction of illumination and a face with a lens or lens attached to detect a fluorescent light emitted by the sample in the detection direction.
    [0038] c) The cuvette is rotatable about an axis perpendicular to said plane, so that it is possible to orient a particular lens or objective in the direction of detection. Obviously, the face configured to receive the flat light beam is at the same time oriented according to the direction of illumination.
    [0039]
    [0040] In fact, it is known that the direction of detection and the direction of illumination form 90 °, so that it is necessary that the face of the cuvette through which the illumination beam enters is perpendicular to the face of the cuvette through from which the fluorescent light emitted by the sample is received. Until now, this had been achieved by configuring the bucket in the simplest possible way: with a cubic shape. That is to say, the cuvette conventionally used up to now has a prism shape with a square cross section. These conventional buckets usually have a single objective that could be fixed to one of their faces. In case of not being fixed, the objective change could be made through complex mechanical systems such as those described in patent application PCT / ES2017 / 070028 of the same inventors as the present application.
    [0041]
    [0042] The inventors of the present application have designed a new cuvette configuration that allows changing lens or objective among several possible ones simply by means of a simple rotation of the cuvette. For this, it is sufficient to increase the number of faces of the tray in such a way that there are pairs of faces formed by mutually perpendicular faces. That is, the shape of the cross section of the bucket changes from being square to adopting a polygon shape of more than four sides where there are several pairs of sides perpendicular to each other. Note that it is not essential that the polygon be regular, but only that it has several pairs of perpendicular sides, for example two or more. Note also that, by increasing the number of sides of the polygon, it is no longer necessary for the two sides that make up a pair of perpendicular sides to be contiguous, as was the case with the conventional cube-shaped cube.
    [0043]
    [0044] Each pair of mutually perpendicular faces is formed by a face configured to receive the flat laser beam and a face provided with a fixed lens or lens. The first allows the passage of the flat beam of illumination used to illuminate the sample in the microscopes of flat laser beam according to the direction of illumination. The second, equipped with a fixed objective lens, allows the fluorescent light emitted by the sample to be collected in the detection direction. In addition, since the rotating cuvette is, there are different positions of use corresponding to the alignment of a particular pair of faces respectively with the directions of detection and illumination. To change lens or objective, it is only necessary to rotate the cuvette determined angle around an axis perpendicular to a plane containing the direction of illumination and the direction of detection. The rotation of the cuvette can be carried out using suitable driving means, such as for example a small geared motor controlled by a processing means.
    [0045]
    [0046] The simplest configuration of this invention implies that the cross section of the prismatic cuvette has a regular poKgono shape with a number of multi-sided faces of four. Although it is not essential, the use of a regular polygon is the simplest and most intuitive alternative. Within regular polygons, it has been proven that those that have a number of multi-sided faces have pairs of perpendicular faces. More preferably, the regular polygon is an octahedron, a dodecahedron, or a hexadecahedron. While it would be possible to use polygons with a larger number of faces, the size of the tray could be excessive in that case.
    [0047]
    [0048] In a preferred embodiment of the invention, the face configured to receive a flat light beam to illuminate a sample according to the lighting direction is a flat transparent face. It could simply be a smooth flat wall made of glass or any other transparent material that would allow the passage of the laser beam flat without altering it. In an alternative embodiment of the invention, the face configured to receive a flat light beam to illuminate a sample according to the lighting direction comprises a lighting objective, which may be immersion or air. The lighting objectives are sometimes used to focus or otherwise treat the flat light beam emitted according to the direction of illumination.
    [0049]
    [0050] Preferably, the front end of at least one objective passes through a wall of the face to which it is attached. This configuration corresponds to the use of so-called "immersion" objectives , which are objectives that require that their front end, that through which light enters, be introduced into the fluid in which the sample is immersed. In this way, the fluid is the only medium that is interposed between objective and sample. Therefore, the front end of the lens passes through the wall of the corresponding face and is immersed in the fluid that supports the sample.
    [0051]
    [0052] In another preferred embodiment of the invention, the leading end of at least one target is externally adjacent to a wall of the face to which it is attached. This configuration corresponds to the use of the so-called "air" objectives , which are targets that may have their front end outside the fluid in which the sample is immersed.
    [0053] In this case, the light that enters the lens passes through the fluid that supports the sample, the wall of the corresponding face of the cuvette, and the air that separates the front end of the lens from the wall of the cuvette.
    [0054]
    [0055] Of course, it is possible to have pairs of faces with different combinations of lenses, "immersion" and "air" lenses on one side and flat transparent faces and faces with an illumination objective on the other, thus providing in the same vat. to the device of the invention of great flexibility. On the other hand, the fixation of the lenses or targets to the faces of the cuvette can be done in any way known in the art.
    [0056]
    [0057] In another preferred embodiment of the invention, the device comprises at least one additional cuvette having the same cross-sectional shape as the cuvette and which is fixed to said cuvette so that each face of the additional cuvette is coplanar with a corresponding face of the cuvette. the bucket That is, the additional cuvette is positioned above or below the original main cuvette, and its faces are oriented in the same way as the faces of the original main cuvette. Thus, the pair of buckets as a whole continues to have the same prism shape that the original main bucket had alone, although with a greater height. In addition, those faces of the additional cuvette which are coplanar with faces with lens or objective of the original main cuvette have also coupled a lens or lens. The cuvette and the additional cuvette are rotatably integral so that it is possible to simultaneously orient, in the direction of detection, a particular lens or objective of the cuvette and a particular lens or objective of the additional cuvette. This configuration allows to take two or more simultaneous images of a large sample.
    [0058]
    [0059] BRIEF DESCRIPTION OF THE FIGURES
    [0060]
    [0061] Figs. 1a and 1b respectively show a perspective view and a top view of the main elements of a conventional flat laser beam microscope.
    [0062]
    [0063] Figs. 2a and 2b respectively show a perspective view and a top view of the main elements of a first example of a device according to the present invention.
    [0064]
    [0065] Figs. 3a-3d show respective top views of the four possible use positions of the first example of device of Figs. 2a and 2b.
    [0066] Figs. 4a and 4b respectively show a perspective view and a top view of the main elements of a second example of device according to the present invention.
    [0067]
    [0068] PREFERRED EMBODIMENT OF THE INVENTION
    [0069]
    [0070] Figs. 2a and 2b show a first example of device (1) according to the present invention comprising a cuvette (2) having the shape of an octahedron. An octahedron is a regular polygon formed by 8 faces where contiguous faces form 45 ° and where alternate faces form 90 °. Therefore, alternate faces (2a, 2a '; 2b, 2b'; 2c, 2c '; 2d, 2d') of the cuvette (2) are perpendicular to each other.
    [0071]
    [0072] Specifically, Fig. 2a shows a perspective view of the trough (2) where a certain pair of faces (2b, 2b ') is in use position. Specifically, the pair of faces (2b, 2b ') comprises a first face (2b) oriented according to the detection direction (DD), which is in the foreground, and a second face oriented according to the direction of illumination (DI) that forms 90 ° with the detection address (DD). The first face (2b) has coupled an "immersion" objective (9b) that passes through the wall of said face (2b) so that its front end is immersed in the fluid that supports the sample (7). The second face (2b ') is a transparent face (2b') free of any obstacle that can prevent the passage of light. In this position, acquisition of images of the sample (7) through the objective (9b) is carried out in the conventional manner: a flat light beam (6) is emitted according to the direction of illumination (DI); the plane light beam (6) traverses the second transparent face (2b ') and reaches the sample (7); the sample emits a fluorescent light (8) in the detection direction (DD); the objective (9b) fixed to the first face (2b) receives the fluorescent light (8).
    [0073]
    [0074] The tray (2) of Figs. 2a and 2b have three more pairs of faces that are not in use, a pair of faces (2a, 2a '), a pair of faces (2c, 2c'), and a pair of faces (2d, 2d '):
    [0075]
    [0076] - Pair of faces (2a, 2a '): The face (2a) is contiguous with the face (2b) according to the clockwise direction, and has a target (9a) also "immersion". The corresponding face (2b) is contiguous with the face (2b) according to the anticlockwise direction, and is transparent to allow the passage of light. The face (2a) forms 90 ° with the face (2a ').
    [0077]
    [0078] - Pair of faces (2b, 2b '): The face (2c) is contiguous to the face (2b') according to the sense counter-clockwise, and has an "immersion" lens (9c) attached . The corresponding face (2c ') is separated from the face (2c) in the clockwise direction by the face (2d), and is transparent to allow the passage of light. The face (2c) forms 90 ° with the face (2c ').
    [0079]
    [0080] - Pair of faces (2d, 2d '): The face (2d) is contiguous with the face (2c) according to the counter-clockwise direction, and has an "immersion" objective (9d). The corresponding face (2d ') is contiguous with the face (2a) in a clockwise direction, and is transparent to allow the passage of light. The face (2d) forms 90 ° with the face (2d ').
    [0081]
    [0082] Figs. 3a-3d show the four possible positions of use of the cuvette (2) shown in Figs. 2a-2b. Each of these positions of use corresponds to an angle of rotation of the cuvette (2) around an axis perpendicular to the plane containing the direction of illumination (DI) and the direction of detection (DD). Fig. 3a shows a top view of the cuvette (2) where the objective (9a) fixed to the wall of the face (2a) is oriented according to the detection direction (DD). The other face (2a) of that pair of faces is oriented according to the direction of illumination (DI). Therefore, the objective (9a) is in active or use position. When the user wishes to use the lens (9d), he only has to rotate the tray (2) 135 ° in the opposite direction to the clockwise. A position is then reached in which the objective (9d) fixed to the wall of the face (2d) is oriented according to the detection direction (DD), as shown in Fig. 3B. Correspondingly, the other face (2d ') of that pair of faces is oriented according to the direction of illumination (DI). Therefore, the objective (9d) is now in active or use position. To use the objective (9c), the cuvette (2) is rotated an additional 45 ° (180 ° relative to the initial position shown in Fig. 3a). The objective (9c) fixed to the face (2c) is now oriented according to the detection direction (DD), and the other face (2c ') of that pair of faces is oriented according to the direction of illumination (DD). Finally, to use the objective (9a), the cuvette (2) is rotated an additional 135 ° (315 ° in relation to the initial position shown in Fig. 3a). The face (2a) in which the objective (9a) is now oriented according to the detection direction (DD).
    [0083]
    [0084] Fig. 4a shows a second example of device (1) according to the invention comprising a basin (2) which is denominated principal and an identical additional basin (20) disposed below the main basin (2). The faces (20a, 20a '; 20b, 20b'; 20c, 20c '; 20d, 20d') of the cuvette (20) are coplanar with the faces (2a, 2a '; 2b, 2b'; 2c, 2c '; 2d, 2d ') of the cuvette (2), and both cuvettes are fixed to each other so that they rotate integrally. The additional basin (20) also has objectives (90a, 90b, 90c, 90d) arranged on the faces (20a, 20b, 20c, 20d) which are coplanar with the faces (2a, 2b, 2c, 2d) provided with objective ( 9a, 9b, 9c, 9d) of the main tank (2). This is seen in greater detail in Fig. 4b, which schematically shows a top view of a cross section of the additional trough (20). Thanks to this configuration, images of a large sample (7) can be acquired simultaneously. For this purpose, two flat illumination sheets (6) can be used which strike the illumination direction (DI) in the sample (7) after passing through the respective faces (2b ', 20b'). The fluorescent light (8) emitted by the sample (7) is received through respective targets (9b, 90b) arranged on the respective other faces (2b, 20b) of said pairs of perpendicular faces. To change the objective, the cuvette assembly (2) - additional cuvette (20) is rotated in the same way as described above with reference to the first example of device (1).
    权利要求:
    Claims (10)
    [1]
    1. A rotary lens change device (1) for a flat laser beam microscope, comprising a cuvette (2) having the shape of an axis prism perpendicular to a plane parallel to a detection direction (DD) and a direction of illumination (DI) that are perpendicular to each other, characterized in that the cross sectional shape of the prism parallel to said plane is a polygon of more than four sides that has pairs of faces (2a, 2a '; 2b, 2b'; 2c, 2c '; 2d, 2d') perpendicular to each other, where each of at least several pairs of faces (2a, 2a '; 2b, 2b'; 2c, 2c '; 2d, 2d') perpendicular to each other comprises a face (2a) ', 2b', 2c ', 2d') configured to receive a flat light beam (6) to illuminate a sample (7) according to the direction of illumination (DI) and one face (2a, 2b, 2c, 2c) with a lens or objective (9a, 9b, 9c, 9d) coupled to detect a fluorescent light (8) emitted by the sample (7) in the detection direction (DD), and where the cuvette (2) is rotating around an axis perpendicular to said plane, so that it is possible to orient a particular lens or objective (9a, 9b, 9c, 9d) according to the detection direction (DD).
    [2]
    2. Device (1) according to claim 1, wherein the polygon is a regular polygon with a number of multiple faces of four.
    [3]
    Device (1) according to claim 2, wherein the regular polygon is an octahedron, a dodecahedron, or a hexadecahedron.
    [4]
    Device (1) according to any of the preceding claims, wherein the face (2a ', 2b', 2c ', 2d') configured to receive a flat light beam (6) to illuminate a sample (7) according to The direction of illumination (DI) is a flat transparent face.
    [5]
    Device (1) according to any of claims 1-3, wherein the face (2a ', 2b', 2c ', 2d') configured to receive a flat light beam (6) to illuminate a sample (7). ) according to the lighting direction (DI) comprises a lighting objective.
    [6]
    6. Device (1) according to claim 5, wherein the illumination target is an immersion lighting target.
    [7]
    7. Device (1) according to claim 5, wherein the lighting objective is an air illumination target.
    [8]
    Device (1) according to any of the preceding claims, wherein the front end of at least one objective (9a, 9b, 9c, 9d) passes through a wall of the face to which it is attached.
    [9]
    Device (1) according to any one of the preceding claims, wherein the front end of at least one objective (9a, 9b, 9c, 9d) is externally adjacent to a wall of the face to which it is attached.
    [10]
    Device (1) according to any of the preceding claims, comprising at least one additional tray (20) having the same cross-sectional shape as the tray (2) and which is fixed to said tray (2) so that each face (20a, 20a '; 20b, 20b'; 20c, 20c '; 20d, 20d') of the additional cuvette (20) is coplanar with one face (2a, 2a '; 2b, 2b'; 2c, 2c '; 2d, 2d') corresponding to the cuvette (2), where faces (20a, 20b, 20c, 20d) of the additional cuvette (20) coplanar with faces (2a, 2b, 2c, 2d) with lens or objective ( 9a, 9b, 9c, 9d) of the cuvette (2) have a lens or objective (90a, 90b, 90c, 90d) coupled to it, and where the cuvette (2) and the additional cuvette (20) are rotatably integral with each other. so that it is possible to simultaneously orient according to the direction of detection (DD) a particular lens or objective (9a, 9b, 9c, 9d) of the cuvette () and a particular lens or objective (90a, 90b, 90c, 90d) of the additional bucket (20).
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    优先权:
    申请号 | 申请日 | 专利标题
    ES201730887A|ES2695798B2|2017-07-04|2017-07-04|Rotary lens shift device for flat laser beam microscope|ES201730887A| ES2695798B2|2017-07-04|2017-07-04|Rotary lens shift device for flat laser beam microscope|
    US16/635,942| US20200218048A1|2017-07-04|2018-07-05|Rotary objective lens switching device for a planar laser beam microscope|
    PCT/ES2018/070483| WO2019008212A1|2017-07-04|2018-07-05|Rotary lens switching device for a planar laser beam microscope|
    EP18827961.6A| EP3650906A4|2017-07-04|2018-07-05|Rotary lens switching device for a planar laser beam microscope|
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