light sheet fluorescence microscopy

Abstract

A light-sheet fluorescence microscope is provided, comprising: a light source device configured to emit excitation light suitable for inducing fluorescence emitted from a sample; a detector device configured to detect fluorescence from the sample; and an optical system configured to illuminate the sample with a light sheet formed by the excitation light and guide the fluorescence from the illuminated sample to the detector device, wherein the optical system includes an objective lens facing the sample, the objective lens being configured to collect the fluorescence emitted from the sample, wherein the light source device is further configured to emit manipulation light suitable for optically manipulating the sample, and wherein the optical system is further configured to guide the manipulation light onto the sample through a spatially confined subregion of the entrance pupil of the objective lens along a light propagation direction different from the light propagation direction of the light sheet.

Description

Technical Field

[0001] This invention relates to a light-sheet fluorescence microscope. Additionally, this invention relates to a method for imaging a sample using a light-sheet fluorescence microscope. Background Technology

[0002] Light-sheet fluorescence microscopy is a technique in which a thin slice of a sample is illuminated with a specific excitation light distribution, which is focused in only one direction, for example, by using a cylindrical lens. Another illumination method uses a collimated beam scanned in one direction to create the light distribution that forms the light sheet. Because only a portion of the sample being observed is illuminated, light-sheet fluorescence microscopy reduces optical damage and stress on living samples. Furthermore, in contrast to confocal laser scanning microscopy, which scans the sample point-by-point, light-sheet fluorescence microscopy is a wide-field-of-view technique that allows for the generation of a three-dimensional stack of images based on optical sections passing through different planes of the sample.

[0003] In conventional methods, light-sheet fluorescence microscopy involves separate objectives used to illuminate the sample with a light sheet and to observe the illuminated object plane. Recently, light-sheet methods have been developed that use only one objective for illumination and detection. For example, a tilted light sheet can be generated by illuminating a small spot in the pupil of a high-NA objective. Therefore, a tilted detector configuration is used to image the tilted object plane. This technique is called "Ope Plane Microscopy" (OPM). An extension of OPM, called "Sweeping Confocal Alignment Plane Excitation Microscopy" (SCAPE), has been developed by simultaneously moving the light sheet and the object plane to be imaged using a scanning mirror.

[0004] Beyond simply imaging the sample, optical manipulation (i.e., manipulating the sample by means of light application) is becoming increasingly important. Therefore, the sample can be optically manipulated during or before actual imaging via methods such as heating, bleaching, photoactivation, and photodeactivation. For this purpose, a laser beam with appropriate intensity and wavelength can be directed onto the sample.

[0005] In the field of light-sheet fluorescence microscopy, for example, a system capable of optically manipulating a sample is disclosed in DE 10 2007 047 464A1. This system includes two objectives for illuminating the sample and for detecting induced fluorescence in the sample. In the optical detection path, a device is provided for guiding the manipulation light onto the sample. However, such known systems are not flexible enough in terms of applicability. For example, the system is not suitable for OPM or SCAPE configurations. Summary of the Invention

[0006] The purpose of this invention is to provide a light-sheet fluorescence microscope and a method for imaging samples, which allows for flexible and efficient optical manipulation of the samples.

[0007] The above-mentioned objectives are achieved using the following light-sheet fluorescence microscopy.

[0008] This light-sheet fluorescence microscope includes a light source, a detector, and an optical system. The light source is configured to emit excitation light suitable for inducing fluorescence emitted from the sample. The detector is configured to detect fluorescence from the sample. The optical system is configured to illuminate the sample with a light sheet formed by the excitation light and guide fluorescence from the illuminated sample to the detector. The optical system includes an objective lens facing the sample, configured to collect fluorescence emitted from the sample. The light source is also configured to emit manipulation light suitable for optically manipulating the sample. The optical system is further configured to guide the manipulation light onto the sample through a spatially confined sub-region of the objective lens's entrance pupil along a light propagation direction different from the light propagation direction of the light sheet.

[0009] Advantageous embodiments are defined in the following description.

[0010] A light-sheet fluorescence microscope includes: a light source device configured to emit excitation light suitable for inducing fluorescence emitted from a sample; a detector device configured to detect fluorescence from the sample; and an optical system configured to illuminate the sample with a light sheet formed by the excitation light and guide fluorescence from the illuminated sample to the detector device. The optical system includes an objective lens facing the sample, wherein the objective lens is configured to collect fluorescence emitted from the sample. The optical system is further configured to guide manipulation light through a spatially confined sub-region of the objective lens onto the sample along a light propagation direction different from the light propagation direction of the light sheet.

[0011] In the following text, excitation light or illumination light should be understood as light applied to a sample to induce emission from it and suitable for detection to image the sample. In other words, excitation light or illumination light, as defined herein, is directly involved in the imaging process. In particular, illumination light or excitation light can be light that excites a sample to emit fluorescence, used to generate an optical image representing structural information of the illuminated sample.

[0012] On the other hand, manipulating light should be understood as light applied to a sample in order to manipulate the sample in a manner beyond pure imaging. For example, manipulating light may include light used for heating, cutting, photoactivation, photodeactivation, bleaching, and triggering chemical reactions, for example, in fields such as optogenetics.

[0013] Light-sheet fluorescence microscopy is operated by partially illuminating the entrance pupil of the objective lens with manipulating light to allow for optical manipulation of the sample in a desired direction under specific experimental conditions. Therefore, partial pupil illumination allows for flexible adjustment of the direction in which the manipulating light propagates into the sample by changing a sub-region of the entrance pupil to which it is guided. When the propagation direction of the manipulating light is adjusted to differ from the direction of propagation into the sample by the light sheet, the region of interest within the object plane illuminated by the light sheet can be freely selected for optical manipulation according to the needs or preferences arising from the specific experimental setup.

[0014] Light-sheet fluorescence microscopy can be used in a wide range of applications. In particular, optical manipulation of the sample through a dedicated sub-region of the entrance pupil allows the microscope to be used advantageously in OPM or SCAPE configurations, but is not limited to these.

[0015] Therefore, the optical system can be configured to illuminate the sample through the objective lens using a light plate. Thus, the sample-facing objective lens is typically used to illuminate the sample with excitation light and to optically manipulate it with manipulation light, as well as to observe the illuminated plane.

[0016] A spatially confined sub-region can be positioned offset from the center of the objective lens's entrance pupil. By changing the offset distance from the pupil center, the propagation direction of light can be easily adjusted to suit the specific experimental requirements.

[0017] The optical system can preferably be configured to guide the excitation light onto the sample through another spatially confined sub-region of the entrance pupil, wherein this other sub-region is positioned offset relative to the sub-region through which the manipulation light is guided onto the sample. This embodiment can be advantageously applied in OPM and SCAPE configurations.

[0018] Preferably, the direction of light propagation of the manipulation light emitted from the objective lens toward the sample forms an angle within the range of 0° to ±45° relative to the direction of light propagation opposite to that of the fluorescence emitted by the sample. Specifically, the aforementioned angle can be set to zero, that is, the manipulation light is irradiated onto the sample in a direction directly opposite to the detection axis. This facilitates accurate optical manipulation of the sample.

[0019] In a preferred embodiment, the optical system includes a scanning mirror device comprising at least one tilting mirror that is tiltable for reflecting excitation light, so that the light sheet as a whole moves through the sample in a light sheet scanning direction that is transverse to the light propagation direction of the light sheet.

[0020] Preferably, at least one scanning mirror is tiltable for reflecting both the excitation light and the manipulation light. Therefore, the excitation light and the manipulation light can be co-scanned through the sample, allowing imaging and optical manipulation to be easily performed in a coordinated manner.

[0021] In a specific embodiment, the scanning mirror device may include a scanning lens and a single tilting mirror located in the rear focal plane of the scanning lens. By arranging the tilting mirror and the scanning lens as mentioned above, it is ensured that the tilting movement of the excitation light is converted into a parallel displacement of the excitation lens on the object side of the scanning lens. Therefore, the light plate can be appropriately scanned through the sample.

[0022] In a preferred embodiment, the scanning mirror device includes two tilting mirrors positioned offset from each other along the optical axis of the optical system. This configuration allows for the omission of a scanning lens. Therefore, using two tilting mirrors instead of a single mirror provides additional degrees of freedom, which can be used to freely adjust the (virtual) axis to tilt the excitation light.

[0023] Furthermore, when the scanning microscope device is equipped with two tilting mirrors, these mirrors can be easily controlled to switch between two tilting states. One tilting state is applied to illuminate the sample with excitation light, while the other tilting state is applied for optical manipulation. The scanning microscope device can rapidly perform the switch between illumination and optical manipulation within milliseconds, which is highly advantageous, for example, in cases where rapid processes should be observed in biological samples.

[0024] Preferably, at least one tilting mirror is tiltable to reflect the fluorescence collected by the objective lens toward the detector device. This allows the microscope to be used in a descan configuration, such as those provided in OPM or SCAPE.

[0025] In another preferred embodiment, an optical shifting unit is provided, configured to shift the incident position of the manipulation light on at least one tiltable mirror to change the light propagation direction of the manipulation light guided onto the sample, independent of the light propagation direction of the fluorescence and / or independent of the light propagation direction of the light sheet. This makes the light manipulation more flexible in terms of its positional relationship relative to the excitation light. For example, the manipulation light can be directed to a location illuminated by the light sheet outside the plane of the object.

[0026] An optical shifting unit may include at least one element that reflects or emits manipulating light, wherein this element is movable to shift the incident position of the manipulating light on at least one tiltable mirror. For example, the optical shifting unit may be formed by a dichroic mirror, which can be used to couple manipulating light into an optical system.

[0027] The light source device can be configured to emit excitation light and manipulation light into an optical system along a common optical path. This allows the use of a single light source that emits both excitation and manipulation light. Alternatively, multiple light sources can be integrated into a common housing. In summary, the light source device includes one or more light sources (e.g., lasers) and optionally a common housing and / or additional optical elements (e.g., lenses, mirrors, filters, and / or beam splitters).

[0028] For example, the light source device includes at least one excitation source (e.g., a laser) configured to emit excitation light along at least one first optical path and at least one manipulation source (e.g., a laser) configured to emit manipulation light along at least one second optical path. The light source device also includes an optical shaping system having an optical input formed by the first and second optical paths and an optical output formed by a common optical path, wherein the optical shaping system is configured to selectively shape at least one of the excitation light and the manipulation light.

[0029] Preferably, the light source device can switch between an excitation operation mode for emitting only excitation light and a manipulation operation mode for emitting only manipulation light.

[0030] The optical shaping system may include an input merging element configured to merge first and second optical paths into a third optical path. The input merging element may be formed, for example, from a dichroic mirror.

[0031] The optical shaping system may also include a beam expander located in the third optical path. Before applying further optical shaping and / or scanning, the beam expander may be used in the first step to shape the excitation and manipulation beams, for example, to dynamically create a light sheet, to shift the focus of the manipulation beam, to shift the excitation and / or manipulation beams in a direction transverse to the direction of light propagation, and so on.

[0032] In this embodiment, the common optical path is formed by a third optical path. Additionally, the optical shaping system may include at least one optical shaping element and a scanner located within the common optical path. In excitation operation mode, the optical 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 to generate an optical sheet. In manipulation operation mode, the optical shaping element is configured to shape the manipulation light, and the scanner is configured to adjust the displacement of the manipulation light in the predetermined direction. According to this embodiment, a single (third) optical path is provided within the optical shaping system to act on both the excitation light and the manipulation light. Furthermore, the optical shaping system can dynamically create an optical sheet, for example, by rapidly scanning collimated excitation light through a sample.

[0033] According to another embodiment, the optical shaping system includes a beam splitter located in a 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 optical shaping system also includes an output merging element configured to merge the fourth and fifth optical paths into a common optical path. Additionally, the optical shaping system includes a deformable optics element located in the fourth optical path, wherein the deformable optics element is configured to generate a light sheet from the excitation light. The optical shaping system also includes at least one optical shaping element and / or a scanner located in the fifth optical path, wherein the optical shaping element is configured to shape the manipulation light and the scanner is configured to adjust the displacement of the manipulation light in a predetermined direction. Here, the optical shaping system provides two separate (fourth and fifth) optical paths to act on the excitation light and the manipulation light respectively. Furthermore, the light sheet is created by means of beam shaping rather than scanning.

[0034] Optical shaping elements may include at least one of electrically adjustable lenses, deformable mirrors, digital mirror devices, and spatial light modulators.

[0035] Preferably, the light shaping element is configured to shift the focus of the manipulating light in its light propagation direction. In this case, the light shaping element can be formed from an electrically adjustable lens.

[0036] According to another aspect, a method for imaging a sample using a light-sheet fluorescence microscope is provided, comprising the steps of: emitting excitation light suitable for inducing fluorescence emitted by the sample; illuminating the sample with a light sheet formed by the excitation light; collecting fluorescence from the illuminated sample using an objective lens facing the sample; and detecting the fluorescence. The method further comprises the steps of: emitting manipulation light suitable for optically manipulating the sample; and guiding the manipulation light onto the sample through a spatially confined sub-region of the objective lens along a light propagation direction different from the light propagation direction of the light sheet. Attached Figure Description

[0037] In the following description, preferred embodiments will be illustrated with reference to the accompanying drawings, wherein:

[0038] Figure 1 This is a schematic diagram illustrating a light-sheet fluorescence microscope according to an embodiment;

[0039] Figure 2 This is a schematic diagram illustrating a light-sheet fluorescence microscope according to another embodiment;

[0040] Figure 3 This is a schematic diagram illustrating a light-sheet fluorescence microscope according to another embodiment;

[0041] Figure 4 It is shown Figure 3 A schematic diagram of the scanning mirror device included in a light-sheet fluorescence microscope;

[0042] Figure 5 It is a diagram. Figure 3 A schematic diagram of the rapid switching between illumination and optical manipulation in a fluorescence microscope;

[0043] Figure 6 It is shown in such Figure 1 and 3 A schematic diagram of a light source device according to an embodiment that can be used in a light-sheet fluorescence microscope; and

[0044] Figure 7 This is a schematic diagram illustrating a light source device according to another embodiment. Detailed Implementation

[0045] exist Figure 1 The schematic diagram illustrates a light-sheet fluorescence microscope 100 according to an embodiment, wherein only those components of the light-sheet fluorescence microscope 100 that are helpful for understanding the embodiment are shown.

[0046] according to Figure 1 The specific embodiment shown includes a light-sheet fluorescence microscope 100 comprising a light source device 102, a detector device 104 which may be formed by a CCD or CMOS camera suitable for wide-field imaging, and an optical system generally designated by reference numeral 106. The optical system 106 may include multiple optical elements, wherein an objective lens 108 faces the sample 110 to be imaged by the light-sheet fluorescence microscope 100. As explained in more detail below, the objective lens 108 can be used for both illumination and detection. Therefore, it can be a common objective through which both fluorescence-stimulated illumination light and fluorescence are guided. Consequently, the light-sheet fluorescence microscope 100 can be operated in a SCAPE or OPM configuration. However, the invention is not limited to this configuration. In particular, the light-sheet fluorescence microscope may also include two separate objectives, one for fluorescence excitation illumination and the other for detection.

[0047] The light source device 102 is configured to emit excitation light E, which is suitable for exciting the sample 110 to emit fluorescence F, which will be detected by the detector device 104. See the following reference. Figure 6 and 7 To explain in more detail, the light source device 102 can be configured to shape the light sheet LS from the excitation light E to illuminate only the sheet of sample 110 at a given point in time. Figure 1 In the specific embodiment shown, the light sheet LS illuminating the sample 110 propagates in a direction inclined relative to the optical axis O of the optical system 106. (See reference...) Figure 1In the orthogonal xyz coordinate system indicated in the diagram, the light sheet LS of the illuminating sample 110 extends in a direction parallel to the x-axis and orthogonal to the propagation direction of the light sheet LS. Therefore, the illumination plane formed by the light sheet LS is defined by two axes, one of which is given by the propagation direction of the light sheet located in the xz plane, and the other is the x-axis.

[0048] The optical system 106 is configured to illuminate the sample 110 with a light sheet LS formed by excitation light E, and to guide fluorescence F from the illuminated sample 110 to the detector device 104. Figure 1 In one embodiment, the optical system 106 includes a first lens 112, a second lens 114, a scanning mirror device 116, a third lens 118, a fourth lens 120, and an objective lens 108 facing the sample 110. Each of lenses 112 and 120 serves as a barrel lens, wherein lens 118 is configured as a scanning lens. Figure 1 In the embodiment shown, the scanning mirror device 116 is formed by a single tilting mirror 122, which reflects the excitation light E passing through lenses 112, 114 and through lenses 118, 120 to the objective lens 108 so as to illuminate the sample 110 with a light sheet LS. The tilting mirror 122 is movable about a tilting axis 124 oriented parallel to the x-axis.

[0049] To illustrate the tilting movement performed by the scanning mirror device 116, Figure 1 A tilting mirror 122 is shown at three different tilt positions at consecutive times t = t1, t = t2, and t = t3. Depending on the corresponding tilt position, the tilting mirror 122 reflects the excitation light E in different directions, thereby causing the light sheet LS to scan within the sample 110 in a direction parallel to the y-axis. In other words, the scanning mirror device 116 is used to make the light sheet LS move as a whole in a direction parallel to the y-axis. Figure 1 The light beam moves through sample 110 in the scanning direction indicated by the double arrow A. For simplicity, the excitation light E performing the aforementioned scanning movement is shown by its principal ray, where E(t=t1) represents the excitation light E reflected by tilting mirror 122 at time t=t1. Similarly, principal ray E(t=t2) represents the excitation light E reflected by tilting mirror 122 at time t=t2, and principal ray E(t=t3) represents the excitation light E reflected by tilting mirror 122 at time t=t3.

[0050] exist Figure 1In the configuration shown, the tilting mirror 122 is located in the rear focal plane of the scanning lens 118, i.e., in the image-side focal plane. By positioning the tilting mirror 122 in the focal plane of the scanning lens 118, it is ensured that the tilting movement of the excitation light E on the tilting mirror 122 is converted into a parallel displacement of the excitation light E on the object side of the scanning lens 118. This parallel displacement of the excitation light E is represented by the principal rays E(t=t1), E(t=t2), and E(t=t3) that propagate parallel to each other between the scanning lens 118 and the barrel lens 120, and when emitted from the objective lens 108 to the sample 110. Thus, the tilting movement performed by the scanning mirror device 116 results in a parallel displacement of the light plate LS in the y-direction within the sample 110.

[0051] The fluorescence F induced by the excitation light E is collected by the objective lens 108 and propagates through the optical system 106 to the barrel lens 112 in the opposite direction to the propagation direction of the excitation light E. Subsequently, the fluorescence F passes through a dichroic mirror 126, which is configured to selectively transmit both the fluorescence F and the excitation light E. Therefore, in Figure 1 In the specific embodiment shown, the dichroic mirror 126 is used as a beam splitter to separate the excitation light E from the fluorescence F. Additionally, a reflector 148 is provided, which serves as an element for coupling the excitation light E into the optical system 106. Needless to say, the configuration formed by the reflectors 126 and 148 for achieving the aforementioned light separation and coupling functions should only be understood as an example. Other configurations can be used, as will become apparent from the other embodiments described later.

[0052] After passing through the dichroic mirror 126, the fluorescence F propagates toward the detector device 104 via an optical detection system formed by two objective lenses 127 and 128 and a barrel lens 130. An intermediate image plane 132 lies between the objective lenses 127 and 128, forming an intermediate image of the plane illuminated by the light filter LS. This intermediate image is imaged onto the detector device 104.

[0053] As from Figure 1 As can be seen, the optical path leading to detector device 104 is tilted relative to the optical path between objective lens 108 and barrel lens 112. This reflects the fact that, in the SCAPE or OPM configuration, the light pane LS is tilted relative to the object-side focal plane P1 of objective lens 108. Accordingly, the plane P2, which is conjugate to the focal plane P1, is tilted relative to the intermediate image plane 132.

[0054] When the fluorescence F emitted from sample 110 is guided to tilting mirror 122, the optical system 106 of the light-sheet fluorescence microscope 100 forms a so-called descan configuration. This descan configuration ensures that the fixed detector device 104 continuously detects the illuminated plane within sample 110 while simultaneously scanning the illuminated plane in the y-direction through sample 110. Just as in the case of excitation light E, in Figure 1 This fact is illustrated by indicating the principal rays F(t=t1), F(t=t2), and F(t=t3) of fluorescence F at different tilt positions of tilt mirror 122 at times t1, t2, and t3.

[0055] As mentioned above, Figure 1 The embodiment shown corresponds to a SCAPE or OPM configuration. Therefore, both the excitation light E and the fluorescence F pass through objective lens 108. Specifically, neither the excitation light E nor the fluorescence F fully utilizes the pupil of objective lens 108. In fact, the excitation light E and the fluorescence F pass through different portions of objective lens 108, which are eccentrically located on opposite sides of the optical axis O.

[0056] The light source device 102 is also configured to emit manipulation light M suitable for optically manipulating the sample 110. As explained above, optically manipulating the sample 110 should be understood broadly as the application of light, which is not only related to imaging, but also to heating, bleaching, etc.

[0057] exist Figure 1 In the specific configuration shown, a dichroic mirror 126 is used to introduce the manipulation light M into the optical system 106. Therefore, the characteristics of the dichroic mirror 126 are selected such that it reflects both the manipulation light M and the excitation light E while transmitting the fluorescence F. Thus, in this embodiment, it is assumed that the manipulation light M has substantially the same wavelength as the excitation light E. In this case, a single laser source can be used to generate both the excitation light E and the manipulation light M.

[0058] like Figure 1 As shown, the manipulation light M passes through a portion of the cylindrical lens 112, which is different from the propagation direction of the excitation light E. Therefore, after passing through lenses 112 and 114, the manipulation light M falls onto the tilting mirror 112 at an incident position different from that of the excitation light E. Consequently, after being reflected by the tilting mirror 122, the manipulation light M propagates towards the objective lens 108 in a direction different from the propagation direction of the excitation light E. Thus, the optical system 106 is configured to guide the manipulation light M into the sample 110 along a light propagation direction different from the propagation direction of the light sheet LS formed by the excitation light E. The manipulation light M passes through a spatially confined sub-region of the entrance pupil EP of the objective lens 108.

[0059] When the manipulation light M is reflected by the tilting mirror 122, the manipulation light M performs a scanning movement in the y-direction, just like the excitation light E. However, for the sake of simplicity, Figure 1 The manipulation light M is indicated by a single principal ray M (t = t2), which relates to the tilt position of the tilt mirror 122 at time t = t2.

[0060] The light-sheet fluorescence microscope 100 may include an optical shifting unit configured to shift the incident position of the manipulation light M on the tilting mirror 122. For example, such an optical shifting unit may be formed by a dichroic mirror 126. Therefore, the dichroic mirror 126 may be configured along the propagation direction of the manipulation light M falling on the dichroic mirror 126 (i.e., according to...). Figure 1 The configuration of the dichroic mirror 126 (in a direction parallel to the z-axis) is movable. By moving the dichroic mirror 126 in this way, the portion through which the manipulating light M passes in the tube lens 122 is displaced perpendicularly to the optical axis O. Therefore, the incident position of the manipulating light M on the tilting mirror 122 is changed by moving the dichroic mirror 126, resulting in a corresponding change in the direction in which the manipulating light M propagates into the sample 110.

[0061] By moving the dichroic mirror 126, the propagation direction of the manipulation light M can be changed independently of the propagation directions of the excitation light E and the fluorescence F.

[0062] By shifting the dichroic mirror 126 in a direction perpendicular to the optical axis O of the optical system 106, the propagation direction of the manipulation light M emitted from the objective lens 108 toward the sample 110 can be varied over a wide range. For example, the angle between the propagation direction of the manipulation light M and the direction opposite to the propagation direction of the fluorescence F emitted by the sample 110 can vary from 0 to ±45°. In particular, it can be advantageous to set the aforementioned angle to 0 (i.e., to illuminate the sample along a direction directly opposite to the direction along which the objective lens 108 collects the fluorescence F). In this regard, it should be noted that the direction of light propagation explained herein refers to the principal ray of the corresponding beam.

[0063] Figure 2 The light-sheet fluorescence microscope 100a is shown, which represents Figure 1 A modified embodiment of the configuration. With Figure 1 Compared to the light-sheet fluorescence microscope 100 shown, this modification involves replacing... Figure 1 The light source device 102 shown is the light source device 202.

[0064] Figure 1 The light source device 102 emits the excitation light E and the manipulation light M into the optical system 106 along a common optical path (i.e., in a collinear manner), while Figure 2The light source device 202 of the light-sheet fluorescence microscope 100a shown outputs excitation light E and manipulation light M along two separate optical paths that are parallel to each other. Therefore, the light source device 202 consists of two separate light sources 240 and 242 that output excitation light E and manipulation light M respectively.

[0065] and Figure 1 In contrast to the configuration, Figure 2 The light-sheet fluorescence microscope 100a shown includes a single component, for example in the form of a dichroic mirror 244, for introducing both excitation light E and manipulation light M into the optical system 106. Figure 2 As can be seen, the dichroic mirror 244 is adapted to the light source device 202, so that both the excitation light E and the manipulation light M are reflected into the optical system 106. Furthermore, regarding... Figure 1 The dichroic mirror 126 shown corresponds to the dichroic mirror 244 of the light sheet fluorescence microscope 100a, which is characterized to transmit fluorescence F toward the detector device 104.

[0066] Since the excitation light E and the manipulation light M are both reflected by the dichroic mirror 244, therefore, with Figure 1 The embodiments shown form a comparison, Figure 2 The light-sheet fluorescence microscope 100a does not provide information on the propagation direction of the manipulation light M being independent of the propagation direction of the excitation light E. Therefore, Figure 1 The dichroic mirror 126 only acts on the manipulating light M, while Figure 2 The dichroic mirror 244 shown reflects both the excitation light E and the manipulation light M. Therefore, even if the dichroic mirror 244 moves in a direction perpendicular to the optical axis O of the optical system 106, the dichroic mirror 244 will not change the positional relationship between the excitation light E and the manipulation light M. In other words, according to... Figure 2 In the embodiment shown, the direction of optical manipulation of the sample 110 by the manipulation light M remains unchanged relative to the direction in which the excitation light E illuminates the sample 110. Therefore, at any given time t1, t2, t3, the corresponding principal ray of the manipulation light M intersects with the corresponding principal ray of the excitation light E in the focal plane P1 of the objective lens 108. (As shown in...) Figure 1 It should be noted that, Figure 2 Only one principal ray M (t = t2) is shown, which intersects with the principal ray E (t = t2) of the excitation light in the focal plane P1. Conversely, according to Figure 1 The configuration shown can change the position where the principal ray M (t = t2) intersects with the focal plane P1, thereby changing the positional relationship between the manipulation light M and the excitation light E within the sample 110.

[0067] It should be noted that even in Figure 2In the illustrated embodiment, it may also be possible to change the propagation direction of the manipulation light M independently of the propagation direction of the excitation light E, i.e., when the manipulation light source 242 includes means for correspondingly shifting the manipulation light M.

[0068] Figure 3 This is a schematic diagram illustrating a light-sheet fluorescence microscope 100b, representing another embodiment. The light-sheet fluorescence microscope 100b and... Figure 1 and 2 The main difference in the embodiment shown is the modified scanning mirror device 318.

[0069] Just like Figure 1 and 2 The microscope shown is the same. Figure 3 The light-sheet fluorescence microscope 100b includes a light source device 302 that emits excitation light E and manipulation light M. The light-sheet fluorescence microscope 100b also includes an optical system 306, which includes a tube lens 305, a scanning mirror device 316, another tube lens 308, and an objective lens 108. The excitation light E and the manipulation light M are coupled into the optical system 306 by means of a mirror 348.

[0070] The fluorescence F emitted from sample 110 propagates through optical system 306 in a direction substantially opposite to the propagation directions of excitation light E and manipulation light M. After passing through tube lens 305, fluorescence F propagates through two objective lenses 327 and 328 and tube lens 330 to reach detector device 304.

[0071] The 100b light-sheet fluorescence microscope is basically the same as... Figure 1 and 2 The microscope shown operates in the same way, while Figure 3 The configuration is advantageously modified in terms of the tilting operation of the scanning mirror device 316, which is used to scan the light sheet LS as a whole through the sample 110 in the y-direction. Therefore, according to Figure 1 and 2 In the configuration shown, a single tilting mirror 122 is located in the rear focal plane of the scanning lens 118 to ensure that the tilting movement of the excitation light E on the tilting mirror 122 is converted into a parallel displacement of the excitation light E on the object side of the scanning lens 118. However, using a scanning lens for the aforementioned purpose may have some disadvantages, particularly in terms of cost, as barrel lenses are quite expensive. Therefore, in such a way... Figure 3 The light-sheet fluorescence microscope 100b shown has been modified to eliminate the need for a scanning lens.

[0072] Specifically, the scanning mirror device 316 includes two tilting mirrors 332 and 334, which are positioned offset from each other along the optical axis O of the optical system 306. Each of the tilting mirrors 332 and 334 is movable about tilt axes 336 and 338, such as... Figure 4 As shown in the diagram. The corresponding tilt axes 336 and 338 are oriented parallel to the x-axis.

[0073] By using two tilting mirrors 332 and 334 instead of... Figure 1 and 2 The configuration provides a single tilting mirror, which enables the desired parallel displacement of the excitation light E emitted from the scanning mirror device 316 without the need for a dedicated scanning lens. Therefore, the two tilting mirrors 332, 334 provide additional degrees of freedom in adjusting the tilting operation, which results in a virtual tilt axis about which the excitation light E tilts when the scanning mirror device 316 is operated. In other words, with a single tilting mirror, the axis used to tilt the excitation light E is fixed (see...). Figure 1 and 2 (In the case of tilt axis 124). Conversely, when using two tilt mirrors 332 and 334 with two physical tilt axes 336 and 338, virtual (i.e., non-physical) tilt axes can be freely created as needed.

[0074] In this configuration, the virtual tilt axis is positioned such that the desired parallel displacement of the excitation light E propagating toward sample 110 is achieved. Figure 4 The example shown illustrates two tilt states of the scanning mirror device 316, the difference between which corresponds to the tilt angle β. The tilt angle β defines the amount of tilt about a virtual tilt axis V, located on the image side of the first tilt mirror 332. The excitation light E tilted about the virtual tilt axis V by the angle β is converted into a parallel displacement d of the excitation light E on the object side of the second tilt mirror 334. According to... Figure 3 and 4 In the configuration shown, the virtual tilt axis V is located in the back focal plane of the objective lens 108, that is, the image-side focal plane (or the plane conjugate thereto).

[0075] It should be noted that, Figure 3 The scanning movement of the manipulation light M, achieved by operating the scanning mirror device 316 as explained above, is not illustrated. However, regarding the basic operation of scanning the excitation light E and the manipulation light M through the sample 110 in the y-direction, Figure 3 The embodiments shown are similar to Figure 1 and 2 The configurations are the same.

[0076] The light source devices 102 and 302 can switch between an excitation operation mode and a manipulation operation mode. In the excitation operation mode, the light source devices 102 and 302 emit only the excitation light E, and the manipulation light M is cut off. Similarly, in the manipulation operation mode, the light source devices 102 and 302 emit only the manipulation light M, and the excitation light E is cut off.

[0077] Figure 5 The schematic diagram shown illustrates how the scanning mirror device 316 of the light-sheet fluorescence microscope 100b can be used to quickly switch between illumination of the sample 110 by means of excitation light E and optical manipulation by directly guiding the manipulation light M into the sample 110.

[0078] Figure 5 The scanning mirror device 316 is shown in two tilt states, indicated by solid and dashed lines, respectively. The tilt state indicated by the solid line refers to the excitation operation mode in which only the excitation light E is emitted into the sample. Similarly, the tilt state indicated by the dashed line refers to the manipulation operation mode in which only the manipulation light M is emitted into the sample 110. Therefore, the light source device 302 can be controlled to generate only the excitation light E in the excitation operation mode and only the manipulation light M in the manipulation operation mode.

[0079] Therefore, in the excitation mode, the planar IP of the sample 110 illuminated by the light sheet LS is imaged onto the detector device 104. By switching to the excitation mode, specific areas of the object planar IP can be optically manipulated along a direction different from the propagation direction of the light sheet LS.

[0080] It should be noted that the scanning mirror device 316 can be switched very quickly between the tilt states mentioned above. In fact, the scanning mirror device 316 can be switched within milliseconds. Therefore, it is possible to quickly change between illumination and light manipulation of the sample 110. In addition, by appropriately controlling the tilt movement of the two tilt mirrors 332, 334, the propagation direction of the manipulation light M can be changed over a wide range.

[0081] Figure 1 , 3 The embodiments shown in Figure 5 are configured to couple the excitation light E and the manipulation light M to the optical systems 106 and 306 in a collinear manner (i.e., along a common optical path). Thus, the light source devices 102 and 302 can be formed from a combined source for emitting both the excitation light E and the manipulation light M. This combined source is advantageous, for example, in terms of a compact design. Therefore, different laser sources that generate the excitation light E and the manipulation light M respectively can be integrated into a common housing.

[0082] However, providing such a combined source that meets the specific requirements arising in light sheet microscopy is a challenge. Therefore, the source should be able to generate the light sheet LS in the desired orientation and to shape the manipulation light M as needed. For example, the focus of the manipulation light M can be shifted in the z-direction. Additionally, the manipulation light M can be deflected in the x and y directions.

[0083] Figure 6 and 7 Different embodiments of the light source devices 102, 302 that can be used to meet the requirements mentioned above are shown.

[0084] according to Figure 6 The light source devices 102 and 302 include an excitation light source 650 that emits excitation light E along a first optical path OP1 and a manipulation light source 652 that emits manipulation light M along a second optical path OP2. The first optical path OP1 and the second optical path OP2 can be offset and positioned parallel to each other. The light source devices 102 and 302 also include a dichroic mirror 654, which is characterized to transmit the excitation light E and reflect the manipulation light M deflected by the mirror 656 onto the dichroic mirror 654. Therefore, the dichroic mirror 654 can be used as an input merging element, which is configured to merge the first optical path OP1 and the second optical path OP2 into a common third optical path in which the excitation light E and the manipulation light M propagate.

[0085] The light source devices 102 and 302 also include a beam expander 658, through 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. Following the beam expander 658, an optical shaping element 660 is located in the third optical path OP3. The optical shaping element 660 can be formed from an electrically adjustable lens (ETF), a deformable mirror (DM), a digital mirror device (DMD), or a spatial light modulator (SLM). Additionally, the third optical path OP3 includes two lenses 662 and 664 and a scanner 666. The scanner 666 can be formed from a mirror that can be tilted about a tilt axis 668 oriented parallel to the z-axis. In this respect, it should be noted that... Figure 6 The plane of the diagram is perpendicular to Figures 1 to 5 The plane of the diagram.

[0086] The aforementioned components 654 to 666 form an optical shaping system 670, whose optical input is defined by a first optical path OP1 and a second optical path OP2, and whose optical output is defined by a third optical path OP3. According to Figure 6 In the configuration shown, the third optical path OP3 serves as a common optical path, along which the light source devices 102 and 302 emit excitation light E and manipulation light M into the optical systems 106 and 306. Figure 6 Only the lens barrels 112 and 305 of the optical systems 106 and 306 are shown.

[0087] The light source devices 102 and 302 can operate in an excitation operation mode for generating only the excitation light E and a manipulation operation mode for generating only the manipulation light M. The light shaping system 670 operates differently depending on whether the excitation or manipulation operation mode is selected. For example, the light shaping system 670 can be controlled in excitation operation mode such that the light shaping member 660 is neutral relative to the excitation light E, i.e., it has no effect on the excitation light E in terms of light shaping. Alternatively, in excitation operation mode, the scanner 666 can be operated to perform very rapid tilting movements about the tilt axis 668 to scan the excitation light E through the sample 110 in the x-direction to generate the light sheet LS. Therefore, in this embodiment, the light sheet LS is dynamically generated.

[0088] Conversely, in the manipulation operation mode, the optical shaping system 670 is controlled such that the optical shaping element 660 acts on the manipulation light M to shape it in a desired manner. For example, the optical shaping element 660 can shift the focus of the manipulation light M in the z-direction. On the other hand, the scanner 666 operates in the manipulation operation mode, thus functioning as a detector element. Therefore, the scanner 666 can provide displacement of the manipulation light M in the x-direction.

[0089] The optical shaping element 670 can be located in a plane conjugate to the object-side focal plane of the objective lens 108. Alternatively, the optical shaping element 607 can be positioned in the image-side focal plane of the objective lens 108. In this case, lenses 662 and 664 can form a telecentric optical system, wherein lenses 662 and 664 are positioned such that the distance between them is equal to the sum of their focal lengths. Therefore, lenses 662 and 664 can form a so-called 4f system.

[0090] The scanner 668 is preferably located in the back focal plane of the barrel lenses 112 and 305, that is, at a distance from the barrel lenses 112 and 305 equal to their back focal length f1. Alternatively, the scanner 666 may be located in a plane conjugate to the back focal plane of the barrel lenses 112 and 305. In any case, the aforementioned plane containing the scanner 666 is conjugate to the back focal plane of the objective lens 108.

[0091] exist Figure 6 In the configuration shown, the scanner 666 can be tilted about a tilt axis 668, causing a beam displacement in the x-direction within the sample 110. Furthermore, the scanner 666 can be tilted about a second tilt axis perpendicular to the tilt axis 668. Figure 6 (Not shown in the image) is tilted to create a beam displacement in the y-direction within the sample 110. Due to this displacement in the y-direction, the focus of the excitation light E or the manipulation light M is no longer located in the object plane IP imaged onto the detector device 104 (see image 110). Figure 5 More precisely, the light focus is offset in the y-direction relative to the plane IP.

[0092] Figure 6 The configurations shown are to be understood as examples only. For example, it is possible to combine elements that cause two optical imaging processes, respectively associated with the excitation light E and the manipulation light M. Furthermore, the order in which the illumination shaping element 660 and the scanner 666 are provided can be reversed, and multiple light shaping elements and scanners can be provided. Similarly, more than two laser sources can be provided, for example, to achieve cascading of light sources.

[0093] Figure 7 Another embodiment of a light source device 102, 302 including a modified light shaping system 770 is shown. According to... Figure 6 In the configuration shown, the third optical path OP3, extending from the beam expander 658, directly forms the optical output for supplying the excitation light E and the manipulation light M to the optical systems 106 and 306, while Figure 7 The embodiment shown provides separation of the excitation light E and the manipulation light M after the beam expander 658, as explained below.

[0094] Figure 7 The beam shaping system 770 includes a beam splitter 772, such as a switchable mirror or dichroic element configured to spatially separate the excitation light E from the manipulation light M. Needless to say, the dichroic element can only be used as a beam splitter if the excitation light E and the manipulation light M output from the light sources 650 and 652 have different wavelengths. Conversely, a switchable mirror can be used regardless of the wavelengths of the excitation light E and the manipulation light M.

[0095] exist Figure 7 In the example, it is assumed that the excitation light E propagates in a fourth optical path OP4 extending from the beam splitter 772. In the fourth optical path OP4, a deformable optical element 773 (e.g., a cylindrical lens) is provided, configured to optically shape the excitation light E such that a focal point with an elliptical cross-section is created in the image-side focal plane of the objective lens 108. Figure 7 In the example shown, the major axis of the elliptical cross-section in the image-side focal plane of objective lens 108 extends in the y-direction, while the minor axis extends in the x-direction. Therefore, the plane illuminated by the light pane LS within sample 110 extends in the x-direction. In this respect, it should be noted that... Figure 7 The plane of the diagram is perpendicular to Figures 1 to 5 The plane of the diagram.

[0096] The manipulation light M propagates in a fifth optical path OP5 extending from the beam splitter 772 to the optical shaping element 760, which may include a DM, DMD, SLM, and / or ETL. For example, the optical shaping element 760 can be used to shift the focus of the manipulation light M in the z-direction. In such an application, an ETL is preferably used.

[0097] The optical shaping system 770 also includes two lenses 762 and 764 and a scanner 766 having a tilt axis 768 oriented in the z-direction. By tilting the scanner 766 about the tilt axis 768, the manipulating light M can be shifted in the x-direction.

[0098] The optical shaping system 770 also includes an output combining element 774, which is configured to combine the fourth optical path OP4 and the fifth optical path OP5 into a sixth optical path OP6 extending to the lens barrels 112 and 305 of the optical systems 106 and 306. Thus, the sixth optical path OP6 represents a common optical path used to output the excitation light E and the manipulation light M to the optical systems 106 and 306 in a collinear manner (i.e., spatially overlapping). Like the beam splitter 772, the output combining element 774 can be formed from a switchable mirror or a dichroic element.

[0099] exist Figure 7 In the illustrated configuration, the scanner 766 is preferably located in the back focal plane of the barrel lenses 112 and 305, which have a back focal length f1. Additionally, the optical shaping element 760 may be located in a plane conjugate to the object-side focal plane of the objective lens 108. For this purpose, lenses 762 and 764, having focal lengths f2 and f3 respectively, form a 4f system. Furthermore, according to an advantageous embodiment, each of the optical shaping element 760 and the scanner 766 may be located in a plane conjugate to the image-side focal plane of the objective lens 108.

[0100] Needless to say, the embodiments of light source devices 102 and 302 mentioned above should be understood as examples only. In particular, the light source devices are not limited to the 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). More precisely, as Figure 2 As shown in the embodiments, two separate optical paths can be provided for coupling the excitation light E and the manipulation light M into the optical system 106. In particular, modifications can be made... Figure 6 and 7 The embodiments shown are adapted to a light source device having two separate optical outputs, with the configuration described therein suitable. For example, refer to... Figure 7 This modification can be achieved by omitting the output merging element 774. In this case, the excitation light E and the manipulation light M are coupled into the optical systems 106 and 306 along two separate optical paths OP4 and OP5, respectively.

[0101] In addition, regarding Figure 6 and 7 Explanation reference for the example shown Figures 1 to 5The coordinate system shown is provided. However, it should be noted that throughout this specification, specific directions, particularly regarding different types of light within a scanning or shifting system, should be understood as merely examples and can be modified as needed. Similarly, Figure 6 and 7 The light source device shown should be understood as an exemplary configuration only, which can be used in an abstract form for implementation. Figures 1 to 5 The light source devices 102 and 302 in the embodiments shown. In other words, when in Figures 1 to 5 In the implementation of the embodiments, Figure 6 and 7 The configuration shown can be modified as appropriate. For example, optical elements (e.g., dichroic mirrors) can be added or removed as needed.

[0102] Although some aspects have been described in the context of the apparatus, it is clear that these aspects also represent a description of the corresponding method, where blocks or devices correspond to method steps or features of method steps. Similarly, aspects described in the context of method steps also represent a description of corresponding blocks, items, or features of the corresponding apparatus. Some or all of the method steps can be performed by (or using) hardware devices (e.g., processors, microprocessors, programmable computers, or electronic circuits). In some embodiments, such devices can perform some of one or more of the most important method steps.

[0103] Depending on certain implementation requirements, embodiments of the present invention can be implemented in hardware or software. This implementation can be carried out using a non-transitory storage medium (such as a digital storage medium, e.g., floppy disk, DVD, Blu-ray disc, CD, ROM, PROM, EPROM, EEPROM, or flash memory) having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating with) a programmable computer system to cause the corresponding method to be executed. Therefore, the digital storage medium can be computer-readable.

[0104] Some embodiments of the invention include a data carrier having electronically readable control signals that are capable of cooperating with a programmable computer system to perform one of the methods described herein.

[0105] Generally, embodiments of the present invention can be implemented as a computer program product having program code that, when run on a computer, is operable to perform one of the methods. The program code may, for example, be stored on a machine-readable medium.

[0106] Other embodiments include a computer program stored on a machine-readable medium for performing one of the methods described herein.

[0107] In other words, therefore, an embodiment of the present invention is a computer program having program code that, when run on a computer, performs one of the methods described herein.

[0108] Therefore, another embodiment of the invention is a storage medium (or data carrier, or computer-readable medium) comprising a computer program stored thereon for performing one of the methods described herein when executed by a processor. Data carriers, digital storage media, or recording media are generally tangible and / or non-transitory. Another embodiment of the invention is an apparatus as described herein, comprising a processor and a storage medium.

[0109] Therefore, another embodiment of the invention represents a data stream or signal sequence for performing one of the methods described herein. The data stream or signal sequence may, for example, be configured to be transmitted via a data communication connection (e.g., via the Internet).

[0110] Another embodiment includes a processing component, such as a computer or programmable logic device, configured or adapted to perform one of the methods described herein.

[0111] Another embodiment includes a computer on which a computer program is installed for performing one of the methods described herein.

[0112] Another embodiment of the invention includes an apparatus or system configured to transmit (e.g., electronically or optically) a computer program for performing one of the methods described herein to a receiver. The receiver may be, for example, a computer, a mobile device, a memory device, etc. The apparatus or system may, for example, include a file server for transmitting the computer program to the receiver.

[0113] In some embodiments, a programmable logic device (e.g., a field-programmable gate array) may be used to perform some or all of the functions of the methods described herein. In some embodiments, the field-programmable gate array may cooperate with a microprocessor to perform one of the methods described herein. Generally, the method is preferably performed by any hardware device.

[0114] List of reference numerals

[0115] 100, 100a, 100b Light-sheet fluorescence microscopy

[0116] 102 Light source equipment

[0117] 104 Detector Equipment

[0118] 106 Optical System

[0119] 108 Objective Lenses

[0120] 110 samples

[0121] 112 Lens tube

[0122] 114 Lenses

[0123] 116 Scanning mirror equipment

[0124] 118 Scanning Lens

[0125] 120mm telescope lens

[0126] 122 Tilting Mirror

[0127] 124 Tilt Axis

[0128] 126 Dichroic mirror

[0129] 127 Objective Lens

[0130] 128 Objective Lens

[0131] 130mm lens

[0132] 132 Intermediate Image Plane

[0133] 202 Light Source Equipment

[0134] 244 Dichroic Mirror

[0135] 302 Light Source Equipment

[0136] 304 detector equipment

[0137] 305 Lens Barrel

[0138] 306 Optical System

[0139] 316 Scanning mirror equipment

[0140] 327 Objective Lens

[0141] 328 Objective Lens

[0142] 330 telescope lens

[0143] 332 Tilting Mirror

[0144] 334 Tilting Mirror

[0145] 336 Inclined Axis

[0146] 338 Inclined Axis

[0147] 348 Reflector

[0148] 650 excitation source

[0149] 652 Manipulating the light source

[0150] 654 Dichroic Mirror

[0151] 656 reflector

[0152] 658 Beam Expander

[0153] 660 Optical Shaping Component

[0154] 662 Lens

[0155] 664 Lens

[0156] 666 Scanner

[0157] 668 Tilt Axis

[0158] 670 Light Shaping System

[0159] 760 Optical Shaping Component

[0160] 768 Scanner

[0161] 772 Spectrometer

[0162] 773 Deformation System

[0163] 774 Output Combining Component

[0164] E Excitation Light

[0165] M Manipulating Light

[0166] F fluorescence

[0167] O optical axis

[0168] A double arrow

[0169] LS film

[0170] EP Entrance Pupil

[0171] P1 Focal plane

[0172] P2 conjugate plane

[0173] V Virtual Tilt Axis

[0174] d Displacement

[0175] IP object plane

[0176] β angle

[0177] f1 focal length

[0178] f2 focal length

[0179] f3 focal length

Claims

1. A light-sheet fluorescence microscope (100, 100a, 100b), comprising: The light source devices (102, 202, 302) are configured to emit excitation light (E) suitable for inducing fluorescence (F) emitted from the sample (110). Detector devices (104, 304) are configured to detect the fluorescence (F) from the sample (110), and An optical system (106, 306) is configured to illuminate the sample (110) with a light sheet (LS) formed by the excitation light (E) and to guide the fluorescence (F) from the illuminated sample (110) to the detector device (104, 304). The optical system (106, 306) includes an objective lens (108) facing the sample (110), the objective lens (108) being configured to collect the fluorescence (F) emitted from the sample (110). The light source devices (102, 202, 302) are also configured to emit manipulation light (M) suitable for optically manipulating the sample (110), and The optical system (106, 306) is further configured to guide the manipulation light (M) through a spatially confined sub-region of the entrance pupil (EP) of the objective lens (108) onto the sample (110) along a light propagation direction different from that of the light sheet (LS). The optical system (106, 306) includes a scanning mirror device (116, 316), which includes at least one tilting mirror (122, 332, 334). The at least one tilting mirror (122, 332, 334) is tiltable and is used to reflect both the excitation light (E) and the manipulation light (M).

2. The light-sheet fluorescence microscope (100, 100a, 100b) according to claim 1, wherein the optical system (106, 306) is configured to illuminate the sample (110) through the objective lens (108) using the light sheet (LS).

3. The light-sheet fluorescence microscope (100, 100a, 100b) according to claim 1, wherein the spatially confined sub-region is positioned offset from the center of the entrance pupil (EP) of the objective lens (108).

4. The light-sheet fluorescence microscope (100, 100a, 100b) according to claim 2, wherein the optical system (106, 306) is configured to guide the excitation light (E) onto the sample (110) through another spatially confined sub-region of the entrance pupil (EP), the other spatially confined sub-region being positioned offset relative to the sub-region through which the manipulation light (M) is guided onto the sample (110).

5. The light-sheet fluorescence microscope (100, 100a, 100b) according to any one of claims 1 to 4, wherein the light propagation direction of the manipulation light (M) emitted from the objective lens (108) toward the sample (110) has an angle in the range of 0 to ±45° relative to the direction opposite to the light propagation direction of the fluorescence emitted by the sample.

6. The light-sheet fluorescence microscope (100, 100a, 100b) according to any one of claims 1 to 4, wherein the at least one tilting mirror (122, 332, 334) is tiltable for reflecting the excitation light (E) so that the light sheet (LS) as a whole moves through the sample (110) in a light sheet scanning direction transverse to the light propagation direction of the light sheet (LS).

7. The light-sheet fluorescence microscope (100, 100a) according to any one of claims 1 to 4, wherein the scanning mirror device (116) comprises a scanning lens (118) and a single tilting mirror (122) located in the back focal plane of the scanning lens (118).

8. The light-sheet fluorescence microscope (100b) according to any one of claims 1 to 4, wherein the scanning mirror device (316) comprises two tilting mirrors (332, 334) positioned offset from each other along the optical axis (O) of the optical system (306).

9. The light-sheet fluorescence microscope (100, 100a, 100b) according to any one of claims 1 to 4, wherein the scanning mirror device (116, 316) is configured to shift the principal ray of the excitation light (E) parallel when the excitation light (E) is reflected by the at least one tilting mirror (122, 332, 334).

10. The light-sheet fluorescence microscope (100, 100a, 100b) according to any one of claims 1 to 4, wherein the at least one tilting mirror (122, 332, 334) is tiltable to reflect the fluorescence (F) collected by the objective lens (108) toward the detector device (104, 304).

11. The light-sheet fluorescence microscope (100) according to any one of claims 1 to 4, comprising an optical shifting unit (126) configured to shift the incident position of the manipulation light (M) on the at least one tilting mirror (122) to change the light propagation direction of the manipulation light (M) directed onto the sample (110) independently of the light propagation direction of the fluorescence (F) and / or independently of the light propagation direction of the light sheet (LS).

12. The light-sheet fluorescence microscope (100) according to claim 11, wherein the optical shifting unit (126) includes at least one element that reflects or emits the manipulation light (M), the element being movable to shift the incident position of the manipulation light (M) on the at least one tilting mirror (122).

13. The light-sheet fluorescence microscope (100, 100b) according to claim 12, wherein the light source device (102, 302) is configured to emit the excitation light (E) and the manipulation light (M) into the optical system (106, 306) along a common optical path.

14. The light-sheet fluorescence microscope (100, 100b) according to claim 13, wherein the light source device (102, 302) comprises at least one excitation source (650) configured to emit the excitation light (E) along at least one first optical path (OP1), and at least one manipulation source (652) configured to emit the manipulation light (M) along at least one second optical path (OP2). The light source device (102, 302) further includes an optical shaping system (670, 770) having an optical input formed by the first optical path and the second optical path (OP1, OP2) and an optical output formed by the common optical path. The optical shaping system (670, 770) is configured to selectively shape at least one of the excitation light (E) and the manipulation light (M).

15. The light-sheet fluorescence microscope (100, 100b) according to claim 14, wherein the light source device (102, 302) is switchable between an excitation operation mode for emitting only the excitation light (E) and a manipulation operation mode for emitting only the manipulation light (M).

16. The light-sheet fluorescence microscope (100, 100b) according to claim 15, wherein the light shaping system (670, 770) includes an input merging element (654) configured to merge the first and second optical paths (OP1, OP2) into a third optical path (OP3).

17. The light-sheet fluorescence microscope (100, 100b) according to claim 16, wherein the light shaping system (670, 770) includes a beam expander (658) located in the third optical path (OP3).

18. The light-sheet fluorescence microscope (100, 100b) according to claim 16, wherein the common optical path is formed by the third optical path (OP3). The optical shaping system (670) includes at least one optical shaping element (660) and a scanner (666) located in the common optical path. in, In the excitation operation mode, the light shaping element (660) is configured not to shape the excitation light (E), and the scanner (666) is configured to cause the excitation light (E) to move in a predetermined direction transverse to the light propagation direction of the excitation light (E) to generate the light sheet (LS). In the manipulation operation mode, the light shaping element is configured to shape the manipulation light (M), and the scanner (666) is configured to adjust the displacement of the manipulation light (M) in the predetermined direction.

19. The light-sheet fluorescence microscope (100, 100b) according to claim 18, wherein the light shaping system (770) includes a beam splitter (772) and an output combining element (774), the beam splitter (772) being located in the third optical path (OP3) and configured to spatially separate the excitation light (E) from the manipulation light (M), the excitation light (E) propagating in a fourth optical path (OP4) and the manipulation light (M) propagating in a fifth optical path (OP5), and the output combining element (774) being configured to combine the fourth and fifth optical paths (OP4, OP5) into the common optical path. The optical shaping system (770) includes a deformable optical element (773) located in the fourth optical path (OP4), the deformable optical element (773) being configured to generate the light sheet from the excitation light (E), and The optical shaping system (770) includes at least one optical shaping element (760) and / or a scanner (766) located in the fifth optical path (OP5), the optical shaping element (760) being configured to shape the manipulation light (M) and the scanner (766) being configured to adjust the displacement of the manipulation light (M) in a predetermined direction.

20. The light-sheet fluorescence microscope (100, 100b) according to claim 18, wherein the light shaping element (660, 760) comprises at least one of an electrically adjustable lens, a deformable mirror, a digital mirror device, and a spatial light modulator.

21. The light-sheet fluorescence microscope (100, 100b) according to claim 18, wherein the light-shaping element is configured to shift the focus of the manipulation light (M) in its light propagation direction.

22. A method for imaging a sample (110) using a light-sheet fluorescence microscope (100, 100a, 100b), comprising the following steps: The emission is adapted to induce excitation light (E) that induces fluorescence (F) emitted by the sample (110). The sample (110) is illuminated by a light plate (LS) formed by the excitation light (E). The fluorescence (F) from the illuminated sample (110) is collected using an objective lens (108) facing the sample (110), and Detect the fluorescence (F), The method also includes the following steps: A manipulation light (M) suitable for optically manipulating the sample (110) is emitted, the excitation light and the manipulation light being scanned together through the sample, and The manipulation light (M) is guided onto the sample (110) through the spatially confined sub-region of the entrance pupil (EP) of the objective lens (108) along a light propagation direction different from that of the light plate (LS).

Images