Patent Publication Number: US-2021172876-A1

Title: Light sheet fluorescence microscope

Description:
CROSS-REFERENCE TO PRIOR APPLICATIONS 
     Priority is claimed to European Patent Application No. EP 19213913.7, filed on Dec. 5, 2019, the entire disclosure of which is hereby incorporated by reference herein. 
     FIELD 
     The present invention relates to a light sheet fluorescence microscope, comprising a light source device configured to emit excitation light suitable for inducing fluorescent light emitted from a specimen, a detector device configured to detect the fluorescent light from the specimen, and an optical system configured to illuminate the specimen with a light sheet formed from the excitation light and to guide the fluorescent light from the illuminated specimen to the detector device, wherein the optical system comprises an objective facing the specimen, the objective being configured to collect the fluorescent light emitted from the specimen. Further, the invention relates to a method for imaging a specimen by means of a light sheet fluorescence microscope. 
     BACKGROUND 
     Light sheet fluorescence microscopy is a technique in which a thin slice of a specimen is illuminated with a specific excitation light distribution which is focused only in one direction for example by using a cylindrical lens. Another illumination method uses a collimated light beam which is scanned in one direction to create a light distribution forming a light sheet. Due to the fact that only a portion of the specimen is illuminated which is actually observed, light sheet fluorescence microscopy reduces photo damage and stress induced on a living specimen. Furthermore, in contrast to confocal laser scanning microscopy where the specimen is scanned point by point, light sheet fluorescence microscopy is a wide field technique which allows to generate a three-dimensional stack of images based on optical sections through different planes of the specimen. 
     In a common approach, a light sheet fluorescence microscope comprises separate objectives for illuminating the specimen with the light sheet and for observing the illuminated object plane. Recently, light sheet methods have been developed which use only one objective both for illumination and detection. For example, by illuminating a small spot of an aperture of a high NA objective, a tilted light sheet may be generated. Thus, an oblique object plane is imaged using a tilted detector configuration. This technique is known as “oblique plane microscopy” (OPM). An extension of OPM called “swept confocally aligned planar excitation microscopy” (SCAPE) has been developed by using a scanning mirror for moving both the light sheet and the object plane to be imaged. 
     Apart from mere imaging a specimen, photomanipulation, i.e. manipulating the specimen by means of light application, has become more and more important. Thus, a specimen may be photomanipulated during or before actual imaging by e.g. heating, bleaching, photoactivating, photodeactivating etc. For this, a laser beam of suitable intensity and wavelength may be directed to the specimen. 
     In the field of light sheet fluorescence microscopy, a system capable of photomanipulating a specimen is e.g. disclosed in DE 10 2007 047 464 A1. This system comprises two objectives for illuminating the specimen and for detecting fluorescent light induced in the specimen. In the optical detection path, means for directing manipulation light onto the specimen are provided. However, this known system is not flexible enough in terms of applicability. For example, the system is not applicable to OPM or SCAPE configurations. 
     SUMMARY 
     In an embodiment, the present invention provides a light sheet fluorescence microscope which includes a light source configured to emit excitation light suitable for inducing fluorescent light emitted from a specimen, a detector configured to detect the fluorescent light from the specimen, and an optical system configured to illuminate the specimen with a light sheet formed from the excitation light, and to guide the fluorescent light from the illuminated specimen to the detector. The optical system comprises an objective facing the specimen, the objective being configured to collect the fluorescent light emitted from the specimen. The light source is further configured to emit manipulation light suitable for photomanipulating the specimen. The optical system is further configured to direct the manipulation light through a spatially limited sub-area of an entrance pupil of the objective onto the specimen along a light propagation direction which is different from a light propagation direction of the light sheet. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments of the present invention will be described in even greater detail below based on the exemplary figures. The present invention is not limited to the exemplary embodiments. All features described and/or illustrated herein can be used alone or combined in different combinations in embodiments of the present invention. The features and advantages of various embodiments of the present invention will become apparent by reading the following detailed description with reference to the attached drawings which illustrate the following: 
         FIG. 1  is a diagram showing a light sheet fluorescence microscope according to an embodiment; 
         FIG. 2  is a diagram showing a light sheet fluorescence microscope according to another embodiment; 
         FIG. 3  is a diagram showing a light sheet fluorescence microscope according to another embodiment; 
         FIG. 4  is a diagram showing a scanning mirror device included in the light sheet fluorescence microscope of  FIG. 3 ; 
         FIG. 5  is a diagram which illustrates rapid switching between illumination and photomanipulation in the fluorescence microscope of  FIG. 3 ; 
         FIG. 6  is a diagram showing a light source device according to an embodiment usable in the light sheet fluorescence microscope as shown in  FIGS. 1 and 3 ; and 
         FIG. 7  is a diagram showing a light source device according to another embodiment. 
     
    
    
     DETAILED DESCRIPTION 
     Embodiments of the present invention provide a light sheet fluorescence microscope and a method for imaging a specimen which allow for a flexible and efficient photomanipulation of the specimen. 
     A light sheet fluorescence microscope comprises a light source device configured to emit excitation light suitable for inducing fluorescent light emitted from a specimen, a detector device configured to detect the fluorescent light from the specimen, and an optical system configured to illuminate the specimen with a light sheet formed from the excitation light and to guide the fluorescent light from the illuminated specimen to the detector device. The optical system comprises an objective facing the specimen, wherein the objective is configured to collect the fluorescent light emitted from the specimen. The optical system is further configured to direct the manipulation light through a spatially limited sub-area of an entrance pupil of the objective onto the specimen along a light propagation direction which is different from a light propagation direction of the light sheet. 
     Hereinafter, excitation light or illumination light is to be understood as light which is applied to the specimen in order to induce light emanating therefrom and being suitable to be detected for imaging the specimen. In other words, excitation or illumination light as defined herein directly refers to the imaging process. In particular, the illumination or excitation light may be light which excites the specimen to emit fluorescent light for generating an optical image which represents structural information of the illuminated specimen. 
     On the other hand, manipulation light is to be understood as light which is applied to the specimen in order to manipulate the same by means of light in a way going beyond pure imaging. For example, manipulation light may comprise light which is used for heating, cutting, photoactivating, photodeactivating, bleaching, triggering chemical reactions e.g. in the field of optogenetics, etc. 
     The light sheet fluorescence microscope operates to partially illuminate the entrance pupil of the objective with manipulation light in order to photomanipulate the specimen along a direction as desired under the specific experimental circumstances. Thus, applying a partial pupil illumination allows a direction along which the manipulation light propagates into the specimen to be adjusted in a flexible manner by varying the sub-area of the entrance pupil onto which the manipulation light is directed. As the propagation direction of the manipulation light is adjusted to be different from the direction in which the light sheet propagates into the specimen, a region of interest within the object plane illuminated with the light sheet can be freely selected for photomanipulation according to the needs or preferences arising from the specific experimental setup. 
     The light sheet fluorescence microscope is usable in a wide range of applications. In particular, photomanipulating the specimen through a dedicated sub-area of the entrance pupil allows the microscope to advantageously be used in OPM or SCAPE configurations without being restricted thereto. 
     Accordingly, the optical system may be configured to illuminate the specimen with the light sheet through the objective. Thus, the objective facing the specimen is commonly used for illuminating the specimen with excitation light and photomanipulating the same with manipulation light as well as for observing the illuminated plane. 
     The spatially limited sub-area may be located offset from a center of the entrance pupil of the objective. By varying the offset distance from the pupil center, the propagation direction of the manipulation light can be easily adjusted as required in the specific experiment. 
     The optical system may be preferably configured to direct the excitation light through another spatially limited sub-area of the entrance pupil onto the specimen, wherein this other sub-area is located offset from the sub-area through which the manipulation light is directed onto the specimen. This embodiment may be advantageously applied in OPM and SCAPE configurations. 
     Preferably, the light propagation direction of the manipulation light emanating from the objective towards the specimen has an angle relative to a direction opposite to a light propagation direction of the fluorescent light emitted by the specimen in an angular range from 0 to ±45°. Specifically, the afore-mentioned angle may be set to zero, i.e. the manipulation light is irradiated onto the specimen in a direction directly opposite to the detection axis. This facilitates to photomanipulate the specimen accurately. 
     In a preferred embodiment, the optical system comprises a scanning mirror device including at least one tilting mirror which is tiltable for reflecting the excitation light to move the light sheet as a whole through the specimen in a light sheet scanning direction transverse to the light propagation direction of the light sheet. 
     Preferably, the at least one scanning mirror is tiltable for reflecting both the excitation light and the manipulation light. Thus, the excitation light and the manipulation light can be commonly scanned through the specimen so that imaging and photomanipulating can be easily performed in a coordinated manner. 
     In a specific embodiment, the scanning mirror device may comprise a scan lens and one single tilting mirror which is located in a rear focal plane of the scanning lens. By arranging the tilting mirror and the scan lens as mentioned above, it is ensured that a tilting movement of the excitation light is converted into a parallel displacement of the excitation lens on the object-side of the scan lens. Thus, the light sheet can be properly scanned through the specimen. 
     In a preferred embodiment, the scanning mirror device comprises two tilting mirrors located offset to each other along an optical axis of the optical system. This configuration allows that a scan lens can be dispensed with. Thus, using two tilting mirrors rather than one single mirror provides for an additional degree of freedom which can be utilized to freely adjust the (virtual) axis for tilting the exaction light. 
     Further, in case that the scanning mirror device is provided with two tilting mirrors, these mirrors can be easily controlled to switch between two tilting states, one of which being applied to illuminate the specimen with the excitation light and the other being applied for photomanipulation. Switching between illumination and photomanipulation can be rapidly performed by the scanning mirror device within several milliseconds which is of significant advantageous e.g. in cases in which fast processes shall be observed in a biological specimen. 
     Preferably, the at least one tilting mirror is tiltable for reflecting the fluorescent light collected by the objective towards the detector device. This allows the microscope to be used a descanned configuration as e.g. provided in OPM or SCAPE. 
     In a further preferred embodiment, an optical shifting unit is provided which is configured to shift an incident position of the manipulation light on the at least one tiltable mirror for varying the light propagation direction of the manipulation light directed onto the specimen independently of the light propagation direction of the fluorescent light and/or independently of the light propagation direction of the light sheet. This renders the photomanipulation even more flexible in terms its positional relationship relative to the excitation light. For instance, the manipulation light may be directed to a site located outside the object plane which is illuminated with the light sheet 
     The optical shifting unit may comprise at least one element reflecting or emitting the manipulation light, wherein this element is movable to shift the incident position of the manipulation light on the at least one tiltable mirror. For example, the optical shifting unit may be formed by a dichroic mirror which may be used to couple the manipulation into the optical system. 
     The light source device may be configured to emit the excitation light and the manipulation light along a common optical path into the optical system. This allows to use one single light source emitting both the excitation light and the manipulation light. Alternatively, a plurality of light sources may be integrated in a common casing. All in all, the light source device comprises one or more light sources (for example lasers) and optionally a common casing and/or further optical elements, for example lenses, mirrors, optical filters and/or beam splitters. 
     For example, the light source device comprises at least one excitation light source (for example a laser) configured to emit the excitation light along at least one first optical path, and at least one manipulation light source (for example a laser) configured to emit the manipulation light along at least one second optical path. The light source device further comprises a light shaping system having an optical input formed by the first and second optical paths and an optical output formed by the common optical path, wherein the light shaping system is configured to selectively shape at least one of the excitation light and the manipulation light. 
     Preferably, the light source device is switchable between an excitation operating mode for emitting only the excitation light and a manipulation operating mode for emitting only the manipulation light. 
     The light shaping system may comprise an input merging element configured to merge the first and second optical paths in a third optical path. The input merging element may be formed e.g. by a dichroic mirror. 
     The light shaping system may further include a beam expander located in the third optical path. The beam expander may be used to shape both the excitation light and the manipulation light in a first step before further light shaping and/or scanning is applied, e.g. for dynamically creating the light sheet, shifting a focus of the manipulation light, displacing the excitation light and/or the manipulation light in directions transverse to the light propagation direction etc. 
     In an embodiment, the common optical path is formed by the third optical path. Further, the light shaping system may comprise at least one light shaping element and a scanner which are located in the common optical path, wherein, in the excitation operating mode, the light shaping element is configured not to shape the excitation light and the scanner is configured to cause a scanning movement of the excitation light in a predetermined direction transverse to the light propagation direction of the excitation light for generating the light sheet. In the manipulation operating mode, the light shaping element is configured to shape the manipulation light and the scanner is configured to adjust a displacement of the manipulation light in the predetermined direction. According to this embodiment, a single (third) optical path is provided inside the light shaping system for taking effect on both the excitation light and the manipulation. Further, the light shaping system the light sheet is created dynamically, e.g. by rapidly scanning the collimated excitation light through the specimen. 
     According to a further embodiment, the light shaping system includes a splitting element located in the third optical path and configured to spatially separate the excitation light and the manipulation light from each other, wherein the excitation light propagates in a fourth optical path and the manipulation light propagates in a fifth optical path. The light shaping system further comprises an output merging element configured to merge the fourth and fifth optical paths in the common optical path. Further, the light shaping system comprises an anamorphic optical element located in the fourth optical path, wherein the anamorphic optical element is configured to generate the light sheet from the excitation light. The light shaping system further comprises at least one light shaping element and/or a scanner located in the fifth optical path, wherein the light shaping element is configured to shape the manipulation light and the scanner is configured to adjust a displacement of the manipulation light in a predetermined direction. Here, the light shaping system provides for two separate (fourth and fifth) optical paths in order take effect on the excitation light and the manipulation light, respectively. Further, the light sheet is created by means of optical beam shaping rather than by scanning. 
     The light shaping element may comprise at least one of an electrically tunable lens, a deformable mirror, a digital mirror device, and a spatial light modulator. 
     Preferably, the light shaping element is configured to shift a focus of the manipulation light in the light propagation direction thereof. In this case, the light shaping element may be formed by an electrically tunable lens. 
     According to another aspect, a method is provided for imaging the specimen by means of a light sheet fluorescence microscope, comprising the following steps: emitting excitation light suitable for inducing fluorescent light emitted by the specimen; illuminating the specimen with a light sheet formed from the excitation light; collecting the fluorescent light from the illuminated specimen by means of an objective facing the specimen; and detecting the fluorescent light. The method further comprises the steps of: emitting manipulation light suitable for photomanipulating the specimen; and directing the manipulation light through a spatially limited sub-area of an entrance pupil of the objective onto the specimen along a light propagation direction which is different from a light propagation direction of the light sheet. 
     A light sheet fluorescence microscope  100  according to an embodiment is illustrated in the diagram of  FIG. 1 , wherein only those components of the light sheet fluorescence microscope  100  are shown which are helpful for understanding the embodiment. 
     According to the specific embodiment shown in  FIG. 1 , the light sheet fluorescence microscope  100  comprises a light source device  102 , a detector device  104  which may be formed by a CCD or CMOS camera suitable for wide field imaging, and an optical system generally designated by reference sign  106 . The optical system  106  may comprise a plurality of optical elements, among which an objective  108  faces a specimen  110  which is to be imaged by the light sheet fluorescence microscope  100 . As explained in more detail below, the objective  108  may be used for both illumination and detection. Thus, it may be a common objective through which both fluorescence-stimulating illumination light and fluorescence light are guided. Accordingly, the light sheet fluorescence microscope  100  may be operated in SCAPE or OPM configuration. However, the invention shall not be restricted to such a configuration. In particular, the light sheet fluorescence microscope may also comprise two separate objectives for fluorescence-exciting illumination and detection, respectively. 
     The light source device  102  is configured to emit excitation light E which is suitable to excite the specimen  110  to emit fluorescent light F which is to be detected by the detector device  104 . As explained below in more detail with reference to  FIGS. 6 and 7 , the light source device  102  may be configured to shape a light sheet LS from the excitation light E for illuminating only a thin slice of the specimen  110  at a given point in time. In the specific embodiment shown in  FIG. 1 , the light sheet LS illuminating the specimen  110  propagates in a direction which is oblique to the optical axis O of the optical system  106 . Referring to an orthogonal xyz coordinate system indicated in  FIG. 1 , the light sheet LS illuminating the specimen  110  is expanded in a direction parallel to the x-axis which is orthogonal to the propagation direction of the light sheet LS. Accordingly, the illumination plane formed by the light sheet LS is defined by two axes, one of which given by the light sheet propagation direction lying in the xz-plane and the other being the x-axis. 
     The optical system  106  is configured to illuminate the specimen  110  with the light sheet LS formed from the excitation light E as well as to guide the fluorescent light F from the illuminated specimen  110  to the detector device  104 . In the embodiment of  FIG. 1 , the optical system  106  comprises a first lens  112 , a second lens  114 , a scanning mirror device  116 , a third lens  118 , a fourth lens  120  and the objective  108  facing the specimen  110 . Each of the lenses  112  and  120  serves as a tube lens, wherein the lens  118  is configured as a scan lens. According to the embodiment shown in  FIG. 1 , the scanning mirror device  116  is formed by a single tilting mirror  122  which reflects the excitation light E passing the lenses  112 ,  114  through the lenses  118 ,  120  into the objective  108  for illuminating the specimen  110  with the light sheet LS. The tilting mirror  122  is movable around a tilt axis  124  which is oriented parallel to the x-axis. 
     In order to illustrate the tilting movement performed by the scanning mirror device  116 ,  FIG. 1  shows the tilting mirror  122  in three different tilting positions at consecutive times t=t 1 , t=t 2 , and t=t 3 . Depending on the respective tilting position, the tilting mirror  122  reflects the excitation light E in different directions resulting in a scanning movement of the light sheet LS within the specimen  110  in a direction parallel to the y-axis. In other words, the scanning mirror device  116  serves to move the light sheet LS as a whole through the specimen  110  in a light sheet scanning direction which is illustrated in  FIG. 1  by a double arrow A. For simplicity, the excitation light E performing the afore-mentioned scanning movement is illustrated by its principle ray, wherein E(t=t 1 ) represents the excitation light E which is reflected by the tilting mirror  122  at the time t=t 1 . Likewise, the principle ray E(t=t 2 ) illustrates the excitation light E reflected by the tilting mirror  122  at the time t=t 2 , and the principle ray E(t=t 3 ) represents the excitation light E reflected by the tilting mirror  122  at the time t=t 3 . 
     In the configuration shown in  FIG. 1 , the tilting mirror  122  is located in a rear, i.e. image-side focal plane of the scan lens  118 . By locating the tilting mirror  122  in the focal plane of the scan lens  118 , it is ensured that a tilting movement of the excitation light E on the tilting mirror  122  is converted into a parallel displacement of the excitation light E on an object side of the scan lens  118 . This parallel displacement of the excitation light E is illustrated by the principle rays E(t=t 1 ), E(t=t 2 ), and E(t=t 3 ) propagating parallel to each other between the scan lens  118  and the tube lens  120  as well as when emanating from the objective  108  into the specimen  110 . Accordingly, the tilting movement performed by the scanning mirror device  116  results in a parallel displacement of the light sheet LS within the specimen  110  in y-direction. 
     The fluorescent light F induced by the excitation light E is collected by the objective  108  and propagates in a direction opposite to the propagation direction of the excitation light E through the optical system  106  up to the tube lens  112 . Subsequently, the fluorescent light F passes a dichroic mirror  126  which is configured to selectively transmit both the fluorescent light F and the excitation light E. Accordingly, in the specific embodiment shown in  FIG. 1 , the dichroic mirror  126  is used as a beam splitter for separating the excitation light E and the fluorescent light F. Further, a mirror  148  is provided serving as an element for coupling the excitation light E into the optical system  106 . Needless to say that the configuration formed by the mirrors  126 ,  148  for implementing the afore-mentioned light separation and coupling functions is to be understood merely as an example. Other configurations may be used as will become evident from further embodiments described later. 
     After passing the dichroic mirror  126 , the fluorescent light F propagates through an optical detection system formed by two objectives  127 ,  128  and a tube lens  130  towards the detector device  104 . Between the objectives  127 ,  128 , an intermediate image plane  132  is located in which an intermediate image of the plane illuminated with the light sheet LS is formed. This intermediate image is imaged onto the detector device  104 . 
     As can be seen from  FIG. 1 , the optical path leading to the detector device  104  is oblique relative to the optical path between the objective  108  and the tube lens  112 . This reflects the fact that in SCAPE or OPM configuration the light sheet LS is oblique relative to an object-side focal plane P 1  of the objective  108 . Correspondingly, a plane P 2  conjugate to the focal plane P 1  is oblique relative to the intermediate image plane  132 . 
     As the fluorescence light F emanating from the specimen  110  is guided to the tilting mirror  122 , the optical system  106  of the light sheet fluorescence microscope  100  forms a so-called descanned configuration. Such a descanned configuration ensures that the illuminated plane within the specimen  110  is continuously detected by the stationary detector device  104  while the illuminated plane is scanned in y-direction through the specimen  110 . As in the case of the excitation light E, this fact is illustrated in  FIG. 1  by indicating the principle rays F(t=t 1 ), F(t=t 2 ), and F(t=t 3 ) of the fluorescent light F for the different tilting positions in which the tilting mirror  122  is located at times t 1 , t 2 , and t 3 . 
     As already mentioned above, the embodiment shown in  FIG. 1  corresponds to a SCAPE or OPM configuration. Therefore, both the excitation light E and the fluorescent light F pass through the objective  108 . Specifically, neither the excitation light E nor the fluorescent light F fully utilize the aperture of the objective  108 . In fact, the excitation light E and the fluorescent light F pass the objective  108  through different portions thereof, these portions being eccentrically located on opposite sides of the optical axis O. 
     The light source device  102  is further configured to emit manipulation light M which is suitable to photomanipulate the specimen  110 . As already explained above, photomanipulating the specimen  110  is to be understood broadly as light application which is not exclusively related to imaging as for example heating, bleaching etc. 
     In the specific configuration shown in  FIG. 1 , the dichroic mirror  126  is used to introduce the manipulation light M into the optical system  106 . Accordingly, the characteristic of the dichroic mirror  126  is selected such that the dichroic mirror  126  reflects both the manipulation light M and the excitation light E and transmits the fluorescent light F. Thus, in the present embodiment it is assumed that the manipulation light M has essentially the same wavelength as the excitation light E. In such a case, a single laser source may be used to generate both the excitation light E and the manipulation light M. 
     As shown in  FIG. 1 , the manipulation light M passes the tube lens  112  through a portion which is different from a portion through which the excitation light E propagates through the tube lens  112 . As a result, after passing the lenses  112 ,  114 , the manipulation light M falls onto the tilting mirror  112  at an incident position which is different from the incident position of the excitation light E. Accordingly, after being reflected by the titling mirror  122 , the manipulation light M propagates towards the objective  108  in a direction which is different from the propagation direction of the excitation light E. Thus, the optical system  106  is configured to direct the manipulation light M into the specimen  110  along a light propagation direction which is different from the propagation direction of the light sheet LS formed from the excitation light E. The manipulation light M passes the objective  108  through a spatially limited sub-area of an entrance pupil EP of the objective  108 . 
     As the manipulation light M is reflected by the tilting mirror  122 , the manipulation light M performs a scanning movement in y-direction as the excitation light E does. However, for simplifying the illustration,  FIG. 1  indicates the manipulation light M only by means of a single principle ray M(t=t 2 ) which belongs to the manipulation light M referring to the tilting position of the tilting mirror  122  at the time t=t 2 . 
     The light sheet fluorescence microscope  100  may comprise an optical shifting unit which is configured to shift the incident position of the manipulation light M on the tilting mirror  122 . For example, such an optical shifting unit may be formed by the dichroic mirror  126 . Thus, the dichroic mirror  126  may be configured to be movable along the propagation direction of the manipulating light M falling onto the dichroic mirror  126 , i.e. according to the configuration of  FIG. 1  in a direction parallel to the z-axis. By moving the dichroic mirror  126  in such a way, the portion of the tube lens  122 , through which the manipulation light M passes, is shifted perpendicular to the optical axis O. Thus, the incident position of the manipulation light M on the tilting mirror  122  changes by moving the dichroic mirror  126  resulting in a corresponding variation of the direction in which the manipulation light M propagates into the specimen  110 . 
     By moving the dichroic mirror  126 , the propagation direction of the manipulation light M can be varied independently of the propagation directions of the excitation light E and the fluorescent light F. 
     By shifting the dichroic mirror  126  in a direction perpendicular to the optical axis O of the optical system  106  the propagation direction of the manipulation light M emanating from the objective  108  towards the specimen  110  can be varied in a wide range. For example, the angle of the propagation direction of the manipulation light M relative to a direction opposite to the propagation direction of the fluorescent light F emitted by the specimen  110  may be varied in an angular range from 0 to ±45°. In particular, it may be advantageous to set the afore-mentioned angle to 0, i.e. to irradiate the manipulation light onto the specimen along a direction which is directly opposite to the direction along which the florescent light F is collected by the objective  108 . In this respect, it is to be noted that the light propagation directions explained herein refer to the principal rays of the corresponding light beams. 
       FIG. 2  shows a light sheet fluorescence microscope  100   a  which represents a modified embodiment of the configuration of  FIG. 1 . The modification compared to the light sheet fluorescence microscope  100  shown in  FIG. 1  relates to a light source device  202  replacing the light source device  102  shown in  FIG. 1 . 
     Whereas the light source device  102  of  FIG. 1  emits the excitation light E and the manipulation light M along a common optical path, i.e. in a collinear manner, into the optical system  106 , the light source device  202  of the light sheet fluorescence microscope  100   a  shown in  FIG. 2  outputs the excitation light E and the manipulation light M along two separate optical paths which may lie parallel to each other. Accordingly, the light source device  202  is comprised of two separate light sources  240  and  242  which output the excitation light E and the manipulation light M, respectively. 
     In contrast to the configuration of  FIG. 1 , the light sheet fluorescence microscope  100   a  shown in  FIG. 2  comprises a single component e.g. in form of a dichroic mirror  244  for commonly introducing the excitation light E and the manipulation light M into the optical system  106 . As can be seen in  FIG. 2 , the dichroic mirror  244  is adapted to the light source device  202  such that both the excitation light E and the manipulation light M are reflected into the optical system  106 . Further, insofar corresponding to the dichroic mirror  126  shown in  FIG. 1 , the characteristic of the dichroic mirror  244  of the light sheet fluorescence microscope  100   a  is adapted to transmit the fluorescent light F towards the detector device  104 . 
     As the excitation light E and the manipulation light M are commonly reflected by the dichroic mirror  244 , in contrast to the embodiment shown in  FIG. 1 , the light sheet fluorescence microscope  100   a  of  FIG. 2  does not provide for a variation of the propagation direction of the manipulation light M independently of the propagation direction of the excitation light E. Thus, whereas the dichroic mirror  126  of  FIG. 1  only acts on the manipulation light M, the dichroic mirror  244  shown in  FIG. 2  reflects both the excitation light E and the manipulation light M. Thus, the positional relationship between the excitation light E and the manipulation light M is not changed by the dichroic mirror  244  even if the dichroic mirror  244  was moved in a direction perpendicular to the optical axis O of the optical system  106 . In other words, according to the embodiment shown in  FIG. 2 , the direction along which the specimen  110  is photomanipulated by the manipulation light M does not change in relation to the direction along which the excitation light E illuminates the specimen  110 . Accordingly, at any given time t 1 , t 2 , t 3 , the corresponding principle ray of the manipulation light M intersects the corresponding principle ray of the excitation light E in the focal plane P 1  of the objective  108 . As in  FIG. 1 , it is to be noted that  FIG. 2  shows only one principle ray M (t=t 2 ) which intersects the principle ray E(t=t 2 ) of the excitation light in the focal plane P 1 . In contrast, according to the configuration shown in  FIG. 1 , it is possible to vary the position in which the principle ray M(t=t 2 ) intersects the focal plane P 1  enabling the positional relationship between the manipulation light M and the excitation light E to be varied within the specimen  110 . 
     It is to be noted that even in the embodiment shown in  FIG. 2 , it may be possible to vary the propagation direction of the manipulation light M independently of the propagation direction of the excitation light E, namely e.g. in case the manipulation light source  242  includes means for shifting the manipulation light M correspondingly. 
       FIG. 3  is a diagram showing a light sheet fluorescence microscope  100   b  representing a further embodiment. The light sheet fluorescence microscope  100   b  differs from the embodiments shown in  FIGS. 1 and 2  mainly by a modified scanning mirror device  318 . 
     As the microscopes shown in  FIGS. 1 and 2 , the light sheet fluorescence microscope  100   b  of  FIG. 3  comprises a light source device  302  emitting the excitation light E and the manipulation light M. The light sheet fluorescence microscope  100   b  further comprises an optical system  306  which includes a tube lens  305 , the scanning mirror device  316 , a further tube lens  308  and the objective  108 . The excitation light E and the manipulation light M are coupled into the optical system  306  by means of a mirror  348 . 
     The fluorescent light F emitted from the specimen  110  propagates through the optical system  306  in a direction essentially opposite to the propagation direction of the excitation light E and the manipulation light M. After passing the tube lens  305 , the fluorescent light F propagates through two objectives  327 ,  328  and a tube lens  330  to a detector device  304 . 
     Whereas the light sheet fluorescence microscope  100   b  is fundamentally operated in the same manner as the microscopes shown in  FIGS. 1 and 2 , the configuration of  FIG. 3  is advantageously modified in terms of the tilting operation of the scanning mirror device  316  which is applied to scan the light sheet LS as a whole in y-direction through the specimen  110 . Thus, according to the configurations shown in  FIGS. 1 and 2 , the single tilting mirror  122  is located in the rear focal plane of the scan lens  118  to ensure that the tilting movement of the excitation light E on the tilting mirror  122  is converted into a parallel displacement of the excitation light E on the object side of the scan lens  118 . However, using a scan lens for the afore-mentioned purpose may have some drawbacks, in particular in terms of costs as a tube lens is rather expensive. Therefore, the light sheet fluorescence microscope  100   b  shown in  FIG. 3  is modified in such a way that a scan lens can be dispensed with. 
     Specifically, the scanning mirror device  316  comprises two tilting mirrors  332 ,  334  which are located offset to each other along the optical axis O of the optical system  306 . Each of the tilting mirrors  332 ,  334  is movable around a tilt axis  336 ,  338  as shown in  FIG. 4 . The respective tilt axis  336 ,  338  is oriented parallel to the x-axis. 
     By using the two tilting mirrors  332 ,  334  instead of one single tilting mirror as provided in the configurations of  FIGS. 1 and 2 , the intended parallel displacement of the excitation light E emanating from the scanning mirror device  316  can be achieved without providing a dedicated scan lens. Thus, the two tilting mirrors  332 ,  334  provide for an additional degree of freedom in terms of adjusting the tilting operation which results in a virtual tilt axis about which the excitation light E is tilted when operating the scanning mirror device  316 . In other words, in case of using one single tilting mirror, the axis for tilting the excitation light E is fixed (see tilt axis  124  in  FIGS. 1 and 2 ). In contrast, when using the two tilting mirrors  332 ,  334  having two physical tilt axes  336 ,  338 , a virtual, i.e. non-physical tilt axis can be freely created as needed. 
     In the present configuration, this virtual tilt axis is located such that the intended parallel displacement of the excitation light E propagating towards the specimen  110  is achieved. In the example shown in  FIG. 4 , two tilting states of the scanning mirror device  316  are illustrated, a difference between these two tilting states corresponding to a tilting angle β. The tilting angle R defines the amount of tilting about a virtual tilt axis V which is located on the image side of the first tilting mirror  332 . Tilting the excitation light E in the amount of angle β about the virtual tilt axis V is converted into a parallel displacement d of the excitation light E on the object side of the second tilting mirror  334 . According to the configuration shown in  FIGS. 3 and 4 , the virtual tilt axis V is located in the rear, i.e. image-side focal plane of the objective  108  (or a plane conjugate thereto). 
     It is to be noted that  FIG. 3  does not illustrate the scanning movement of the manipulation light M achieved by operating the scanning mirror device  316  as explained above. However, in terms of the fundamental operation for scanning the excitation light E and the manipulation light M in y-direction through the specimen  110 , the embodiment shown in  FIG. 3  does not differ from the configurations of  FIGS. 1 and 2 . 
     The light source device  102 ,  302  may be switchable between an excitation operating mode and a manipulation operating mode. In the excitation operating mode, the light source device  102 ,  302  emits only the excitation light E, and the manipulation light M is shut off. Likewise, in the manipulating operating mode, the light source device  102 ,  302  emits only the manipulation light M, and the excitation light E is shut off. 
     The diagram shown in  FIG. 5  illustrates how the scanning mirror device  316  of the light sheet fluorescence microscope  100   b  can be used to rapidly switch between illumination of the specimen  110  by means of the excitation light E and photomanipulation by directing the manipulation light M into the specimen  110 . 
       FIG. 5  shows two tilting states of the scanning mirror device  316  indicated by solid and dashed lines, respectively. The tilting state illustrated by solid lines refers to the excitation operating mode in which only the excitation light E is emitted into the specimen. Likewise, the tilting state illustrated by dashed lines refers to the manipulation operating mode in which only the manipulation light M is emitted into the specimen  110 . Accordingly, the light source device  302  may be controlled in such a manner that in the excitation operating mode only the excitation light E is generated, and in the manipulation operating mode only the manipulation light M is generated. 
     Thus, in the excitation operating mode, a plane IP of the specimen  110 , which is illuminated by the light sheet LS, is imaged onto the detector device  104 . By switching to the excitation mode, a certain area of the object plane IP may be photomanipulated along a direction which is different from the propagation direction of the light sheet LS. 
     It is to be noted that it is possible to switch the scanning mirror device  316  very fast between the afore-mentioned tilting states. Indeed, the scanning mirror device  316  can be switched within a few milliseconds. Accordingly, it is possible to rapidly change between illumination and photomanipulation of the specimen  110 . Further, by appropriately controlling the tilting movement of the two tilting mirrors  332 ,  334 , the propagation direction of the manipulation light M can be varied in a wide range. 
     The embodiments shown in  FIGS. 1, 3 and 5  are configured to couple the excitation light E and the manipulation light M in a collinear manner, i.e. along a common optical path into the optical system  106 ,  306 . Accordingly, the light source device  102 ,  302  may be formed by a combined source for emitting both the excitation light E and the manipulation light M. Such a combined source is advantageous e.g. in terms of a compact design. Thus, different laser sources generating the excitation light E and the manipulation light M, respectively, may be integrated in a common casing. 
     However, it is a challenge to provide such a combined source meeting specific requirements arising in light sheet microscopy. Thus, the source should be capable of generating the light sheet LS in a desired orientation as well as of shaping the manipulation light M as needed. For example, a focus of the manipulation light M may be shifted in z-direction. Further, the manipulation light M may be deflected in x- and y-direction. 
       FIGS. 6 and 7  show different embodiments of the light source device  102 ,  302  which may be used to satisfy the afore-mentioned requirements. 
     The light source device  102 ,  302  according to  FIG. 6  comprises an excitation light source  650  emitting the excitation light E along a first optical path OP 1  and a manipulation light source  652  emitting the manipulation light M along a second optical path OP 2 . The first and second optical paths OP 1 , OP 2  may be located offset from each other in parallel. The light source device  102 ,  302  further comprises a dichroic mirror  654 , the characteristic thereof being adapted to transmit the excitation light E and to reflect the manipulation light M which is deflected by a mirror  656  onto the dichroic mirror  654 . Thus, the dichroic mirror  654  may serve as an input merging element which is configured to merge the first and second optical paths OP 1 , OP 2  in a common third optical path in which the excitation light E and the manipulation light M propagate. 
     The light source device  102 ,  302  further comprises a beam expander  658  by which the cross-sections of the excitation light E and the manipulation light M are expanded in a direction perpendicular to the light propagation direction. Subsequent to the beam expander  658 , an optical light shaping element  660  is located in the third optical path OP 3 . The light shaping element  660  may be formed by an electrically tunable lens (ETF), a deformable mirror (DM), a digital mirror device (DMD) or a spatial light modulator (SLM). Further, two lenses  662 ,  664  and a scanner  666  are included in the third optical path OP 3 . The scanner  666  may be formed by a mirror which is tiltable around a tilt axis  668  which is oriented parallel to the z-axis. In this respect, it is to be noted that the plane of drawing in  FIG. 6  is perpendicular to the plane of drawing in  FIGS. 1 to 5 . 
     The afore-mentioned elements  654  to  666  form a light shaping system  670 , an optical input thereof being defined by the first and second optical paths OP 1 , OP 2  and an optical output thereof being defined by the third optical path OP 3 . According to the configuration shown in  FIG. 6 , the third optical path OP 3  is used as the common optical path along which the light source device  102 ,  302  emits the excitation light E and the manipulation light M into the optical system  106 ,  306 . In  FIG. 6 , only the tube lens  112 ,  305  of the optical system  106 ,  306  is shown. 
     The light source device  102 ,  302  can be operated in the excitation operating mode for generating only excitation light E and in the manipulation operating mode for generating only the manipulation light M. The light shaping system  670  is operated in different ways, depending on whether the excitation operating mode or the manipulation operating mode is selected. For example, the light shaping system  670  may be controlled in the excitation operating mode such that the light shaping member  660  is neutral with respect to the excitation light E, i.e. does not have any effect on the excitation light E in terms of light shaping. Further, in the excitation operating mode, the scanner  666  may be operated to perform a very fast tilting movement around the tilt axis  668  in order to scan the excitation light E in x-direction through the specimen  110  for generating the light sheet LS. Accordingly, the light sheet LS is dynamically generated in this embodiment. 
     In contrast, in the manipulating operating mode, the light shaping system  670  is controlled such that the light shaping element  660  acts on the manipulation light M in order to shape the same in a desired manner. For example, the light shaping element  660  may shift the focus of the manipulation light M in z-direction. On the other hand, the scanner  666  is operated in the manipulation operating mode such that it serves as a detector element. Thus, the scanner  666  may provide for a displacement of the manipulation light M in x-direction. 
     The light shaping element  670  may be located in a plane which is conjugate to an object-side focal plane of the objective  108 . Alternatively, the light shaping element  607  may be positioned in an image-side focal plane of the objective  108 . In this case, the lenses  662 ,  664  may form a telecentric optical system in which the lenses  662 ,  664  are located at a distance from each other being equal to the sum of their focal lengths. Thus, the lenses  662 ,  664  may form a so-called  4   f  system. 
     The scanner  668  is preferably located in a rear focal plane of the tube lens  112 ,  305 , i.e. at a distance from the tube lens  112 ,  305  which equals the rear focal length f 1  thereof. Alternatively, the scanner  666  may be located in a plane conjugate to the rear focal plane of the tube lens  112 ,  305 . In any case, the afore-mentioned plane, in which the scanner  666  is located, in conjugate to the rear focal plane of the objective  108 . 
     In the configuration shown in  FIG. 6 , the scanner  666  is tiltable about the tilt axis  668  such that a light beam displacement in x-direction is caused inside the specimen  110 . In addition, the scanner  666  may be tiltable about a second tilt axis perpendicular to the tilt axis  668  in order to create a beam displacement in y-direction within the specimen  110 . As a result of such a displacement in y-direction, the focus of the excitation light E or the manipulation light M is not located anymore in the object plane IP imaged onto the detector device  104  (see  FIG. 5 ). Rather, the light focus is offset from the plane IP in y-direction. 
     The configuration shown in  FIG. 6  is to be understood merely as an example. For instance, it is possible to combine the elements causing the two optical imaging processes related to the excitation light E and the manipulation light M, respectively. Furthermore, the sequence in which the lighting shaping element  660  and the scanner  666  are provided may be reversed, and multiple of light shaping elements and scanners by be provided. Likewise, more than two laser sources may be provided, e.g. for implementing a cascade of light sources. 
       FIG. 7  shows another embodiment of the light source device  102 ,  302  comprising a modified light shaping system  770 . Whereas according to the configuration shown in  FIG. 6 , the third optical path OP 3  extending from the beam expander  658  directly forms the optical output for supplying the excitation light E and the manipulation light M to the optical system  106 ,  306 , the embodiment shown in  FIG. 7  provides for a separation of the excitation light E and the manipulation light M subsequent to the beam expander  658  as explained hereinafter. 
     The beam shaping system  770  of  FIG. 7  comprises a splitting element  772 , e.g. a switchable mirror or a dichroic element which is configured to spatially separate the excitation light E and the manipulation light M. Needless to say that a dichroic element may only be used as splitting element in case that the excitation light E and the manipulation light M output from the light sources  650 ,  652  have different wavelengths. In contrast, a switchable mirror may be used irrespectively of the wavelengths of the excitation light E and the manipulation light M. 
     In the example of  FIG. 7 , it is assumed that the excitation light E propagates in a fourth optical path OP 4  extending from the splitting element  772 . In the fourth optical path OP 4 , an anamorphic optical element  773 , e.g. a cylinder lens, is provided which is configured to optically shape the excitation light E such that a focus with elliptical cross-section is created in the image-side focal plane of the objective  108 . In the example shown in  FIG. 7 , a long axis of the elliptical cross-section in the image-side focal plane of the objective  108  extends in y-direction, whereas a short axis extends in x-direction. Accordingly, the plane illuminated with the light sheet LS within the specimen  110  extends in x-direction. In this respect, it is to be noted that the plane of drawing in  FIG. 7  is perpendicular to the plane of drawing in  FIGS. 1 to 5 . 
     The manipulation light M propagates in a fifth optical path OP 5  extending from the splitting element  772  to the light shaping element  760  which may comprise a DM, DMD, SLM and/or an ETL. For example, the light shaping elements  760  may be used to shift the focus of the manipulation light M in z-direction. In such an application, an ETL may preferably be used. 
     The light shaping system  770  further comprises two lenses  762 ,  764  and a scanner  766  having a tilt axis  768  which is oriented in z-direction. By tilting the scanner  766  about the tilt axis  768 , the manipulation light M can be displaced in x-direction. 
     The light shaping system  770  further comprises an output merging element  774  which is configured to merge the fourth optical path OP 4  and the fifth optical path OP 5  in a sixth optical path OP 6  which extends to the tube lens  112 ,  305  of the optical system  106 ,  306 . Accordingly, the sixth optical path OP 6  represents the common optical path which is used to output the excitation light E and the manipulation light M in a collinear, i.e. spatially coinciding manner to the optical system  106 ,  306 . As the splitting element  772 , the output merging element  774  may be formed by a switchable mirror or a dichroic element. 
     In the configuration shown in  FIG. 7 , the scanner  766  is preferably located in the rear focal plane of the tube lens  112 ,  305  having the rear focal length f 1 . Further, the light shaping element  760  may be located in a plane which is conjugate to the object-side focal plane of the objective  108 . For this, the lenses  762 ,  764  having focal lengths f 2 , f 3 , respectively, form a 4f-system. Further, according to an advantageous embodiment, each of the light shaping element  760  and the scanner  766  may be located in a plane which is conjugated to the image-side focal plane of the objective  108 . 
     Needless to say that the afore-mentioned embodiments of the light source device  102 ,  302  are to be understood merely as examples. In particular, the light source device is not limited to a configuration in which the excitation light E and the manipulation light M are output in a collinear manner, i.e. along a common optical path. Rather, as shown in the embodiment of  FIG. 2 , two separate optical paths for coupling the excitation light E and the manipulation light M into the optical system  106  may be provided. In particular, the embodiments shown in  FIGS. 6 and 7  may be modified to adapt the configurations shown therein to a light source device having two separate optical outputs. For example, referring to  FIG. 7 , such a modification may be achieved by omitting the output merging element  774 . In this case, the excitation light E and the manipulation light M is coupled into the optical system  106 ,  306  along the two separate optical paths OP 4 , OP 5 , respectively. 
     Further, the explanations regarding the examples shown in  FIGS. 6 and 7  refer to the coordinate systems shown in  FIGS. 1 to 5 . However, it is to be noted that the specific directions in particular in terms of scanning or shifting the different types of light within the system are to be understood throughout this specification merely as examples, and may be modified as needed. Likewise, the light source devices shown in  FIGS. 6 and 7  should only be understood as exemplary configurations which may be used in abstracted form for implementing the light source devices  102 ,  302  in the embodiments shown in  FIGS. 1 to 5 . In other words, the configurations shown in  FIGS. 6 and 7  may be suitably adapted when being implemented in the embodiments of  FIGS. 1 to 5 . For example, optical elements (as e.g. a dichroic mirror) may be added or removed as needed. 
     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. Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some embodiments, some one or more of the most important method steps may be executed by such an apparatus. 
     Depending on certain implementation requirements, embodiments of the invention can be implemented in hardware or in software. The implementation can be performed using a non-transitory storage medium such as a digital storage medium, for example a floppy disc, a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable. 
     Some embodiments according to the invention comprise a data carrier having electronically readable control signals, which are capable of cooperating with a programmable computer system, such that one of the methods described herein is performed. 
     Generally, embodiments of the present invention can be implemented as a computer program product with a program code, the program code being operative for performing one of the methods when the computer program product runs on a computer. The program code may, for example, be stored on a machine readable carrier. 
     Other embodiments comprise the computer program for performing one of the methods described herein, stored on a machine readable carrier. 
     In other words, an embodiment of the present invention is, therefore, a computer program having a program code for performing one of the methods described herein, when the computer program runs on a computer. 
     A further embodiment of the present invention is, therefore, a storage medium (or a data carrier, or a computer-readable medium) comprising, stored thereon, the computer program for performing one of the methods described herein when it is performed by a processor. The data carrier, the digital storage medium or the recorded medium are typically tangible and/or non-transitionary. A further embodiment of the present invention is an apparatus as described herein comprising a processor and the storage medium. 
     A further embodiment of the invention is, therefore, a data stream or a sequence of signals representing the computer program for performing one of the methods described herein. The data stream or the sequence of signals may, for example, be configured to be transferred via a data communication connection, for example, via the internet. 
     A further embodiment comprises a processing means, for example, a computer or a programmable logic device, configured to, or adapted to, perform one of the methods described herein. 
     A further embodiment comprises a computer having installed thereon the computer program for performing one of the methods described herein. 
     A further embodiment according to the invention comprises an apparatus or a system configured to transfer (for example, electronically or optically) a computer program for performing one of the methods described herein to a receiver. The receiver may, for example, be a computer, a mobile device, a memory device or the like. The apparatus or system may, for example, comprise a file server for transferring the computer program to the receiver. 
     In some embodiments, a programmable logic device (for example, a field programmable gate array) may be used to perform some or all of the functionalities of the methods described herein. In some embodiments, a field programmable gate array may cooperate with a microprocessor in order to perform one of the methods described herein. Generally, the methods are preferably performed by any hardware apparatus. 
     While embodiments of the invention have 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. It will be understood that changes and modifications may be made by those of ordinary skill within the scope of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below. Additionally, statements made herein characterizing the invention refer to an embodiment of the invention and not necessarily all embodiments. 
     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 ,  100   a ,  100   b  light sheet fluorescence microscope 
           102  light source device 
           104  detector device 
           106  optical system 
           108  objective 
           110  specimen 
           112  tube lens 
           114  lens 
           116  scanning mirror device 
           118  scan lens 
           120  tube lens 
           122  tilting mirror 
           124  tilt axis 
           126  dichroic mirror 
           127  objective 
           128  objective 
           130  tube lens 
           132  intermediate image plane 
           202  light source device 
           244  dichroic mirror 
           302  light source device 
           304  detector device 
           305  tube lens 
           306  optical system 
           316  scanning mirror device 
           327  objective 
           328  objective 
           330  tube lens 
           332  tilting mirror 
           334  tilting mirror 
           336  tilt axis 
           338  tilt axis 
           348  mirror 
           650  excitation light source 
           652  manipulation light source 
           654  dichroic mirror 
           656  mirror 
           658  beam expander 
           660  light shaping elements 
           662  lens 
           664  lens 
           666  scanner 
           668  tilt axis 
           670  light shaping system 
           760  light shaping element 
           768  scanner 
           772  splitting element 
           773  anamorphic system 
           774  output merging element 
         E excitation light 
         M manipulation light 
         F fluorescent light 
         O optical axis 
         A double arrow 
         LS light sheet 
         EP entrance pupil 
         P 1  focal plane 
         P 2  conjugate plane 
         V virtual tilt axis 
         d displacement 
         IP object plane 
         β angle 
         f 1  focal length 
         f 2  focal length 
         f 3  focal length