Patent Description:
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 <CIT>. 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.

Document <CIT> discloses a microscope comprising an objective through which fluorescent light emitted from a specimen is collected. The microscope further comprises two illumination channels through which two parallel illumination beams are directed onto the sample from opposite lateral directions. The illumination channels are arranged outside the objective.

Document <CIT> discloses a light sheet fluorescence microscope including an optical system which is configured to illuminate a specimen with a light sheet formed from excitation light. The optical system is further configured to illuminate the specimen with manipulation light. Both, the excitation light and the manipulation light are directed through an illumination objective onto the specimen. An entrance pupil of the illumination objective is completely illuminated with the manipulation light. On the other side of the specimen, a detection objective is arranged which directs fluorescence light emerging from the specimen onto a detector.

Document <CIT> discloses a light sheet fluorescence microscope according to the preamble of claim <NUM>. The microscope includes separate light sources for emitting illumination light and manipulation light. Both, the illumination light and the manipulation light are directed through a common objective lens into the specimen. A spatial light manipulator is provided outside the beam path of the illumination light and serves to direct the manipulation light onto a beam splitter which reflects the manipulation light into the objective lens. A similar configuration is disclosed in the document <CIT>.

It is an object of the present invention to provide a light sheet fluorescence microscope and a method for imaging a specimen which allow for a flexible and efficient photomanipulation of the specimen.

The afore-mentioned object is achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

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 is 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 <NUM> to ± <NUM>°. 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.

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.

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 the light sources may be integrated in a common casing.

For example, the light source device comprises at least one excitation light source configured to emit the excitation light along at least one first optical path, and at least one manipulation light source 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.

Hereinafter, preferred embodiments are described with reference to the drawings, in which:.

A light sheet fluorescence microscope <NUM> according to an embodiment is illustrated in the diagram of <FIG>, wherein only those components of the light sheet fluorescence microscope <NUM> are shown which are helpful for understanding the embodiment.

According to the specific embodiment shown in <FIG>, the light sheet fluorescence microscope <NUM> comprises a light source device <NUM>, a detector device <NUM> which may be formed by a CCD or CMOS camera suitable for wide field imaging, and an optical system generally designated by reference sign <NUM>. The optical system <NUM> may comprise a plurality of optical elements, among which an objective <NUM> faces a specimen <NUM> which is to be imaged by the light sheet fluorescence microscope <NUM>.

As explained in more detail below, the objective <NUM> 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 <NUM> 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 <NUM> is configured to emit excitation light E which is suitable to excite the specimen <NUM> to emit fluorescent light F which is to be detected by the detector device <NUM>. As explained below in more detail with reference to <FIG> and <FIG>, the light source device <NUM> may be configured to shape a light sheet LS from the excitation light E for illuminating only a thin slice of the specimen <NUM> at a given point in time. In the specific embodiment shown in <FIG>, the light sheet LS illuminating the specimen <NUM> propagates in a direction which is oblique to the optical axis O of the optical system <NUM>. Referring to an orthogonal xyz coordinate system indicated in <FIG>, the light sheet LS illuminating the specimen <NUM> 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 <NUM> is configured to illuminate the specimen <NUM> with the light sheet LS formed from the excitation light E as well as to guide the fluorescent light F from the illuminated specimen <NUM> to the detector device <NUM>. In the embodiment of <FIG>, the optical system <NUM> comprises a first lens <NUM>, a second lens <NUM>, a scanning mirror device <NUM>, a third lens <NUM>, a fourth lens <NUM> and the objective <NUM> facing the specimen <NUM>. Each of the lenses <NUM> and <NUM> serves as a tube lens, wherein the lens <NUM> is configured as a scan lens. According to the embodiment shown in <FIG>, the scanning mirror device <NUM> is formed by a single tilting mirror <NUM> which reflects the excitation light E passing the lenses <NUM>, <NUM> through the lenses <NUM>, <NUM> into the objective <NUM> for illuminating the specimen <NUM> with the light sheet LS. The tilting mirror <NUM> is movable around a tilt axis <NUM> which is oriented parallel to the x-axis.

In order to illustrate the tilting movement performed by the scanning mirror device <NUM>, <FIG> shows the tilting mirror <NUM> in three different tilting positions at consecutive times t = t1, t = t2, and t = t3. Depending on the respective tilting position, the tilting mirror <NUM> reflects the excitation light E in different directions resulting in a scanning movement of the light sheet LS within the specimen <NUM> in a direction parallel to the y-axis. In other words, the scanning mirror device <NUM> serves to move the light sheet LS as a whole through the specimen <NUM> in a light sheet scanning direction which is illustrated in <FIG> 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 = t1) represents the excitation light E which is reflected by the tilting mirror <NUM> at the time t = t1. Likewise, the principle ray E(t = t2) illustrates the excitation light E reflected by the tilting mirror <NUM> at the time t = t2, and the principle ray E(t = t3) represents the excitation light E reflected by the tilting mirror <NUM> at the time t = t3.

In the configuration shown in <FIG>, the tilting mirror <NUM> is located in a rear, i.e. image-side focal plane of the scan lens <NUM>. By locating the tilting mirror <NUM> in the focal plane of the scan lens <NUM>, it is ensured that a tilting movement of the excitation light E on the tilting mirror <NUM> is converted into a parallel displacement of the excitation light E on an object side of the scan lens <NUM>. This parallel displacement of the excitation light E is illustrated by the principle rays E(t = t1), E(t = t2), and E(t=t3) propagating parallel to each other between the scan lens <NUM> and the tube lens <NUM> as well as when emanating from the objective <NUM> into the specimen <NUM>. Accordingly, the tilting movement performed by the scanning mirror device <NUM> results in a parallel displacement of the light sheet LS within the specimen <NUM> in y-direction.

The fluorescent light F induced by the excitation light E is collected by the objective <NUM> and propagates in a direction opposite to the propagation direction of the excitation light E through the optical system <NUM> up to the tube lens <NUM>. Subsequently, the fluorescent light F passes a dichroic mirror <NUM> which is configured to selectively transmit both the fluorescent light F and the excitation light E. Accordingly, in the specific embodiment shown in <FIG>, the dichroic mirror <NUM> is used as a beam splitter for separating the excitation light E and the fluorescent light F. Further, a mirror <NUM> is provided serving as an element for coupling the excitation light E into the optical system <NUM>. Needless to say that the configuration formed by the mirrors <NUM>, <NUM> 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 <NUM>, the fluorescent light F propagates through an optical detection system formed by two objectives <NUM>, <NUM> and a tube lens <NUM> towards the detector device <NUM>. Between the objectives <NUM>, <NUM>, an intermediate image plane <NUM> 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 <NUM>.

As can be seen from <FIG>, the optical path leading to the detector device <NUM> is oblique relative to the optical path between the objective <NUM> and the tube lens <NUM>. This reflects the fact that in SCAPE or OPM configuration the light sheet LS is oblique relative to an object-side focal plane P1 of the objective <NUM>. Correspondingly, a plane P2 conjugate to the focal plane P1 is oblique relative to the intermediate image plane <NUM>.

As the fluorescence light F emanating from the specimen <NUM> is guided to the tilting mirror <NUM>, the optical system <NUM> of the light sheet fluorescence microscope <NUM> forms a so-called descanned configuration. Such a descanned configuration ensures that the illuminated plane within the specimen <NUM> is continuously detected by the stationary detector device <NUM> while the illuminated plane is scanned in y-direction through the specimen <NUM>. As in the case of the excitation light E, this fact is illustrated in <FIG> by indicating the principle rays F(t = t1), F(t = t2), and F(t = t3) of the fluorescent light F for the different tilting positions in which the tilting mirror <NUM> is located at times t1, t2, and t3.

As already mentioned above, the embodiment shown in <FIG> corresponds to a SCAPE or OPM configuration. Therefore, both the excitation light E and the fluorescent light F pass through the objective <NUM>. Specifically, neither the excitation light E nor the fluorescent light F fully utilize the aperture of the objective <NUM>. In fact, the excitation light E and the fluorescent light F pass the objective <NUM> through different portions thereof, these portions being eccentrically located on opposite sides of the optical axis O.

The light source device <NUM> is further configured to emit manipulation light M which is suitable to photomanipulate the specimen <NUM>. As already explained above, photomanipulating the specimen <NUM> 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>, the dichroic mirror <NUM> is used to introduce the manipulation light M into the optical system <NUM>. Accordingly, the characteristic of the dichroic mirror <NUM> is selected such that the dichroic mirror <NUM> 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>, the manipulation light M passes the tube lens <NUM> through a portion which is different from a portion through which the excitation light E propagates through the tube lens <NUM>. As a result, after passing the lenses <NUM>, <NUM>, the manipulation light M falls onto the tilting mirror <NUM> at an incident position which is different from the incident position of the excitation light E. Accordingly, after being reflected by the titling mirror <NUM>, the manipulation light M propagates towards the objective <NUM> in a direction which is different from the propagation direction of the excitation light E. Thus, the optical system <NUM> is configured to direct the manipulation light M into the specimen <NUM> 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 <NUM> through a spatially limited sub-area of an entrance pupil EP of the objective <NUM>.

As the manipulation light M is reflected by the tilting mirror <NUM>, the manipulation light M performs a scanning movement in y-direction as the excitation light E does. However, for simplifying the illustration, <FIG> indicates the manipulation light M only by means of a single principle ray M(t = t2) which belongs to the manipulation light M referring to the tilting position of the tilting mirror <NUM> at the time t = t2.

The light sheet fluorescence microscope <NUM> may comprise an optical shifting unit which is configured to shift the incident position of the manipulation light M on the tilting mirror <NUM>. For example, such an optical shifting unit may be formed by the dichroic mirror <NUM>. Thus, the dichroic mirror <NUM> may be configured to be movable along the propagation direction of the manipulating light M falling onto the dichroic mirror <NUM>, i.e. according to the configuration of <FIG> in a direction parallel to the z-axis. By moving the dichroic mirror <NUM> in such a way, the portion of the tube lens <NUM>, 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 <NUM> changes by moving the dichroic mirror <NUM> resulting in a corresponding variation of the direction in which the manipulation light M propagates into the specimen <NUM>.

By moving the dichroic mirror <NUM>, 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 <NUM> in a direction perpendicular to the optical axis O of the optical system <NUM> the propagation direction of the manipulation light M emanating from the objective <NUM> towards the specimen <NUM> 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 <NUM> may be varied in an angular range from <NUM> to ±<NUM>°. In particular, it may be advantageous to set the afore-mentioned angle to <NUM>, 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 <NUM>. 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> shows a light sheet fluorescence microscope 100a which represents a modified embodiment of the configuration of <FIG>. The modification compared to the light sheet fluorescence microscope <NUM> shown in <FIG> relates to a light source device <NUM> replacing the light source device <NUM> shown in <FIG>.

Whereas the light source device <NUM> of <FIG> 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 <NUM>, the light source device <NUM> of the light sheet fluorescence microscope 100a shown in <FIG> 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 <NUM> is comprised of two separate light sources <NUM> and <NUM> which output the excitation light E and the manipulation light M, respectively.

In contrast to the configuration of <FIG>, the light sheet fluorescence microscope 100a shown in <FIG> comprises a single component e.g. in form of a dichroic mirror <NUM> for commonly introducing the excitation light E and the manipulation light M into the optical system <NUM>. As can be seen in <FIG>, the dichroic mirror <NUM> is adapted to the light source device <NUM> such that both the excitation light E and the manipulation light M are reflected into the optical system <NUM>. Further, insofar corresponding to the dichroic mirror <NUM> shown in <FIG>, the characteristic of the dichroic mirror <NUM> of the light sheet fluorescence microscope 100a is adapted to transmit the fluorescent light F towards the detector device <NUM>.

As the excitation light E and the manipulation light M are commonly reflected by the dichroic mirror <NUM>, in contrast to the embodiment shown in <FIG>, the light sheet fluorescence microscope 100a of <FIG> 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 <NUM> of <FIG> only acts on the manipulation light M, the dichroic mirror <NUM> shown in <FIG> 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 <NUM> even if the dichroic mirror <NUM> was moved in a direction perpendicular to the optical axis O of the optical system <NUM>. In other words, according to the embodiment shown in <FIG>, the direction along which the specimen <NUM> is photomanipulated by the manipulation light M does not change in relation to the direction along which the excitation light E illuminates the specimen <NUM>. Accordingly, at any given time t1, t2, t3, the corresponding principle ray of the manipulation light M intersects the corresponding principle ray of the excitation light E in the focal plane P1 of the objective <NUM>. As in <FIG>, it is to be noted that <FIG> shows only one principle ray M (t = t2) which intersects the principle ray E(t = t2) of the excitation light in the focal plane P1. In contrast, according to the configuration shown in <FIG>, it is possible to vary the position in which the principle ray M(t = t2) intersects the focal plane P1 enabling the positional relationship between the manipulation light M and the excitation light E to be varied within the specimen <NUM>.

It is to be noted that even in the embodiment shown in <FIG>, 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 <NUM> includes means for shifting the manipulation light M correspondingly.

<FIG> is a diagram showing a light sheet fluorescence microscope 100b representing a further embodiment. The light sheet fluorescence microscope 100b differs from the embodiments shown in <FIG> and <FIG> mainly by a modified scanning mirror device <NUM>.

As the microscopes shown in <FIG> and <FIG>, the light sheet fluorescence microscope 100b of <FIG> comprises a light source device <NUM> emitting the excitation light E and the manipulation light M. The light sheet fluorescence microscope 100b further comprises an optical system <NUM> which includes a tube lens <NUM>, the scanning mirror device <NUM>, a further tube lens <NUM> and the objective <NUM>. The excitation light E and the manipulation light M are coupled into the optical system <NUM> by means of a mirror <NUM>.

The fluorescent light F emitted from the specimen <NUM> propagates through the optical system <NUM> in a direction essentially opposite to the propagation direction of the excitation light E and the manipulation light M. After passing the tube lens <NUM>, the fluorescent light F propagates through two objectives <NUM>, <NUM> and a tube lens <NUM> to a detector device <NUM>.

Whereas the light sheet fluorescence microscope 100b is fundamentally operated in the same manner as the microscopes shown in <FIG> and <FIG>, the configuration of <FIG> is advantageously modified in terms of the tilting operation of the scanning mirror device <NUM> which is applied to scan the light sheet LS as a whole in y-direction through the specimen <NUM>. Thus, according to the configurations shown in <FIG> and <FIG>, the single tilting mirror <NUM> is located in the rear focal plane of the scan lens <NUM> to ensure that the tilting movement of the excitation light E on the tilting mirror <NUM> is converted into a parallel displacement of the excitation light E on the object side of the scan lens <NUM>. 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 100b shown in <FIG> is modified in such a way that a scan lens can be dispensed with.

Specifically, the scanning mirror device <NUM> comprises two tilting mirrors <NUM>, <NUM> which are located offset to each other along the optical axis O of the optical system <NUM>. Each of the tilting mirrors <NUM>, <NUM> is movable around a tilt axis <NUM>, <NUM> as shown in <FIG>. The respective tilt axis <NUM>, <NUM> is oriented parallel to the x-axis.

By using the two tilting mirrors <NUM>, <NUM> instead of one single tilting mirror as provided in the configurations of <FIG> and <FIG>, the intended parallel displacement of the excitation light E emanating from the scanning mirror device <NUM> can be achieved without providing a dedicated scan lens. Thus, the two tilting mirrors <NUM>, <NUM> 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 <NUM>. In other words, in case of using one single tilting mirror, the axis for tilting the excitation light E is fixed (see tilt axis <NUM> in <FIG> and <FIG>). In contrast, when using the two tilting mirrors <NUM>, <NUM> having two physical tilt axes <NUM>, <NUM>, 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 <NUM> is achieved. In the example shown in <FIG>, two tilting states of the scanning mirror device <NUM> are illustrated, a difference between these two tilting states corresponding to a tilting angle β. The tilting angle β defines the amount of tilting about a virtual tilt axis V which is located on the image side of the first tilting mirror <NUM>. 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 <NUM>. According to the configuration shown in <FIG> and <FIG>, the virtual tilt axis V is located in the rear, i.e. image-side focal plane of the objective <NUM> (or a plane conjugate thereto).

It is to be noted that <FIG> does not illustrate the scanning movement of the manipulation light M achieved by operating the scanning mirror device <NUM> 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 <NUM>, the embodiment shown in <FIG> does not differ from the configurations of <FIG> and <FIG>.

The light source device <NUM>, <NUM> may be switchable between an excitation operating mode and a manipulation operating mode. In the excitation operating mode, the light source device <NUM>, <NUM> emits only the excitation light E, and the manipulation light M is shut off. Likewise, in the manipulating operating mode, the light source device <NUM>, <NUM> emits only the manipulation light M, and the excitation light E is shut off.

The diagram shown in <FIG> illustrates how the scanning mirror device <NUM> of the light sheet fluorescence microscope 100b can be used to rapidly switch between illumination of the specimen <NUM> by means of the excitation light E and photomanipulation by directing the manipulation light M into the specimen <NUM>.

<FIG> shows two tilting states of the scanning mirror device <NUM> 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 <NUM>. Accordingly, the light source device <NUM> 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 <NUM>, which is illuminated by the light sheet LS, is imaged onto the detector device <NUM>. 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 <NUM> very fast between the afore-mentioned tilting states. Indeed, the scanning mirror device <NUM> can be switched within a few milliseconds. Accordingly, it is possible to rapidly change between illumination and photomanipulation of the specimen <NUM>. Further, by appropriately controlling the tilting movement of the two tilting mirrors <NUM>, <NUM>, the propagation direction of the manipulation light M can be varied in a wide range.

The embodiments shown in <FIG>, <FIG> and <FIG> 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 <NUM>, <NUM>. Accordingly, the light source device <NUM>, <NUM> 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.

<FIG> and <FIG> show different embodiments of the light source device <NUM>, <NUM> which may be used to satisfy the afore-mentioned requirements.

The light source device <NUM>, <NUM> according to <FIG> comprises an excitation light source <NUM> emitting the excitation light E along a first optical path OP1 and a manipulation light source <NUM> emitting the manipulation light M along a second optical path OP2. The first and second optical paths OP1, OP2 may be located offset from each other in parallel. The light source device <NUM>, <NUM> further comprises a dichroic mirror <NUM>, the characteristic thereof being adapted to transmit the excitation light E and to reflect the manipulation light M which is deflected by a mirror <NUM> onto the dichroic mirror <NUM>. Thus, the dichroic mirror <NUM> may serve as an input merging element which is configured to merge the first and second optical paths OP1, OP2 in a common third optical path in which the excitation light E and the manipulation light M propagate.

The light source device <NUM>, <NUM> further comprises a beam expander <NUM> 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 <NUM>, an optical light shaping element <NUM> is located in the third optical path OP3. The light shaping element <NUM> 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 <NUM>, <NUM> and a scanner <NUM> are included in the third optical path OP3. The scanner <NUM> may be formed by a mirror which is tiltable around a tilt axis <NUM> which is oriented parallel to the z-axis. In this respect, it is to be noted that the plane of drawing in <FIG> is perpendicular to the plane of drawing in <FIG>.

The afore-mentioned elements <NUM> to <NUM> form a light shaping system <NUM>, an optical input thereof being defined by the first and second optical paths OP1, OP2 and an optical output thereof being defined by the third optical path OP3. According to the configuration shown in <FIG>, the third optical path OP3 is used as the common optical path along which the light source device <NUM>, <NUM> emits the excitation light E and the manipulation light M into the optical system <NUM>, <NUM>. In <FIG>, only the tube lens <NUM>, <NUM> of the optical system <NUM>, <NUM> is shown.

The light source device <NUM>, <NUM> 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 <NUM> 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 <NUM> may be controlled in the excitation operating mode such that the light shaping member <NUM> 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 <NUM> may be operated to perform a very fast tilting movement around the tilt axis <NUM> in order to scan the excitation light E in x-direction through the specimen <NUM> 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 <NUM> is controlled such that the light shaping element <NUM> acts on the manipulation light M in order to shape the same in a desired manner. For example, the light shaping element <NUM> may shift the focus of the manipulation light M in z-direction. On the other hand, the scanner <NUM> is operated in the manipulation operating mode such that it serves as a detector element. Thus, the scanner <NUM> may provide for a displacement of the manipulation light M in x-direction.

The light shaping element <NUM> may be located in a plane which is conjugate to an object-side focal plane of the objective <NUM>. Alternatively, the light shaping element <NUM> may be positioned in an image-side focal plane of the objective <NUM>. In this case, the lenses <NUM>, <NUM> may form a telecentric optical system in which the lenses <NUM>, <NUM> are located at a distance from each other being equal to the sum of their focal lengths. Thus, the lenses <NUM>, <NUM> may form a so-called 4f system.

The scanner <NUM> is preferably located in a rear focal plane of the tube lens <NUM>, <NUM>, i.e. at a distance from the tube lens <NUM>, <NUM> which equals the rear focal length f1 thereof. Alternatively, the scanner <NUM> may be located in a plane conjugate to the rear focal plane of the tube lens <NUM>, <NUM>. In any case, the afore-mentioned plane, in which the scanner <NUM> is located, in conjugate to the rear focal plane of the objective <NUM>.

In the configuration shown in <FIG>, the scanner <NUM> is tiltable about the tilt axis <NUM> such that a light beam displacement in x-direction is caused inside the specimen <NUM>. In addition, the scanner <NUM> may be tiltable about a second tilt axis (not shown in <FIG>) perpendicular to the tilt axis <NUM> in order to create a beam displacement in y-direction within the specimen <NUM>. 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 <NUM> (see <FIG>). Rather, the light focus is offset from the plane IP in y-direction.

The configuration shown in <FIG> 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 <NUM> and the scanner <NUM> 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> shows another embodiment of the light source device <NUM>, <NUM> comprising a modified light shaping system <NUM>. Whereas according to the configuration shown in <FIG>, the third optical path OP3 extending from the beam expander <NUM> directly forms the optical output for supplying the excitation light E and the manipulation light M to the optical system <NUM>, <NUM>, the embodiment shown in <FIG> provides for a separation of the excitation light E and the manipulation light M subsequent to the beam expander <NUM> as explained hereinafter.

The beam shaping system <NUM> of <FIG> comprises a splitting element <NUM>, 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 <NUM>, <NUM> 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>, it is assumed that the excitation light E propagates in a fourth optical path OP4 extending from the splitting element <NUM>. In the fourth optical path OP4, an anamorphic optical element <NUM>, 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 <NUM>. In the example shown in <FIG>, a long axis of the elliptical cross-section in the image-side focal plane of the objective <NUM> extends in y-direction, whereas a short axis extends in x-direction. Accordingly, the plane illuminated with the light sheet LS within the specimen <NUM> extends in x-direction. In this respect, it is to be noted that the plane of drawing in <FIG> is perpendicular to the plane of drawing in <FIG>.

The manipulation light M propagates in a fifth optical path OP5 extending from the splitting element <NUM> to the light shaping element <NUM> which may comprise a DM, DMD, SLM and/or an ETL. For example, the light shaping elements <NUM> 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 <NUM> further comprises two lenses <NUM>, <NUM> and a scanner <NUM> having a tilt axis <NUM> which is oriented in z-direction. By tilting the scanner <NUM> about the tilt axis <NUM>, the manipulation light M can be displaced in x-direction.

The light shaping system <NUM> further comprises an output merging element <NUM> which is configured to merge the fourth optical path OP4 and the fifth optical path OP5 in a sixth optical path OP6 which extends to the tube lens <NUM>, <NUM> of the optical system <NUM>, <NUM>. Accordingly, the sixth optical path OP6 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 <NUM>, <NUM>. As the splitting element <NUM>, the output merging element <NUM> may be formed by a switchable mirror or a dichroic element.

In the configuration shown in <FIG>, the scanner <NUM> is preferably located in the rear focal plane of the tube lens <NUM>, <NUM> having the rear focal length f1. Further, the light shaping element <NUM> may be located in a plane which is conjugate to the object-side focal plane of the objective <NUM>. For this, the lenses <NUM>, <NUM> having focal lengths f2, f3, respectively, form a 4f-system. Further, according to an advantageous embodiment, each of the light shaping element <NUM> and the scanner <NUM> may be located in a plane which is conjugated to the image-side focal plane of the objective <NUM>.

Needless to say that the afore-mentioned embodiments of the light source device <NUM>, <NUM> 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>, two separate optical paths for coupling the excitation light E and the manipulation light M into the optical system <NUM> may be provided. In particular, the embodiments shown in <FIG> and <FIG> may be modified to adapt the configurations shown therein to a light source device having two separate optical outputs. For example, referring to <FIG>, such a modification may be achieved by omitting the output merging element <NUM>. In this case, the excitation light E and the manipulation light M is coupled into the optical system <NUM>, <NUM> along the two separate optical paths OP4, OP5, respectively.

Further, the explanations regarding the examples shown in <FIG> and <FIG> refer to the coordinate systems shown in <FIG>. 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 <FIG> and <FIG> should only be understood as exemplary configurations which may be used in abstracted form for implementing the light source devices <NUM>, <NUM> in the embodiments shown in <FIG>. In other words, the configurations shown in <FIG> and <FIG> may be suitably adapted when being implemented in the embodiments of <FIG>. For example, optical elements (as e.g. a dichroic mirror) may be added or removed as needed.

Claim 1:
A light sheet fluorescence microscope (<NUM>, 100a, 100b), comprising:
a light source device (<NUM>, <NUM>, <NUM>) configured to emit excitation light (E) suitable for inducing fluorescent light (F) emitted from a specimen (<NUM>),
a detector device (<NUM>, <NUM>) configured to detect the fluorescent light (F) from the specimen (<NUM>), and
an optical system (<NUM>, <NUM>) configured to illuminate the specimen (<NUM>) with a light sheet (LS) formed from the excitation light (E) and to guide the fluorescent light (F) from the illuminated specimen (<NUM>) to the detector device (<NUM>, <NUM>),
wherein the optical system (<NUM>, <NUM>) comprises an objective (<NUM>) facing the specimen (<NUM>), the objective (<NUM>) being configured to collect the fluorescent light (F) emitted from the specimen (<NUM>),
wherein the optical system (<NUM>, <NUM>) is configured to illuminate the specimen (<NUM>) with the light sheet (LS) through the objective (<NUM>), and
wherein the light source device (<NUM>, <NUM>, <NUM>) is further configured to emit manipulation light (M) suitable for photomanipulating the specimen (<NUM>),
wherein the optical system (<NUM>, <NUM>) is further configured to direct the manipulation light (M) through the objective (<NUM>) through an entrance pupil (EP) thereof onto the specimen (<NUM>) along a light propagation direction which is different from a light propagation direction of the light sheet (LS), and
wherein the optical system (<NUM>, <NUM>) comprises a scanning mirror device (<NUM>, <NUM>) including at least one tilting mirror (<NUM>, <NUM>, <NUM>) which is tiltable for reflecting the excitation light (E) to move the light sheet (LS) as a whole through the specimen (<NUM>) in a light sheet scanning direction transverse to the light propagation direction of the light sheet (LS),
characterized in that the optical system (<NUM>, <NUM>) is configured to direct the manipulation light (M) through a spatially limited sub-area of the entrance pupil (EP) of the objective (<NUM>) on the specimen (<NUM>);
wherein the at least one scanning mirror (<NUM>, <NUM>, <NUM>) is tiltable for reflecting both the excitation light (E) and the manipulation light (M).