Patent Description:
In some known microscopy methods, a sample is manipulated by means of targeted light irradiation, so called photo-manipulation techniques including "Fluorescence Recovery after Photobleaching" (FRAP), a local activation of so called "caged compounds", and an activation of transmembrane pumps within the context of optogenetics. The light typically used for photo-manipulation differs from the light used for illuminating the sample, e.g. by scattering or exciting fluorophores located within the sample, in that it has a much higher intensity.

Document <CIT> discloses a scanning oblique plane microscope. Further, document <CIT> discloses a scanning oblique plane microscope having a unit for photo-manipulation. However, the oblique plane microscope according to said document requires an additional light source for creating manipulation light and means for coupling said manipulation light into an optical system of the microscope. The need for these additional elements make the microscope less compact and increases the cost of manufacturing. Further reference is made to documents <CIT>, <CIT> and <CIT> each disclosing a light sheet microscope.

It is an objective of the present invention to provide a light sheet microscope suitable for manipulating a sample that is compact and can be manufactured cost efficiently.

The aforementioned object is achieved by the subject-matter according to the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

A light sheet microscope comprises a light source configured to emit illumination light, and an optical system configured to form a light sheet from the illumination light in a sample space. The light sheet is focused in a thickness direction perpendicular to a light propagation direction thereof to form a beam waist in said thickness direction. The optical system has a field diaphragm adjustable to vary a width of the light sheet in a width direction being perpendicular to both the light propagation direction and the thickness direction, a scanning element configured to move the light sheet a scanning distance in the sample space along a scanning direction, and a control unit configured to control the field diaphragm for adjusting the width of the light sheet and to control the scanning element for moving the light sheet by the scanning distance in order to manipulate a target area of a sample by scanning the target area with the beam waist of the light sheet. The target area is defined by the width of the light sheet and the scanning distance.

In the present application manipulating the sample in particular means to photo-manipulate the sample, i.e. manipulating the sample by means of targeted irradiation with light, i.e. the illumination light or manipulation light. This manipulation of the sample is distinguished from illuminating the sample, e.g. by scattering of the illumination light or exciting fluorophores located within the sample with the illumination light, in that higher intensity illumination light is used, that typically induces an effect lasting several orders of magnitude longer than effects caused by illuminating the sample. Further, the target area for manipulation the sample is typically only a small part of a field of view of the light sheet microscope. In contrast hereto illuminating the sample means illuminating the majority of the field of view of the light sheet microscope.

The beam waist of light sheet, i.e. the part of the light sheet where its thickness is minimal, defines a scan line. This scan line is moved by the scanning distance in the sample space along the scanning direction by means of the scanning element. Thereby, the target area is scanned. The exact geometry of the target area is further defined by the angle enclosed by the width direction and the scanning direction. If the width direction and the scanning direction are perpendicular to each other, the target area is a rectangle. Otherwise, the target are is a parallelogram. The length of one side of the target area is defined by the width of the light sheet and the length of another side of the target area is defined by the scanning distance. Thus, by adjusting the width of the light sheet and the scanning distance as well as the angle enclosed by the width direction and the scanning direction the geometry of the target area can be adjusted flexibly. Since the intensity density of the light sheet is highest at is beam waist, only the parts of the specimen that intersect the target area are manipulated.

The optical system used for forming the light sheet for manipulating the sample has a very simple configuration. The optical system may further be used for forming a light sheet for illuminating the sample. In particular, the light source used for forming the light sheet for manipulating the sample may be the same light source used for forming the light sheet for illuminating the sample. Thus, eliminating the need for optical switches or additional beam splitters used for coupling the illumination light into the optical system. Thereby, the light sheet microscope is very compact and can be manufactured cost efficiently.

According to the claimed invention the control unit is configured to control the field diaphragm and the scanning element such that the width of the light sheet is varied while the light sheet is moved along the scanning direction. The target area may be e.g. a simple polygon. Thus, a greater number of geometries can be chosen for the target area. Thereby, the flexibility of the light sheet microscope is greatly increased.

In a preferred embodiment the field diaphragm is configured to adjust a position of a first end of the width of the light sheet and a position of a second end of the width of the light sheet independently of each other. In particular, in this embodiment it is possible to move the scan line sideways with respect to the scanning direction. Thus, the target area may be a parallelogram although the width direction and the scanning direction are perpendicular to each other. In this preferred embodiment, an even greater number of geometries can be chosen for the target area further increasing the flexibility of the light sheet microscope. Also, since there is no need to adjust the angle enclosed by the width direction and the scanning direction in order to adjust the geometry of the target area, fewer elements may be used, thus, making the light sheet microscope more cost effective.

In another preferred embodiment the optical system is configured to form an intermediate image of the light sheet in an intermediate image space, wherein the optical system comprises an optical transport system configured to image the intermediate image of the light sheet from the intermediate image space into the sample space. In particular, in this embodiment the light sheet microscope might be configured to be an oblique plane microscope. The geometry of the light sheet within the sample, in particular the light propagation direction, are defined by the geometry of the light sheet in the intermediate image space. Thus, removing the need for having optical elements such as light deflection elements in the sample space. Thereby, more space is available in the sample space.

In another preferred embodiment the optical transport system comprises a first objective facing the intermediate image space, wherein the optical system comprises an optical detection system for detecting detection light emitted by the sample, said optical detection system having a detector element and a second objective facing the intermediate image space, and wherein the optical axes of the first and second objectives are oblique to each other. Preferably, the optical axis of the second objective and the light propagation direction of the light sheet in the intermediate image space are perpendicular to each other. In this embodiment, the optical transport system transports the light sheet formed into the sample space and the detection light back from the sample space into the intermediate image space. This embodiment is a very simple configuration realizing an oblique plane microscope.

In another preferred embodiment the optical system comprises an optical illumination system configured to form the light sheet from the illumination light in the intermediate image space, and wherein the optical axis of the first objective and the optical axis of the optical illumination system are oblique to each other. The geometry of the light sheet in the sample space is defined by the optical illumination system, i.e. in the intermediate image space. In particular, the angle enclosed by the light propagation direction and the optical axis of the first objective, i.e. the obliqueness of the light sheet in the sample space, is defined by the angle enclosed by the optical axis of the second objective and the optical axis of the optical illumination system. No further light deflection elements are needed in the sample space in order to realize an oblique light sheet. Thus, more of the sample space can be dedicated to receiving the sample, increasing the versatility of the light sheet microscope.

In an alternative embodiment the optical transport system comprises a light deflection element configured to direct illumination light towards the sample and to direct detection light the detection light towards the intermediate image space. In this alternative embodiment, the illumination light is coupled directly into the optical transport system.

Preferably, the optical illumination system comprises a light sheet forming element for forming the light sheet, said light sheet forming element being arranged in an optical path between the intermediate image space and the light source. This light sheet forming element may be e.g. a cylindrical lens or a movable mirror configured to form a quasi-static light sheet from a light beam or any other means known from the prior art.

According to another aspect, a light sheet microscope is provided. The light sheet microscope comprising a light source configured to emit illumination light, a manipulation light source configured to emit manipulation light for manipulating a target area of a sample, and an optical system configured to form a light sheet from the illumination light in a sample space. The optical system has an optical transport system configured to transport an intermediate image of the light sheet from an intermediate image space into the sample space, an optical detection system configured to detect detection light emitted by the sample, and an optical erecting unit comprising at least first and second objectives facing the intermediate image space, the first objective being configured to direct light into the optical transport system, and the second objective being configured to direct light into the optical detection system. The optical axes of the first and second objectives are oblique to each other. The optical detection system comprises a light deflection element configured to direct the manipulation light into the second objective.

In this embodiment, the light sheet microscope is configured as an oblique plane microscope.

The manipulation light is coupled into a detection light path of the optical system by the light deflection element. The manipulation light is then directed into the intermediate image space by the second objective. The first objective receives the manipulation light and directs it into the optical transport system. The manipulation light is then transported by the optical transport system into the sample space, where it is used to manipulate the target area of the sample. The optical system used for transporting the manipulation light into the sample space shares many components with an optical system used for forming a light sheet for illuminating the sample already present in typical light sheet microscopes. Thereby, the light sheet microscope is very compact and can be manufactured cost efficiently.

In a preferred embodiment the optical detection system comprises a detector and the light deflection element is configured to direct the detection light to the detector. The light deflecting element is e.g. a dichroitic beam splitter. In this embodiment the detection light path leading to the detector is used to couple the manipulation light into the optical system. Thereby, no additional light path need to be created which makes this embodiment even more compact. Alternatively or additionally, the light deflection element is configured such that it can be removed from the detection light path. Thereby, allowing to switch between a detection mode in which the detector can receive the detection light and a manipulating mode in which the manipulation light is directed into the sample space.

In another preferred embodiment the optical system comprises a light forming element for forming a light pattern from the manipulation light, said light forming element being arranged in an optical path between the light deflection element of the optical detection system and the light source. The light forming element might e.g. be a digital mirror device. The light pattern is imaged into the sample space by the optical system, thereby defining the target area. This allows for a very precise and flexible definition of the target area.

Alternatively, the light forming element is a lens or a lens group configured to focus the manipulation light into a manipulation light beam. The target area is scanned with the manipulation light beam by means of a scanning element. Said scanning element e.g. being arranged in the light path of the optical transport system. In this alternative embodiment the light pattern is a light spot and the target area is defined as the area scanned by the light pattern.

In another preferred embodiment the optical transport system comprises an optical zoom system, which is adjustable for adapting the magnification of the optical transport system to a ratio between two refractive indices, one of which being associated with the sample space and the other being associated with the intermediate image space. The zoom system may be used to move the position of the focal plane of the first objective along its optical axis without moving the sample itself, which might disturb it. In order for this remote focusing to work, the magnification of the optical transport system must be equal to the ratio of the two refractive indices.

By moving the position of the focal plane of the first or second objective along its optical axis, the position of the beam waist of the light sheet, i.e. the target area, is moved along the optical axis of the first objective. This allows e.g. manipulating more than one target area located at different positions along the optical axis of the first objective. Further, this allows to tilt the target area by shifting the focal plane of the first objective while the light sheet is moved along the scanning direction. Thereby making additional geometric configurations of the target area possible and further enhancing to flexibility of the light sheet microscope.

In another preferred embodiment the optical system comprises an objective facing the sample space, and wherein the opening angle of said objective has a value between <NUM>° and <NUM>°, in particular between <NUM>° and <NUM>°. In this embodiment a high numerical aperture can be achieved which is advantageous for most light sheet microscopy techniques, in particular oblique plane microscopy.

In another preferred embodiment the optical system comprises a single objective facing the sample space. The single objective is used for imaging the light sheet into the sample space and for receiving the detection light emitted by the sample. Thereby, most of the sample space can be dedicated towards receiving the sample. Further, in this embodiment the optical axis of the single objective may be perpendicular to a cover slip holding the sample. This greatly reduces light loss and aberrations caused by reflections on the cover slip.

In another preferred embodiment the scanning direction is perpendicular to an optical axis of the single objective. This allows for a simple geometric configuration of the optical system, since the scanning direction is located in or parallel to the focal plane of the single objective.

In another preferred embodiment the target area is located in a focal plane of the single objective. In this embodiment, no remote focusing is necessary in order to focus the light sheet in the target area. This greatly simplifies the optical design of the optical system.

In another preferred embodiment the illumination light source and/or the manipulation light sources comprises a pulsed laser. Pulsed laser can efficiently achieve the high intensities needed for photo-manipulating the sample.

According to another aspect, a method for manipulating a target area of a sample using a light sheet microscope is provided. The methods comprises the following steps: Forming a light sheet from illumination light in a sample space, said light sheet being focused in a thickness direction perpendicular to a light propagation direction thereof to form a beam waist in said thickness direction. Adjusting the width of the light sheet during moving the light sheet by a scanning distance in order to manipulate the target area of the sample by scanning the target area with the beam waist of the light sheet, said target area being defined by the width of the light sheet and the scanning distance.

The method has the same advantages as the microscope claimed and can be supplemented using the features of the dependent claims directed at the microscope.

<FIG> shows a schematic diagram of a light sheet microscope <NUM> according to an embodiment. <FIG> also shows a coordinate cross <NUM> defining coordinates in a sample space <NUM> of the light sheet microscope <NUM> (c. The light sheet microscope <NUM> comprises a light source <NUM>, an optical system <NUM>, and a control unit <NUM>.

The light source <NUM> is configured to emit illumination light, in particular laser light. In the present embodiment, the light source <NUM> is exemplarily configured to be a pulsed laser. However, the light source <NUM> may also be a continuous laser or an assembly of two or more lasers and a beam combining element configured to combine the laser light beams emitted by the two or more lasers into a single laser light beam. Also, the light source <NUM> may be a source of incoherent light.

The illumination light is then formed into a light sheet by the optical system <NUM> in the sample space <NUM> of the light sheet microscope <NUM> in order to manipulate a sample <NUM>. The optical system <NUM> comprising an optical illumination system <NUM> and an optical transport system <NUM>. The optical illumination system <NUM> is configured to form the light sheet from the illumination light in an intermediate image space <NUM>. The optical transport system <NUM> is configured to form an image of the light sheet in the sample space <NUM> and to form an image of an object plane in the sample space <NUM> in the intermediate image space <NUM>. The optical system <NUM> further comprises an optical detection system <NUM> configured to detect the image formed by the optical transport system <NUM>.

The optical illumination system <NUM> comprises a light sheet forming element <NUM>, for example a cylindrical lens or a scanning element, an illumination objective <NUM> configured to direct the light sheet into the intermediate image space <NUM>. The light sheet is focused in a thickness direction perpendicular to a light propagation direction thereof and forms a beam waist in said thickness direction. The optical illumination system <NUM> further comprises an adjustable field diaphragm <NUM> which is configured to be adjustable in order vary a width of the light sheet in a width direction being perpendicular to both the light propagation direction and the thickness direction.

The optical transport system <NUM> forms a transport system in the sense that it is configured to transport the light sheet from the intermediate image space <NUM> into the sample space <NUM> and the image of the object plane from the sample space <NUM> into the intermediate image space <NUM>. In other words, the optical system <NUM> transports the illumination light and detection light emitted by the sample <NUM> from the intermediate image space <NUM> to the sample space <NUM> and back, respectively.

In the present embodiment, the optical transport system <NUM> is telecentric. The optical system <NUM> comprises an image side objective <NUM>, a first tube lens <NUM>, a first ocular <NUM>, a second ocular <NUM>, a second tube lens <NUM>, and an object side objective <NUM>, in this order from the intermediate image space <NUM>. The optical transport system <NUM> further comprises a scanning element <NUM> arranged between the first and second oculars. Said scanning element <NUM> being configured to move the light sheet through the sample space <NUM> along a scanning direction perpendicular to the optical axis O of the objective. In an alternative embodiment the scanning element <NUM> may be an e.g. piezo driven objective actuator configured to drive the object side objective <NUM>.

The optical detection system <NUM> comprises a detection objective <NUM>, a tube lens <NUM>, and a detector <NUM>. The detection objective <NUM> and the tube lens <NUM> are configured to image the intermediate image space <NUM> onto the detector <NUM>. This means that the image of the sample space <NUM> formed by the optical transport system <NUM> in the intermediate image space <NUM> is imaged onto the detector <NUM>. In other words, the image is detected by the detector <NUM>.

The control unit <NUM> comprises an input device <NUM> for inputting the geometry of the target area and is connected to the scanning element <NUM>, the field diaphragm <NUM>, the light source <NUM>, and the detector <NUM>. The control unit <NUM> is further configured to manipulate a target area <NUM> (see <FIG>) of the sample <NUM> by controlling the scanning element <NUM> and the field diaphragm <NUM>. The scanning element <NUM> is controlled for moving the light sheet along a scanning direction by a scanning distance. The field diaphragm <NUM> is adjusted for setting the width of the light sheet. The field diaphragm <NUM> may be adjusted once before the light sheet is moved along the scanning direction. According to the claimed invention the field diaphragm <NUM> is adjusted continuously while the light sheet is moved in order to determine the exact geometry of the target area <NUM>. How the geometry of the target area <NUM> is defined by adjusting the field diaphragm <NUM> is explained in more detail below with reference to <FIG>.

<FIG> shows a schematic diagram of the sample space <NUM> of the light sheet microscope <NUM> according to <FIG>. <FIG> also shows a coordinate grid defining coordinates in the sample space <NUM>. A first coordinate axis X is parallel to the scanning direction, a second coordinate axis Y is perpendicular to the scanning direction and the optical axis O of the objective directed at the sample space <NUM>, and a third coordinate axis Z is parallel the optical axis O of said objective.

The target area <NUM> is shown in <FIG> as a polygon with a solid outline. The sample <NUM> is manipulated in the target area <NUM> by moving a beam waist of light sheet, i.e. the part of the light sheet where its thickness is minimal and the intensity density of the light sheet is highest, along the scanning direction in a scanning motion. The scanning direction is shown in <FIG> as an arrow S. The light sheet is indicated by dotted lines <NUM> crossing each other at the position of the beam waist. The position of the beam waist at the start of the scanning motion is shown in <FIG> as a first thick line <NUM> and the position of the beam waist at the end of the scanning motion is shown in <FIG> as a second thick line <NUM>. The width of the light sheet is varied during the scanning motion, as is shown in <FIG> by two double-headed arrows P1, P2. This allows the target area <NUM> to be the polygon instead of a rectangle or parallelogram.

<FIG> shows a schematic diagram of a light sheet microscope <NUM> according to an embodiment not covered by the claimed invention. The light sheet microscope <NUM> according to <FIG> is distinguished from the light sheet microscope <NUM> according to <FIG> in a manipulation light source <NUM>. The manipulation light source <NUM> is configured to emit manipulation light for manipulating a target area <NUM> (see <FIG>) of the sample <NUM>.

The optical system <NUM> comprises a light forming element <NUM> configured to form the manipulation light into a light pattern, e.g. a digital mirror device, a spatial light modulator, or a scanning mirror. The light pattern is either static or quasi-static, e.g. a light pattern created by a fast moving scanning mirror. Alternatively, the light forming element <NUM> is a lens or a set of lenses and the light pattern may be a focused light spot. After leaving the light forming element <NUM>, the formed manipulation light is directed into the optical detection system <NUM>.

The optical detection system <NUM> comprises a light deflection element <NUM>, which is exemplarily formed as a beam splitter. The light deflection element <NUM> is configured to direct the detection light to the detector <NUM> and to direct the manipulation light to the objective <NUM> of the optical detection system <NUM>. The manipulation light is then directed by the objective <NUM> into the intermediate image space <NUM> and imaged into the sample space <NUM> by means of the optical transport system <NUM>.

The objective <NUM> of the optical transport system <NUM> and the objective <NUM> of the optical detection system <NUM> define an optical erecting unit <NUM> configured to image an oblique image plane of the sample <NUM>. Due to the geometry of the optical erecting unit <NUM> the target area <NUM> is tilted as is shown in <FIG>.

The optical system <NUM> of the light sheet microscope <NUM> further comprises a light sheet illumination system <NUM>, that is configured to form a light sheet for illuminating the sample <NUM> in an intermediate image space <NUM>. Said light sheet illumination system <NUM> comprising a light sheet forming element <NUM>, for example a cylindrical lens or a scanning element, and an illumination objective <NUM> configured to direct the light sheet into the intermediate image space <NUM>.

<FIG> shows a schematic diagram of the sample space <NUM> of the light sheet microscope <NUM> according to <FIG>. The plane <NUM> in the which the target area <NUM> is located is indicated by a dashed rectangle. As can be seen in <FIG> the target area <NUM> are tilted with respect to the y axis.

By adjusting the scanning element <NUM>, the whole plane in the which the target area <NUM> is located is moved along the x axis. This is indicated in <FIG> by a double-headed arrow P3 By adjusting the scanning element <NUM> each time the scanning motion is completed, multiple target areas located in different planes arranged along the x axis in sequence are manipulated. Thus, a volume or stack formed by the target areas is manipulated. Alternatively, it is possible to adjust the scanning element <NUM> during the scanning motion. Thereby, the target area <NUM> may be non-flat.

Claim 1:
A light sheet microscope (<NUM>), comprising:
a light source (<NUM>) configured to emit illumination light,
an optical system (<NUM>) configured to form a light sheet from the illumination light in a sample space (<NUM>), said light sheet being focused in a thickness direction perpendicular to a light propagation direction thereof to form a beam waist in said thickness direction,
wherein the optical system (<NUM>) has a field diaphragm (<NUM>) adjustable to vary a width of the light sheet in a width direction being perpendicular to both the light propagation direction and the thickness direction,
a scanning element (<NUM>) configured to move the light sheet a scanning distance in the sample space (<NUM>) along a scanning direction, and
a control unit (<NUM>) configured to control the field diaphragm (<NUM>) for adjusting the width of the light sheet and to control the scanning element (<NUM>) for moving the light sheet by the scanning distance in order to photo-manipulate a target area (<NUM>) of a sample (<NUM>) by scanning the target area (<NUM>) with the beam waist of the light sheet, said target area (<NUM>) being defined by the width of the light sheet and the scanning distance.
characterized in that,
the control unit (<NUM>) is configured to control the field diaphragm (<NUM>) and the scanning element (<NUM>) such that the width of the light sheet is varied while the light sheet is moved along the scanning direction.