Multi-modal microscope and method for microscopy

Abstract

The invention relates to a multi-modal microscope and a method for microscopy. The multi-mode microscope has: a light source for emitting excitation light; an illumination beam path having a microscope objective for guiding excitation light onto and / or into the sample to be examined, at least one first spatial light modulator being present in the illumination beam path in order to manipulate the excitation light; the detector is used for detecting the detection light emitted by the sample under the illumination of the exciting light; comprising a microscope objective or a further microscope objective for guiding the probe light onto the probe; and a control unit at least for actuating the first spatial light modulator. According to the invention, there is a switchable optical functional component which can be switched at least into a first switching state and into a second switching state, and depending on which switching state the first spatial light modulator is located in or near the intermediate image plane of the illumination beam path, and wherein the second spatial light modulator is located in or near the intermediate image plane of the illumination beam path. Or is located in or near the pupil plane of the illumination light path.

Description

Technical Field

[0001] The invention relates to a multimodal microscope according to the preamble of claim 1 and to a method for microscopy according to the preamble of claim 17 . Background Art

[0002] A multimodal microscope according to the category has at least 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, wherein at least one first spatial light modulator is arranged in the illumination light path for manipulating the excitation light; 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; and a control unit, which is used at least to drive the first spatial light modulator.

[0003] The method for microscopy according to the category performs at least the following steps: guiding excitation light onto a sample to be examined and / or into the sample to be examined via an illumination light path having a microscope objective; manipulating the excitation light using at least a first spatial light modulator in the illumination light path; guiding detection light emitted from the sample due to illumination with the excitation light onto a detector via a detection light path including the microscope objective or another microscope objective and detecting the detection light by the detector.

[0004] Microscopes of the generic type and methods of the generic type are known in numerous embodiments and variants, for example from DE 10 2022103 051 A1.

[0005] In this microscope, it is known to adjust the excitation light to the required conditions in the illumination beam path by using a spatial light modulator (SLM) which can be arranged in the pupil plane or the intermediate image plane of the illumination beam path, so as to be suitable for the respectively desired microscopy technique. In particular, it is known to adjust the phase of the excitation light in the intermediate image plane and / or in the pupil plane in a respectively desired manner in order to realize different microscopy measures. Microscopes are also known in which a plurality of different microscopy measures can be carried out in principle with one and the same device, see references

[20] ,

[21] . In this context, these microscopy measures can also be referred to as microscopy techniques or microscopy modes. However, in order to change from one microscopy measure to another, relatively complex replacement measures are required in the known microscopes, at least for certain pairs of microscopy measures between which switching is to be performed. Summary of the Invention

[0006] The object of the present invention is to provide a multimodal microscope of the aforementioned type that is suitable for carrying out a variety of different microscopy methods. Furthermore, a microscopy method that can be carried out in a variety of variants should be described. The switching or conversion between these methods should be as simple as possible.

[0007] This object is achieved by a multimodal microscope having the features of claim 1 and a method for microscopy having the features of claim 17 .

[0008] According to the present invention, a multimodal microscope of the above-mentioned type is improved in the following manner, namely, there is a switchable optical functional component, which can be switched into at least a first switching state and a second switching state, and depending on which switching state the optical functional component is in, the first spatial light modulator is either located in or near the intermediate image plane of the illumination light path, or in the pupil plane of the illumination light path or near the pupil plane.

[0009] According to the invention, a method for microscopy of the type described above is improved in that, before performing a microscopic measurement, a switchable optical functional component is switched either into a first switching state or into a second switching state, wherein, depending on which switching state the optical functional component is in, a first spatial light modulator is located either in or near an intermediate image plane of the illumination beam path or in or near a pupil plane of the illumination beam path, and at least one of the spatial light modulators is then controlled to provide the desired illumination pattern.

[0010] Preferred embodiments of the multimodal microscope according to the invention and advantageous variants of the method according to the invention are explained below, in particular in conjunction with the dependent claims and the drawings.

[0011] The multimodal microscope according to the invention can be particularly suitable for carrying out the method for microscopy according to the invention. The method according to the invention can be carried out particularly with the multimodal microscope according to the invention.

[0012] The main idea of ​​the present invention is to provide a switchable or convertible component in the illumination beam path, which allows the position in which the first spatial light modulator is arranged to be selectively switched to the pupil plane function or the intermediate image plane function, respectively. This switchable component is referred to in the present application as an optical functional component.

[0013] In a more complex variant, the optical functional component can be used such that for other positions, at which further second spatial light modulators are arranged, the function of the exit pupil plane or the function of the intermediate image plane, respectively, can also be selectively set.

[0014] The present invention provides a multimodal microscope that can be configured to perform a variety of different microscopy modes, with easy switching between the different microscopy modes. Essentially, this requires only manipulating the optical functional components and selecting a suitable control method for the first spatial light modulator and / or any additional spatial light modulators. The microscopy method according to the present invention can also be implemented in a large number of design variants, easily switching between them. The equipment expenditure is relatively low.

[0015] For the microscope according to the present invention, lasers are primarily considered as light sources, but other light sources are also feasible. The excitation light is preferably electromagnetic radiation in the visible 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.

[0016] 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). These optical components are used to direct the excitation light from the light source onto the specimen to be examined. Beam-modifying components also include dispersive and, in particular, diffractive elements. In principle, commercially available microscope objectives can be used.

[0017] The manipulation of the excitation light in the illumination beam path by means of the first spatial light modulator or a possibly present further spatial light modulator, in particular the second light modulator, can be used in particular to provide a desired illumination pattern.

[0018] In principle, the first spatial light modulator can be the only spatial light modulator in the illumination beam path. However, it is also possible to arrange a second spatial light modulator in the illumination beam path, and when the first spatial light modulator is located in or near the intermediate image plane, the second spatial light modulator is located in or near the pupil plane, and when the first spatial light modulator is located in or near the pupil plane, the second spatial light modulator is located in or near the intermediate image plane.

[0019] 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.

[0020] In principle, it is possible for at least one, a plurality of, or each of the first, second, and third spatial light modulators to be formed by an amplitude modulation spatial light modulator. Advantageously, however, at least one, a plurality of, or each of the first, second, and third spatial light modulators to be formed by a phase modulation spatial light modulator. Phase modulation spatial light modulators are generally preferred due to their lower optical loss.

[0021] Then, at least one, multiple, or each of the first, second, and third spatial light modulators can be formed by a reflective spatial light modulator. However, it is also feasible that at least one, multiple, or each of the first, second, and third spatial light modulators can be formed by a transmissive spatial light modulator.

[0022] For example, the first spatial light modulator, the second spatial light modulator, and the spatial light modulator, at least one, multiple, or each of these components 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 = Deformable Mirror).

[0023] The term "pupil plane" refers to a plane, in particular 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, in particular 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.

[0024] 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.

[0025] In order to change the location on or in the specimen that is to be illuminated by the excitation light, a scanner can be present in the illumination beam path. The scanner can, for example, comprise a galvanometer mirror or a MEMS mirror, which can operate in a quasi-static or resonant manner. It is particularly preferred that the scanning mirror be arranged in or near the pupil plane of the illumination beam path. However, a scanner is not absolutely necessary for implementing the present invention.

[0026] The term "control unit" refers to all hardware and software components that cooperate with the components of the microscope according to the invention to realize the prescribed functions of these components. In particular, the control unit can include a computing device, such as a PC, and a camera control unit. The computing resources of the control unit can be distributed among multiple computers and, if necessary, distributed 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 image data from the detector and can also be used and set up to drive the light source. According to the invention, the control unit is set up to drive the first spatial light modulator. If further spatial light modulators are present, the control unit can also be suitably set up to drive these further light modulators.

[0027] Probe light is electromagnetic radiation emitted by the sample when illuminated by the excitation light. "Emitted" means that the probe light originates from the sample. The probe light can be reflected from the sample or transmitted through the illuminated sample. The probe light is typically red-shifted fluorescence from a fluorescent marker used in sample preparation, compared to the excitation light.

[0028] Commercially available detectors can be used as detectors. Semiconductor detectors are particularly preferred. The detector can particularly be a two-dimensional spatially resolved detector, that is, a camera, such as a CCD, CMOS, or SPAD array camera. However, for certain microscopy techniques, such as those in which an illumination point or line is scanned over or through a specimen, it is feasible to use point detectors, such as photomultiplier tubes, or line detectors, such as CCD or CMOS arrays or linear SPAD arrays.

[0029] 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), with which the detection 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, in which 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, in reflected light microscopes in which the specimen is illuminated obliquely, or in light-sheet microscopes.

[0030] In particular, a switchable optical functional component can be switched between exactly two switching states. However, it is also possible that further switching states may exist. Examples of switchable optical functional components will be explained below. Particularly preferably, the control unit can be configured to switch or convert the optical functional component.

[0031] The optical functional component can realize a switchable relay system, optionally in cooperation with other components installed in the illumination beam path.

[0032] The optical functional component may be arranged in the illumination light path downstream of the beam of the first spatial light modulator or may be activatable in the illumination light path downstream of the beam of the first spatial light modulator. Preferably, in the first switching state and in the second switching state, at least a portion of the optical functional component is arranged in the illumination light path downstream of the beam of the first spatial light modulator.

[0033] In a first preferred embodiment, the optical functional component comprises a transformation device comprising a first lens and a second lens, or the optical functional component is implemented by a transformation device comprising a first lens and a second lens. The transformation device allows the first lens or the second lens, in particular either the first lens or the second lens, to be introduced into the illumination beam path. Advantageously, in a first switching state, the first lens introduced into the illumination beam path is spaced approximately twice the focal length of the lens from the first spatial light modulator and from an intermediate image plane downstream of the beam of the first lens. Furthermore, in a second switching state, the second lens is spaced approximately as far from the first spatial light modulator and from an intermediate image plane downstream of the beam of the second lens as the focal length of the second lens, and the second lens, together with the tube lens, can form a 4f system that images the plane of the first spatial light modulator onto the back focal plane of the microscope objective.

[0034] In a further preferred embodiment, the optical functional component comprises an adjustable lens, or the optical functional component can be realized by an adjustable lens. In this case, in a first switching state, the adjustable lens is advantageously spaced apart from the first spatial light modulator and from an intermediate image plane downstream of the beam of the adjustable lens, each approximately equal to twice the adjusted focal length of the adjustable lens. Furthermore, in a second switching state, the adjustable lens is spaced apart from the first spatial light modulator and from an intermediate image plane downstream of the beam of the adjustable lens, each approximately equal to the adjusted focal length of the adjustable lens, and the adjustable lens can form a 4f system together with the tube lens, which images the plane of the first spatial light modulator onto the back focal plane of the microscope objective.

[0035] These two variants can be combined in a preferred variant, which is characterized in that the optical functional component has an adjustable lens assembly, which has an adjustable lens and a lens with a fixed focal length, or is implemented by a lens assembly having an adjustable lens and a lens with a fixed focal length. In this case, in a first switching state, the distance between the adjustable lens assembly and the first spatial light modulator and the distance between the adjustable lens assembly and the intermediate image plane downstream of the beam of the adjustable lens assembly are each approximately twice the adjusted focal length of the adjustable lens assembly. In addition, in a second switching state, the distance between the adjustable lens assembly and the first spatial light modulator and the distance between the adjustable lens assembly and the intermediate image plane downstream of the beam of the adjustable lens assembly are each approximately the same as the adjusted focal length of the adjustable lens assembly, and the adjustable lens assembly can form a 4f system together with the tube lens, which images the plane of the first spatial light modulator onto the back focal plane of the microscope objective.

[0036] In the case of a first switching state in which the distance between the first lens introduced into the illumination beam path and the first spatial light modulator and the intermediate image plane downstream of the beam of the first lens is approximately twice the focal length of the lens, this is also referred to as 2f-2f imaging.

[0037] This also applies to the case of the first switching state in which the distance between the adjustable lens and the first spatial light modulator and the distance between the adjustable lens and the intermediate image plane downstream of the beam of the adjustable lens is approximately twice as large as the adjusted focal length of the adjustable lens; and the same applies to the case in which the distance between the adjustable lens assembly and the first spatial light modulator and the distance between the adjustable lens assembly and the intermediate image plane downstream of the beam of the adjustable lens assembly is approximately twice as large as the adjusted focal length of the adjustable lens assembly.

[0038] Due to the 2f-2f imaging, a quadratic phase is introduced, which can be corrected, for example, by means of a lens with a positive focal length (i.e., a field lens) in the intermediate image plane. This lens with a positive focal length is not absolutely necessary, but can be advantageous if vignetting of the beam downstream of the intermediate image plane is to be avoided.

[0039] In a further preferred embodiment of the microscope according to the invention, the optical functional component is arranged at least partially, in particular completely, in the illumination beam path downstream of the beam of the spatial light modulator in either the first switching state or the second switching state, while the optical functional component is not part of the illumination beam path in the respective other state. For example, the optical functional component can form an alternative beam path having at least one lens, the alternative beam path having a pupil plane and an intermediate image plane, wherein the excitation light is guided via this alternative beam path in either the first switching state or the second switching state in the direction of the microscope objective.

[0040] In order to selectively guide the excitation light via an alternative beam path of the illumination beam path, the optical functional component can have a first switching device, in particular a first adjustable reflector, and in order to couple the excitation light from the alternative beam path back into the main part of the illumination beam path, the optical functional component can have a second switching device, in particular a second adjustable reflector. These reflectors can be ordinary reflectors, but can also be formed by reflecting prisms.

[0041] The first reflector and the second reflector can be introduced into and removed from the illumination light path together or separately, while other components of the optical functional assembly can remain unchanged when the optical functional assembly is switched. For example, the reflector can be selectively pushed into and pushed out of the illumination light path, or selectively swung into and out of the illumination light path.

[0042] However, a particularly preferred embodiment of the microscope according to the invention is characterized in that the optically active components of the optical component are rigidly connected to one another when the optical component is introduced into the illumination beam path and when the optical component is removed. This has the advantage that the integrity of the optical component itself, and therefore its optical properties, is not affected when the optical component is replaced.

[0043] A mechanical drive may be advantageously provided for switching the optical functional component. The control unit may be configured to control the drive. The drive may be a rotary drive, which allows at least a portion of the optical functional component to be moved into or out of the illumination light path in an oscillating motion. Particularly preferably, the drive may be a linear drive, which allows at least a portion of the optical functional component to be moved into or out of the illumination light path in a linear motion.

[0044] In an advantageous improvement of the microscope according to the invention, the optical functional component can be introduced into the illumination beam path at the location of the pupil plane and is located between the following two lenses: when the optical functional component is located outside the illumination beam path, the two lenses form an optical relay device, wherein the relay device images the plane in which the first spatial light modulator or the second spatial light modulator is arranged into an intermediate image plane of the illumination beam path.

[0045] Alternatively, the optical functional component can be capable of being introduced into the illumination light path at the location of an intermediate image plane and be located between the tube lens and the lens, wherein, when the optical functional component is located outside the illumination light path, the tube lens and the lens form an optical relay device that images the plane in which the first spatial light modulator or the second spatial light modulator is arranged into the pupil plane of the illumination light path.

[0046] In a first particularly preferred variant of the method according to the invention, the optical functional component is actuated in such a way that the first spatial light modulator is located in or near the pupil plane.

[0047] The first spatial light modulator can then be driven to behave like a lens with a positive focal length. When this driving is performed, it is advantageous if 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. The wavefront distortions to be compensated for are, in particular, quadratic phase terms of the excitation light in the transverse spatial directions (x, y). The axial spatial direction (z) is defined by the optical axis of the illumination beam path, while the two transverse spatial directions are spatial directions perpendicular to each other and to the axial spatial direction. Preferably, at least one wavefront manipulator can have a positive focal length.

[0048] For example, to compensate for the quadratic phase term of the excitation light in the transverse spatial directions (x, y), a lens with a positive focal length can be arranged in or near an intermediate image plane of the illumination beam path. To selectively introduce or remove a glass lens from the illumination beam path at the location of the intermediate image plane, a switching device can be provided, such as one having a suitable mechanical displacement or pivoting mechanism. Removing the glass lens from the illumination beam path may be desirable or necessary when no lens is present on the first spatial light modulator.

[0049] Additionally or alternatively, the wavefront manipulator may comprise one or more of the following components, or be partially or completely implemented by one or more of the following components: an adjustable lens with a positive focal length, a Fresnel lens. The transformation device may also comprise a plurality of glass lenses with different focal lengths, and one of the glass lenses may be selectively introduced into or near the intermediate image plane using the transformation device. When an adjustable lens is used as a wavefront manipulator for compensating for the secondary phase, it is advantageous if the adjustable lens can be adjusted to a neutral position. When no lens is represented in the pupil plane using the first spatial light modulator, the adjustable lens can be suitably adjusted to a neutral position. Additionally or alternatively, it is also feasible that the control unit is configured to drive a second spatial light manipulator located in the intermediate image plane downstream of the beam of the first spatial light manipulator, in order to at least partially implement the Fresnel lens for compensating for the secondary phase.

[0050] Point-shaped or multi-point illumination can be achieved by displaying a so-called tilt and defocus phase pattern on the first spatial light modulator in the pupil plane. For line illumination and, if necessary, light sheet illumination, a cylindrical lens phase pattern can be displayed on the first spatial light modulator in the pupil plane.

[0051] For example, a first spatial light modulator in the pupil plane can be driven to manipulate particles in the sample to realize light trapping and / or optical tweezers. The basic principle of these technologies is that by strongly focusing light in the sample, a strong field gradient is generated. Under the strong field gradient, the movable particles in the sample will move towards the light-intensive area. In this way, micro-manipulation of particles can be achieved. For example, a first spatial light modulator can be used to generate multiple focal volumes laterally and axially within the sample. Related examples and details are described in [1] and [2].

[0052] In other microscopy techniques, a first spatial light modulator in the pupil plane can be controlled so that only specific parts of the specimen are illuminated. Through targeted optical manipulation or stimulation, biological and / or chemical processes in the specimen can be triggered. For example, in the so-called FRAP method (Fluorescence Recovery After Photobleaching), regions of the specimen are selectively bleached through optical manipulation, and the temporal course of fluorescence in the bleached region is subsequently examined. This allows inferences about biological processes. The microscopy technique known as optogenetics uses targeted stimulation of neurons to examine processes in the brain (e.g., the mouse brain). In these microscopy methods, the illumination pattern must be matched to the region to be manipulated / stimulated, the “region of interest” (ROI). By setting an appropriate phase pattern on the first spatial light modulator, the desired intensity pattern in the intermediate image plane and, therefore, in the specimen plane, can be achieved in essentially any desired manner. The phase pattern required in each case can be calculated, for example, using the Gerchberg-Saxton algorithm (Computer Generated Holograms) or the GPC method (GPC = Generalized Phase Contrast). Examples and further details can be found in the literature [3], [4] and [5].

[0053] Furthermore, it is possible to control the first spatial light modulator in the pupil plane to correct aberrations caused by the optical components of the microscope and / or by the sample. This makes it possible to flexibly cope with deteriorations in the optical imaging quality of the microscope caused by system-induced aberrations, for example caused by optical functional components but especially also caused by different samples. For this aberration correction, the first spatial light modulator can be controlled using a linear combination of appropriate Zernike modes. Details and examples are described in the literature [6] and [7]. Even when the first spatial light modulator is primarily used for other illumination methods, this aberration correction can be performed due to the linear superposition of the contributions made by the electromagnetic field.

[0054] Another variant of the method according to the invention is characterized in that the first spatial light modulator is driven to defocus the excitation light in the sample. As an alternative to mechanically moving the microscope objective and / or the sample in order to change the focal plane of the microscope objective within the sample, defocusing is performed optically with the aid of a first spatial light modulator in the pupil plane. This technique is also known as remote focusing. For example, for this purpose, a phase pattern that essentially corresponds to the phase pattern of a Fresnel lens can be displayed on the first spatial light modulator. In addition or as an alternative, it is also possible to drive the first spatial light modulator to display a Kinoform lens. Relevant details and examples are described in the document [8].

[0055] In another variant of the method according to the invention, a first spatial light modulator in the pupil plane can be driven for STED microscopy in order to shape the excitation light into a doughnut-shaped beam in the sample. In the STED method (STED = Stimulation Emission Depletion), an optical resolution greater than the resolution given by the diffraction limit (i.e., the Abbe limit) can be achieved. The main principle here is to switch the fluorescent molecules on and off in a targeted manner. In the STED method, a laser is then scanned over the sample, which excites the dye molecules with a doughnut-shaped beam superimposed on the excitation laser, so that all molecules except those in the center of the doughnut-shaped beam are transferred to a non-fluorescent ground state due to stimulated emission. The doughnut-shaped beam necessary for the STED method can be generated using a spatial light modulator in the pupil plane, wherein a vortex-type phase pattern must then be present on the light modulator. Details and examples are described in [9].

[0056] In a further variant of the method according to the invention, the optical functional component is driven in such a way that the first spatial light modulator is located in or near an intermediate image plane.

[0057] For example, a first spatial light modulator in the intermediate image plane can be driven to produce an illumination pattern in a region of the back focal plane of the microscope objective that is so far from the optical axis that the TIRF condition is satisfied for the excitation light in the specimen. In the TIRF method (TIRF = Total Internal Reflection Fluorescence), the excitation light is incident on the specimen at an angle of incidence relative to the optical axis that is greater than the angle at which total reflection occurs when the light passes from an optically dense medium (i.e. a medium with a higher refractive index n) to an optically rarefaction medium (i.e. a medium with a lower refractive index n). This results in fluorescence being excited in a thinner region near the coverslip and thus achieving a high surface sensitivity. This is also called sectioning. A large angle of incidence relative to the optical axis corresponds to a point in the pupil of the objective lens that is relatively far from the optical axis. In a microscope, the TIRF condition can be achieved by flipping the mirror or flipping the linear phase pattern in a plane conjugate to the intermediate image. Details and examples are described in

[11] .

[0058] In another variant of the method according to the invention, a first spatial light modulator in the intermediate image plane is driven to display a phase grating for performing SIM microscopy. The SIM method (SIM = Structured Illumination Microscopy) uses grating illumination and subsequent computer-based calculations to achieve resolution improvement. The grating illumination is generated by a phase grating in a plane conjugate to the intermediate image. Details and examples are described in

[12] .

[0059] Due to the linear superposition of the contributions of the electromagnetic field, a variant is also possible in which the TIRF method is combined with the SIM method. This method is called TIRF-SIM and uses structured illumination to achieve resolution improvement and exploits the surface sensitivity of TIRF, i.e., slicing. Specifically, a first spatial light modulator is driven to represent a phase grating in the intermediate image plane, wherein the diffraction orders of the grating are located far enough from the optical axis in the back focal plane of the microscope objective that the TIRF condition is met in the specimen. Details and examples are described in

[13] and

[14] .

[0060] Light sheet illumination can also be achieved using a spatial light modulator, in particular a phase modulator, arranged in the intermediate image plane. This light sheet illumination is called a lattice light sheet if the phase modulator is driven with a checkerboard-like phase pattern whose rows taper towards the edges.

[0061] In another advantageous variant of the method according to the invention, it is possible to arrange a second spatial light modulator in the illumination beam path and to either drive the optical functional component such that the first spatial light modulator is located in or near the pupil plane and the second spatial light modulator is located in or near the intermediate image plane, or to drive the optical functional component such that the first spatial light modulator is located in or near the intermediate image plane and the second spatial light modulator is located in or near the pupil plane. The second spatial light modulator can be arranged downstream of the beam of the first spatial light modulator or vice versa.

[0062] The intensity of the excitation light in the specimen can be varied in such a way that a first spatial light modulator or a second spatial light modulator positioned in or near the pupil plane or in or near the intermediate image plane diffracts at least a certain portion of the excitation light into an area outside the area propagated by the optical system, wherein the portion is varied by varying the diffraction efficiency of the spatial light modulator used in each case, and wherein the diffraction efficiency is varied by varying the contrast of the phase grating set in the spatial light modulator used in each case. Details and examples are described in

[10] ,

[15] ,

[16] . Due to the linear superposition of the contributions of the electromagnetic field, such intensity modulation can be performed even when the first spatial light modulator or the second spatial light modulator, respectively, is primarily used for other illumination methods.

[0063] Finally, the method according to the invention can also be used to implement the GPC method (GPC = General-Phase-Contrast). In this case, a first spatial light modulator in the intermediate image plane can be driven to represent the phase pattern of the image to be imaged, and a second spatial light modulator can be driven to represent a phase contrast filter, for example a rectangular or circular phase shift mask, which has a phase deviation of π for low spatial frequencies. The intensity distribution of the image to be imaged can then be recovered from the interference of the phase-shifted low spatial frequencies and the unmodulated higher spatial frequencies. Examples and details are described in

[18] ,

[19] .

[0064] The intensity and phase of the excitation light in the specimen can be manipulated using a first spatial light modulator, for example, in the pupil plane and / or using a second spatial light modulator, for example, in the intermediate image plane. Phase modulation can be performed in the illumination beam path first in the pupil plane and then downstream in the intermediate image plane. This is described, for example, in the literature

[17] . It is also possible to perform phase modulation in the illumination beam path first in the intermediate image plane and then downstream in the pupil plane. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Further advantages and features of the present invention are explained below with reference to the accompanying drawings, in which:

[0066] Figure 1 : shows a schematic diagram of a first embodiment of a microscope according to the present invention;

[0067] Figure 2 : shows a schematic diagram of a second embodiment of a microscope according to the present invention;

[0068] Figure 3 : shows a schematic diagram of a third embodiment of a microscope according to the present invention;

[0069] Figure 4 : shows a schematic diagram of a fourth embodiment of a microscope according to the present invention in a first operating state;

[0070] Figure 5 :shows the second operating state Figure 4 microscope;

[0071] Figure 6 : shows a schematic diagram of a fifth embodiment of a microscope according to the present invention in a first operating state;

[0072] Figure 7 :shows the second operating state Figure 6 microscope;

[0073] Figure 8 : shows a schematic diagram of a sixth embodiment of a microscope according to the present invention in a first operating state; and

[0074] Figure 9 :shows the second operating state Figure 8 Microscope in.

[0075] In the figures, identical and identically acting parts are generally provided with the same reference symbols. DETAILED DESCRIPTION

[0076] The first embodiment of the multimodal microscope 100 according to the present invention refers to Figure 1 Then, the variant of microscope 100 is referred to Figure 2 and Figure 3 describe.

[0077] Figure 1 The multimodal microscope 100 schematically shown in FIG is used to examine a sample 1 and first has a light source 10 for emitting excitation light 13 and an illumination beam path for guiding the excitation light 13 onto the sample 1 to be examined. In order to manipulate the excitation light 13, at least one first spatial light modulator 15 is present in the illumination beam path. Figure 1In the embodiment shown in FIG, the first spatial light modulator 15 is the only spatial light modulator and can preferably be a reflective phase-modulated SLM, such as an LCOS display. According to the present invention, the illumination light path ultimately comprises a microscope objective 30. In the embodiment shown, the illumination light path also comprises a tube lens 23 and a primary beam splitter 24.

[0078] The detection light 32 emitted by the sample 1 due to the illumination by the excitation light 13 is detected by the detector 40 according to the present invention. In the embodiment shown, the detector 40 can be a camera, that is, a pixelated two-dimensional spatial resolution detector. However, this is not mandatory. A detector composed of a single pixel (such as a photomultiplier tube or SPAD) or a row of pixels (such as a CMOS, CCD or SPAD linear array) can also be used. The detection light 32 is guided to the detector 40 via a detection light path. In the embodiment shown, the microscope objective 30 is part of the detection light path, but this is not mandatory. The detection light path can also have a separate microscope objective. In the embodiment shown, the detection light path also has a main beam splitter 24, a radiation filter 34 and a tube lens 36. The detector 40 can be located in the intermediate image plane of the detection light path.

[0079] A control unit 90, such as a PC, is provided to control at least the first spatial light modulator 15. The control unit 90 can also be used to control other components of the microscope 100 and to evaluate the measurement data of the camera 40. The control unit 90 is connected in an effective manner, for example via cables, to the components that need to be controlled by the control unit and whose measurement data need to be evaluated by the control unit (not shown in the figure).

[0080] According to the present invention, Figure 1 The microscope 100 includes a switchable optical functional component 70, which can be switched to at least a first switching state and a second switching state. Figure 1 In the embodiment shown in , the optical functional component is realized by a transformation device 70 having a first lens 71 and a second lens 72. In the optical functional component 70, the two switching states according to the invention are realized in the following manner, that is, the first lens 71 or the second lens 72 is introduced into the illumination beam path by means of the transformation device.

[0081] According to the invention, the first spatial light modulator 15 is located either in or near an intermediate image plane or in or near a pupil plane of the illumination beam path, depending on the switching state of the optical functional component 70 .

[0082] The excitation light 13 from the light source 10 first reaches the lens 12 via the optical fiber 11, which is used to collimate the excitation light. The collimated excitation light 13 then falls on the first spatial light modulator 15 and is reflected by the first spatial light modulator toward the first lens 71 located in the optical path. It then passes through the intermediate image plane 22 and is guided via the tube lens 23 and the main beam splitter 24 toward the microscope objective 30. The microscope objective guides the excitation light 13, for example, focusing it into the specimen plane 28 of the specimen 1. A specimen holder 26 is used to hold the specimen 1. The microscope structural component is indicated by the reference numeral 60.

[0083] exist Figure 1 In the situation shown in , the first lens 71 is located in the illumination beam path and a first switching state is achieved. The optical axis of the illumination beam path in the region of the first lens 71 is denoted by z. The distance between the first lens 71 and the first spatial light modulator 15 and the intermediate image plane 22 downstream of the beam from the first lens 71 are each approximately twice the focal length f1 of the lens 71. This means that the intermediate image plane 22 and the plane of the spatial light modulator 15 are optically conjugated in the first switching state, and therefore the spatial light modulator 15 is located in the intermediate image plane of the illumination beam path. In this switching state, all of the above-described methods can then be performed in which the excitation light is manipulated in the intermediate image plane. For example, the excitation light can be manipulated to implement a TIRF method or a SIM method or a combination thereof. Since the distance between the plane of the spatial light modulator 15 and the lens 71 and the distance between the intermediate image plane 22 and the lens are each approximately twice the focal length f1 of the lens 71, this is also referred to as 2f-2f imaging. A quadratic phase is introduced here, which can be corrected with the aid of a lens with a positive focal length in this intermediate image plane (i.e., a field lens). A lens with a positive focal length is not absolutely necessary, but can be advantageous if vignetting of the beam downstream of the intermediate image plane is to be avoided.

[0084] exist Figure 1 In the case where the second lens 72 is located in the illumination beam path (not shown), a second switching state is achieved. The distance between the second lens 72 and the first spatial light modulator 15 and the intermediate image plane 22 downstream of the second lens 72 is approximately the same as the focal length f2 of the second lens 72, and the second lens 72, together with the tube lens 23, forms a 4f system that images the plane of the first spatial light modulator 15 onto the back focal plane 25 of the microscope objective 30. This means that the back focal plane 25 of the microscope objective 30 and the plane of the spatial light modulator 15 are optically conjugated in the second switching state, and the spatial light modulator 15 is therefore located in the pupil plane of the illumination beam path.

[0085] The first spatial light modulator can then be controlled to exhibit a lens with a positive focal length. When this control is performed, as described above for 2f-2f imaging, a glass lens with a positive focal length is preferably positioned in the intermediate image plane 22, for example, to compensate for the quadratic phase term of the excitation light caused by the lens with a positive focal length on the first spatial light modulator in the transverse spatial directions x and y. Details are provided in the invention summary of the specification. When no lens is present on the first spatial light modulator 15, it may be desirable or necessary to remove the glass lens from the intermediate image plane 22.

[0086] Thus, in the second switching state, when the first spatial light modulator 15 is located in the pupil plane, optical tweezers or optical trapping can be realized, for example, and FRAP, STED and many other methods can be implemented. Details can be found in the summary of the description.

[0087] exist Figure 2 The second embodiment of the microscope 200 according to the present invention is shown in FIG. Figure 1 The microscope 100 is different only in the optical functional components. Instead of the transformation device 70 having two lenses 71 and 72, Figure 2 In the example, the optical functional component 73 is realized by an adjustable lens 74.

[0088] In the first switching state, i.e., the first setting state, the adjustable lens is set to a focal length f1. The distance between the adjustable lens 74 and the first spatial light modulator 15 and the intermediate image plane 22 downstream of the beam path of the adjustable lens 74 is approximately twice the set focal length f1 of the adjustable lens 74. This means that the intermediate image plane 22 and the plane of the spatial light modulator 15 are optically conjugated in the first switching state, and the spatial light modulator 15 is therefore located in the intermediate image plane of the illumination beam path. In this switching state, all of the above-described methods can be performed again, in which the excitation light is manipulated in the intermediate image plane.

[0089] In the second switching state, i.e., the second setting state, the adjustable lens is set to a focal length f2, which is twice as large as the focal length f1 set for the first switching state. The distance between the adjustable lens 74 and the first spatial light modulator 15 and the intermediate image plane 22 downstream of the beam path of the adjustable lens 74 is then approximately the same as the set focal length f2 of the adjustable lens 74. Together with the tube lens 23, the adjustable lens 74 forms a 4f system that images the plane of the first spatial light modulator 15 into the back focal plane 25 of the microscope objective 30. This means that in the second switching state, the back focal plane 25 of the microscope objective 30 and the plane of the spatial light modulator 15 are optically conjugated, so that the spatial light modulator 15 is located in the pupil plane of the illumination beam path. In this switching state, all of the above-described methods can then be performed again, in which the excitation light is manipulated in the pupil plane.

[0090] exist Figure 3 The third embodiment of the microscope 300 according to the present invention schematically shown in FIG. Figure 1 The microscope 100 is different only in the optical functional components. Instead of the transformation device 70 having two lenses 71 and 72, Figure 3 In the example of FIG. 5 , the optical function component is implemented by an adjustable lens assembly 75 having an adjustable lens 76 and a lens 77 with a fixed focal length.

[0091] In a first switching state, i.e., the first setting state, the adjustable lens 76 is set so that the lens assembly 75 as a whole achieves a focal length f1. The distance between the adjustable lens assembly 75 and the first spatial light modulator 15 and the intermediate image plane 22 downstream of the beam path of the adjustable lens assembly 75 is approximately twice the set focal length f1 of the adjustable lens assembly 75. This means that the intermediate image plane 22 and the plane of the spatial light modulator 15 are optically conjugated in the first switching state, and the spatial light modulator 15 is therefore located in the intermediate image plane of the illumination beam path. In the first switching state, all of the above-described methods can be performed in which the excitation light is manipulated in the intermediate image plane.

[0092] In the second switching state, i.e., the second setting state, the adjustable lens 76 is set so that the lens assembly 75 as a whole achieves a focal length f2, which is twice the focal length f1 set for the first switching state. For example, the fixed-focus lens 77 can have a focal length f2. In the second switching state, the adjustable lens 76 is then adjusted to a neutral position, so that only the focal length of lens 77 is effective. The distance between the adjustable lens assembly 75 and the first spatial light modulator 15 and the intermediate image plane 22 downstream of the adjustable lens assembly 75 is then approximately the same as the set focal length f2 of the adjustable lens assembly 75. The adjustable lens assembly 75, together with the tube lens 23, forms a 4f system that images the plane of the first spatial light modulator 15 into the back focal plane 25 of the microscope objective 30. This means that in the second switching state, the rear focal plane 25 of the microscope objective 30 and the plane of the spatial light modulator 15 are optically conjugate, and the spatial light modulator 15 is therefore located in the pupil plane of the illumination beam path. In the second switching state, all the above-described methods can then be performed in which the excitation light is manipulated in the pupil plane. The fixed-focal-length lens 77 can preferably be an achromatic lens. This achieves the advantage that color effects can be corrected, which is Figure 2 In the example, this is not possible.

[0093] Further embodiments of the multimodal microscope according to the present invention refer to Figures 4 to 9 describe.

[0094] exist Figures 1 to 3 In an embodiment of the present invention, in each switching state, at least one component of the optical functional component is always part of the illumination light path, and in Figures 4 to 9 In the exemplary embodiment shown there, the optical functional component is arranged completely in the illumination beam path downstream of the beam of the spatial light modulator 15 in each case in the first switching state or in the second switching state, whereas in the respective other switching state, the optical functional component is not part of the illumination beam path.

[0095] Figures 4 to 9 The components of the structural assembly 60 are combined with the above Figure 1 The components explained above are not described again here. Figures 4 to 9 Not all components of the assembly 60 are shown.

[0096] First, combine Figure 4 and Figure 5 A fourth embodiment of a multimodal microscope 400 according to the present invention is described. Figure 4The microscope 400 is shown in a first switching state, in which the optical functional component 50 is part of the illumination light path, and Figure 5 The same microscope 400 is shown in a second switching state, in which the optically functional component 50 is removed from the illumination beam path.

[0097] exist Figure 4 In the multimodal microscope 400 according to the present invention in the first switching state shown in FIG, the excitation light 13 of the light source 10 is first collimated by the lens 12 and then guided to the first spatial light modulator 15 by the lens 14. The lenses 12 and 14 are used to expand the laser beam to the aperture of the first spatial light modulator 15. Figure 4 and Figure 5 In the embodiment shown in , the first spatial light modulator 15 is again the only spatial light modulator and may preferably be a transmissive phase-modulating SLM, such as an LCOS display. Figure 4 In FIG. 1 , the first spatial light modulator 15 is followed by a lens 16 , an optical functional component 50 according to the invention and a lens 21 .

[0098] The optical function component 50 provides an alternative beam path having an intermediate image plane 52 and a pupil plane 56. Viewed in the direction of the illumination beam path, the optical function component has a first mirror 51, with which the excitation light 13 from the lens 16 is guided into the alternative beam path. The alternative beam path is then guided via the intermediate image plane 52 to a first internal mirror 53, which guides the excitation light via a lens 54 and a second internal mirror 55 to a second mirror 57. The excitation light 13 is coupled into the main part of the illumination beam path via the second mirror and guided in the direction of the lens 21.

[0099] In the illustrated embodiment, the optically active components 51, 53, 54, 55, and 57 of the optical functional assembly 50 are rigidly connected to one another. In other words, the optical functional assembly can be introduced into and removed from the illumination beam path as a whole. However, this is not mandatory. For example, it is also possible to provide that only the reflectors 51 and 57 are rigidly coupled to one another, wherein the structural unit can then be selectively introduced into and removed from the illumination beam path. Components 53 to 55 can then be arranged in a fixed spatial arrangement.

[0100] For switching the optical functional component, ie, for moving the optical functional component 50 into and out of the illumination beam path, a mechanical drive of a generally known nature can be expediently provided. This mechanical drive can preferably be controlled by the control unit 90 .

[0101] exist Figure 5In the situation shown in , the optical functional component 50 is in a second switching state, which is achieved by positioning the optical functional component 50 outside the illumination beam path. In this situation, the lenses 16 and 21 form a relay optical system, by which the plane in which the first light modulator 15 is arranged is imaged into the intermediate image plane 22 of the illumination beam path. Between the lenses 16 and 21, essentially where the optical functional component 50 can be introduced, a pupil plane 19 is formed, in which further light modulators can be arranged if necessary. The first light modulator 15 is therefore located in the intermediate image plane. In this switching state, all of the above-described methods can be performed, in which the excitation light is manipulated in the intermediate image plane.

[0102] exist Figure 4 In the situation shown in , the optical functional component 50 is in a first switching state, which is achieved by placing the optical functional component 50 in the illumination light path. In this situation, the rear focal plane 25 of the microscope objective 30 is imaged by the tube lens 23 and the lens 21 into a plane 56 within the optical functional component 50, that is, the plane 56 is the pupil plane. In addition, the lens 16 and the lens 54 of the optical functional component 50 form a relay optical device, which images the plane in which the first light modulator is arranged into the pupil plane 56. Therefore, in Figure 4 In the switching state, the first light modulator 15 is located in the pupil plane. In this switching state, all the methods described above can be performed in which the excitation light is manipulated in the pupil plane.

[0103] The fifth embodiment of the multimodal microscope 500 according to the present invention refers to Figure 6 and Figure 7 Description. The optical functional component 50 used therein is combined with Figure 4 and Figure 5 Same as described. Figure 6 The microscope 500 is shown in a first switching state, in which the optical functional component 50 is part of the illumination beam path, and Figure 7 The same microscope 500 is shown in a second switching state, in which the optically functional component 50 is removed from the illumination beam path.

[0104] The fifth embodiment of the microscope 500 is Figure 4 and Figure 5 The fourth embodiment differs only in that a second spatial light modulator 17 and a further lens 18 are arranged immediately downstream of the lens 16. The second spatial light modulator 17 is preferably a transmissive phase modulator, for example an LCOS display.

[0105] exist Figure 6In the state shown in FIG5 , in which the optical functional component 50 is located in the illumination light path, the tube lens 23 again forms a relay optical device together with the lens 21, which images the rear focal plane 25 of the microscope objective 30 into the plane 56, thereby making the plane 56 a pupil plane. In addition, the lens 18 forms a relay optical device together with the lens 54 of the optical functional component 50, which images the plane in which the second spatial light modulator 17 is arranged into the pupil plane 56. Therefore, in Figure 6 , the second spatial light modulator 17 is located in the pupil plane. Thus, lens 21 together with lens 54 forms relay optics, which images the intermediate image plane 22 onto plane 52. Plane 52 is also the intermediate image plane. Finally, lens 16 together with lens 18 form relay optics, which images the plane in which the first spatial light modulator 15 is arranged onto the intermediate image plane 52. The first spatial light modulator 15 is therefore arranged in the intermediate image plane. Thus, with this arrangement, all method variants for microscopy are possible, in which the excitation light 13 in the illumination beam path is first manipulated in the intermediate image plane, i.e., in the example, by the first spatial light manipulator 15, and then in the pupil plane, i.e., by the second spatial light manipulator 17.

[0106] exist Figure 7 In the state shown in , the optical functional component 50 is not in the illumination light path. Thus, the tube lens 23 together with the lens 21 forms a relay optical device that images the back focal plane 25 of the microscope objective 30 into the pupil plane 19. In addition, the lens 18 together with the lens 21 forms a relay optical device that images the plane in which the second spatial light modulator 17 is arranged into the intermediate image plane 22. Therefore, in Figure 7 Finally, lens 16 together with lens 18 form relay optics that images the plane in which the first spatial light modulator 15 is arranged into pupil plane 19. Thus, the first spatial light modulator 15 is located in the intermediate image plane. Figure 7 In this arrangement, all method variants for microscopy are therefore possible in which the excitation light 13 in the illumination beam path is first manipulated in the pupil plane, i.e. in the example by means of a first spatial light manipulator 15, and then in an intermediate image plane, i.e. by means of a second spatial light manipulator 17. Figure 6 and Figure 7 The lens 21 can be eliminated. This will result in the roles of the intermediate image plane and the pupil plane in the illumination beam path before the intermediate image plane 22 being interchanged, that is, the intermediate image plane when the lens 21 is used will become the pupil plane when the lens 21 is not present, and vice versa.

[0107] The sixth embodiment of the multimodal microscope 600 according to the present invention refers to Figure 8 and Figure 9 The optical functional component 50 used therein is again combined with Figure 4 and Figure 5 Same as described. Figure 8 The microscope 600 is shown in a first switching state, in which the optical functional component 50 is part of the illumination beam path and Figure 9 The same microscope 600 is shown in a second switching state in which the optical functional component 50 is removed from the illumination light path. Figure 4 and Figure 5 The fourth embodiment differs only in that the lens 21 is not present.

[0108] exist Figure 8 In the state shown in FIG in which the optical functional component 50 is located in the illumination light path, the tube lens 23 and the lens 54 of the optical functional component 50 together form a relay optical device that images the rear focal plane 25 of the microscope objective 30 into the plane 52, thereby making Figure 8 Plane 52 in is the pupil plane. Plane 56 in Figure 8 In the figure, there is an intermediate image plane before the tube lens 23. In addition, the lens 16 forms together with the lens 54 of the optical functional component 50 a relay optical device that images the plane in which the first spatial light modulator 15 is arranged into the intermediate image plane 56. Therefore, in Figure 8 In the embodiment, the first spatial light modulator 15 is arranged in the intermediate image plane. In this arrangement, all method variants for microscopy are therefore possible in which the excitation light 13 is manipulated in the intermediate image plane in the illumination beam path.

[0109] exist Figure 9 In the state shown in , the optical functional component 50 is not in the illumination light path. Thus, the tube lens 23 together with the lens 16 forms a relay optical device that images the rear focal plane 25 of the microscope objective 30 into the plane in which the first spatial light modulator 15 is arranged. Figure 9 In the embodiment, the first spatial light modulator 15 is arranged in the pupil plane. In this arrangement, all method variants for microscopy are therefore possible in which the excitation light 13 is manipulated in the pupil plane in the illumination beam path.

[0110] exist Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 and Figure 9 In the case of [ 0 ], as described above, the spatial light modulators arranged in the pupil planes can be driven to exhibit lenses with positive focal lengths. When performing this driving, it is advantageous to position glass lenses in the intermediate image plane 22 to compensate for the quadratic phase term of the excitation light caused by the lenses having positive focal lengths on the first spatial light modulators in the transverse spatial directions x and y. Details are provided in the invention summary of the specification. If no lenses are present on the spatial light modulators in the pupil planes, it may be advantageous or necessary to remove the glass lenses from the intermediate image plane 22.

[0111] Reference Signs List

[0112] 1. Sample

[0113] 10 Light source, laser

[0114] 11 Fiber Optic

[0115] 12 Lenses, collimating lenses

[0116] 13 Excitation light

[0117] 14 Lens

[0118] 15. First spatial light modulator, first phase modulator

[0119] 16 lenses

[0120] 17 Second spatial light modulator, second phase modulator

[0121] 18 lenses

[0122] 19 Pupil plane

[0123] 21 Lens

[0124] 22 Intermediate image plane

[0125] 23 Tube lens

[0126] 24 Main beam splitter

[0127] 25 Back focal plane, pupil plane, objective pupil of microscope objective 30

[0128] 26 Specimen Holder

[0129] 28 Focal plane in specimen 1

[0130] 30 microscope objectives

[0131] 32 Light reflected from sample 1

[0132] 34 Emission Filters

[0133] 36 Tube lens in the detection light path

[0134] 40 detectors, cameras

[0135] 50 Optical functional components that can be moved in and out as a whole

[0136] 51 First Reflector

[0137] 52 Intermediate image plane or pupil plane

[0138] 53 First internal reflector

[0139] 54 Lens

[0140] 55 Second internal reflector

[0141] 56 Pupil plane or intermediate image plane

[0142] 57 Second Reflector

[0143] 60 Microscope Structural Components

[0144] 70 Transformers, optical functional components

[0145] 71 A first lens having a focal length f1

[0146] 72 A second lens having a focal length of f2

[0147] 73 Optical functional components

[0148] 74 Adjustable lens

[0149] 75 Optical functional components

[0150] 76 Adjustable lens

[0151] 77 Lens with fixed focal length, e.g. f2

[0152] 90 Control unit, PC

[0153] 100 First embodiment of the microscope according to the present invention

[0154] 200 Second embodiment of the microscope according to the present invention

[0155] 300 A third embodiment of the microscope according to the present invention

[0156] 400 A fourth embodiment of the microscope according to the present invention

[0157] 500 Fifth embodiment of the microscope according to the present invention

[0158] 600 Sixth embodiment of the microscope according to the present invention

[0159] f1 focal length of lens 71

[0160] f2 Focal length of lens 72 = 2 × f1

[0161] References

[0162] [1] US7835051B2

[0163] [2] Alexander Jesacher, Severin Furhapter, Stefan Bernet, and Monika Ritsch-Marte; Diffractive optical tweezers in the Fresnel regime. 12, 10 OPTICS EXPRESS 2243 (2004)

[0164] [3] US10568516B2

[0165] [4] L Golan, I Reutsky, N Farah and S Shoham; Design and characteristics of holographic neural photo-stimulation systems. J. NeuralEng. 6 066004 (2009)

[0166] [5] Emiliano Ronzitt, Cathie Ventalon, Marco Canepari, Benoît CForget, Eirini Papagiakoumou and Valentina Emiliani; Recent advances inpatterned photostimulation for optogenetics. J. Opt. 19 113001 (2017)

[0167] [6] US9891172B

[0168] [7] Jinhan Ren, Kyu Young Han; 2.5D microscopy with polarizationindependent SLM for enhanced detection efficiency and aberration correction. 29, 17 / 16 / Optics Express 27530 (2021)

[0169] [8] US11665324B2

[0170] [9] Pierre Mahou, Nathan Curry, Dorothea Pinotsi, Gabriele KaminskiSchierle, and Clemens Kaminski "Stimulated emission depletion microscopy tostudy amyloid fibril formation", Proc. SPIE 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII, 93310U (2015)

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[10] Jeffrey A. Davis, Keevin Olea Valade´z, and Don M. Cottrell; Encoding amplitude and phase information onto a binary phase-only spatial light modulator. 42, 11 APPLIED OPTICS (2003)

[0172]

[11] US9915815B2

[0173]

[12] US9829690B2

[0174]

[13] Reto Fiolka, Markus Beck, and Andreas Stemmer; Structured illumination in total internal reflection fluorescence microscopy using aspatial light modulator. 33, 14 OPTICS LETTERS 1629 (2008)

[0175]

[14] Young, LJ, Ströhl, F., Kaminski, CF A Guide to Structured Illumination TIRF Microscopy at High Speed ​​with Multiple Colors. J. Vis. Exp. (111), e53988, doi:10.3791 / 53988 (2016)

[0176]

[15] Clark, “Comparison of beam generation techniques using a phase-only spatial light modulator”, Opt.Expr. 24 / 6, 6249, 2016

[0177]

[16] Yoshiki Nakata, Kazuhito Osawa and Noriaki Miyanaga; Utilizationof the high spatial-frequency component in adaptive beam shaping by using avirtual diagonal phase grating. Scientific Reports 9, 4640 (2019)

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[17] US9778573B2

[0179]

[18] Andrew Bañas and Jesper Glückstad; Light Shaping withHolography, GPC and Holo-GPC. Opt. Data Process. Storage 3:20–40 (2017)

[0180]

[19] Alexander Jesacher, Christian Maurer, Andreas Schwaighofer,Stefan Bernet and Monika Ritsch-Marte; Near-perfect hologram reconstruction with a spatial light modulator. 16, 4 OPTICS EXPRESS 2597 (2008)

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[20] CN106094189B

[0182]

[21] Christian Maurer, Alexander Jesacher, Stefan Bernet, and Monika Ritsch-Marte; What spatial light modulators can do for optical microscopy. Laser Photonics Rev. 5, No. 1, 81–101 (2011)

Claims

1. A multimodal 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 (30) for guiding the excitation light (13) onto and / or into a sample (1) to be examined, wherein: At least one first spatial light modulator (15) is present in the illumination beam path for manipulating the excitation light (13). a detector (40) for detecting detection light (32) emitted by the sample (1) due to illumination by the excitation light (13), a detection beam path having the microscope objective (30) or a further microscope objective, the detection beam path being used to guide the detection light (32) to the detector (40), a control unit (90), the control unit being at least used to drive and control the first spatial light modulator (15), It is characterized by: There is a switchable optical functional component (50; 70; 73; 75), which can be switched into at least a first switching state and a second switching state, and Depending on which switching state the optical functional component (50; 70; 73; 75) is in, the first spatial light modulator (15) is located either in or near an intermediate image plane of the illumination light path, or in or near a pupil plane of the illumination light path.

2. The microscope according to claim 1, It is characterized by: The optical functional component includes a transformation device (70) having a first lens (71) and a second lens (72) or is realized by the transformation device (70) having a first lens (71) and a second lens (72).

3. The microscope according to claim 1 or 2, It is characterized by: The optical functional component (73) has an adjustable lens (74) or is realized by an adjustable lens (74).

4. The microscope according to any one of claims 1 to 3, It is characterized by: The optical functional component comprises an adjustable lens assembly (75) having an adjustable lens (76) and a lens (77) with a fixed focal length, or is realized by a lens assembly having an adjustable lens (76) and a lens (77) with a fixed focal length.

5. The microscope according to any one of claims 2 to 4, It is characterized by: In the first switching state, one of the following features is achieved: • the distance between the first lens (71) and the first spatial light modulator (15) and the intermediate image plane (22) downstream of the beam of the first lens (71) is in each case approximately twice as great as the focal length (f1) of the lens (71); • the distance between the adjustable lens (74) and the first spatial light modulator (15) and the distance between the adjustable lens (74) and the intermediate image plane (22) downstream of the beam of the adjustable lens (74) is approximately twice the adjusted focal length (f1) of the adjustable lens (74); • The distance between the adjustable lens assembly (75) and the first spatial light modulator (15) and the intermediate image plane (22) downstream of the beam of the adjustable lens assembly (75) is approximately twice the adjusted focal length (f1) of the adjustable lens assembly (75).

6. The microscope according to any one of claims 2 to 5, It is characterized by: In the second switching state, one of the following characteristics is achieved: • the distance between the second lens (72) and the first spatial light modulator (15) and the intermediate image plane (22) downstream of the second lens (72) is approximately as large as the focal length (f2) of the second lens (72), and the second lens (72) together with the tube lens (23) forms a 4f system which images the plane of the first spatial light modulator (15) into the back focal plane (25) of the microscope objective (30); • the distance between the adjustable lens (74) and the first spatial light modulator (15) and the intermediate image plane (22) downstream of the beam of the adjustable lens (74) is approximately the same as the adjusted focal length (f2) of the adjustable lens (74), and the adjustable lens (74) together with the tube lens (23) forms a 4f system, which images the plane of the first spatial light modulator (15) into the rear focal plane (25) of the microscope objective (30), • The distance between the adjustable lens assembly (75) and the first spatial light modulator (15) and the intermediate image plane (22) downstream of the beam of the adjustable lens assembly (75) are approximately the same as the adjusted focal length (f2) of the adjustable lens assembly (75), and the adjustable lens assembly (75) together with the tube lens (23) forms a 4f system, which images the plane of the first spatial light modulator (15) into the rear focal plane (25) of the microscope objective (30).

7. The microscope according to any one of claims 1 to 6, It is characterized by: The optical functional component (50) is arranged at least partially, in particular completely, in the illumination beam path downstream of the beam of the spatial light modulator (15) in either a first switching state or a second switching state, and The optical functional component (50) is not part of the illumination light path in the respective other switching state.

8. The microscope according to claim 7, It is characterized by: An alternative beam path (51-57) having at least one lens (54) is formed by the optical functional component (50), the alternative beam path having a pupil plane (52, 56) and an intermediate image plane (56, 52), The excitation light (13) is guided in the direction of the microscope objective (30) via the alternative beam paths (51-57) either in a first switching state or in a second switching state.

9. The microscope according to claim 7 or 8, It is characterized by: In order to guide the excitation light (13) via an alternative beam path (51-57) of the illumination light path, the optical functional component (50) has a first switching device, in particular an adjustable first reflector (51). In order to couple the excitation light (13) from the alternative beam paths (51-57) back into the main part of the illumination beam path, the optical functional component (50) has a second switching device, in particular a second adjustable mirror (57).

10. The microscope according to any one of claims 7 to 9, It is characterized by: The first reflector (51) and the second reflector (57) can be introduced into and removed from the illumination light path together, and when the optical functional component (50) is switched, other components of the optical functional component remain unchanged.

11. The microscope according to any one of claims 7 to 9, It is characterized by: When the optical functional component (50) is introduced into the illumination beam path and when the optical functional component (50) is removed, the optically active components of the optical functional component (50) are rigidly connected to one another.

12. The microscope according to any one of claims 1 to 11, It is characterized by: A second spatial light modulator (17) is arranged in the illumination optical path. When the first spatial light modulator (15) is located in or near the intermediate image plane, the second spatial light modulator is located in or near the pupil plane, and when the first spatial light modulator (15) is located in or near the pupil plane, the second spatial light modulator is located in or near the intermediate image plane.

13. The microscope according to claim 12, It is characterized by: The first spatial light modulator (15) is formed by a first sub-region of a spatial light modulator, and the second spatial light modulator (17) is formed by a second sub-region of the same spatial light modulator.

14. The microscope according to any one of claims 7 to 13, It is characterized by: The optical functional component (50) can be introduced into the illumination light path at the location of the pupil plane and is located between the following two lenses (16, 21): when the optical functional component (50) is located outside the illumination light path, the two lenses form an optical relay device, wherein the optical relay device images the plane in which the first spatial light modulator (15) or the second spatial light modulator (17) is arranged into an intermediate image plane of the illumination light path.

15. The microscope according to any one of claims 7 to 14, It is characterized by: The optical functional component (50) can be introduced into the illumination light path at the location of the intermediate image plane and is located between the tube lens (23) and the lens (16), wherein, when the optical functional component (50) is located outside the illumination light path, the tube lens (23) and the lens (16) form an optical relay device, which images the plane in which the first spatial light modulator (15) or the second spatial light modulator (17) is arranged into the pupil plane of the illumination light path.

16. The microscope according to any one of claims 1 to 15, It is characterized by: In order to compensate for the quadratic phase term of the excitation light (13) in the transverse spatial direction (x, y), a lens with a positive focal length is arranged or can be arranged in or near an intermediate image plane of the illumination beam path.

17. A method for microscopy, wherein: guiding the excitation light (13) via an illumination beam path with a microscope objective (30) onto and / or into the sample (1) to be examined, The excitation light (13) is manipulated by at least a first spatial light modulator (15) in the illumination light path, guiding the detection light (32) emitted from the sample (1) due to illumination by the excitation light (13) to a detector (40) via a detection light path including the microscope objective (30) or another microscope objective, and detecting the detection light by the detector, It is characterized by: Before performing a microscopic measurement, the switchable optical functional component (50; 70; 73; 75) is switched either into a first switching state or into a second switching state. wherein, depending on which switching state the optical functional component (50; 70; 73; 75) is in, the first spatial light modulator (15) is either located in or near an intermediate image plane of the illumination light path, or in or near a pupil plane of the illumination light path, and Then, at least one of the spatial light modulators (15) is driven to provide a desired illumination pattern.

18. The method according to claim 17, It is characterized by: The optical functional component (50; 70; 73; 75) is driven so that the first spatial light modulator (15) is located in or near the pupil plane.

19. The method according to claim 18, It is characterized by: The first spatial light modulator (15) is driven to manipulate particles in the sample (1) to realize optical trapping and / or optical tweezers.

20. The method according to claim 18 or 19, It is characterized by: The first spatial light modulator (15) is driven so that only a specific part of the sample (1) is illuminated.

21. The method according to any one of claims 18 to 20, It is characterized by: The first spatial light modulator (15) is driven to correct aberrations caused by optical components of the microscope and / or by the sample (1).

22. The method according to any one of claims 18 to 21, It is characterized by: The first spatial light modulator (15) is driven to defocus the excitation light (13) in the sample (1).

23. The method according to any one of claims 18 and 20 to 22, It is characterized by: The first spatial light modulator (15) is driven for STED microscopy to shape the excitation light (13) into a donut-shaped beam in the sample (1).

24. The method according to claim 17, It is characterized by: The optical functional component (50; 70; 73; 75) is driven so that the first spatial light modulator (15) is located in or near the intermediate image plane.

25. The method according to claim 24, It is characterized by: The first spatial light modulator (15) is driven to be far enough from the optical axis (z) at the rear focal plane (25) of the microscope objective (30) to produce an illumination pattern in a region of the sample (1) that satisfies TIRF conditions for the excitation light (13).

26. The method according to claim 24 or 25, It is characterized by: The first spatial light modulator (15) is driven to exhibit a phase grating in an intermediate image plane to perform SIM microscopy.

27. The method according to claim 18 or 24, It is characterized by: A second spatial light modulator (17) is arranged in the illumination light path, and the optical functional components (50; 70; 73; 75) are either driven so that the first spatial light modulator (15) is located in or near the pupil plane, and the second spatial light modulator (17) is located in or near the intermediate image plane. Either the optical functional components are driven so that the first spatial light modulator (15) is located in or near the intermediate image plane, and the second spatial light modulator (17) is located in or near the pupil plane.

28. The method according to claim 27, It is characterized by: The intensity of the excitation light (13) in the sample (1) is changed by causing a first spatial light modulator (15) or a second spatial light modulator (17) positioned in or near a pupil plane to diffract at least a portion of the excitation light (13) into an area outside the area propagated by the optical system, wherein the portion is changed by changing the diffraction efficiency of the respective spatial light modulator used, and wherein the diffraction efficiency is changed by changing the contrast of a phase grating set in the respective spatial light modulator used.

29. The method according to claim 27 or 28, It is characterized by: A first spatial light modulator (15) in the intermediate image plane is driven to exhibit a phase pattern of the image to be imaged, and The second spatial light modulator (17) is driven to represent a phase contrast filter, such as a rectangular or circular phase shift mask, the phase contrast filter having a phase deviation of π for low spatial frequencies, and The intensity distribution of the image to be imaged is recovered from the interference of the phase-shifted low spatial frequency and the unmodulated higher spatial frequency.

30. The method according to any one of claims 27 to 29, It is characterized by: The intensity and phase of the excitation light (13) in the sample (1) are manipulated by using the first spatial light modulator (15) and / or by using the second spatial light modulator (17).

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