Patent Publication Number: US-2023152562-A1

Title: Light sheet microscope and method for manipulating a target area of a sample

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a U.S. National Phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2020/059130, filed on Mar. 31, 2020. The International Application was published in English on Oct. 7, 2021 as WO 2021/197587 A1 under PCT Article 21(2). 
    
    
     FIELD 
     The invention relates to a light sheet microscope. The invention further relates to a method for manipulating a target area of a sample using a light sheet microscope. 
     BACKGROUND 
     In some known microscopy methods, a sample is manipulated by means of targeted light irradiation, so called photo-manipulation techniques including “Fluorescence Recovery after Photobleaching” (FRAP), a local activation of so called “caged compounds”, and an activation of transmembrane pumps within the context of optogenetics. The light typically used for photo-manipulation differs from the light used for illuminating the sample, e.g. by scattering or exciting fluorophores located within the sample, in that it has a much higher intensity. 
     Document DE 10 2016 119 268 B3 discloses a scanning oblique plane microscope. Further, document WO 2015 109 323 A2 discloses a scanning oblique plane microscope having a unit for photo-manipulation. However, the oblique plane microscope according to said document requires an additional light source for creating manipulation light and means for coupling said manipulation light into an optical system of the microscope. The need for these additional elements make the microscope less compact and increases the cost of manufacturing. 
     SUMMARY 
     In an embodiment, the present disclosure provides a light sheet microscope, comprising a light source configured to emit illumination light, an optical system configured to form a light sheet from the illumination light in a sample space, the light sheet being focused in a thickness direction perpendicular to a light propagation direction thereof to form a beam waist in the thickness direction, wherein the optical system has a field diaphragm adjustable to vary a width of the light sheet in a width direction being perpendicular to both the light propagation direction and the thickness direction, a scanning element configured to move the light sheet a scanning distance in the sample space along a scanning direction, and a control unit configured to control the field diaphragm for adjusting the width of the light sheet and to control the scanning element for moving the light sheet by the scanning distance in order to manipulate a target area of a sample by scanning the target area with the beam waist of the light sheet, the target area being defined by the width of the light sheet and the scanning distance. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Subject matter of the present disclosure will be described in even greater detail below based on the exemplary figures. All features described and/or illustrated herein can be used alone or combined in different combinations. The features and advantages of various embodiments will become apparent by reading the following detailed description with reference to the attached drawings, which illustrate the following: 
         FIG.  1    is a schematic diagram of a light sheet microscope according to an embodiment; 
         FIG.  2    is a schematic diagram of a sample space of the light sheet microscope according to  FIG.  1   ; 
         FIG.  3    is a schematic diagram of a light sheet microscope according to an embodiment; and 
         FIG.  4    is a schematic diagram of a sample space of the light sheet microscope according to  FIG.  3   . 
     
    
    
     DETAILED DESCRIPTION 
     In an embodiment, the present invention provides a light sheet microscope suitable for manipulating a sample that is compact and can be manufactured cost efficiently. 
     A light sheet microscope comprises a light source configured to emit illumination light, and an optical system configured to form a light sheet from the illumination light in a sample space. The light sheet is focused in a thickness direction perpendicular to a light propagation direction thereof to form a beam waist in said thickness direction. The optical system has a field diaphragm adjustable to vary a width of the light sheet in a width direction being perpendicular to both the light propagation direction and the thickness direction, a scanning element configured to move the light sheet a scanning distance in the sample space along a scanning direction, and a control unit configured to control the field diaphragm for adjusting the width of the light sheet and to control the scanning element for moving the light sheet by the scanning distance in order to manipulate a target area of a sample by scanning the target area with the beam waist of the light sheet. The target area is defined by the width of the light sheet and the scanning distance. 
     In the present disclosure, manipulating the sample in particular means to photo-manipulate the sample, i.e. manipulating the sample by means of targeted irradiation with light, i.e. the illumination light or manipulation light. This manipulation of the sample is distinguished from illuminating the sample, e.g. by scattering of the illumination light or exciting fluorophores located within the sample with the illumination light, in that higher intensity illumination light is used, that typically induces an effect lasting several orders of magnitude longer than effects caused by illuminating the sample. Further, the target area for manipulation the sample is typically only a small part of a field of view of the light sheet microscope. In contrast hereto illuminating the sample means illuminating the majority of the field of view of the light sheet microscope. 
     The beam waist of light sheet, i.e. the part of the light sheet where its thickness is minimal, defines a scan line. This scan line is moved by the scanning distance in the sample space along the scanning direction by means of the scanning element. Thereby, the target area is scanned. The exact geometry of the target area is further defined by the angle enclosed by the width direction and the scanning direction. If the width direction and the scanning direction are perpendicular to each other, the target area is a rectangle. Otherwise, the target are is a parallelogram. The length of one side of the target area is defined by the width of the light sheet and the length of another side of the target area is defined by the scanning distance. Thus, by adjusting the width of the light sheet and the scanning distance as well as the angle enclosed by the width direction and the scanning direction the geometry of the target area can be adjusted flexibly. Since the intensity density of the light sheet is highest at is beam waist, only the parts of the specimen that intersect the target area are manipulated. 
     The optical system used for forming the light sheet for manipulating the sample has a very simple configuration. The optical system may further be used for forming a light sheet for illuminating the sample. In particular, the light source used for forming the light sheet for manipulating the sample may be the same light source used for forming the light sheet for illuminating the sample. Thus, eliminating the need for optical switches or additional beam splitters used for coupling the illumination light into the optical system. Thereby, the light sheet microscope is very compact and can be manufactured cost efficiently. 
     In an embodiment the control unit is configured to control the field diaphragm and the scanning element such that the width of the light sheet is varied while the light sheet is moved along the scanning direction. In an embodiment, the target area may be e.g. a simple polygon. Thus, a greater number of geometries can be chosen for the target area. Thereby, the flexibility of the light sheet microscope is greatly increased. 
     In an embodiment the field diaphragm is configured to adjust a position of a first end of the width of the light sheet and a position of a second end of the width of the light sheet independently of each other. In particular, in an embodiment it is possible to move the scan line sideways with respect to the scanning direction. Thus, the target area may be a parallelogram although the width direction and the scanning direction are perpendicular to each other. In an embodiment, an even greater number of geometries can be chosen for the target area further increasing the flexibility of the light sheet microscope. Also, since there is no need to adjust the angle enclosed by the width direction and the scanning direction in order to adjust the geometry of the target area, fewer elements may be used, thus, making the light sheet microscope more cost effective. 
     In an embodiment the optical system is configured to form an intermediate image of the light sheet in an intermediate image space, wherein the optical system comprises an optical transport system configured to image the intermediate image of the light sheet from the intermediate image space into the sample space. In particular, in this embodiment the light sheet microscope might be configured to be an oblique plane microscope. The geometry of the light sheet within the sample, in particular the light propagation direction, are defined by the geometry of the light sheet in the intermediate image space. Thus, removing the need for having optical elements such as light deflection elements in the sample space. Thereby, more space is available in the sample space. 
     In an embodiment the optical transport system comprises a first objective facing the intermediate image space, wherein the optical system comprises an optical detection system for detecting detection light emitted by the sample, said optical detection system having a detector element and a second objective facing the intermediate image space, and wherein the optical axes of the first and second objectives are oblique to each other. Preferably, the optical axis of the second objective and the light propagation direction of the light sheet in the intermediate image space are perpendicular to each other. In an embodiment, the optical transport system transports the light sheet formed into the sample space and the detection light back from the sample space into the intermediate image space. This embodiment is a very simple configuration realizing an oblique plane microscope. 
     In an embodiment the optical system comprises an optical illumination system configured to form the light sheet from the illumination light in the intermediate image space, and wherein the optical axis of the first objective and the optical axis of the optical illumination system are oblique to each other. The geometry of the light sheet in the sample space is defined by the optical illumination system, i.e. in the intermediate image space. In particular, the angle enclosed by the light propagation direction and the optical axis of the first objective, i.e. the obliqueness of the light sheet in the sample space, is defined by the angle enclosed by the optical axis of the second objective and the optical axis of the optical illumination system. No further light deflection elements are needed in the sample space in order to realize an oblique light sheet. Thus, more of the sample space can be dedicated to receiving the sample, increasing the versatility of the light sheet microscope. 
     In an embodiment the optical transport system comprises a light deflection element configured to direct illumination light towards the sample and to direct detection light the detection light towards the intermediate image space. In an embodiment, the illumination light is coupled directly into the optical transport system. 
     Preferably, the optical illumination system comprises a light sheet forming element for forming the light sheet, said light sheet forming element being arranged in an optical path between the intermediate image space and the light source. This light sheet forming element may be e.g. a cylindrical lens or a movable mirror configured to form a quasi-static light sheet from a light beam or any other means known from the prior art. 
     According an aspect, a light sheet microscope is provided. The light sheet microscope comprising a light source configured to emit illumination light, a manipulation light source configured to emit manipulation light for manipulating a target area of a sample, and an optical system configured to form a light sheet from the illumination light in a sample space. The optical system has an optical transport system configured to transport an intermediate image of the light sheet from an intermediate image space into the sample space, an optical detection system configured to detect detection light emitted by the sample, and an optical erecting unit comprising at least first and second objectives facing the intermediate image space, the first objective being configured to direct light into the optical transport system, and the second objective being configured to direct light into the optical detection system. The optical axes of the first and second objectives are oblique to each other. The optical detection system comprises a light deflection element configured to direct the manipulation light into the second objective. 
     In an embodiment, the light sheet microscope is configured as an oblique plane microscope. 
     The manipulation light is coupled into a detection light path of the optical system by the light deflection element. The manipulation light is then directed into the intermediate image space by the second objective. The first objective receives the manipulation light and directs it into the optical transport system. The manipulation light is then transported by the optical transport system into the sample space, where it is used to manipulate the target area of the sample. The optical system used for transporting the manipulation light into the sample space shares many components with an optical system used for forming a light sheet for illuminating the sample already present in typical light sheet microscopes. Thereby, the light sheet microscope is very compact and can be manufactured cost efficiently. 
     In an embodiment the optical detection system comprises a detector and the light deflection element is configured to direct the detection light to the detector. The light deflecting element is e.g. a dichroitic beam splitter. In this embodiment the detection light path leading to the detector is used to couple the manipulation light into the optical system. Thereby, no additional light path need to be created which makes this embodiment even more compact. Alternatively or additionally, the light deflection element is configured such that it can be removed from the detection light path. Thereby, allowing to switch between a detection mode in which the detector can receive the detection light and a manipulating mode in which the manipulation light is directed into the sample space. 
     In an embodiment the optical system comprises a light forming element for forming a light pattern from the manipulation light, said light forming element being arranged in an optical path between the light deflection element of the optical detection system and the light source. The light forming element might e.g. be a digital mirror device. The light pattern is imaged into the sample space by the optical system, thereby defining the target area. This allows for a very precise and flexible definition of the target area. 
     Alternatively, the light forming element is a lens or a lens group configured to focus the manipulation light into a manipulation light beam. The target area is scanned with the manipulation light beam by means of a scanning element. Said scanning element e.g. being arranged in the light path of the optical transport system. In this alternative embodiment the light pattern is a light spot and the target area is defined as the area scanned by the light pattern. 
     In an embodiment the optical transport system comprises an optical zoom system, which is adjustable for adapting the magnification of the optical transport system to a ratio between two refractive indices, one of which being associated with the sample space and the other being associated with the intermediate image space. The zoom system may be used to move the position of the focal plane of the first objective along its optical axis without moving the sample itself, which might disturb it. In order for this remote focusing to work, the magnification of the optical transport system must be equal to the ratio of the two refractive indices. 
     By moving the position of the focal plane of the first or second objective along its optical axis, the position of the beam waist of the light sheet, i.e. the target area, is moved along the optical axis of the first objective. This allows e.g. manipulating more than one target area located at different positions along the optical axis of the first objective. Further, this allows to tilt the target area by shifting the focal plane of the first objective while the light sheet is moved along the scanning direction. Thereby making additional geometric configurations of the target area possible and further enhancing to flexibility of the light sheet microscope. 
     In an embodiment the optical system comprises an objective facing the sample space, and wherein the opening angle of said objective has a value between 17° and 72°, in particular between 45° and 72°. In an embodiment a high numerical aperture can be achieved which is advantageous for most light sheet microscopy techniques, in particular oblique plane microscopy. 
     In an embodiment the optical system comprises a single objective facing the sample space. The single objective is used for imaging the light sheet into the sample space and for receiving the detection light emitted by the sample. Thereby, most of the sample space can be dedicated towards receiving the sample. Further, in an embodiment the optical axis of the single objective may be perpendicular to a cover slip holding the sample. This greatly reduces light loss and aberrations caused by reflections on the cover slip. 
     In an embodiment the scanning direction is perpendicular to an optical axis of the single objective. This allows for a simple geometric configuration of the optical system, since the scanning direction is located in or parallel to the focal plane of the single objective. 
     In an embodiment the target area is located in a focal plane of the single objective. In this embodiment, no remote focusing is necessary in order to focus the light sheet in the target area. This greatly simplifies the optical design of the optical system. 
     In an embodiment the illumination light source and/or the manipulation light sources comprises a pulsed laser. Pulsed laser can efficiently achieve the high intensities needed for photo-manipulating the sample. 
     According to an aspect, a method for manipulating a target area of a sample using a light sheet microscope is provided. The methods comprises the following steps: Forming a light sheet from illumination light in a sample space, said light sheet being focused in a thickness direction perpendicular to a light propagation direction thereof to form a beam waist in said thickness direction. Adjusting the width of the light sheet. Moving the light sheet by a scanning distance in order to manipulate the target area of the sample by scanning the target area with the beam waist of the light sheet, said target area being defined by the width of the light sheet and the scanning distance. 
     The method has the same advantages as the microscope. 
       FIG.  1    shows a schematic diagram of a light sheet microscope  100  according to an embodiment.  FIG.  1    also shows a coordinate cross  102  defining coordinates in a sample space  104  of the light sheet microscope  100  (c.f.  FIG.  2   ). The light sheet microscope  100  comprises a light source  106 , an optical system  108 , and a control unit  110 . 
     The light source  106  is configured to emit illumination light, in particular laser light. In the present embodiment, the light source  106  is exemplarily configured to be a pulsed laser. However, the light source  106  may also be a continuous laser or an assembly of two or more lasers and a beam combining element configured to combine the laser light beams emitted by the two or more lasers into a single laser light beam. Also, the light source  106  may be a source of incoherent light. 
     The illumination light is then formed into a light sheet by the optical system  108  in the sample space  104  of the light sheet microscope  100  in order to manipulate a sample  146 . The optical system  108  comprising an optical illumination system  112  and an optical transport system  114 . The optical illumination system  112  is configured to form the light sheet from the illumination light in an intermediate image space  116 . The optical transport system  114  is configured to form an image of the light sheet in the sample space  104  and to form an image of an object plane in the sample space  104  in the intermediate image space  116 . The optical system  108  further comprises an optical detection system  136  configured to detect the image formed by the optical transport system  114 . 
     The optical illumination system  112  comprises a light sheet forming element  118 , for example a cylindrical lens or a scanning element, an illumination objective  119  configured to direct the light sheet into the intermediate image space  116 . The light sheet is focused in a thickness direction perpendicular to a light propagation direction thereof and forms a beam waist in said thickness direction. The optical illumination system  112  further comprises an adjustable field diaphragm  120  which is configured to be adjustable in order vary a width of the light sheet in a width direction being perpendicular to both the light propagation direction and the thickness direction. 
     The optical transport system  114  forms a transport system in the sense that it is configured to transport the light sheet from the intermediate image space  116  into the sample space  104  and the image of the object plane from the sample space  104  into the intermediate image space  116 . In other words, the optical system  108  transports the illumination light and detection light emitted by the sample  146  from the intermediate image space  116  to the sample space  104  and back, respectively. 
     In the present embodiment, the optical transport system  114  is telecentric. The optical system  108  comprises an image side objective  132 , a first tube lens  130 , a first ocular  128 , a second ocular  126 , a second tube lens  124 , and an object side objective  122 , in this order from the intermediate image space  116 . The optical transport system  114  further comprises a scanning element  134  arranged between the first and second oculars. Said scanning element  134  being configured to move the light sheet through the sample space  104  along a scanning direction perpendicular to the optical axis O of the objective. In an embodiment the scanning element  134  may be an e.g. piezo driven objective actuator configured to drive the object side objective  132 . 
     The optical detection system  136  comprises a detection objective  138 , a tube lens  140 , and a detector  142 . The detection objective  138  and the tube lens  140  are configured to image the intermediate image space  116  onto the detector  142 . This means that the image of the sample space  104  formed by the optical transport system  114  in the intermediate image space  116  is imaged onto the detector  142 . In other words, the image is detected by the detector  142 . 
     The control unit  110  comprises an input device  144  for inputting the geometry of the target area and is connected to the scanning element  134 , the field diaphragm  120 , the light source  106 , and the detector  142 . The control unit  110  is further configured to manipulate a target area  200  (see  FIG.  2   ) of the sample  146  by controlling the scanning element  134  and the field diaphragm  120 . The scanning element  134  is controlled for moving the light sheet along a scanning direction by a scanning distance. The field diaphragm  120  is adjusted for setting the width of the light sheet. The field diaphragm  120  may be adjusted once before the light sheet is moved along the scanning direction or continuously while the light sheet is moved in order to determine the exact geometry of the target area  200 . How the geometry of the target area  200  is defined by adjusting the field diaphragm  120  is explained in more detail below with reference to  FIG.  2   . 
       FIG.  2    shows a schematic diagram of the sample space  104  of the light sheet microscope  100  according to  FIG.  1   .  FIG.  2    also shows a coordinate grid defining coordinates in the sample space  104 . A first coordinate axis X is parallel to the scanning direction, a second coordinate axis Y is perpendicular to the scanning direction and the optical axis O of the objective directed at the sample space  104 , and a third coordinate axis Z is parallel the optical axis O of said objective. 
     The target area  200  is shown in  FIG.  2    as a polygon with a solid outline. The sample  146  is manipulated in the target area  200  by moving a beam waist of light sheet, i.e. the part of the light sheet where its thickness is minimal and the intensity density of the light sheet is highest, along the scanning direction in a scanning motion. The scanning direction is shown in  FIG.  2    as an arrow S. The light sheet is indicated by dotted lines  202  crossing each other at the position of the beam waist. The position of the beam waist at the start of the scanning motion is shown in  FIG.  2    as a first thick line  204  and the position of the beam waist at the end of the scanning motion is shown in  FIG.  2    as a second thick line  206 . The width of the light sheet is varied during the scanning motion, as is shown in  FIG.  2    by two double-headed arrows P 1 , P 2 . This allows the target area  200  to be the polygon instead of a rectangle or parallelogram. 
       FIG.  3    shows a schematic diagram of a light sheet microscope  300  according to another embodiment. The light sheet microscope  300  according to  FIG.  3    is distinguished from the light sheet microscope  100  according to  FIG.  1    in a manipulation light source  302 . The manipulation light source  302  is configured to emit manipulation light for manipulating a target area  400  (see  FIG.  4   ) of the sample  146 . 
     The optical system  318  comprises a light forming element  308  configured to form the manipulation light into a light pattern, e.g. a digital mirror device, a spatial light modulator, or a scanning mirror. The light pattern is either static or quasi-static, e.g. a light pattern created by a fast moving scanning mirror. Alternatively, the light forming element  308  is a lens or a set of lenses and the light pattern may be a focused light spot. After leaving the light forming element  308 , the formed manipulation light is directed into the optical detection system  304 . 
     The optical detection system  304  comprises a light deflection element  306 , which is exemplarily formed as a beam splitter. The light deflection element  306  is configured to direct the detection light to the detector  142  and to direct the manipulation light to the objective  138  of the optical detection system  304 . The manipulation light is then directed by the objective  136  into the intermediate image space  116  and imaged into the sample space  104  by means of the optical transport system  114 . 
     The objective  132  of the optical transport system  114  and the objective  138  of the optical detection system  304  define an optical erecting unit  312  configured to image an oblique image plane of the sample  146 . Due to the geometry of the optical erecting unit  310  the target area  400  is tilted as is shown in  FIG.  4   . 
     The optical system  318  of the light sheet microscope  300  further comprises a light sheet illumination system  312 , that is configured to form a light sheet for illuminating the sample  146  in an intermediate image space  116 . Said light sheet illumination system  312  comprising a light sheet forming element  314 , for example a cylindrical lens or a scanning element, and an illumination objective  316  configured to direct the light sheet into the intermediate image space  116 . 
       FIG.  4    shows a schematic diagram of the sample space  104  of the light sheet microscope  300  according to  FIG.  3   . The plane  402  in the which the target area  400  is located is indicated by a dashed rectangle. As can be seen in  FIG.  4    the target area  400  are tilted with respect to the y axis. 
     By adjusting the scanning element  134 , the whole plane in the which the target area  400  is located is moved along the x axis. This is indicated in  FIG.  4    by a double-headed arrow P 3  By adjusting the scanning element  134  each time the scanning motion is completed, multiple target areas located in different planes arranged along the x axis in sequence are manipulated. Thus, a volume or stack formed by the target areas is manipulated. Alternatively, it is possible to adjust the scanning element  134  during the scanning motion. Thereby, the target area  400  may be non-flat. 
     As used herein the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”. 
     Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus. 
     While subject matter of the present disclosure has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. Any statement made herein characterizing the invention is also to be considered illustrative or exemplary and not restrictive as the invention is defined by the claims. It will be understood that changes and modifications may be made, by those of ordinary skill in the art, within the scope of the following claims, which may include any combination of features from different embodiments described above. 
     The terms used in the claims should be construed to have the broadest reasonable interpretation consistent with the foregoing description. For example, the use of the article “a” or “the” in introducing an element should not be interpreted as being exclusive of a plurality of elements. Likewise, the recitation of “or” should be interpreted as being inclusive, such that the recitation of “A or B” is not exclusive of “A and B,” unless it is clear from the context or the foregoing description that only one of A and B is intended. Further, the recitation of “at least one of A, B and C” should be interpreted as one or more of a group of elements consisting of A, B and C, and should not be interpreted as requiring at least one of each of the listed elements A, B and C, regardless of whether A, B and C are related as categories or otherwise. Moreover, the recitation of “A, B and/or C” or “at least one of A, B or C” should be interpreted as including any singular entity from the listed elements, e.g., A, any subset from the listed elements, e.g., A and B, or the entire list of elements A, B and C. 
     LIST OF REFERENCE SIGNS 
     
         
         
           
               100  Microscope 
               102  Coordinate cross 
               104  Sample space 
               106  Light source 
               108  Optical system 
               110  Control unit 
               112  Optical illumination system 
               114  Optical transport system 
               116  Intermediate image space 
               118  Light sheet forming element 
               119  Objective 
               120  Field diaphragm 
               122  Objective 
               124  Tube lens 
               126 ,  128  Ocular 
               130  Tube lens 
               132  Objective 
               134  Scanning element 
               136  Optical detection system 
               138  Objective 
               140  Tube lens 
               142  Detector 
               144  Input device 
               146  Sample 
               200  Target area 
               202  Dotted line 
               204 ,  206  Thick line 
               300  Microscope 
               302  Manipulation light source 
               304  Optical detection system 
               306  Light deflection element 
               308  Light forming element 
               310  Optical erecting system 
               312  Light sheet illumination system 
               314  Light sheet forming element 
               316  Objective 
               400  Target area 
               402  Plane 
             P 1 , P 2 , P 3  Double-headed arrow 
             S Arrow