Microscopes and methods with light-sheet excitation for light-field microscopy and confocal microscopy

Abstract

The invention relates to a microscope comprising an excitation light beam path, in which there are: a light source; an objective which illuminates a sample with excitation radiation and which captures detection radiation; and means for switching between a first and a second operating mode of the microscope. In the excitation light beam path there is a dichroic mirror which is reflective for the excitation radiation, as a result of which the beam of excitation radiation is reflected along a further excitation light beam path as a result of the action of the dichroic mirror. Furthermore, - a first device is present in the excitation light beam path between the light source and the dichroic mirror, in the first operating state of the microscope a first cylindrical optical element can be introduced into the excitation light beam path by means of the first device; - a second device is present in the excitation light beam path between the dichroic mirror and the scanning device, in the first operating state of the microscope a second cylindrical optical element can be introduced into the excitation light beam path by means of the second device. The invention also relates to a method of operating a microscope having two operating modes.

Description

Technical Field

[0001] This invention relates to a microscope and a method for operating the microscope for light field microscopy with light sheet excitation and confocal microscopy. Background Technology

[0002] Modern microscopy increasingly emphasizes the rapid capture of three-dimensional sample volumes. In this context, a crucial application is measuring neural signals within neural networks, which extend over hundreds or thousands of micrometers in the brain. To understand the brain's fundamental capabilities, it is essential to capture these networks, or at least a large portion of their responses, with high temporal resolution. Here, these methods are also combined in critical functional imaging, as understanding not only morphological structures but also functional processes is crucial.

[0003] In the field of microscopy, many different methods are known that can approximate functional imaging. Method-based approaches, such as rapid axial scanning of 2D records, are generally too slow for the aforementioned applications. Methods that rely purely on algorithmic evaluation (computational imaging) are often susceptible to artifacts.

[0004] The disadvantage of confocal scanning methods with low parallelism is that they are relatively slow and operate sequentially. Increased speed is often associated with increased emission power in the sample, where higher emission power can saturate the fluorescent label used and damage the sample. Another point-scanning method is multiphoton microscopy. Parallelism is also low in this case.

[0005] For example, parallelism can be improved using rotating disk microscopy. In this case, a relatively large number of scanning beams are simultaneously guided onto the sample, and the separately induced detection radiation is captured through so-called apertures located in the rotating disk. This confocal method allows for, for example, parallel scanning of hundreds of focal volumes.

[0006] Methods and apparatuses using so-called light sheet illumination also exhibit relatively high parallelism. For this purpose, static or dynamic light sheets are generated and introduced into the sample. Because the thickness of the light sheet across its two-dimensional extent (the light sheet thickness) is very small, detection radiation, particularly fluorescence radiation, is induced only in the plane of the current illumination.

[0007] In addition to selectively illuminating the sample area, wide-field illumination can also be used when applying light field microscopy. For microscopy purposes, detection using light field techniques allows for rapid data acquisition with larger volume and good depth resolution. The disadvantages are the lack of possibility for optical sectioning and strong background radiation.

[0008] By using a microlens array upstream of the detector, a relatively large volume of capture can be achieved while simultaneously improving resolution. A microlens array with microlenses of different focal lengths was proposed in the publication of Cong et al. (Cong, L et al., 2017; eLife, 6:e28158). However, a drawback is that only a portion of the aperture is used in each microlens group.

[0009] Light field imaging methods in the form of Fourier integral microscopy utilize an array of microlenses arranged in the pupil plane of the microscope. Multiple imaged presentations of the sample space appear in the focal plane of the microlens array. Here, the focal plane of each individual microlens is conjugate to the same object plane. In the simplest case, the detector used for image recording is located in this intermediate image plane. This improvement in light field microscopy will also be referred to below as light field imaging in the Fourier domain.

[0010] The goal of light field microscopy imaging is to capture the spatial structure (3D structure) of a sample using only a single recording. To this end, imaged presentations induced by a microlens array are computationally combined. Each of these presentations displays the same sample but at a different angle, resulting in the sharp rendering of different planes within the depth of field of these presentations, for example, using a simple "shift-plus" algorithm. While the lateral resolution is significantly limited due to the reduced numerical aperture of each imaging channel, the resolution of the axial structure of the sample volume is increased.

[0011] When light field microscopy is combined with wide-field illumination, contrast is significantly reduced due to the defocused object structure. To illuminate large volumes and achieve high-contrast imaging, samples can be illuminated with a thicker light sheet than is typically used in light sheet microscopy (selective planar illumination microscopy; SPIM). The advantage of a thicker light sheet is that only the volume of the sample to be imaged is illuminated. Areas not illuminated by the thicker light sheet do not experience a reduction in contrast (Truong, TV et al., (2020), Commun Biol 3:74).

[0012] As is known from existing technology, in order to radiate in a tilted manner in a thick light film, an objective lens that simultaneously performs illumination and detection can be used. In addition to illumination, the common objective lens also allows for collimation of the radiation to be detected from the sample. Using this technical solution, equipment costs are controlled, and the production cost of the equipment is limited (e.g., German Patent 10 2014 102215A1, German Patent 10 2011 000835B4).

[0013] Another embodiment of the microscope is known from German Patent 10 2016 011 227B3. The latter describes a microscope system that can switch between two operating modes. This microscope system includes a light-sheet microscopy unit that, in a first operating state of the microscope system, illuminates and images the sample using a light-sheet type illumination light distribution. Furthermore, a scanning microscopy unit is presented that, in a second operating state of the microscope system, illuminates and images the sample using a point-type illumination light distribution. A control unit is used to switch between the first and second operating states. Summary of the Invention

[0014] One object of the present invention is to provide a flexible option for imaging samples using light field techniques, which in particular allows for the use of other microscopic methods. Another object of the present invention is to provide a method for operating a microscope that has a variety of possible uses.

[0015] This objective is achieved by the microscope described below and the method for operating the microscope having a first operating mode and a second operating mode. Advantageous developments are mentioned below.

[0016] This invention relates to a microscope including an excitation beam path, wherein:

[0017] A light source, used to provide excitation radiation;

[0018] A scanning device for controlled deflection of the excitation radiation;

[0019] An objective lens for illuminating a sample located in the sample space with the excitation radiation and for capturing detection radiation induced by the excitation radiation; and

[0020] A switching device for switching between a first operating mode and a second operating mode of the microscope;

[0021] Its features

[0022] - A dichroic element is present in the excitation beam path, which is reflective to the excitation radiation, and due to the action of the dichroic element, the beam of the excitation radiation is reflected along another excitation beam path;

[0023] - A first device is provided in the excitation beam path located between the light source and the beam splitter, the first device being used to introduce an optical element into the excitation beam path, wherein, in a first operating state of the microscope, a first cylindrical optical element can be introduced into the excitation beam path by means of the first device;

[0024] - A second device is provided in the excitation beam path located between the dichroic device and the scanning device, the second device being used to introduce an optical element into the excitation beam path, wherein, in the first operating state of the microscope, a second cylindrical optical element can be introduced into the excitation beam path by means of the second device.

[0025] In one embodiment, an adjustable aperture is provided in the excitation beam path located between the first device for introducing optical elements into the excitation beam path and the dichroic separator.

[0026] In one embodiment, an adjustable mirror is provided in the excitation beam path and upstream of the dichroic separator, and the excitation radiation is redirected to the dichroic separator due to the action of the adjustable mirror.

[0027] In one embodiment, the dichroic device has a reflector tilted at an angle, wherein the tilt angle causes the reflected beam of excitation radiation to deviate from the optical axis of the microscope.

[0028] In one embodiment, the detection radiation induced in the illuminated sample by the excitation radiation is guided in a first detection path, and a first detector having an upstream microlens array comprising multiple microlenses is present in the first detection path, wherein each microlens is optically assigned multiple detector elements of the first detector, as a result, multiple angular information of the detection radiation can be captured in addition to spatial information of the origin of the detection radiation.

[0029] In one embodiment, the excitation radiation reflected by the microscope can be deflected in a controlled manner by the scanning device.

[0030] In one embodiment, in the second operating state, the cylindrical optical elements of the first device and the second device are removed from the excitation beam path, the excitation radiation is directed at the sample with at least one focal point, and the focal point can be guided over the sample by the scanning device.

[0031] In one embodiment, in the detection beam path of the microscope, in the second operating state, detection radiation induced in the sample by the excitation radiation pointing to the focal point is guided in the second detection path, and a second detector for capturing the detection radiation is present in the second detection path.

[0032] In one embodiment, a controllable optical element for influencing the intensity distribution of the excitation radiation deflected in a controlled manner by the scanning device is arranged in the sample space or in the sample (13) located in the excitation beam path between the light source and the dichroic device.

[0033] The present invention also relates to a method for operating a microscope having a first operating mode and a second operating mode, wherein,

[0034] In the first operating mode

[0035] - By using a first cylindrical optical element, a cross section of the excitation radiation transverse to the propagation direction of the excitation radiation is restricted in a first direction, thereby shaping the excitation radiation in the excitation beam path;

[0036] - The resulting excitation radiation is reflected by a reflective element arranged at an angle, wherein the angle causes the reflected excitation radiation to deviate from the optical axis of the microscope;

[0037] - By means of the second cylindrical optical element, the cross section of the reflected excitation radiation, which is transverse to the propagation direction of the excitation radiation, extends in a second direction orthogonal to the first direction, thereby shaping the reflected excitation radiation by means of the second cylindrical optical element;

[0038] - The excitation radiation, which has already been shaped in two directions, is radiated into the image-side entrance pupil of the objective lens in a manner that is not on the optical axis, thereby producing a tilted light sheet in the object-side sample space;

[0039] - The detection radiation induced by the excitation radiation in the sample located in the sample space is collected through the objective lens; and

[0040] - Detection is performed using a detector with an upstream microlens array, and the collected detection radiation is captured using light field microscopy;

[0041] and,

[0042] In the second operating mode,

[0043] - Remove the first and second cylindrical optical elements from the excitation beam path.

[0044] - The excitation radiation is directed toward the sample with at least one focal point, and the focal point is guided over the sample in a grid-like manner;

[0045] - The detection radiation induced in the sample by the focused excitation radiation is collected through the objective lens; and

[0046] - The collected detection radiation is captured in the second detection path of the confocal array.

[0047] In one embodiment, the excitation radiation in the path of the excitation beam is configured by optical elements that influence the intensity distribution of the excitation radiation so that the resulting light sheet has a uniform intensity distribution along its cross-section.

[0048] In one embodiment, image data captured during the first and second operating modes is evaluated and calculated.

[0049] The microscope has an excitation beam path containing a light source, particularly a laser source, for providing excitation radiation. The excitation beam path also includes: a scanning device by which the reflected excitation radiation can be deflected in a controlled manner; an objective lens for illuminating the sample with the excitation radiation and capturing the detection radiation induced by the excitation radiation; and a switching device for switching between a first operating mode and a second operating mode of the microscope.

[0050] According to the present invention, the microscope is characterized in that a dichroic device is arranged in the excitation beam path, the dichroic device being reflective to the excitation radiation, and through the action of the dichroic device, the beam of excitation radiation is reflected along another excitation beam path. Furthermore, a first device for introducing optical elements into the excitation beam path is located in the excitation beam path between the light source and the dichroic device, wherein, in a first operating state of the microscope, a first cylindrical optical element can be introduced into the excitation beam path via the first device. Similarly, a second device for introducing optical elements into the excitation beam path is located in the excitation beam path between the dichroic device and the scanning device, wherein, in the first operating state of the microscope, a second cylindrical optical element can be introduced into the excitation beam path via the second device.

[0051] The first or second cylindrical optical element can be introduced, for example, by pivoting or pushing into the excitation beam path. In the second operating mode, the first and second cylindrical optical elements are removed from the excitation beam path. To enable pivoting, pushing in, or pulling out the associated cylindrical optical element, the first and second devices are implemented, for example, in the form of wheels or turntables, and have different positions for receiving optical elements, such as filters, cylindrical optical elements, or possibly free light paths for excitation radiation. In a further embodiment, the first and / or second devices may also be implemented in the form of sliders or boxes.

[0052] Due to the action of the first cylindrical optical element, the cross-section of the excitation radiation beam transverse to the direction of excitation radiation propagation, that is, the cross-section transverse to the microscope optical axis, is restricted in the first direction. In this way, the thickness of the light plate to be generated is roughly set.

[0053] Furthermore, a settable stop may be present in the excitation beam path between the first device (hereinafter referred to as the first device) for introducing optical elements into the excitation beam path and the dichroic separator. This settable stop is used to finely set the thickness of the light sheet to be generated. For example, the settable stop may be implemented in the form of a turntable wheel with multiple positions for receiving stops with different apertures. In a further improvement, the settable stop is a slit stop with variable dimensions. The stop may also be a movable hopper or a strip with different aperture sizes. The strip with the aperture can, for example, move transversely to the excitation beam path.

[0054] To illustrate the settings for the thickness and width of the slit, the following values ​​are provided as examples: When the aperture is open, the width of the slit is approximately 3 mm, which corresponds to almost one-fifth of the field of view that can be scanned using a laser scanning microscope (LSM). In the intermediate image (ZB) of the microscope, the thickness of the slit is approximately 0.7 mm. The aperture can be used to reduce the slit thickness. For example, using a slit width of 1 mm, the slit thickness in the intermediate image is approximately 0.32 mm, with an efficiency of approximately 40%. The limitations on field scanning using the obtained slit width are determined by the position of the second cylindrical optics, fixed in the LSM design, and the distance between the plane conjugate to the objective pupil (incident pupil) in the scanner space. In alternative configurations of the device design, this limitation can be omitted. For example, the second cylindrical optics, particularly a cylindrical lens, can have a shortened focal length, resulting in focusing in the scanner plane with a larger NA. Here, focusing can be performed such that, for example, one-fifth, one-quarter, one-third, half, or the entire field of view can be scanned with the obtained slit thickness.

[0055] In the first operating mode, the excitation radiation reflected by the dichroic separator is shaped by means of a second cylindrical optical element by extending, i.e., widening, the cross-section of the excitation radiation transverse to its propagation direction in a second direction orthogonal to the first direction. In other words, the width of the light plate to be generated is set by means of the second cylindrical optical element. The excitation radiation shaped in this way in both directions radiates into the image-side entrance pupil of the objective lens in a manner not on the optical axis, thus producing a tilted light plate in the object-side sample space. The excitation radiation induces detection radiation in the sample located in the sample space, which is collected by the objective lens.

[0056] In one possible embodiment of the invention, the dichroic separator and the second device for introducing the element into the path of the excitation beam (hereinafter referred to as the second device) can each be a master dichroic separator. For example, the dichroic separator can be an RGB master dichroic separator (RGB = red, green, blue), and the second device can be a so-called invisible master dichroic separator (invisible = not visible; wavelengths not belonging to visible light).

[0057] Detection radiation is understood to refer to radiation induced in a sample by excitation radiation. Excitation radiation can in particular induce fluorescence, which is then captured as detection radiation. Therefore, an excitable fluorescent label can be applied to the sample or its regions or structures.

[0058] In an advantageous embodiment of the invention, a reversible element, such as a manually or automatically adjustable mirror, is advantageously positioned upstream of the dichroic separator in the excitation beam path. Due to its function, the excitation radiation is directed to the dichroic separator. The reversible element allows for correction of the focal position of the excitation radiation in the pupil plane, particularly in the entrance pupil of the objective lens. Thus, the tilt angle of the plate relative to the objective optical axis can be finely adjusted by means of the adjustable mirror. This correction may be necessary, for example, if an objective change occurs, and if compensation is to be made for the altered pupil aperture boundary resulting from that change. This option to change the objective is one of the advantages of light field microscopy, which differs in this respect from methods such as SCAPE (scanning confocal aligned-plane excitation) or OPM (tilting plane microscopy). It is possible to use different microscope objectives without changing the arrangement of the equipment, especially if the pupil size is approximately the same. Advantageously, objectives with the same M / NA ratio can be used (e.g., 40x / 1.2NA; 20x / 0.6NA, 10x / 0.3NA). This makes it possible to observe volumes of different sizes at potentially different resolutions.

[0059] Because dichroics are used for reflection, they are well-suited for setting the desired tilt angle of the resulting light sheet. For this purpose, the invention can be further developed in a embodiment where the dichroic separator has a mirror tilted at a tilt angle. The tilt angle of the mirror causes the reflected beam of excitation radiation to deviate from the optical axis. Due to this deviation from the optical axis, the focal point of the reflected excitation radiation, induced by the action of the first and second cylindrical optical elements in the (incident) pupil, shifts towards the periphery of the incident pupil. As a result of this displacement, the shaped excitation radiation departs from the optical axis and radiates into the incident pupil, resulting in a light sheet tilted relative to the optical axis in the object-side sample space. For example, the tilt angle is ±0.5°.

[0060] In another advantageous embodiment, two or more locations of the first device can be occupied by mirrors tilted, for example, by 0.5° in different directions. This allows the orientation of the light sheet to be generated in the sample space to be varied, resulting in the entire sample volume being scanned by light sheets with different orientations. Data generated using different illumination directions is advantageously combined. This is particularly important because a portion of the image along the light sheet orientation, while viewed, contributes very little information to subsequent calculations of the image data, but introduces unwanted background signals. Thus, the contribution of the lenses can be excluded as long as the difference between the tilt angle of the light sheet and the viewing angle of the microlens is small, for example, less than 10°.

[0061] To capture the detection radiation captured in the first operating mode of the microscope, a first detection path exists in the detection beam path of the microscope. A first detector is present in the first detection path, having an upstream microlens array with multiple microlenses, each microlens being optically assigned multiple detector elements of the first detector. As a result, in addition to spatial information about the origin of the detection radiation, multiple angular information of the detection radiation can also be captured.

[0062] The microlens array is preferably located in the pupil upstream of the detector. The detector itself, particularly its detector surface having multiple detector elements, is arranged in the image plane of the microlens array.

[0063] In another possible embodiment of the microscope according to the invention, a microlens array is arranged in the nominal image plane and images the detected radiation onto the detector element of the first detector. In a first operating mode, the microscope according to the invention can therefore optionally be used for light field detection in both the spatial and Fourier domains.

[0064] In an advantageous embodiment, the detected radiation can be diverted away from the excitation beam path downstream of the objective lens, thus avoiding the passage of a second device, dichroist, and any scanning equipment that may be present. This deflection can be achieved using a dichroic mirror or beam splitter, which is additionally controllable and can be moved into or out of the (excitation) beam path depending on the operating mode. This embodiment allows for the retrofitting of, for example, existing microscopes without requiring careful adjustment of the optics of the excitation beam path.

[0065] The microscope according to the invention has a scanning device in the excitation beam path. The reflected excitation radiation can be deflected in a controlled manner by the scanning device. In a first operating mode, the generated light plate can be pushed through the sample space and correspondingly through the sample located therein. Thus, the sample can be scanned entirely or partially by the light plate. In a second operating mode, the scanning device is used to direct the reflected excitation radiation into the entrance pupil of the objective lens, which serves as both an illumination objective and a detection objective, and in a further process, a spot (focal point) is specifically used to scan the sample located in the sample space.

[0066] In another embodiment of the invention, optical zoom may exist between the color separator and the scanning device.

[0067] To allow alternating operation of the microscope according to the invention in a first or second operating mode, there are means for switching between the two operating modes. These means include: first and second devices for introducing cylindrical optical elements, and, if appropriate, controllable actuators thereof; optionally, a dichroic separator adjustable in a controlled manner; and a switching element by which selection can be made between a first and a second detection path in the detection beam path, and the captured detection radiation can be diverted accordingly to the respective selected detection path. The switching element is, for example, a folding mirror or filter wheel equipped with different dichroic beam splitters, etc.

[0068] The operating modes are switched and the light source can be optionally controlled using control commands generated by the control unit. The control unit is, for example, a computer, connected to the controlled technical element in a manner suitable for data transmission. The first and second detectors can also be connected to the control unit and / or evaluation unit for data transmission. For example, the control unit can be configured to match the light field detection with the currently existing illumination state, such as the current alignment, tilt, and / or intensity of the generated light sheet.

[0069] In a further embodiment of the invention, the light source can be controlled by a control unit in such a way that the intensity of the respective adapted excitation radiation (especially laser radiation) is available in both operating modes.

[0070] In the second operating mode of the microscope, the first cylindrical optical element of the first device and the second cylindrical optical element of the second device are removed from the excitation beam path. The excitation radiation is directed at the sample with at least one focal point, and the one or more focal points can be guided over the sample by means of a scanning device, for example, in a grid manner.

[0071] If the microscope is in the second operating mode, that is, if the microscope is in the second operating state, the detection radiation induced in the illuminated sample by the excitation radiation directed to the focal point is guided along the second detection path. A second detector for capturing this detection radiation is present in the second detection path.

[0072] Advantageously, switching between the two operating modes can be achieved in a short time. For this purpose, the aforementioned switching device and its driver can be controlled in a coordinated manner by a control unit, resulting in, for example, switching in less than one second.

[0073] In another embodiment of the microscope, controllable optical elements for influencing the intensity distribution of the excitation radiation can be arranged in the sample space or within the sample, along the excitation beam path between the light source and the dichroic separator. In this case, for a given moment, only the intensity of the excitation radiation is affected. The modulation of the intensity distribution in the sample space or sample occurs only at specific time intervals and at multiple locations of the excitation radiation, particularly as a result of the scanning motion of the scanning device. In other words, the modulation of the spatial intensity distribution is achieved through temporal modulation associated with the scanning operation, and the actual incident intensity of the excitation light depends on the sample position.

[0074] The object of the invention is also achieved by a method for operating a microscope having two operating modes. In the first operating mode, the excitation radiation is constrained in a first direction by means of a first cylindrical optical element, the cross section of which is transverse to its propagation direction (i.e., transverse to the optical axis), thereby shaping the excitation radiation in the path of the excitation beam. This is preferably achieved by means of the first cylindrical optical element. The excitation radiation thus formed is reflected by means of a reflective element (e.g., a dichroic separator) set at an angle, wherein the angle causes the reflected excitation radiation to deviate from the optical axis. The cross section of the reflected excitation radiation, transverse to the propagation direction of the excitation radiation, is extended in a second direction orthogonal to the first direction by means of a second cylindrical optical element, thereby shaping the reflected excitation radiation by means of the second cylindrical optical element. The excitation radiation, which has been shaped in both directions, radiates into the image-side entrance pupil of the objective lens in a manner not on the optical axis. Since the light is radiated outside the optical axis, a tilted light plate is produced in the sample space on the object side. Excitation radiation induces detection radiation in the sample located in the sample space. This detection radiation is captured in a collecting manner using light field microscopy through an objective lens and detected by a detector having an upstream microlens array.

[0075] In the second operating mode, the microscope has been switched to a second operating state. For this purpose, the first and second cylindrical optical elements are removed from the excitation beam path, and the excitation radiation is directed at the sample with at least one focal point. The focal point is guided over the sample, particularly in a grid pattern. The detection radiation induced in the sample by the focused excitation radiation is collected through the objective lens and captured in a confocal second detection path.

[0076] In a further improvement to the method according to the invention, in a first operating mode, an optical element is used to set the excitation radiation in the path of the excitation beam, the optical element being used to influence the intensity distribution of the excitation radiation in such a way that the resulting light sheet has at least a substantially uniform intensity distribution, particularly along its cross-section.

[0077] In an advantageous improvement to the method according to the invention, image data captured during the first and second operating modes can be evaluated and computationally combined. For example, image data already computationally combined in this manner can be combined into a common presentation. This improvement makes it possible to combine the advantages of light field detection, particularly confocal detection, and to provide the user with increased information content. If the image data from the two operating modes are appropriately distributed among themselves, they can also be used for further evaluation. Attached Figure Description

[0078] The invention will now be explained in more detail with reference to exemplary embodiments and accompanying drawings. In the drawings:

[0079] Figure 1 A schematic diagram of a first exemplary embodiment of the invention in a first operating mode is shown, wherein light field detection in the Fourier domain occurs;

[0080] Figure 2 A schematic diagram of a second exemplary embodiment of the invention in a first operating mode is shown, wherein light field detection in the spatial domain occurs;

[0081] Figure 3 A schematic diagram of a third exemplary embodiment of the invention in a second operating mode is shown; and

[0082] Figure 4 A detailed schematic diagram of a color separator in the form of a filter wheel with different optical elements is shown. Detailed Implementation

[0083] exist Figure 1The basic setup of a microscope 1 according to the invention is schematically shown. For clarity, only the technical elements necessary to explain the invention are shown. A light source 3 is arranged in the excitation beam path 2 of the microscope 1 and is specifically implemented in the form of a laser light source 3, which emits excitation radiation ER along the optical axis 4 of the excitation beam path 2. In the first operating state of the microscope 1 shown (the microscope 1 is used in a first operating mode), there is a first device 5 for introducing optical elements into the excitation beam path 2, and a first cylindrical optical element 5.1 in the form of a first cylindrical lens is introduced into the excitation beam path 2. Due to the action of the first cylindrical optical element 5.1, the excitation radiation ER is shaped by limiting the cross section transverse to its propagation direction in a first direction, that is, transverse to the optical axis 4. In this way, the thickness of the light plate 14 to be produced is substantially set. The thickness of the light plate 14 can be set or changed more precisely by using an aperture 6 optionally arranged downstream of the excitation beam path 2. For this purpose, the aperture 6 can be implemented in the form of an adjustable slit aperture, a wheel, or a slider with different apertures. The thickness of the light plate 14 is affected by which available apertures are introduced into the excitation beam path 2. The actuation movement of the aperture 6 can be performed by the driver 26, which can be controlled by control commands from the control unit 23.

[0084] The shaped excitation radiation travels to the adjustable mirror 7, which reflects the excitation radiation ER onto the dichroic separator 8. For example, if the objective lens has been changed and the illumination is about to be adjusted to accommodate the changing pupil boundary as the objective lens changes, the beam position can be adjusted via the mirror 7.

[0085] Dichroic separator 8 is reflective to the excitation radiation ER, but transparent to the detection radiation DR captured in the second operating mode (see [link]). Figure 3 The dichroic separator 8 is implemented as a so-called dichroic wheel or beam splitter and may have at least one tilted mirror 8.1, which, due to its action, reflects the excitation radiation ER at a tilt angle and slightly deviates from the optical axis 4 along another excitation beam path 2. Other tilted mirrors 8.2 (of which only the second is shown) may be present on the beam splitter of the dichroic separator 8, whose action may alter the position and / or orientation of the light slice 14 to be generated. The beam splitter also includes mirrors 8.3 without a tilt angle (see also...). Figure 4 ).

[0086] The reflected excitation radiation ER is incident on the second cylindrical optical element 9.1, which is introduced by pivoting in the second device 9 for introducing the optical element into the excitation beam path 2. The second cylindrical optical element 9.1 causes a widening of the shaped excitation radiation ER in a second direction orthogonal to the first direction. In other words, the width of the light plate 14 is set by the second cylindrical optical element 9.1.

[0087] The excitation radiation ER, or more precisely the beam of the excitation radiation ER, which is shaped in the first and second directions and focused in one direction in each case, arrives at the scanning device 10, through which the beam of the excitation radiation ER can be turned in a controlled manner in two directions perpendicular to each other (xy scanner).

[0088] To control the light source 3, the first device 5, the optional aperture 6 and / or the adjustable reflector 7, the beam splitter wheel of the dichroic separator 8, the optional zoom optical unit 27, the second device 9, the scanning device 10, and the switching element 25, there is a control unit 23. The control unit 23 is connected to the aforementioned technical units in a manner suitable for exchanging data, or is connected to the driver 26 assigned to it via a data connection. The control unit 23 is also additionally connected to an evaluation unit 24, which is configured to evaluate image data from the first detector 17 and the second detector 21, and optionally to combine the image data computationally. The evaluation unit 24 and detectors 17 and 21 are interconnected to transmit data (shown as illustrated).

[0089] The scanning device 10, controlled by the control unit 23, transfers the excitation light ER, particularly a laser, in a controlled manner along the x-direction and / or y-direction (scanning motion). The scanning device 10 can be used to change the incident angle and incident position of the excitation light ER in the incident pupil EP (objective pupil) of the objective lens 11, which is used for both illumination and detection, and thus set the position of the light plate 14.

[0090] The excitation radiation ER is directed towards the incident position in the entrance pupil EP, which is not located on the optical axis 4 of the objective lens 11. Due to the incident position and scanning motion, an optical sheet 14, tilted relative to the optical axis 4, is generated by the objective lens 18 on the object side in a correspondingly tilted optical sheet plane. If the sample 13 is located in the sample space 12 upstream of the objective lens 11, the optical sheet 14 can be directed towards the sample.

[0091] The detection radiation DR induced in the sample 13 is collected by objective lens 11. This detection radiation DR is diverted to the first detection path 16 (shown by dashed lines) of the detection beam path 15 by the action of switching element 25 introduced into the beam path. Switching element 25 is implemented in the form of a dichroic beam splitter and is placed in or removed from the optical path by driver 26. Microlens array 18 is arranged in the pupil of the first detection path 16. Each microlens (shown only) focuses the detection radiation DR captured by it onto an image plane in which multiple detector elements (not shown) of the first detector 17 are arranged. Detector elements optically assigned to each microlens capture the image recorded by that particular microlens. The image data thus obtained is transmitted to evaluation unit 24 and evaluated in the Fourier domain according to the principles of light field technology in terms of light field detection.

[0092] Between the light source 3 and the dichroic separator 8, a controllable optical element 29 for influencing the intensity distribution of the excitation radiation ER can optionally be arranged in the sample space 12 or in the sample 13 located in the sample space 12. The optical element 29 can be controlled, for example, by a control unit 23, and can be implemented, for example, in the form of an AOTF (acousto-optic tunable filter). For example, if it is not necessary to specifically influence the intensity distribution of the excitation radiation ER in the second operating mode (as described further below), the optical element 29 can enter a neutral or time-constant switching state or can be removed from the excitation beam path 2. The purpose of this influence on the intensity distribution of the excitation radiation ER is to provide the most uniform intensity distribution possible across the cross-section of the light sheet 14 produced as a result of the scanning motion. In particular, the intention here is to at least reduce the effect of peripheral decreases in intensity distribution.

[0093] Figure 2 The exemplary embodiments shown are substantially corresponding to Figure 1 An exemplary embodiment of the microscope 1 differs in that at least one optical lens 28 is located in the first detection path 16, and due to its function, the detection radiation DR is focused onto the nominal image plane where the microlens array 18 is located. In this embodiment of the microscope 1, light field detection occurs in the spatial domain.

[0094] Figure 3 The diagram schematically illustrates the operating state of microscope 1 in its second operating mode. A first device 5 and a second device 9 are positioned relative to the excitation beam path 2 such that the excitation radiation ER can pass through the unoccupied positions of the respective devices 5 and 9. In an alternative embodiment, the first and second devices 5 and 9 can be removed from the excitation beam path 2. The cross-section of the excitation radiation ER beam is affected only by the current size and possible aperture shape of the aperture 6. The dichroic separator 8 is positioned such that the reflector 8.3 is positioned in the excitation beam path 2 without tilt angle.

[0095] In the second operating mode, the excitation radiation ER is reflected along the optical axis 4 starting from the reflector 8.3. If one of the tilted reflectors 8.1 and 8.2 of the dichroic separator 8 should remain in the optical path, the deviation from the optical axis 4 can be compensated by the correspondingly controlled scanning device 10. In the exemplary embodiment shown, an optional zoom optical unit 27, controllable by the control unit 23, is located between the dichroic separator 8 and the second device 9.

[0096] Excitation radiation ER is directed through objective lens 11 to focal point 19, which is deflected along the x and y axes of a Cartesian coordinate system in a manner controlled by scanning device 10, thereby scanning sample 13. Detection radiation DR, induced in sample 13 by the focused excitation radiation ER, is then collected by objective lens 11, converted into a stationary beam (“de-scanning”) by scanning device 10, and transmitted via second device 9 and optional zoom optics unit 27 to dichroic separator 8, which is transparent to detection radiation DR. Detection radiation DR is then redirected to second detection path 20, configured as a confocal detection path.

[0097] At least one optical lens 28 is arranged in the detection path, which focuses the detection radiation DR onto an intermediate image plane ZB. A pinhole 22 is located in the intermediate image plane ZB to shield the defocused portion of the detection radiation DR. A second detector 21 is located downstream of the pinhole 22. Image data captured by the second detector 21 is transmitted to the evaluation unit 24. Furthermore, the evaluation unit 24 receives the current position data of the scanning device 10 from the control unit 23 in each case, resulting in the ability to assign the origin position in the sample 13 to the captured image data in each case.

[0098] Figure 4 An embodiment using a color wheel as the color separator 8 is schematically illustrated. In the four positions shown, a first tilted mirror 8.1, a second tilted mirror 8.2, a mirror 8.3 without tilt angle, and a free position 8.4 are arranged around the optical axis 4. The first tilted mirror 8.1 has a tilt angle of 0.5°, while the second tilted mirror 8.2 has a tilt angle of -0.5°. The beam of excitation radiation ER is thus deflected by the second tilted mirror 8.2 in the opposite direction to that used with the first tilted mirror 8.1. Mirror 8.3 has no tilt angle and reflects the excitation radiation ER substantially along the optical axis 4.

[0099] Reference marker:

[0100] 1: Microscope

[0101] 2: Excitation beam path

[0102] 3: (Laser) light source

[0103] 4: Optical axis

[0104] 5: First Equipment

[0105] 5.1: First cylindrical optical element

[0106] 6: Aperture (fast)

[0107] 7: Adjustable reflector

[0108] 8: Color Separator

[0109] 8.1: Tilted Reflector

[0110] 8.2: Tilted Reflector

[0111] 8.3: Reflector (without tilt angle)

[0112] 84.: Free position

[0113] 9: Second equipment

[0114] 9.1: Second cylindrical optical element

[0115] 10: Scanning equipment

[0116] 11: Objective lens

[0117] 12: Sample Space

[0118] 13: Sample

[0119] 14: Light film

[0120] 15: Detect the beam path

[0121] 16: First detection path

[0122] 17: First Detector

[0123] 18: Microlens array

[0124] 19: Focus

[0125] 20: Second detection path

[0126] 21: Second detector

[0127] 22: Small hole

[0128] 23: Control Unit

[0129] 24: Evaluation Unit

[0130] 25: Switching Components

[0131] 26: Driver

[0132] 27: Zoom optical unit

[0133] 28: Optical Lens

[0134] 29: Optical elements [used to influence the intensity distribution of excitation radiation (ER)]

[0135] EP: Entrance Pupil

[0136] ER: Excited radiation

[0137] DR: Radiation Detection

[0138] ZB: Middle Image

Claims

1. A microscope (1) including an excitation beam path (2), wherein: Light source (3) is used to provide excitation radiation; A scanning device (10) for controlled deflection of the excitation radiation; Objective lens (11) for illuminating sample (13) located in sample space (12) with said excitation radiation and for capturing detection radiation induced by said excitation radiation; as well as A switching device for switching between a first operating mode and a second operating mode of the microscope (1); Its features - A dichroic device (8) is present in the excitation beam path (2), the dichroic device (8) is reflective to the excitation radiation, and due to the action of the dichroic device (8), the beam of the excitation radiation is reflected along another excitation beam path; - A first device (5) is provided in the excitation beam path (2) located between the light source (3) and the dichroic device (8), the first device (5) being used to introduce an optical element into the excitation beam path (2), wherein, in a first operating mode of the microscope (1), a first cylindrical optical element (5.1) can be introduced into the excitation beam path (2) by means of the first device (5); - A second device (9) is present in the excitation beam path (2) located between the dichroic device (8) and the scanning device (10), the second device (9) being used to introduce an optical element into the excitation beam path (2), wherein, in the first operating mode of the microscope (1), a second cylindrical optical element (9.1) can be introduced into the excitation beam path (2) by means of the second device (9); In the second operating mode, the cylindrical optical element (5.1) of the first device (5) and the cylindrical optical element (9.1) of the second device (9) are removed from the excitation beam path (2), the excitation radiation is directed at the sample (13) with at least one focal point (19), and the focal point (19) can be guided over the sample (13) by the scanning device (10).

2. The microscope (1) according to claim 1, characterized in that, In the excitation beam path (2) located between the first device (5) for introducing optical elements into the excitation beam path (2) and the dichroic device (8), there is a settable aperture (6).

3. The microscope (1) according to claim 1 or 2, characterized in that, In the excitation beam path (2) and upstream of the dichroic separator (8), there is an adjustable mirror (7) that, due to the action of the adjustable mirror (7), directs the excitation radiation toward the dichroic separator (8).

4. The microscope (1) according to claim 1 or 2, characterized in that, The dichroic device (8) has mirrors (8.1, 8.2) tilted at an angle, wherein the tilt angle causes the reflected beam of excitation radiation to deviate from the optical axis (4) of the microscope (1).

5. The microscope (1) according to claim 1 or 2, characterized in that, The detection radiation induced by the excitation radiation in the illuminated sample (13) is guided in the first detection path (16), and a first detector (17) having an upstream microlens array (18) including multiple microlenses is present in the first detection path (16), wherein each microlens is optically assigned multiple detector elements of the first detector (17), as a result, multiple angular information of the detection radiation can be captured in addition to the spatial information of the origin of the detection radiation.

6. The microscope (1) according to claim 1, characterized in that, The excitation radiation reflected by the microscope (1) can be deflected in a controlled manner by the scanning device (10).

7. The microscope (1) according to claim 1 or 2, characterized in that, In the detection beam path (15) of the microscope (1), in the second operating mode, the detection radiation induced in the sample (13) by the excitation radiation pointing to the focal point (19) is guided in the second detection path (20), and a second detector (21) for capturing the detection radiation is present in the second detection path (20).

8. The microscope (1) according to claim 1 or 2, characterized in that, Controllable optical elements (29) for influencing the intensity distribution of the excitation radiation deflected in a controlled manner by the scanning device (10) are arranged in the sample space (12) or in the sample (13) located in the excitation beam path (2) between the light source (3) and the dichroic device (8).

9. A method for operating a microscope (1) having a first operating mode and a second operating mode and being capable of switching between the first operating mode and the second operating mode, wherein, In the first operating mode - By means of a first cylindrical optical element (5.1), a cross section of the excitation radiation that is transverse to the propagation direction of the excitation radiation is restricted in a first direction, thereby shaping the excitation radiation in the excitation beam path (2); - The resulting excitation radiation is reflected by reflective elements (8.1, 8.2) arranged at an angle, wherein the angle causes the reflected excitation radiation to deviate from the optical axis (4) of the microscope (1). -The cross section of the reflected excitation radiation, which is transverse to the propagation direction of the excitation radiation, is extended in a second direction orthogonal to the first direction by means of the second cylindrical optical element (9.1), thereby shaping the reflected excitation radiation by means of the second cylindrical optical element (9.1); - The excitation radiation, which has already been formed in two directions, is radiated into the image-side entrance pupil of the objective lens (11) in a manner that is not on the optical axis (4), thereby producing a tilted light sheet (14) in the object-side sample space (12). - The detection radiation induced by the excitation radiation in the sample (13) located in the sample space (12) is collected through the objective lens (11); as well as - Detection is performed by a detector (17) with an upstream microlens array (18), and the collected detection radiation is captured using light field microscopy; and, In the second operating mode, - Remove the first and second cylindrical optical elements (5.1, 9.1) from the excitation beam path (2). - The excitation radiation is directed at the sample (13) with at least one focal point (19) and the focal point (19) is guided over the sample (13) in a grid manner. - The detection radiation induced in the sample (13) by the focused excitation radiation is collected through the objective lens (11); as well as - The collected detection radiation is captured in the second detection path (20) of the confocal path.

10. The method according to claim 9, characterized in that, The excitation radiation in the excitation beam path (2) is set by an optical element (29), which is used to influence the intensity distribution of the excitation radiation so that the resulting light sheet (14) has a uniform intensity distribution along its cross-section.

11. The method according to claim 9, characterized in that, Evaluate and calculate the combined image data captured during the first and second operating modes.

Images