Microscope and method for microscopy

Abstract

The invention relates to a microscope and a method for microscopy, the microscope having: a light source for emitting excitation light; an illumination light path having a micromirror objective for guiding the excitation light onto and / or into the sample to be inspected; the detector is used for detecting the detection light emitted by the sample under the illumination of the exciting light; a probe light path for guiding probe light onto a detector, comprising a microscope or a further microscope objective; a first spatial light modulator arranged in or near a pupil plane of the illumination light path; and a control unit at least for actuating the first spatial light modulator, the control unit being configured to actuate the first spatial light modulator to at least partially implement a lens function. According to the invention, the microscope is characterized in that a wavefront manipulator is arranged and / or formed in or near at least one intermediate image plane of the illumination beam path in order to compensate for wavefront distortions of the excitation light caused by the first spatial light modulator.

Description

Technical Field

[0001] The invention relates to a microscope according to the preamble of claim 1 and to a method for microscopy according to the preamble of claim 15 . Background Art

[0002] A microscope according to the category has the following components: a light source, which is used to emit excitation light; an illumination light path having a microscope objective, which is used to guide the excitation light onto a sample to be examined and / or into the sample to be examined; a detector, which is used to detect detection light emitted by the sample due to illumination by the excitation light; a detection light path for guiding the detection light to the detector, which includes the microscope objective or another microscope objective; a first spatial light modulator, which is arranged in or near the pupil plane of the illumination light path; and a control unit for driving at least the first spatial light modulator, which is configured to drive the first spatial light modulator to at least partially realize a lens function.

[0003] In a generic method for microscopy, the following steps are performed: excitation light is directed onto and / or into a sample to be examined via an illumination light path having a microscope objective; detection light emitted by the sample as a result of illumination by the excitation light is directed onto a detector via the microscope objective or another microscope objective and the detection light is detected by the detector; the excitation light is directed through a first spatial light modulator arranged in or near a pupil plane of the illumination light path, and the first spatial light modulator is driven to at least partially realize a lens function.

[0004] A generic microscope and a generic method are known, for example, from DE 10 2022 103 051 A1.

[0005] A phase-modulating light modulator, such as a nematic LCoS (Liquid Crystal on Silicon) modulator, is known. This phase-modulating light modulator, arranged in or near the pupil plane of the illumination beam path, can be used to generate a spatially structured illumination pattern in an intermediate image plane located downstream of the beam in the illumination beam path (i.e., viewed in the direction of the specimen). The pupil plane and the intermediate image plane are connected to each other via a lens that performs a Fourier transform. This lens, also known as a Fourier lens, is arranged between the pupil plane and the intermediate image plane and has a spacing from each of these planes that corresponds to the focal length f of the lens.

[0006] By controlling the phase pattern of a light modulator that modulates the phase in the pupil plane, a virtually arbitrary amplitude pattern can be generated in the intermediate image plane. A light modulator that modulates the phase can also be referred to as a phase modulator, a variable phase plate, a spatial light modulator, or an SLM (Spatial Light Modulator). Phase patterns suitable for generating a defined amplitude pattern can be obtained, for example, using the iterative Gerchberg-Saxton algorithm, also known as IFTA (Iterative Fourier Transform Algorithm).

[0007] It is also known to use a phase-modulating optical modulator to generate the Fourier lens itself, see [1]. The phase function used to drive the phase-modulating optical modulator So for

[0008]

[0009] in, represents the phase pattern of the desired amplitude distribution generated in the intermediate image plane, and is the phase pattern used to generate the Fourier lens.

[0010] When the phase-modulating light modulator generates a Fourier lens, a true conventional Fourier lens is no longer required, and the spacing between the pupil plane in which the phase-modulating light modulator is arranged and the intermediate image plane is reduced to the simple focal length of the Fourier lens generated by the phase-modulating light modulator Thus, a more compact structure of the optical system can be achieved. The Fourier lens generated by the phase modulator can also be called an SLM lens. The illumination light or excitation light from the phase-modulating light modulator is selected so as to match the optical parameters (such as field number and aperture) of the subsequent optical components in the microscope frame and, for example, the intermediate image.

[0011] The following fact needs to be taken into account: when a phase-modulating light modulator generates a Fourier lens, the complex light distribution of the plane in which the phase-modulating light modulator is arranged is not subjected to a pure Fourier transformation, but rather there is a The product of the Fourier transform of with a complex amplitude term that has quadratic phase in the imaginary exponent.

[0012] Specifically, the complex amplitude in the range plane is given by:

[0013]

[0014]

[0015] where x and y are the coordinates in the intermediate image after the quasi-pupil SLM. It is the Fourier transform of the light distribution immediately after interaction with the optical modulator that modulates the phase.

[0016] For illumination light, the secondary phase The presence of means that, for example, diverging beam cones emanating from off-axis illumination points have a centroid angle pointing away from the optical axis, so that subsequent optics may poorly capture such beams or may even no longer capture them. This means that vignetting and other field-related aberrations may occur, and the field of view transmitted toward the specimen is reduced.

[0017] Furthermore, the secondary phase causes the actual pupil position to deviate from the desired pupil position. Specifically, this can result in the back focal plane of the microscope objective, also known as the objective pupil, not being accessible. This is disadvantageous for applications such as structured illumination microscopy (SIM) and TIRF microscopy (TIRF), in which a defined light distribution in the back focal plane of the microscope objective is required. Summary of the Invention

[0018] It is an object of the present invention to specify a microscope and a method for microscopy in which vignetting and field-related aberrations can be reduced or avoided.

[0019] This object is achieved by a microscope having the features of claim 1 and a method having the features of claim 15 .

[0020] According to the present invention, a microscope of the above type is improved in the following manner, that is, a wavefront manipulator is arranged and / or formed in at least one intermediate image plane or near at least one intermediate image plane of the illumination light path to compensate for the wavefront distortion of the excitation light caused by the first spatial light modulator.

[0021] According to the invention, a method for microscopy of the type described above is improved in that the wavefront distortion produced by the first spatial light modulator is compensated by means of at least one wavefront manipulator arranged or formed in or near an intermediate image plane.

[0022] Preferred exemplary embodiments of the microscope according to the invention and advantageous variants of the method according to the invention are described below, in particular in conjunction with the dependent claims and the accompanying drawings.

[0023] The microscope according to the invention can advantageously be set up to carry out the method for microscopy according to the invention, and the method for microscopy according to the invention can advantageously be carried out using the microscope according to the invention.

[0024] For the microscope according to the present invention, a laser is primarily considered as the light source. However, other light sources are also feasible. The excitation light is preferably electromagnetic radiation in the visible light range and the adjacent range. There are generally no restrictions on the sample to be examined. Typically, the sample to be examined is a biological sample.

[0025] The term "illumination beam path" refers to all optical components that guide and modify the beam, such as microscope objectives, lenses, mirrors, prisms, gratings, filters, apertures, beam splitters, and modulators, such as spatial light modulators (SLMs), with which the excitation light from the light source is directed onto the specimen to be examined. Beam-modifying components also include dispersive and, in particular, diffractive elements.

[0026] The probe light is the electromagnetic radiation emitted by the specimen in response to illumination with the excitation light. It can be reflected by the specimen or transmitted through the illuminated specimen. It is typically fluorescence from fluorescent markers used in specimen preparation that is red-shifted compared to the excitation light. The detector can, in particular, be a two-dimensional spatially resolved detector, thus a camera. Other detectors are possible, such as line detectors for line scanning systems and point detectors for point scanning systems. The term "detection beam path" includes all optical components that guide and modify the beam, such as objectives, lenses, mirrors, prisms, gratings, filters, apertures, beam splitters, and modulators, such as spatial light modulators (SLMs), by means of which the probe light is guided from the specimen to be examined to the detector. The microscope objective of the illumination beam path and the microscope objective of the detection beam path can be one and the same microscope objective. This is the case, for example, in reflected light microscopes, where the specimen is illuminated and observed from one and the same direction. However, the microscope objective of the illumination beam path can also be different from the microscope objective of the detection beam path. This is the case, for example, in transmitted light microscopes and in reflected light microscopes in which the specimen is illuminated obliquely, or in light sheet microscopes.

[0027] The term "pupil plane" refers to a plane perpendicular to the optical axis of the illumination or detection beam path, which is optically conjugate to the back focal plane of the respective microscope objective. The term "intermediate image plane" refers to a plane perpendicular to the optical axis of the illumination or detection beam path, which is optically conjugate to the image plane of the respective microscope objective.

[0028] In this specification, when it is mentioned that a component is located in the pupil plane or in the intermediate image plane, it is always implied that the relevant component is located in the vicinity of the respective pupil plane or in the vicinity of the respective intermediate image plane. This is already clear because neither the pupil plane nor the intermediate image plane is a plane in the mathematical sense, and because the components of interest here, such as the spatial light modulator and the lens, each have a finite extension in the direction of the optical axis.

[0029] The term "wavefront manipulator" refers to any optical component that changes (thus manipulates) the wavefront of light transmitted through or reflected at the relevant component. The manipulation may for example be based on diffraction and / or refraction.

[0030] The term "control unit" refers to all hardware and software components that interact with the components of the microscope according to the invention to realize their prescribed functions. In particular, the control unit can include a computing device, such as a PC, and a camera control unit that can quickly read out the measurement signals. The computing resources of the control unit can be distributed among several computers and, if necessary, over a computer network, in particular also via the Internet. The control unit can, in particular, have common operating devices and peripherals such as a mouse, keyboard, screen, storage medium, joystick, Internet connection. The control unit can, in particular, read out measurement data from the detector, in particular image data, and can also be used and set up for driving a light source. According to the invention, the control unit is set up for driving a first spatial light modulator to at least partially realize the lens function.

[0031] The basic concept of the present invention is to provide a wavefront manipulator in at least one intermediate image plane to compensate for or correct the wavefront distortion of the excitation light caused by the first spatial light modulator. This wavefront manipulator can, in principle, be simple in nature. The terms "compensation" and "correction" are used synonymously in this specification.

[0032] The present invention provides a microscope and a method for microscopy that are characterized by a multiplicity of application possibilities and different possibilities for illumination modes. The microscope and the method can be implemented with a relatively compact optical design.

[0033] The first spatial light modulator can, in principle, be the only spatial light modulator in the illumination beam path. In advantageous embodiments of the microscope according to the invention, a second spatial light modulator is arranged downstream of the beam of the first spatial light modulator in an intermediate image plane of the illumination beam path or in the vicinity of this intermediate image plane, and the control unit can advantageously also be configured to drive the second spatial light modulator, in particular to achieve an illumination pattern for the specimen. Accordingly, a preferred variant of the method according to the invention is characterized in that the excitation light is guided downstream of the beam of the first spatial light modulator through a second spatial light modulator, which is arranged or formed in the intermediate image plane of the illumination beam path or in the vicinity of this intermediate image plane.

[0034] In a particularly preferred configuration, the first spatial light modulator may be formed by a first subregion of a spatial light modulator, and the second spatial light modulator may be formed by a second subregion of the same spatial light modulator.

[0035] In principle, it is possible for at least one, multiple, or each component of the first spatial light modulator, the second spatial light modulator, or the spatial light modulator to be formed by an amplitude-modulating spatial light modulator. However, it is advantageous for at least one, multiple, or each component of the first spatial light modulator, the second spatial light modulator, or the spatial light modulator to be formed by a phase-modulating spatial light modulator. Phase-modulating spatial light modulators are generally preferred due to their lower optical losses.

[0036] Then, at least one, a plurality of, or each component of the first spatial light modulator, the second spatial light modulator, or the spatial light modulator may be formed by a reflective spatial light modulator. However, it is also feasible that at least one, a plurality of, or each component of the first spatial light modulator, the second spatial light modulator, or the spatial light modulator is formed by a transmissive spatial light modulator.

[0037] For example, at least one, multiple or each component of the first spatial light modulator, the second spatial light modulator, or the spatial light modulator can be formed by one or more of the following components: DMD (Digital Mirror Device), a nematic SLM, an LCOS display (LCOS = Liquid Chrystal on Silicon), a variable phase plate, and a controllable deformable mirror (DM = DeformableMirror).

[0038] The control unit can be configured to control the first spatial light modulator to achieve an illumination pattern for the sample. If a second spatial light modulator is present, the control unit can additionally or alternatively be configured to control the second spatial light modulator to achieve an illumination pattern for the sample. For example, multi-point illumination of the sample can be achieved.

[0039] When beam shaping is performed on the first spatial light modulator in the pupil plane, for example using a phase pattern, a desired intensity distribution is generated on the second spatial light modulator in the intermediate image plane. Inaccuracies in the optical structure, inaccuracies in the calibration of the first spatial light modulator, and / or inaccuracies in the generation and representation of the pattern used to drive the first spatial light modulator, particularly discretization errors, can cause some of the excitation light from the first spatial light modulator to fall outside the desired region of interest on the second spatial light modulator.

[0040] The basic idea of ​​a further advantageous improvement of the invention can now be to counteract these inaccuracies by means of a second spatial light modulator representing a grating in an area outside the illumination area of ​​interest, the period of which is chosen such that light diffracted at this grating cannot propagate to the sample from other illumination beam paths.

[0041] This improves the accuracy of the illumination pattern. In particular, for many illumination modes, a lower intensity background in the specimen can be achieved.

[0042] Specifically, in a particularly preferred embodiment of the microscope according to the present invention, the control unit can be set up to drive the second spatial light modulator to exhibit a grating outside the desired illumination area, the period of which is selected so that the excitation light of the +1st and / or -1st order diffraction is located outside the area propagating to the specimen in at least one pupil plane.

[0043] Accordingly, a preferred variant of the method according to the invention is characterized in that the second spatial light modulator is driven to exhibit a grating outside the desired illumination area, the period of which is chosen so that the +1st and / or -1st order diffracted light is located outside the area propagating to the specimen in at least one pupil plane.

[0044] An axial spatial direction and two lateral spatial directions can be given by the optical axis of the illumination beam path, and the wavefront distortion to be compensated can be, in particular, the quadratic phase term of the excitation light in the coordinates of the lateral spatial directions.

[0045] In a preferred embodiment of the method and microscope according to the present invention, multiple wavefront manipulators cooperate in the compensation step. These different wavefront manipulators can be arranged or formed in different intermediate image planes or near different intermediate image planes. However, it is also feasible to arrange or form multiple wavefront manipulators in a single intermediate image plane or near the same intermediate image plane.

[0046] The aforementioned vignetting caused by the use of the first spatial light modulator in the described manner can be at least partially reduced in a design of the microscope according to the invention in which at least one wavefront manipulator achieves a positive focal length. The aforementioned disadvantages can be avoided, for example, by introducing the field lens into an intermediate image plane or in the vicinity of this intermediate image plane.

[0047] In a relatively simple solution for compensating the wavefront distortion of the excitation light caused by the first spatial light modulator, in particular for compensating the quadratic phase of the excitation light in the lateral coordinate, the wavefront manipulator comprises or is implemented by a glass lens with a positive focal length. This lens, in particular the glass lens, provides a positive corrective field lens that provides an inverse quadratic phase to the quadratic phase remaining due to the first spatial light modulator.

[0048] For an intermediate image plane downstream of the phase modulator in the illumination beam path, such a field lens can again implement the concept of imaging from the exit pupil of the upstream optics to the entrance pupil of the downstream optics. This can reduce field vignetting, edge light falloff, and general aberrations of the optical system.

[0049] In a simple embodiment of the method, the compensation step is partially or completely provided by a glass lens acting as a wavefront manipulator. For example, a conventional positive glass lens can be arranged in or near an intermediate image plane of the illumination beam path. Since a single field lens with a typical focal length (e.g., 200 mm) has a relatively severe influence on the wavefront of the excitation light, it is disadvantageous to permanently position this positive glass lens in the illumination beam path, as certain illumination modes may no longer be possible. In particular, illumination modes that require collimated light in the intermediate image plane, such as TIRF (Total Internal Reflection Fluorescence) or SIM (Structured Illumination Microscopy), would not be possible, which is detrimental to multimodal illumination concepts. TIRF and SIM can, for example, be implemented using a spatial light modulator arranged in or near an intermediate image plane in the illumination beam path. Therefore, in a preferred embodiment of the microscope according to the invention, a transformation device is present for introducing or removing the glass lens into or from the illumination beam path.

[0050] The conversion device can include multiple glass lenses with different focal lengths, and the conversion device can be used to introduce one of the glass lenses into or near the intermediate image plane. Suitably, the control device can be configured to control the conversion device. The conversion device is preferably designed so that the glass lens can be removed from the illumination beam path or moved into the illumination beam path using a motor.

[0051] Another advantageous embodiment of the microscope according to the invention is characterized in that the wavefront manipulator has an adjustable lens or is implemented by an adjustable lens. The compensation step can be provided partially or completely by the adjustable lens as a wavefront manipulator. In addition, within the meaning of multimodal illumination concepts, in particular in the case of illumination modes that require collimated light in the intermediate image plane, as described above, it is advantageous if the adjustable lens can be adjusted into a neutral position. Advantageously, the control device can be configured to drive the adjustable lens.

[0052] The adjustable lens can also advantageously be used and configured to adjust different pupil positions of different microscope objectives.

[0053] Another advantageous embodiment of the microscope according to the invention is characterized in that the wavefront manipulator alternatively or additionally comprises or is implemented by a Fresnel lens. The compensation step can be provided partially or completely by driving the second spatial light modulator to generate a Fresnel lens with a positive focal length. Alternatively or additionally, the first spatial light modulator and / or the second spatial light modulator can be driven to adjust different pupil positions of different microscope objectives to implement the Fresnel lens.

[0054] The control device can preferably be configured to control the second spatial light modulator such that the quadratic phase term of the excitation light in the lateral spatial direction coordinate is partially or completely compensated by the second spatial light modulator. For example, the second spatial light modulator can be controlled to at least partially implement a Fresnel lens.

[0055] The first spatial light modulator and / or the second spatial light modulator can be controlled to generate different illumination patterns for the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Further advantages and features of the present invention will be explained below with reference to the accompanying drawings, in which:

[0057] Figure 1 : shows a first embodiment of a microscope according to the present invention; and

[0058] Figure 2 : shows a second embodiment of the microscope according to the present invention.

[0059] Identical components and components having the same effect are generally denoted by the same reference numerals in the figures. DETAILED DESCRIPTION

[0060] A first embodiment of the microscope according to the present invention refers to Figure 1 be explained. Figure 1The microscope 100 according to the present invention, schematically shown in the figure, first has a light source 10 for emitting excitation light 13 and an illumination beam path with a microscope objective 29 for guiding the excitation light onto the sample 1 to be examined. Then, there is a detector 50 for detecting the detection light 32 emitted by the sample 1 as a result of being illuminated by the excitation light 13. In order to guide the detection light 32 to the detector 50, there is a detection beam path, which, in the embodiment shown, includes the microscope objective 29. It is also possible that, for example in a transmitted light assembly, the detection beam path has its own microscope objective. According to the present invention, there is a first spatial light modulator 15 in the illumination beam path, which is arranged in the pupil plane of the illumination beam path or in the vicinity of the pupil plane. In the embodiment of the accompanying drawings, there is also a second spatial light modulator 19 downstream of the beam of the first spatial light modulator 15, which is arranged in the intermediate image plane of the illumination beam path or in the vicinity of the intermediate image plane.

[0061] In the embodiment shown in the figures, the first spatial light modulator 15 is formed by a first sub-area of ​​the spatial light modulator 14, and the second spatial light modulator 19 is formed by a second sub-area of ​​the same spatial light modulator 14. The light modulator 14 may be, for example, an SLM from the German company HOLOEYE Photonics AG.

[0062] In addition, there is a control unit 90, which is used at least to drive the first spatial light modulator 15 and, in the embodiment of the accompanying drawings, also drives the second spatial light modulator 19, and is configured according to the present invention to drive the first spatial light modulator 15 so that the lens function for the excitation light is at least partially realized by the first spatial light modulator 15.

[0063] In the exemplary embodiment shown, the control unit 90 is then configured to control the first spatial light modulator 15 and / or the second spatial light modulator 19 in such a way that a desired illumination pattern is achieved in the intermediate image plane and in particular in the specimen 1. For example, the specimen 1 can be illuminated from multiple points.

[0064] Finally, in Figure 1 In the example of the microscope 100 according to the present invention shown in , there is a wavefront manipulator formed by a glass lens 24a in the intermediate image plane 25, which wavefront manipulator is used according to the present invention to compensate or correct the wavefront distortion of the excitation light generated by the first spatial light modulator 15.

[0065] In detail, the excitation light 13 is transmitted from the light source 10 (e.g., a laser) via the optical fiber 11 and the lens 12 for collimating the beam to the phase modulator 14. Specifically, the excitation light 13 falls on the first spatial light modulator 15 which is arranged in or near the pupil plane of the illumination light path and is driven by the control unit 90 to realize the lens function or partial lens function. Figure 1 In the embodiment shown in , the pupil plane is a plane that is optically conjugate to the back focal plane 28 of the microscope objective 29 .

[0066] The excitation light 16 manipulated in this way then falls on a mirror 17 which is arranged at a distance from the phase modulator 14 so that the second sub-region 19 and therefore the second spatial light modulator 19 is located in the intermediate image plane of the illumination beam path or at least in the vicinity of the intermediate image plane. Figure 1 In the embodiment shown in , the intermediate image plane is a plane optically conjugate to the focal plane 30 of the microscope objective 29. Since the focal plane lies within the specimen 1 in the example shown, it can also be called the specimen plane or object plane.

[0067] The light reflected from the second spatial light modulator 19 reaches the glass lens 24a in the intermediate image plane 25 via the relay optical device formed by the lenses 21 and 23. A pupil plane 22 is formed between the lenses 21 and 23, in which a further spatial light modulator, in particular a phase modulator ( Figure 1 The double arrow in the glass lens 24a indicates a changeover device, by means of which the glass lens 24a can be removed from the optical path or can be replaced by a Figure 1 It is replaced by another glass lens not shown in the figure.

[0068] After the excitation light 13 passes through a tube lens 26 downstream of the intermediate image plane 25, the excitation light 13 falls on a primary beam splitter 27 and is directed by the primary beam splitter toward a microscope objective 29. The microscope objective 29 ultimately directs the excitation light 13 into a specimen plane 30 of the specimen 1, which is mounted on a schematically shown specimen holder 31. For example, the excitation light 13 is focused into the specimen plane 30 of the specimen 1.

[0069] Because the sample 1 is illuminated by the excitation light 13, the sample 1 emits a detection light 32, which is in particular a red-shifted fluorescence compared to the excitation light 13. Via the microscope objective 29, the detection light 32 returns to the main beam splitter 27, is transmitted by the main beam splitter, is subsequently filtered by the radiation filter 33, and is then imaged by the tube lens 41 as filtered radiation light 34 onto the detector 50. The detector 50 can be, for example, a camera. The detection light 34 detected by the camera 50 can be evaluated with a control unit 90. In order to match different microscope objectives and / or to achieve different image sizes on the camera 50, a tube lens converter 40 is provided in the example shown, which is used to introduce different tube lenses into the detection light path.

[0070] The key to the present invention lies in the glass lens 24a with a positive focal length arranged in the intermediate image plane 25. The glass lens with a positive focal length realizes a wavefront manipulator, and the glass lens with a positive focal length is used according to the present invention to compensate for or correct the wavefront distortion of the excitation light caused by the first sub-region of the phase modulator 14, that is, the first spatial light modulator 15.

[0071] The wavefront distortion to be compensated is, in particular, the quadratic phase term in the lateral spatial directions x and y of the excitation light 13. The lateral spatial directions x and y are given by two spatial directions perpendicular to each other and respectively perpendicular to the axial spatial direction, wherein the axial spatial direction is given by the optical axis z of the illumination beam path.

[0072] In combination Figure 1 In the first embodiment explained, compensation for the wavefront distortion caused by driving the first spatial light modulator 15 to realize the lens function can be substantially completely performed by the glass lens 24a serving as the wavefront manipulator.

[0073] Alternatively, it is also possible to compensate for the wavefront distortion generated by the first spatial light modulator 15 entirely by driving the second spatial light modulator 19 to generate a Fresnel lens with a positive focal length. In this case, the glass lens 24a can be removed from the illumination beam path using a transformation device. The control device 90 is then configured to drive the second spatial light modulator 19 such that the quadratic phase term of the excitation light 13 in the lateral spatial coordinates is entirely compensated by the second spatial light modulator 19.

[0074] It should be noted that the pixels of a phase modulator 14, such as an SLM, each allow only a maximum phase range, i.e., a phase dynamic range less than or equal to 2π. This means that if the value of the quadratic phase to be corrected is greater than 2π, the phase value must be mapped to the specific possible phase dynamic range of the relevant pixel in the phase modulator 14 used. This means that an integer multiple of 2π must be subtracted in each case. This method is known as "phase wrapping" and can be mathematically described using the modulo function:

[0075]

[0076] in, is the phase value used to drive the phase modulator 14, and The quadratic phase to be corrected is the value of an integer multiple of 2π minus 2π. The quadratic phase to be corrected is also called the quadratic field lens phase. The phase shift increases rapidly towards the edges of the intermediate image, which may require the introduction of a large number of phase jumps, similar to a Fresnel lens or a zone plate. This can result in the field lens, also known as the SLM field lens, implemented by the second spatial light modulator 19, i.e., in this example, by the second sub-region 19 of the phase modulator 14, being less effective towards the edges of the field. This can mean, for example, that the reduction of field vignetting decreases towards the edges.

[0077] Therefore, when the second spatial light modulator 19 is also used to compensate for the distortion introduced by the first spatial light modulator 15, the possibility of using the second spatial light modulator 19 to manipulate the excitation light 13, for example, to generate an illumination pattern for the specimen 1, is limited. In short, the phase adjustment power of the second spatial light modulator 19 is consumed by the generation of the Fresnel lens.

[0078] However, the second spatial light modulator 19 is preferably also controlled such that, although a Fresnel lens and thus an additional wavefront manipulator are implemented there, this additional manipulator still cooperates with the glass lens in the intermediate image plane to compensate for the distortion introduced by the first spatial light modulator 15. This means that the glass lens remains in the illumination beam path. The control device 90 is then configured to control the second spatial light modulator 19 such that the quadratic phase term of the excitation light 13 in the coordinates of the lateral spatial direction is only partially compensated by the second spatial light modulator 19. Thus, in this variant, compensation is provided by distributing the function of the correction field lens to a certain extent between the two intermediate image planes: the plane in which the second spatial light modulator 19 is arranged and the intermediate image plane 25. For example, a weaker first Fresnel field lens can initially be generated in the second spatial light modulator 19. This is preferably done without phase jumps in the edge regions of the second spatial light modulator 19. It is then sufficient to use a glass lens having a lower refractive power than the glass lens 24a, which alone is sufficient to perform the compensation, as the glass lens in the intermediate image plane 25. The two field lenses, namely the field lens provided by the second spatial light modulator 19 and the glass lens in the intermediate image plane 25, then jointly produce the necessary compensation function according to the present invention, which in the previously described variants was provided only by the glass lens 24a or only by the field lens of the second spatial light modulator 19.

[0079] A second embodiment of the microscope 200 according to the present invention is Figure 2 The microscope 200 is shown in FIG. Figure 1The only difference compared to the microscope 100 is that an adjustable lens 24b with a positive focal length is provided as a wavefront manipulator in the intermediate image plane 25 instead of the glass lens 24a with a positive focal length. The adjustable lens 24b, which can be an adaptive lens from, for example, Optotune Switzerland AG, DYNAMIC OPTICS srl, Italy, or Phaseform GmbH, Germany, can be suitably controlled by the control unit 90.

[0080] The adjustable lens 24b with a positive focal length can also achieve compensation for the secondary phase when necessary. Preferably, the adjustable lens 24b can also achieve a neutral position in which the excitation light 13 is substantially unaffected, so that, as described above, illumination modes such as TIRF and SIM can also be implemented, in which a collimated light path is required in the intermediate image plane 25.

[0081] As in Figure 1 As in the embodiment of Figure 2 In the embodiment of FIG, the second spatial light modulator 19 can be driven to generate a Fresnel lens with a positive focal length to fully compensate for the wavefront distortion generated by the first spatial light modulator 15. Then, the glass lens 24b can be adjusted to its neutral position.

[0082] In addition, Figure 2 In the embodiment of Figure 1 As described in the embodiment of the present invention, the second spatial light modulator 19 is driven to implement a Fresnel lens. This Fresnel lens, acting as a first wavefront modulator, cooperates with the adjustable lens 24b, acting as a second wavefront modulator, to compensate for the distortion introduced by the first spatial light modulator 15. Compared to a case where the adjustable lens 24b alone performs compensation, the adjustable lens 24b only needs to be adjusted to a lower refractive power. Advantageously, less phase adjustment power is consumed in the second spatial light modulator 19 to provide the Fresnel lens.

[0083] The advantages of using an adjustable lens 24b are that fewer mechanically movable components are required and, given appropriate dimensions, no phase adjustment power is consumed in the second spatial light modulator 19 to create a Fresnel lens. Potential disadvantages of using an adjustable lens include its aging characteristics, relatively high cost, and possible slight scattering of the excitation light at the control electrode in the optical window. For example, TIRF mode can be adversely affected by such scattering.

[0084] The advantage of combining the Fresnel lens provided by the second spatial light modulator 19 with the adjustable lens 24b is that the adjustable lens 24b requires only a lower refractive power and is therefore mechanically simpler and more cost-effective to implement.

[0085] When both field lenses are closed, that is, when the second spatial light modulator 19 and the adjustable lens 24b are adjusted to neutral, the illumination light 13 is no longer affected by the lens effect and other illumination modes such as TIRF and / or SIM can be implemented.

[0086] When a field lens is not required in the intermediate image plane 25 in one of the illumination modes, then Figure 1 In the case of the embodiment of the present invention, the glass lens 24a needs to be removed from the illumination light path, and Figure 2 In the embodiment of FIG. 2 , the adjustable lens 24 b needs to be adjusted to its neutral position. The glass lens 24 a can only remain in the illumination beam path, or the adjustable lens 24 b can only remain in the final refractive power setting, if the influence of these components on the respective illumination mode is small or negligible.

[0087] Alternatively, it is also possible to replace the glass lens 24a ( Figure 1 ) is kept in the illumination light path, or the adjustable lens 24b ( Figure 2 ) is kept in the driving state with the final refractive power, and the second spatial light modulator 19 is additionally driven to realize a negative Fresnel lens, so that the glass lens 24a ( Figure 1 ) or adjustable lens 24b ( Figure 2 ) to compensate for the impact caused.

[0088] Different microscope objectives also typically have different pupil positions. To correct for these pupil position distortions when changing microscope objectives, movable optical components, such as movable collimators, are typically used. The solutions proposed here, which are primarily based on compensating for quadratic phases using, in particular, variable field lenses, can also be used to perform the necessary corrections to the pupil position. For example, the adjustable lens 24b is preferably also used and can be configured to adjust the pupil position of different microscope objectives.

[0089] Likewise, in both embodiments, the first spatial light modulator 15 and / or the second spatial light modulator 19 can also be driven to adjust the pupil position of different microscope objectives to realize a Fresnel lens. In the first spatial light modulator 15, this may be limited only in terms of optical quality.

[0090] The present invention provides a flexible multimodal illumination system which is based on the use of a spatial light modulator and has improved properties with regard to its optical performance, without compromising the multimodality.

[0091] Reference Signs List

[0092] 1 sample

[0093] 10 Light source, laser

[0094] 11 optical fiber

[0095] 12 lenses

[0096] 13 Excitation light

[0097] 14Phase modulator = phase mask, SLM

[0098] 15 first spatial light modulator, first half of phase modulator 14, pupil plane

[0099] 16 The excitation light 13 is emitted from the phase modulator 14 toward the direction of the reflector 17

[0100] 17 reflector

[0101] 18 The excitation light 13 reflected from the reflector 17

[0102] 19 Second spatial light modulator 19, second half of phase modulator 14, intermediate image plane

[0103] 20 The excitation light 13 emitted from the phase modulator 14 toward the microscope

[0104] 21 lenses

[0105] 22 pupil plane

[0106] 23 lens, relay optical device formed together with lens 21

[0107] 24a glass lens, can be moved into and out of the light path

[0108] 24b Adjustable lens, adaptive lens

[0109] 25 Intermediate image plane

[0110] 26 tube lens

[0111] 27 Main beam splitter

[0112] 28 Back focal plane of microscope objective 29

[0113] 29 microscope objectives

[0114] 30 sample plane, object plane, focal plane of microscope objective 29

[0115] 31 Sample holding part

[0116] 32 Light emitted from pattern 1, here reflected back

[0117] 33 radiation filter

[0118] 34 Radiated light, filtered by radiation filter 33

[0119] 40 Tube lens converter in the detection light path

[0120] 41 tube lens

[0121] 50 cameras

[0122] 90 Control Unit (PC)

[0123] 100 Embodiments of the microscope according to the present invention

[0124] 200 Embodiments of the microscope according to the present invention

[0125] References

[0126] [1]Paolo Pozzi et al.: High-throughput spatial light modulation two-photon microscopy for fast functional imaging, Pozzi; In Neurophotonics Vol.2, S. 015005-1 (2015)

[0127] [Paolo Pozzi et al., “High-throughput spatially light-modulated two-photon microscopy for rapid functional imaging,” Neurophotonics, vol. 2, pp. 015005–1 (2015)]

[0128] [2]Reto Fiolka: Clearer view for TIRF and oblique illumination; InOptics Express Vol. 24, S. (2016)

[0129] [By Reto Fiolka: "A Clearer View of TIRF and Oblique Illumination," Optics Express, Vol. 24 (2016)]

[0130] [3]Reto Fiolka, Markus Beck, and Andreas Stemmer: Structurediillumination in total internal reflection fluorescence microscopy using aspatial light modulator; In Optics Letters Vol. 33, S. 1629 (2008)

[0131] [Reto Fiolka, Markus Beck, and Andreas Stemmer: "Structured-Illumination Microscopy in Total Internal Reflection Fluorescence Microscopy Using a Spatial Light Modulator," Optics Express, vol. 33, p. 1629 (2008)]

[0132] [4]Shao L.; Kner P.; Hesper E.; Gustafsson Mats G. L: Super-resolution 3D-microscopy of live whole cells using structured illumination; In Nature Methods, Vol. 8 (2011)

[0133] [Shao L.; Kner P.; Hesper E.; Gustafsson Mats G. L., “Super-resolution three-dimensional microscopy of living whole cells using structured light illumination,” Nature Methods, Vol. 8 (2011)]

Claims

1. A microscope (100), comprising: a light source (10), the light source being configured to emit excitation light (13), an illumination beam path having a microscope objective (29), which serves to guide the excitation light onto and / or into the sample (1) to be examined, a detector (50) for detecting detection light (32) emitted by the sample (1) due to illumination by the excitation light (13), a detection beam path for guiding the detection light (32) to the detector (50), the detection beam path comprising the microscope objective (29) or a further microscope objective, a first spatial light modulator (15), which is arranged in or near a pupil plane of the illumination light path, and a control unit (90) for driving at least the first spatial light modulator (15), the control unit being configured to drive the first spatial light modulator (15) to at least partially realize a lens function, It is characterized in that A wavefront manipulator (14, 24a, 24b) is arranged and / or formed in at least one intermediate image plane (19, 25) of the illumination light path or near at least one intermediate image plane (19, 25) to compensate for the wavefront distortion of the excitation light caused by the first spatial light modulator (15).

2. The microscope according to claim 1, It is characterized in that A second spatial light modulator (19) is arranged downstream of the beam of the first spatial light modulator (15) in or near an intermediate image plane of the illumination light path, and The control unit (90) is also configured to actuate the second spatial light modulator (19), in particular to realize an illumination pattern for the specimen (1).

3. The microscope according to claim 2, It is characterized in that The control unit (90) is configured to drive the second spatial light modulator (19) to exhibit a grating outside a desired illumination area, the period of the grating being selected so that the +1st and / or -1st order diffracted excitation light is located outside the area propagating to the specimen (1) in at least one pupil plane (22, 28).

4. The microscope according to any one of claims 1 to 3; It is characterized in that The optical axis of the illumination beam path defines an axial spatial direction (z) and two lateral spatial directions (x, y), and The wavefront distortion to be compensated is a quadratic phase term of the excitation light (13) in the coordinates of the lateral spatial direction (x, y).

5. The microscope according to any one of claims 1 to 4, It is characterized in that At least one wavefront manipulator (14, 24a, 24b) achieves a positive focal length.

6. The microscope according to any one of claims 1 to 5, It is characterized in that The wavefront manipulator has a glass lens (24a) with a positive focal length, or is realized by a glass lens (24a) with a positive focal length.

7. The microscope according to claim 6, It is characterized in that A changeover device is provided for introducing the glass lens (24a) into the illumination beam path or removing it from the illumination beam path.

8. The microscope according to claim 7, It is characterized in that The conversion device has a plurality of glass lenses with different focal lengths, and The transformation device can be used to introduce one of the glass lenses into the intermediate image plane (25) or in the vicinity of the intermediate image plane (25).

9. The microscope according to any one of claims 1 to 8, It is characterized in that The wavefront manipulator has an adjustable lens (24b) or is realized by an adjustable lens (24b).

10. The microscope according to claim 9, It is characterized in that The adjustable lens (24b) can be adjusted into a neutral position.

11. The microscope according to any one of claims 1 to 10, It is characterized in that The wavefront manipulator has a Fresnel lens or is realized by a Fresnel lens.

12. The microscope according to any one of claims 2 to 11, It is characterized in that The control device (90) is configured to drive the second spatial light modulator (19) to at least partially realize a Fresnel lens.

13. The microscope according to any one of claims 2 to 12, It is characterized in that The control device (90) is configured to drive the second spatial light modulator (19) so that a quadratic phase term of the excitation light (13) in the coordinates of the lateral spatial direction is partially or completely compensated by the second spatial light modulator (19).

14. The microscope according to any one of claims 2 to 13, It is characterized in that The first spatial light modulator (15) is formed by a first sub-area of ​​a spatial light modulator (14), and the second spatial light modulator (19) is formed by a second sub-area of ​​the same spatial light modulator (14).

15. A method for microscopy, wherein: • guiding the excitation light (13) via an illumination beam path with a microscope lens (29) onto and / or into the specimen (1) to be examined, • guiding the detection light (32) emitted by the sample (1) due to illumination by the excitation light (13) to a detector (50) via the microscope objective (29) or another microscope objective and detecting the detection light using the detector, • directing the excitation light (13) through a first spatial light modulator (15) arranged in or near a pupil plane of the illumination light path, and • driving the first spatial light modulator (15) to at least partially realize the lens function, It is characterized in that Wavefront distortion produced by the first spatial light modulator (15) is compensated by means of at least one wavefront manipulator (14, 24a, 24b) arranged or formed in or near an intermediate image plane (19, 25).

16. The method according to claim 15, It is characterized in that Downstream of the beam of the first spatial light modulator (15), the excitation light (13) is guided through a second spatial light modulator (19) arranged in or near an intermediate image plane of the illumination light path.

17. The method according to claim 16, It is characterized in that The second spatial light modulator (19) is driven to display a grating outside the desired illumination area, the period of the grating being selected so that the excitation light of the +1st and / or -1st order diffraction is located outside the area propagating to the sample (1) in at least one pupil plane (22, 28).

18. The method according to any one of claims 15 to 17, It is characterized in that In the step of compensating, a plurality of wavefront manipulators (14, 24a, 24b) act in coordination.

19. The method according to claim 18, It is characterized in that The wavefront manipulators are respectively arranged or formed in different intermediate image planes or near different intermediate image planes.

20. The method according to any one of claims 15 to 19, It is characterized in that The compensation step is provided partly or completely by a glass lens (24a) acting as a wavefront manipulator.

21. The method according to any one of claims 15 to 20, It is characterized in that The compensation step is provided partly or completely by an adjustable lens (24b) acting as a wavefront manipulator.

22. The method according to claim 21, It is characterized in that The adjustable lens (24b) is used to adjust different pupil positions of different microscope objectives.

23. The method according to any one of claims 15 to 22, It is characterized in that The step of compensating is provided partly or completely by driving the second spatial light modulator (19) to produce a Fresnel lens with a positive focal length.

24. The method according to any one of claims 15 to 23, It is characterized in that In order to adjust different pupil positions of different microscope objectives, the first spatial light modulator (15) and / or the second spatial light modulator (19) is driven to realize a Fresnel lens.

25. The method according to any one of claims 15 to 24, It is characterized in that The first spatial light modulator (15) and / or the second spatial light modulator (19) are driven to generate an illumination pattern for the sample (1).

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