Technical field

The invention relates to a light sheet microscope. The invention further relates to a method for manipulating a target area of a sample using a light sheet microscope.

Background

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

Document DE 10 2016 119 268 B3 discloses a scanning oblique plane microscope. Further, document WO 2015 109 323 A2 discloses a scanning oblique plane micro scope having a unit for photo-manipulation. However, the oblique plane microscope according to said document requires an additional light source for creating manipula tion 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 com pact and increases the cost of manufacturing.

Summary 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 inde pendent 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 perpendicu lar to a light propagation direction thereof to form a beam waist in said thickness di rection. 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 di rection 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 con trol 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-ma- nipulate 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 illumi nation 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 mi croscope.

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 form ing 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 man ufactured cost efficiently. In a preferred embodiment the control unit is configured to control the field dia phragm and the scanning element such that the width of the light sheet is varied while the light sheet is moved along the scanning direction. In this embodiment, the target area may be e.g. a simple polygon. Thus, a greater number of geometries can be chosen for the target area. Thereby, the flexibility of the light sheet microscope is greatly increased.

In another preferred embodiment the field diaphragm is configured to adjust a posi tion 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 embodi ment it is possible to move the scan line sideways with respect to the scanning direc tion. Thus, the target area may be a parallelogram although the width direction and the scanning direction are perpendicular to each other. In this preferred embodi ment, an even greater number of geometries can be chosen for the target area fur ther 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 inter mediate 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 ob jective facing the intermediate image space, wherein the optical system comprises an optical detection system for detecting detection light emitted by the sample, said op tical 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 objec tives 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 illumina tion system configured to form the light sheet from the illumination light in the inter mediate image space, and wherein the optical axis of the first objective and the opti cal 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 propa gation 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 fur ther 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 deflec tion element configured to direct illumination light towards the sample and to direct detection light the detection light towards the intermediate image space. In this al ternative 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 op tical 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 mi croscope comprising a light source configured to emit illumination light, a manipula tion 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 illumina tion light in a sample space. The optical system has an optical transport system con figured 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 be ing configured to direct light into the optical transport system, and the second objec tive 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 sys tem 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 mi croscope.

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 interme diate image space by the second objective. The first objective receives the manipula tion 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 sys tem used for forming a light sheet for illuminating the sample already present in typi cal 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 de tection 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 re ceive 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 ele ment for forming a light pattern from the manipulation light, said light forming ele ment 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 ele ment e.g. being arranged in the light path of the optical transport system. In this al ternative 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 associ ated with the sample space and the other being associated with the intermediate im age 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 op tical 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 be tween 17° and 12°, in particular between 45° and 12°. In this embodiment a high nu merical aperture can be achieved which is advantageous for most light sheet micros copy techniques, in particular oblique plane microscopy.

In another preferred embodiment the optical system comprises a single objective fac ing 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 opti cal 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 fo cus 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 manipula tion 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 us ing a light sheet microscope is provided. The methods comprises the following steps: Forming a light sheet from illumination light in a sample space, said light sheet being focused in a thickness direction perpendicular to a light propagation direction thereof to form a beam waist in said thickness direction. Adjusting the width of the light sheet. Moving the light sheet by a scanning distance in order to manipulate the target area of the sample by scanning the target area with the beam waist of the light sheet, said target area being defined by the width of the light sheet and the scanning dis tance.

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

Short Description of the Figures

Hereinafter, specific embodiments are described referring to the drawings, wherein:

Figure 1 is a schematic diagram of a light sheet microscope according to an embodi ment.

Figure 2 is a schematic diagram of a sample space of the light sheet microscope ac cording to Figure 1. Figure 3 is a schematic diagram of a light sheet microscope according to another em bodiment.

Figure 4 is a schematic diagram of a sample space of the light sheet microscope ac cording to Figure 3.

Detailed description

Figure 1 shows a schematic diagram of a light sheet microscope 100 according to an embodiment. Figure 1 also shows a coordinate cross 102 defining coordinates in a sample space 104 of the light sheet microscope 100 (c.f. Figure 2). The light sheet mi croscope 100 comprises a light source 106, an optical system 108, and a control unit 110.

The light source 106 is configured to emit illumination light, in particular laser light. In the present embodiment, the light source 106 is exemplarily configured to be a pulsed laser. However, the light source 106 may also be a continuous laser or an as sembly of two or more lasers and a beam combining element configured to combine the laser light beams emitted by the two or more lasers into a single laser light beam. Also, the light source 106 may be a source of incoherent light.

The illumination light is then formed into a light sheet by the optical system 108 in the sample space 104 of the light sheet microscope 100 in order to manipulate a sample 146. The optical system 108 comprising an optical illumination system 112 and an optical transport system 114. The optical illumination system 112 is config ured to form the light sheet from the illumination light in an intermediate image space 116. The optical transport system 114 is configured to form an image of the light sheet in the sample space 104 and to form an image of an object plane in the sample space 104 in the intermediate image space 116. The optical system 108 further comprises an optical detection system 136 configured to detect the image formed by the optical transport system 114.

The optical illumination system 112 comprises a light sheet forming element 118, for example a cylindrical lens or a scanning element, an illumination objective 119 config ured to direct the light sheet into the intermediate image space 116. The light sheet is focused in a thickness direction perpendicular to a light propagation direction thereof and forms a beam waist in said thickness direction. The optical illumination system 112 further comprises an adjustable field diaphragm 120 which is configured to be adjustable in order vary a width of the light sheet in a width direction being perpen dicular to both the light propagation direction and the thickness direction.

The optical transport system 114 forms a transport system in the sense that it is con figured to transport the light sheet from the intermediate image space 116 into the sample space 104 and the image of the object plane from the sample space 104 into the intermediate image space 116. In other words, the optical system 108 transports the illumination light and detection light emitted by the sample 146 from the interme diate image space 116 to the sample space 104 and back, respectively.

In the present embodiment, the optical transport system 114 is telecentric. The optical system 108 comprises an image side objective 132, a first tube lens 130, a first ocular 128, a second ocular 126, a second tube lens 124, and an object side objective 122, in this order from the intermediate image space 116. The optical transport system 114 further comprises a scanning element 134 arranged between the first and second oc ulars. Said scanning element 134 being configured to move the light sheet through the sample space 104 along a scanning direction perpendicular to the optical axis O of the objective. In an alternative embodiment the scanning element 134 may be an e.g. pi ezo driven objective actuator configured to drive the object side objective 132. The optical detection system 136 comprises a detection objective 138, a tube lens 140, and a detector 142. The detection objective 138 and the tube lens 140 are configured to image the intermediate image space 116 onto the detector 142. This means that the image of the sample space 104 formed by the optical transport system 114 in the intermediate image space 116 is imaged onto the detector 142. In other words, the image is detected by the detector 142.

The control unit 110 comprises an input device 144 for inputting the geometry of the target area and is connected to the scanning element 134, the field diaphragm 120, the light source 106, and the detector 142. The control unit 110 is further configured to manipulate a target area 200 (see Figure 2) of the sample 146 by controlling the scanning element 134 and the field diaphragm 120. The scanning element 134 is con trolled for moving the light sheet along a scanning direction by a scanning distance. The field diaphragm 120 is adjusted for setting the width of the light sheet. The field diaphragm 120 may be adjusted once before the light sheet is moved along the scan ning direction or continuously while the light sheet is moved in orderto determine the exact geometry of the target area 200. How the geometry of the target area 200 is defined by adjusting the field diaphragm 120 is explained in more detail below with reference to Figure 2.

Figure 2 shows a schematic diagram of the sample space 104 of the light sheet micro scope 100 according to Figure 1. Figure 2 also shows a coordinate grid defining coor dinates in the sample space 104. A first coordinate axis X is parallel to the scanning direction, a second coordinate axis Y is perpendicular to the scanning direction and the optical axis O of the objective directed at the sample space 104, and a third coor dinate axis Z is parallel the optical axis O of said objective. The target area 200 is shown in Figure 2 as a polygon with a solid outline. The sample 146 is manipulated in the target area 200 by moving a beam waist of light sheet, i.e. the part of the light sheet where its thickness is minimal and the intensity density of the light sheet is highest, along the scanning direction in a scanning motion. The scan ning direction is shown in Figure 2 as an arrow S. The light sheet is indicated by dotted lines 202 crossing each other at the position of the beam waist. The position of the beam waist at the start of the scanning motion is shown in Figure 2 as a first thick line 204 and the position of the beam waist at the end of the scanning motion is shown in Figure 2 as a second thick line 206. The width of the light sheet is varied during the scanning motion, as is shown in Figure 2 by two double-headed arrows PI, P2. This allows the target area 200 to be the polygon instead of a rectangle or parallelogram.

Figure 3 shows a schematic diagram of a light sheet microscope 300 according to an other embodiment. The light sheet microscope 300 according to Figure 3 is distin guished from the light sheet microscope 100 according to Figure 1 in a manipulation light source 302. The manipulation light source 302 is configured to emit manipula tion light for manipulating a target area 400 (see Figure 4) of the sample 146.

The optical system 318 comprises a light forming element 308 configured to form the manipulation light into a light pattern, e.g. a digital mirror device, a spatial light mod ulator, or a scanning mirror. The light pattern is either static or quasi-static, e.g. a light pattern created by a fast moving scanning mirror. Alternatively, the light forming element 308 is a lens or a set of lenses and the light pattern may be a focused light spot. After leaving the light forming element 308, the formed manipulation light is di rected into the optical detection system 304.

The optical detection system 304 comprises a light deflection element 306, which is exemplarily formed as a beam splitter. The light deflection element 306 is configured to direct the detection light to the detector 142 and to direct the manipulation light to the objective 138 of the optical detection system 304. The manipulation light is then directed by the objective 136 into the intermediate image space 116 and im aged into the sample space 104 by means of the optical transport system 114.

The objective 132 of the optical transport system 114 and the objective 138 of the optical detection system 304 define an optical erecting unit 312 configured to image an oblique image plane of the sample 146. Due to the geometry of the optical erect ing unit 310 the target area 400 is tilted as is shown in Figure 4.

The optical system 318 of the light sheet microscope 300 further comprises a light sheet illumination system 312, that is configured to form a light sheet for illuminating the sample 146 in an intermediate image space 116. Said light sheet illumination sys tem 312 comprising a light sheet forming element 314, for example a cylindrical lens or a scanning element, and an illumination objective 316 configured to direct the light sheet into the intermediate image space 116.

Figure 4 shows a schematic diagram of the sample space 104 of the light sheet micro scope 300 according to Figure 3. The plane 402 in the which the target area 400 is located is indicated by a dashed rectangle. As can be seen in Figure 4 the target area 400 are tilted with respect to the y axis.

By adjusting the scanning element 134, the whole plane in the which the target area 400 is located is moved along the x axis. This is indicated in Figure 4 by a double headed arrow P3 By adjusting the scanning element 134 each time the scanning mo tion is completed, multiple target areas located in different planes arranged along the x axis in sequence are manipulated. Thus, a volume or stack formed by the target areas is manipulated. Alternatively, it is possible to adjust the scanning element 134 during the scanning motion. Thereby, the target area 400 may be non-flat.

As used herein the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".

Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analo gously, 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.

List of Reference Signs

100 Microscope

102 Coordinate cross

104 Sample space

106 Light source

108 Optical system

110 Control unit

112 Optical illumination system

114 Optical transport system

116 Intermediate image space

118 Light sheet forming element

119 Objective

120 Field diaphragm 122 Objective 124 Tube lens

126, 128 Ocular 130 Tube lens 132 Objective 134 Scanning element 136 Optical detection system 138 Objective 140 Tube lens 142 Detector 144 Input device 146 Sample 200 Target area 202 Dotted line

204, 206 Thick line

300 Microscope

302 Manipulation light source

304 Optical detection system

306 Light deflection element

308 Light forming element

310 Optical erecting system

312 Light sheet illumination system

314 Light sheet forming element

316 Objective

400 Target area

402 Plane

PI, P2, P3 Double-headed arrow S Arrow