Microscope and method for microscopy

Abstract

The invention relates to a microscope and a method for microscopy. The microscope has a light source, an illumination beam path for guiding excitation light into a sample region, a detector for detecting emitted light emitted from the sample, a detection beam path having a microscope objective for guiding the emitted light onto the detector, and a control unit for evaluating the emitted light detected by the detector. The microscope is characterized in that the illumination beam path has at least one relay optic for providing an intermediate image plane, the illumination beam path has a multimode light guide, the output of which is arranged in or near the intermediate image plane.

Description

Technical Field

[0001] The present invention relates to a microscope according to the preamble of claim 1 and a method for microscopy according to the preamble of claim 25. Background Technology

[0002] Microscopes of this type have at least the following components: a light source for providing excitation light; an illumination optical path for guiding the excitation light to a sample region; a two-dimensional spatially resolved detector for detecting emitted light emitted from a sample in the sample region; a detection optical path having a microscope objective for guiding the emitted light to the detector; and a control unit for evaluating the emitted light detected by the detector.

[0003] The method according to the class shall perform at least the following steps: guiding excitation light from a light source to a sample via an illumination optical path; guiding emitted light from the sample via a probe optical path having a microscope objective to a two-dimensional spatially resolved detector, and detecting the emitted light using the detector.

[0004] Microscopes by category and by category methods are known in many design schemes and variations, for example, as known by [1].

[0005] For many microscopy techniques, it is desirable to illuminate areas in the sample plane or pupil plane with the most uniform (i.e., the same) intensity distribution possible. This illumination mode is also known as flat-top illumination.

[0006] Several techniques are known for laser beam shaping, which are used to shape spatially coherent Gaussian modes into at least partially coherent, uniform flat-top illumination.

[0007] First, techniques based on spatial cutting of laser beams exist. Furthermore, techniques based on beam superposition or beam integration are known. Finally, techniques essentially based on beam redistribution have also been described.

[0008] Cutting out portions of a laser beam with the desired intensity distribution from the laser beam is readily achievable. However, these techniques, based on spatial cutting of the laser beam, are not commonly used because they can only utilize a relatively small number of light portions. For example, an aperture (such as a mechanical component or a coated optics device) can be used to cut out the desired portion from the Gaussian beam profile of the laser. Alternatively or supplementarily, a diffraction element can be used to cut out the desired beam profile from the Gaussian beam to be shaped. The desired and undesired portions in the Gaussian beam can be spatially separated using the wedge-shaped phase of the diffraction element [Salter]. Uniformity can be improved by spatially matching the wedge-shaped phase pattern to the Gaussian beam to be shaped [Nakata]. In this method, light outside the aperture (i.e., outside the desired laser shape) is lost and unusable. Due to significant power losses, beam shaping by cutting the spatial beam profile is generally impractical.

[0009] In methods using beam superposition and beam integration, flat-top beam shaping is achieved by spatially dividing the beam and then using focusing optics to superimpose the individual components in a smaller plane. For this purpose, microlens arrays (MLAs) can be used, for example. Alternatively, reflective optics can be employed. In this case, a mirror performs the function of the MLA.

[0010] Because many subbeams overlap spatially, a flat wavefront cannot be guaranteed in such a beam-shaping module, and therefore spatial coherence cannot be ensured either. If such a beam-shaping module is used to superimpose coherent light, a speckled structure will appear due to interference. This is called a "fleck" or a speckled flat top.

[0011] Finally, beam shaping can also be achieved using multimode fibers by, for example, coupling a Gaussian mode into the multimode fiber, thereby exciting a large number of spatial modes in the multimode fiber. By mixing the modes in the multimode fiber, a beam profile shaped according to the fiber core appears at the fiber end. Due to the superposition of the various optical modes, a speckled beam profile appears. Here, the shape of the beam cross section is given by the shape of the cross section of the multimode fiber, especially by the geometry of the core guiding the light. Reducing the speckled structure, i.e., “de-spewing”, can be achieved by rotating and / or oscillating diffusers. In the case of multimode fibers, the multimode fiber itself can also be oscillated. Multimode fibers with integrated de-spewing devices are commercially available from Molex [3]. The light at the end of the multimode fiber may be spatially incoherent or may be partially coherent. Instead of multimode fibers, optical guide rods can also be used, for example, see [2]. A flat-top beam profile appears near the exit region of the multimode fiber or optical guide rod.

[0012] Cizmar et al. [Cizmar] pointed out that the process of mode superposition can be modeled and predicted with a very high degree of accuracy. They specifically noted that when individual modes are coupled into a multimode fiber at once, strong coupling does not occur between the modes. These modes are also known as propagation-invariant modes (PIMs), fiber modes, or fiber-intrinsic modes. Spotting only occurs when these modes move at different speeds. This can even be approximated as the case when the fiber is arched. This again means that only the excitation of some of these modes results in a spot pattern with specific characteristics, independent of fiber bending. These characteristics are related to the type of waveguide. Abruptly refractive index waveguides have modes corresponding to different spatial frequencies. Therefore, if a multimode waveguide is excited with a beam that excites only a portion of these modes, a beam with angular scattering corresponding to the angular scattering of illumination will be obtained. If the waveguide is excited at its center using a finite numerical aperture, a spotted beam with a similar numerical aperture is output. If the incoming beam is coupled at an angle, it typically results in a ring-shaped angular distribution [Farley].

[0013] Beam shaping through redistribution can be achieved by redistributing the intensity distribution of the incident Gaussian beam using optics to produce the desired beam shape at the output. This redistribution of optical power can be achieved using either diffractive elements (e.g., DOEs and / or SLMs) or refractive optics (e.g., lenses). Laser beam modules based on beam redistribution using refractive optics are commercially available from ADL-Optics [Stehr] in Berlin and Asphericon [Khaw] in Jena. Diffractive optics can be implemented not only transmissively (e.g., with transmissive SLMs or DOEs) but also reflectively (e.g., with LCOS-SLMs (Liquid Crystal on Silicon-SLMs) or reflective DOEs, such as those from Midel Photonics).

[0014] The phase function required to transform a Gaussian beam into the desired shape can be determined, for example, using the Gerchberg-Saxton algorithm. However, a drawback of this algorithm is that the phase is not flat after beam shaping, but rather rather messy. This phase can be corrected using a second phase element in the conjugate Fourier plane [Jesacher]. Furthermore, algorithms for reducing speckles in holograms have been described [Schmidt]. These algorithms are relatively complex and have poor optical efficiency. Finally, it is feasible, for example, to shape the beam shaper to the end of the optical fiber using a 3D printer [Printoptix, Schmidt, Plidschun].

[0015] Spatially coherent beam shaping can be achieved solely through spatial trimming of the laser beam profile, beam redistribution using refractive optics, and / or through a combination of two diffractive optical elements in the optical conjugate Fourier plane. Spatial coherence here refers to the flat phase of the shaped beam after beam shaping.

[0016] Microscopic methods that achieve resolutions better than the Abbe limit are considered high-resolution microscopy. In addition to laser-based scanning techniques (such as STED (Stimulated Emission Depletion), RESOLFT (Reversible Saturable Optical Linear Fluorescence Transitions), MINFLUX (Minimal Fluorescence Photon Fluxes microscop), and Airy scanning (Zeiss), there are also techniques based on wide-field illumination. The relevant technologies include SIM (Structured light microscopy) and SMLM (Single Molecules Localization Microscopy). Examples of SMLM include PALM (Photo-Activated Localization Microscopy), (d)STORM ((direct) Stochastic Optical Reconstruction Microscopy), and PAINT (Points Accumulation In Nanoscale Topography).

[0017] SIM measures are based on structured illumination of a sample stained with a fluorescent dye. It has proven practical to utilize, for example, the diffraction order of a phase grating generated by static lighting or by SLM. The diffraction order of the grating is imaged as a point into the pupil of the objective lens. The grating structure is generated in the sample plane by the interference of the beam. Only the lowest diffraction order of the grating is used. Alternatively, an amplitude grating can be imaged into the sample plane. If incoherent light is used for SIM / Apotom, an amplitude grating must be used. Due to light loss at the amplitude grating, a phase grating is generally preferred. To obtain a high-resolution image, calculations must be performed on the original image.

[0018] In the SMLM procedure, wide-field illumination is applied to the sample stained with a fluorescent dye. Depending on the procedure, the scintillation mechanism of the molecules can be activated via different wavelengths (PALM), illumination intensity (dSTORM), or the sample itself (PAINT).

[0019] Detection of scintillation events and localization of fluorescent molecules through computer-based evaluation of image data are possible. TIRF illumination (Total Internal Reflection Fluorescence) can also be employed to avoid interfering background and / or achieve better partitioning. A method for achieving SMLM and TIRF using multimode fibers is described in [Lamm], [Kwakwa], and [Ries]. Here, the angular spectrum of the multimode fiber is imaged onto the sample plane. SIM using multimode fibers is described in [Gustafsson]. A TIRF method using rectangular multimode fibers is disclosed in US 2022 / 0326498 A1, wherein the fiber ends are arranged in a plane conjugate to the sample plane. Summary of the Invention

[0020] The objective of this invention is to describe a microscope and a method for microscopy in which particularly well-defined illumination of the sample can be achieved.

[0021] This task is accomplished by a microscope having the features of claim 1 and a method having the features of claim 25.

[0022] According to the present invention, the microscope of the above type is improved in that the illumination optical path has at least one relay optics to provide an intermediate image plane; the illumination optical path has a multimode optical guide; and the output end of the multimode optical guide is arranged in or near the intermediate image plane; and the illumination optical path has a variable wavefront manipulation device arranged in or near the intermediate image plane.

[0023] According to the present invention, the method of the above type is improved by uniformizing the intensity distribution of the excitation light on the beam cross section by guiding the excitation light provided by the light source via a multimode optical guide, wherein the output end of the multimode optical guide is arranged in or near the intermediate image plane, and especially for performing the SIM method, the excitation light is manipulated by a variable wavefront manipulation device arranged in or near the intermediate image plane in the illumination optical path.

[0024] Preferred embodiments of the microscope according to the invention and advantageous variations of the method according to the invention will be explained below, in particular, in connection with the dependent claims and illustrations.

[0025] For the microscope and method according to the invention, a laser is primarily considered as the light source, but other light sources, such as LEDs, are also feasible. For SIM, light that is generally at least partially coherent is preferred. The excitation light, also known as the illumination light, is preferably electromagnetic radiation in the visible light range and its vicinity. There are no restrictions in principle regarding the sample to be examined. Typically, the sample to be examined is a biological sample.

[0026] The term "illumination optical path" refers to all optical components that guide and modify the light beam, such as illumination objectives, lenses, mirrors, prisms, gratings, filters, apertures, polarizers, beam splitters, and modulators, such as spatial light modulators (SLMs), which guide the excitation light from the light source onto the sample to be examined. Components that modify the light beam also include dispersive elements and, in particular, diffractive elements. Commercially available microscope objectives can be used in principle. Preferably, the excitation light is linearly polarized. For this purpose, at least one polarizer may be present in the illumination optical path, for example. The microscope objectives used preferably have a high numerical aperture, for example, greater than 1, preferably greater than 1.4.

[0027] The spatial region on the object side of the illumination objective in which the sample can be arranged, such as the spatial region in the sample holder supported on the xy-stage, is also referred to as the sample region in this specification. The terms "sample" and "sample region" are used synonymously in this sense.

[0028] The term "intermediate image plane" refers, in particular, to a plane perpendicular to the optical axis of the illumination or probe path, which is optically conjugate to the image plane of the respective microscope objective. The term "pupil plane" refers, in particular, to a plane perpendicular to the optical axis of the illumination or probe path, which is optically conjugate to the back focal plane of the respective microscope objective.

[0029] In this specification, when a component is mentioned as being located in the pupil plane or the intermediate image plane, it always means that the relevant component is located near its respective pupil plane or its respective intermediate image plane. This is self-evident because neither the pupil plane nor the intermediate image plane is a plane in the mathematical sense, and because the components of interest herein (e.g., spatial light modulators and lenses) have finite extensions along their respective optical axes.

[0030] Two-dimensional spatial resolution detectors can be, for example, CCD, CMOS, or SPAD array cameras. Cameras operating in event-based modes can also be used.

[0031] The emitted light from a sample is the electromagnetic radiation emitted by the sample illuminated by the excitation light. "Emitted" means that the probe light originates from the sample. The emitted light can also be called the probe light. The emitted light can be reflected back from the sample, or it can be light transmitted through the illuminated sample. Typically, the emitted light can be fluorescence from a fluorescent label used to prepare the sample that has undergone a redshift compared to the excitation light.

[0032] The term "probe path" refers to all optical components that guide and modify the beam, such as objectives, lenses, mirrors, prisms, gratings, filters, apertures, beam splitters, polarizers, and modulators, such as spatial light modulators (SLMs), which guide the probe light from the sample to be examined to the detector.

[0033] The illumination objective for the illumination path and the probe objective for the probe path can be the same microscope objective. This may be the case, for example, in an epi-irradiation microscope where the sample is illuminated and observed from one and the same direction. However, the illumination objective for the illumination path can also be different from the probe objective. This is the case, for example, in a transmission microscope and an epi-irradiation microscope where the sample is obliquely illuminated and observed, such as in a sheet microscope.

[0034] The term "control unit" refers to all hardware and software components that work in conjunction with the components of the microscope according to the invention to enable these components to function as intended. In particular, the control unit may include a computing device (e.g., a PC) and a camera control unit. The computing resources of the control unit can be allocated to multiple computers, and, if necessary, to a computer network, especially via the Internet. The control unit may include commonly used operating devices and peripherals such as a mouse, keyboard, screen, storage medium, joystick, and Internet connection. The control unit may, in particular, read image data from the detector and may also be used and configured to drive a light source. The control unit may also be configured, for example, to drive a spatial light modulator.

[0035] The flat-top region should be understood as a region in the illumination path with finite axial and finite lateral extension, in which the illumination light has a substantially uniform lateral light distribution.

[0036] The term "relay optics" refers to an optical assembly used to achieve optical imaging, particularly 4f imaging, from one plane, especially an intermediate image plane or pupil plane, to another intermediate image plane or pupil plane. For example, a relay optics device can be formed by two lenses arranged at a distance from each other equivalent to the sum of the focal lengths of the two lenses. Through these two lenses, a first plane, for example, a first intermediate image plane located on the side of the first lens away from the second lens (the distance between the first intermediate image plane and the first lens is equivalent to the focal length of the first lens), is optically imaged onto a second plane, for example, a second intermediate image plane located on the side of the second lens away from the first lens (the distance between the second intermediate image plane and the second lens is equivalent to the focal length of the second lens). Thus, the distance between the first and second intermediate image planes is twice the sum of the focal lengths of the two lenses, i.e., 4f.

[0037] The term "multimode optical guide" refers to an optical guide that can propagate multiple optical modes, unlike single-mode fibers, in which only one optical mode can propagate in each polarization direction.

[0038] The main concept of this invention can be considered as generating a uniform light distribution (especially at least partially coherent light) using at least one multimode optical guide, and arranging the at least one multimode optical guide in the illumination optical path such that its output end is located in or near the intermediate image plane of the illumination optical path. Therefore, the output end of the multimode optical guide is arranged in the illumination optical path to achieve critical illumination.

[0039] A first major advantage of this invention is that, when using a Gaussian illumination profile, periodic modulation of detected fluorescence can be avoided in stitched images of large sample areas because the illumination intensity decreases at the edges of the image field (i.e., outside the maximum part of the Gaussian profile). A second advantage of this invention is that illumination light can still be provided at least partially coherently, thus making SIM particularly feasible.

[0040] The method according to the invention can be performed, in particular, using a microscope according to the invention; that is, using a microscope according to the invention in advantageous variations of the method according to the invention. The microscope according to the invention can be specifically configured to perform the method according to the invention and / or its variations.

[0041] In principle, it is feasible for the multimode optical guide to be formed from a rigid component. For example, the multimode optical guide may have at least one multimode optical guide rod. In the preferred design of the microscope according to the invention and in advantageous variations of the method according to the invention, at least one multimode fiber is used as the multimode optical guide. The multimode fiber may be, in particular, a step-index multimode fiber. A step-index multimode fiber is characterized in that its core has a higher refractive index than its cladding, i.e., it has a higher refractive index than its coating.

[0042] The term "wavefront manipulation device" refers to an optical device that alters the wavefront of light passing through or being reflected (i.e., excitation light). "Variable" means that the wavefront manipulation device can be altered in some way, such as through manipulation. Manipulation of the excitation light means changing the wavefront of the excitation light in some way.

[0043] According to the invention (especially for performing the SIM method), a variable wavefront manipulation device is used to manipulate the excitation light, which is arranged in or near the intermediate image plane in the illumination optical path. For method variants where wavefront manipulation is not required, such as in the SMLM method, the variable wavefront manipulation device can be removed from the optical path or switched to an operating mode that does not manipulate the light. For this purpose, a suitable, controllable driver can be appropriately provided.

[0044] The variable wavefront manipulation device may, for example, have at least one diffraction element and / or at least one refractive element. The refractive element may, for example, have a laterally movable transmission grating and / or at least one laterally movable reflection grating. The transmission grating and / or reflection grating may be a phase grating and / or an amplitude grating. A driver may be present for laterally moving the transmission grating and / or the reflection grating. Here, the term "lateral direction" is understood to mean a direction that extends laterally or vertically relative to the optical axis at the location of the grating. A control unit may be suitably configured to drive the driver to laterally move the transmission grating or the reflection grating. Alternatively or additionally, the wavefront manipulation device may have at least one spatial light manipulator. The control unit may be suitably configured to drive one or more spatial light manipulators. In principle, it is feasible for the spatial light manipulator to be an amplitude-modulated spatial light manipulator. However, due to lower optical loss, a phase-modulated spatial light modulator is generally preferred. The spatial light manipulator may be a reflective spatial light manipulator or a transmission spatial light manipulator.

[0045] Spatial light modulators can also be used to perform corrections and adjustments to the illumination path, for example, to match different axial positions with different microscope objectives, especially their respective back focal planes.

[0046] As mentioned above, spotting, i.e., localized uniformity deviations, may occur at the output end of a multimode optical guide. A relatively simple and feasible solution to reduce this spotting is to use relatively long multimode fibers. For example, multimode fibers with a length of 2 meters or more are preferably used.

[0047] In another feasible method for suppressing speckle, at least a portion of the multimode fiber is moved back and forth in the illumination optical path. Since the geometry of the multimode fiber changes at least slightly during this movement, the combination of optical modes propagating in the multimode fiber at relevant time points changes accordingly. The result is a more uniform light distribution at the exit end of the multimode fiber. To achieve this, the illumination optical path can advantageously have means for mechanically manipulating the multimode fiber. For example, at least a portion of the multimode fiber can be agitated or excited to vibrate within the illumination optical path. For this purpose, the means for mechanically manipulating the multimode fiber can have a device for agitating or vibrating the multimode fiber.

[0048] For example, when the frequency of motion (especially the frequency of vibration) of a portion of the multimode fiber is greater than 500 Hz, preferably greater than 1 kHz, good results are achieved in terms of light distribution uniformity.

[0049] Alternatively or supplementally, in another variation of the method according to the invention, a diffuser arranged downstream or upstream of the beam of the multimode optical guide in the illumination optical path can be used to homogenize the excitation light. Therefore, in an advantageous embodiment where the illumination optical path has a diffuser downstream of the beam of the multimode optical guide, the uniformity of the excitation light distribution can be improved. Alternatively or supplementally, the diffuser or a single diffuser can also be arranged between the light source and the multimode optical guide, i.e., upstream of the beam of the multimode optical guide.

[0050] Here, when the diffuser moves in the illumination path, further improvement in uniformity can be achieved. For this purpose, a device for moving the diffuser is advantageously available. For example, the diffuser can move back and forth in the illumination path. For this purpose, the device for moving the diffuser can have a rocking mechanism. Alternatively or supplementarily, it is also feasible to rotate the diffuser in the illumination path. For this purpose, the device for moving the diffuser can have a rotating mechanism.

[0051] Finally, good results can also be obtained in terms of light distribution uniformity when the diffuser is oscillating, especially relative to the output end of the multimode fiber. For example, the diffuser can be oscillating back and forth, especially relative to the output end of the multimode fiber, using a device for moving the diffuser.

[0052] In principle, it is feasible for multimode light guides to have a circular cross-section. This is sufficient for capturing a single image. However, since the camera chips of available cameras are typically rectangular or at least not circular, even with illumination having a circular cross-section, the surface of the camera chip cannot be optimally utilized.

[0053] However, the cross-section of a multimode light guide can also have a polygonal shape, especially an equilateral one. For example, the cross-section of a multimode light guide can have an equilateral hexagonal shape, especially. For capturing stitched images, it is particularly preferable to use a multimode light guide with a rectangular or square cross-section.

[0054] In a particularly preferred embodiment of the microscope according to the invention, the sensing surface of the detector and the cross-section of the multimode optical guide have the same type of geometry.

[0055] For example, the sensing surface of the detector and the cross-section of the multimode optical guide can each have, in particular, regular polygonal, regular hexagonal, rectangular, or square shapes. It is also advantageous when the shapes of the sensing surface of the detector and the cross-section of the multimode optical guide are geometrically transferable to each other through similar imaging.

[0056] In a particularly preferred embodiment of the microscope according to the invention, the probe optical path is configured to image the illuminated surface in the sample region onto the sensing plane of the camera, such that the overlap between the image of the sensing area of ​​the camera and the image of the illuminated surface in the sample region is maximized. Therefore, the excitation light provided at the exit end of the multimode optical guide and the sensing area of ​​the camera are utilized as effectively as possible.

[0057] In a preferred design of the microscope according to the invention, the illumination optical path has means for adjusting the incident angle of the excitation light onto the sample. Preferably, the incident angle of the excitation light can be adjusted between excitation light incident parallel to the optical axis and excitation light incident satisfying the TIRF condition.

[0058] The apparatus for adjusting the incident angle of the excitation light may have a pivotable mirror or a pivotable, plane-parallel glass plate arranged in or near the intermediate image plane of the illumination path. A mechanical actuator may be present to pivot the pivotable mirror or the plane-parallel glass plate. A control unit may be suitably configured to drive the mechanical actuator for the pivotable mirror or the plane-parallel glass plate. Such a pivotable mirror is also referred to as a TIRF mirror.

[0059] Alternatively or supplementarily, the device for adjusting the incident angle of the excitation light may have a device arranged in or near the pupil plane of the illumination path for generating an adjustable lateral beam deflection. The device for generating the adjustable lateral beam deflection may, for example, have a biprism capable of laterally variable positioning in the illumination path. A mechanical actuator may be present for laterally adjusting the laterally variable biprism. A control unit may be suitably configured to drive the mechanical actuator for the laterally variable biprism. This device for generating an adjustable lateral beam deflection is also referred to as a TIRF slider.

[0060] In an advantageous variant of the method according to the invention, the HILO method (HILO = Highly Inclined and Laminated Optical sheet) is performed.

[0061] Advantageously, at least one of the following methods can be performed: SIM method, SMLM method, TIRF method, TIRF-SIM method, TIRF-SMLM method.

[0062] In both HILO and TIRF methods, the excitation light is deflected at an angle and imaged off-axis into the pupil of the illumination objective (i.e., in the back focal plane) through a lens (e.g., a barrel lens). In the case of TIRF, the excitation light is directed to one or more points at the edge of the pupil. In the case of HILO, the excitation light also illuminates the sample at an angle deviating from the normal direction. However, the angle of incidence is not large enough to achieve total internal reflection.

[0063] The illumination point for TIRF and SIM in the back focal plane of the microscope objective is generated in a manner known in principle by the wavefront modulation device or a wavefront modulation device (i.e., by means of a static grating or spatial light modulator) arranged in the intermediate image plane of the illumination optical path.

[0064] Since the excitation light is no longer completely coherent at the output of the multimode optical guide, interference effects in the sample plane are reduced or minimized. However, for the SIM method to be performed, the excitation light must still possess sufficient partial coherence so that the wavefront manipulation device (especially the phase grating) can still function effectively. When observed over a period much shorter than the camera exposure time, the light at the output of the multimode optical guide is coherent. The coherence of the light is also evident in the speckle pattern. Therefore, SIM is also feasible. Compared to illumination using single-mode fibers, even under optimal collimation, the multimode optical guide itself provides an angular spectrum that causes the projected, so-called speckled image to be distributed across the illumination pattern, thus minimizing or eliminating the problem altogether.

[0065] In a particularly preferred embodiment, at least one means for changing the numerical aperture of the excitation beam coupled from the multimode optical guide is provided in the illumination optical path. This numerical aperture is also referred to as the exit aperture.

[0066] The numerical aperture of the light coupled out of the multimode optical guide can be affected by the numerical aperture of the excitation beam coupled into the multimode optical guide. Therefore, in a preferred design, the means for changing the numerical exit aperture can include means for changing the numerical aperture of the excitation beam coupled into the multimode optical guide. The numerical aperture of the beam coupled into the multimode optical guide is also referred to as the entrance aperture.

[0067] The device for changing the numerical exit aperture can have at least one variable optical element. In principle, this variable optical element is arranged at the exit end of the multimode optical guide. In a preferred variant, the variable optical element is arranged at the entrance end of the multimode optical guide. The variable optical element can advantageously have a variable lens group, particularly a zoom optics device or other optical elements with variable focal length, or a lens changer.

[0068] In principle, it is also feasible for the device for changing the numerical exit aperture to have a non-variable optical component at the inlet end of the multimode optical guide. Alternatively or supplementarily, the device for changing the numerical exit aperture may have an optical component disposed at the outlet end of the multimode optical guide. This optical component may have light diffraction and / or refraction components, such as lenses. This optical component may be material-locked to the output end of the multimode optical guide.

[0069] In a preferred variation, the optical component is formed at the output end of the multimode optical guide. This can be achieved, for example, by 3D printing. Alternatively or complementary, the output end of the multimode optical guide may have a polished surface through which a lensing effect is provided at least partially.

[0070] In another variation, the optical component at the output end of the multimode optical guide has a tapered tip. This tapered tip allows for the alteration of the exit opening diameter. The diameter of the tapered tip can be increased in the beam direction, thereby enlarging the exit opening, or decreased, thereby reducing the exit opening.

[0071] The microscope's control unit may preferably be configured to evaluate the detector's measurement data, drive a variable wavefront modulation device and / or drive a device for adjusting the incident angle of the excitation light onto the sample, and perform at least one of the following methods: SIM method, SMLM method, TIRF method, TIRF-SIM method, TIRF-SMLM method. Attached Figure Description

[0072] Other features and advantages of the present invention will be explained below in conjunction with the accompanying drawings. Wherein:

[0073] Figure 1 A first embodiment of the microscope according to the present invention is shown schematically.

[0074] Figure 2 A second embodiment of the microscope according to the present invention is shown schematically.

[0075] Figure 3 An embodiment of a shaking device for multimodal fibers is shown schematically.

[0076] Figure 4 The components of a diffuser that can move in the illumination path are shown in schematic form.

[0077] Figure 5 This shows a first example of an illuminated area of ​​a sample imaged on the sensing surface of a detector.

[0078] Figure 6 This shows a second example of an illuminated area of ​​a sample imaged on the sensing surface of a detector.

[0079] Figure 7 A third example is shown, on which the illuminated area of ​​the sample is imaged on the sensing surface of the detector.

[0080] Figure 8 This diagram illustrates how excitation light illuminates the sample.

[0081] Figure 9 This diagram illustrates the pupil plane used to explain the phenomenon of excitation light illuminating the sample.

[0082] Figure 10 This diagram illustrates another method for explaining how excitation light is irradiated onto a sample.

[0083] Figure 11 A schematic diagram showing the excitation beam emitted from the multimode optical guide 20 and the rear optical components is shown.

[0084] Figure 12 : A schematic diagram showing the influence of the numerical aperture of the excitation beam emitted from the multimode optical guide on the numerical aperture of the excitation beam entering the multimode optical guide.

[0085] Figure 13 A first example of a device for changing the numerical aperture of an excitation beam coupled from a multimode optical guide is shown; and

[0086] Figure 14A second example of a device for changing the numerical aperture of an excitation beam coupled from a multimode optical guide is shown. Detailed Implementation

[0087] Identical and functionally equivalent components are typically given the same reference numerals in the drawings. The first embodiment of the microscope 100 according to the present invention is combined with... Figure 1 To describe. Figure 1 The microscope 100, schematically shown in the figure, according to the present invention, firstly has a light source 10 (e.g., a laser) for providing excitation light 11 and an illumination optical path for guiding the excitation light 11 into a sample region 1. A sample 2, schematically shown, is present in the sample region 1. The sample plane is indicated by reference numeral 70. In the illustrated embodiment, the illumination optical path has a variable wavefront manipulation device 42 arranged in or near an intermediate image plane 71. In the illustrated embodiment, the wavefront manipulation device 42 is a transmission grating that can be moved laterally (i.e., perpendicular to the optical axis 15) by a driver not shown. The transmission grating 42 may in particular be a phase grating. Furthermore, according to the present invention, the microscope 100 also has a two-dimensional spatially resolved detector 95 (e.g., a CMOS camera) for detecting emitted light 16 emitted from the sample 2 in the sample region 1, and a detection optical path with a microscope objective 93 for guiding the emitted light 16 onto the detector 95. Finally, the microscope 100 also has a control unit 90 for evaluating the emitted light 16 detected by the detector 95. In the illustrated embodiment, the control unit 90 is also configured to drive a driver (not shown) for laterally moving the transmission grating 42. Then, according to the invention, the illumination optical path has at least one relay optics 22, 23 to provide an intermediate image plane 73, and the illumination optical path has a multimode optical guide 20 between the light source 10 and the wavefront manipulation device 42 (i.e., the transmission grating 42). According to the invention, the output end 21 of the multimode optical guide 20 is arranged in or near the intermediate image plane 73.

[0088] Alternatively, the wavefront manipulation device 42 can also be implemented by, for example, a reflective and phase-modulated spatial light modulator, which is driven by the control unit 90.

[0089] Specifically, the excitation light 11 provided by the light source 10 enters the multimode optical guide 20 in Gaussian mode via a beam-shaping optics 12 having lenses 13 and 14. In the illustrated embodiment, this multimode optical guide should be a multimode fiber 20. According to the invention, the output end 21 of the multimode fiber 20 is located in an intermediate image plane 73, which is generated by imaging an intermediate image plane 72 by lenses 22 and 23. Lenses 22 and 23 form a relay optics. The intermediate image plane 72 itself is generated by another relay optics 30 formed by lenses 31 and 32. The relay optics 30 images the intermediate image plane 71, where the transmission grating 42 is located, onto the intermediate image plane 72.

[0090] In the illustrated embodiment, the microscope objective 93 is also part of the illumination optical path. To separate the illumination light 11 and the redshifted emission light 16, there is a main beam splitter 92 (e.g., a dichroic beam splitter) at which the excitation light 11 is reflected, while the redshifted emission light 16 is transmitted toward the detector 95.

[0091] The excitation light 11 thus enters the microscope objective 93 via lenses 22 and 23, via lenses 31 and 32, and via the microscope tube lens 91 and the main beam splitter 92, and is guided from the microscope objective to the sample plane 70 in the sample 2, and is focused. The sample plane 70 is imaged onto the intermediate image plane 71 by the microscope objective 93 and the microscope tube lens 91.

[0092] The emitted light 16, caused by the radiation of the excitation light 11, is collected by the microscope objective 93 and guided toward the main beam splitter 92. The emitted light 16 then passes through the main beam splitter 92 as specified and is guided by the lens 94 in the probe path to the sensing plane 96 of the camera 95, where it is then spatially resolved as specified by the camera 95. The measurement data from the camera 95 is evaluated using the control unit 90.

[0093] In the illustrated embodiment, a pivotable mirror is present in the intermediate image plane 72, which allows for adjusting the angle of incidence of the excitation light 11 onto the sample 2. The device, and which may be referred to as TIRF reflector 41. To enable TIRF reflector 41 to pivot in the direction of the curved double arrow, there exists a device not in... Figure 1 The mechanical actuator shown can be controlled by the control unit 90. Here, as... Figure 10 As explained, the angle of incidence is measured relative to the optical axis 15, which extends collinearly with the z-axis. .

[0094] exist Figure 1In the schematic diagram, the multimode fiber 20 should actually have a length of more than 2 m, so that there is a sufficiently uniformly distributed excitation light 11 at the output end 21 of the multimode fiber 20.

[0095] Furthermore, in the embodiments, in Figure 1 Not shown in the diagram, the sensing surface 297 of the detector 95 and the cross-section of the multimode fiber 20 both have rectangular shapes, and the detection optical path is configured to image the illuminated surface in the sample region 1 onto the sensing plane 96 of the camera 95, such that the overlap between the sensing area of ​​the camera 95 and the image of the illuminated surface in the sample region 1 in the sensing plane 96 is maximized. Figure 7 To illustrate this, the figure shows that an image 298 of the region in sample region 1 illuminated by the excitation light 11, transmitted by the probe optical path, is precisely imaged onto the sensing surface 297 of the camera 95. The image 298 of the region illuminated by the excitation light 11 substantially corresponds to the cross-section of the multimode fiber 20.

[0096] Figure 5 A variation is shown in which the sensing area 97 of the camera 95 has a regular hexagonal shape, and the multimode fiber 20 has a circular cross-section. The probe optical path is configured to image the illuminated surface in the sample area 1 (which is also circular due to the circular cross-section of the multimode fiber 20) onto the sensing plane 96 of the camera 95, such that in the sensing plane 96, the circular image 98 of the illuminated surface in the sample area 1 is completely located on the hexagonal sensing area 97 of the camera 95, and the overlap between the circular image 98 of the illuminated surface in the sample area 1 and the hexagonal sensing area 97 is maximized.

[0097] Figure 6 Another variant is shown, which is related to Figure 5 The difference between the variant described in the text and the variant is that the multimode fiber 20 has a hexagonal cross-section, and therefore the image 198 of the area illuminated in the sample region 1 in the sensing plane 96 also has a hexagonal cross-section.

[0098] Combination Figure 2 Further embodiments of the microscope 200 according to the present invention have been explained. Only those related to... Figure 1 The differences between the microscope 100 and the microscope 100.

[0099] and Figure 1 compared to, Figure 2 In this embodiment, there is no relay optics 30. This means that the first intermediate image plane 71 is imaged onto the intermediate image plane 72 through lenses 22 and 23, and the exit end 21 of the multimode fiber 20 is located in the intermediate image plane 72.

[0100] replace Figure 1 The TIRF reflector 41, in Figure 2 In this embodiment, a device 43 for generating an adjustable lateral beam deflection is arranged in the pupil plane 81. This device may be referred to as a TIRF slider, and it enables the adjustment of the incident angle of the excitation light 11 onto the sample 2. The device, schematically shown, includes a TIRF slider 43, which may, for example, have a biprism capable of laterally variable positioning in the illumination path. For laterally adjusting the laterally variable biprism, a mechanical actuator (not shown) is provided, which can be controlled by a control unit.

[0101] To improve the uniformity of the beam profile at the output end 21 of the multimode fiber 20, a device for shaking or vibrating a portion of the multimode fiber 20 can be used. This device... Figure 3 The multimode fiber 20 is schematically shown. The main portion of the multimode fiber 20 is housed within a housing 61, in which an actuator 60, mechanically coupled to the multimode fiber 20, is located. The multimode fiber 20 is rigidly held relative to the housing 61 at both its input and output ends. The actuator 60 allows vibrations to be transmitted to the multimode fiber 20, thereby altering its geometry and thus the composition of the currently propagating optical mode. For example, the multimode fiber 20 can be... Figure 3 The double arrows indicate that the light oscillates back and forth. This improves the uniformity of light distribution at the exit end 21 of the multimode fiber 20. For example, good results can be obtained in terms of light distribution uniformity when the vibration frequency is on the order of 1 kHz.

[0102] Alternatively or supplementarily, such as Figure 4 The diagram schematically illustrates that a diffuser 62 is present in the region of the exit end 21 of the multimode fiber 20, which can be oscillating or rotating by means of an actuator 64 (e.g., a rocking device and / or a rotating device). A very uniform light distribution, i.e., a largely spotless light distribution, can thus be achieved at the exit end of the diffuser 62.

[0103] Combination Figure 8 and Figure 9 The geometric relationship of the light spot properly positioned in the pupil plane 80 in the back focal plane 80 of the microscope objective 93 is explained.

[0104] Here, Figure 8 The microscope objective 93, sample area 1, sample 2, immersion fluid 3, and pupil plane 80 (i.e., the back focal plane of microscope objective 93) are schematically shown. The optical axis of microscope objective 93 extends collinearly with the z-axis. This represents the refractive index of sample 2, and It is the refractive index of the immersion liquid 3. Figure 9 A cross-section of the pupil plane 80 with light spot 86 is shown. The pupil plane 80 extends in the xy plane. The x, y, and z axes form a right-handed rectangular coordinate system. This indicates the radial extension of the TIRF region (i.e., the region that satisfies the TIRF condition) in the pupil plane 80. It is the inner radius, and It is the maximum outer radius of the TIRF region in the pupil plane 80. The minimum angle equivalent to total internal reflection .

[0105] According to Snell's formula in classical electrodynamics, the critical angle for total internal reflection is expressed as follows:

[0106]

[0107] Using a microscope objective lens with a focal length of 93. and the refractive index of immersion liquid 3 To obtain the minimum radius This minimum radius is equivalent to the minimum angle of total internal reflection. :

[0108]

[0109] The radial width of the ring that satisfies the TIRF condition is

[0110] ,

[0111] It utilizes the length of the lens tube Numerical aperture of illumination objective lens and the magnification of the illumination path It can be described as:

[0112]

[0113] If we substitute the typical values ​​of aqueous samples and typical objectives into this expression, for Conclusion:

[0114]

[0115]

[0116] While spatially coherent light is not necessarily required for SMLM procedures, at least partially coherent light is required in SIM because the variable wavefront manipulation device (especially a static or SLM-generated phase grating) is illuminated and imaged into the sample plane 70. Illuminating the phase grating with excitation light that reduces coherence results in a reduced axial modulation depth of the structured light, and thus a reduced axial resolution. If flat-top illumination is used on the SIM microscope, beam shaping therefore does not completely destroy spatial coherence. In order for light of different diffraction orders to interfere in the sample plane 70, linearly polarized light must be used to illuminate the phase grating. Coherent illumination light leads to interference effects in the illumination field.

[0117] For TIRF, the beam diameter of the excitation light in the objective pupil 80 of the illumination objective 93 should be small to ensure that all portions of the excitation light 11 meet the TIRF conditions. When using multimode fibers for critical illumination, there is a trade-off between the achievable field of view (FOV) and the spot size in the objective pupil 80.

[0118] To achieve the TIRF condition and a sufficiently large field of view (FOV) in the illumination, the relay optics should meet the following conditions. First, the beam diameter of the excitation light 11 in the pupil 80 of the illumination objective 93 should be... It should be less than a specified value to satisfy the TIRF condition. Then, the lateral dimensions of the FOV in the sample plane should include a specified minimum parameter. The FOV dimensions can, for example, refer to the FOV of an existing system in SIM mode. These values ​​are derived based on the following considerations: To satisfy the TIRF condition, the following should be achieved first:

[0119]

[0120] In a preferred embodiment of the invention, the characteristics of multimode optical guides, especially multimode fibers, are fully utilized such that, to a certain extent, the numerical aperture of the coupled light can be influenced by the numerical aperture of the coupled light. This characteristic can be used, for example, to realize a TIRF microscope with a wide field of view formed by multimode fibers. This will be explained below. For a high-resolution microscope with TIRF, such as a SIM or SMLM, the achievable FOV should be as large as possible. When the output end of the multimode optical guide 20 is imaged onto the sample plane, the numerical aperture of the light at the end of the multimode optical guide 20 (especially the multimode fiber) is utilized. The focal length of the telescope lens 91 The focal length of lens 22 The focal length of the illumination objective lens is 93. and the focal length of relay lens 23 The following conditions were derived, which characterize the trade-off between the beam diameter in the pupil and the field of view (FOV) of illumination:

[0121] Formula 1:

[0122]

[0123] Formula 2:

[0124]

[0125] in:

[0126] The diameter of the illumination beam immediately downstream (i.e., after) the beam from lens 22

[0127] The diameter of the illumination beam immediately downstream (i.e., after) the relay lens 23

[0128] The beam diameter of the excitation light 11 in the pupil 80 of the illumination objective lens 93

[0129] The diameter of the multimode optical guide 20 at the exit end 26

[0130] focal length of lens 22

[0131] Focal length of the illumination objective lens 93

[0132] The focal length of the telescope lens 91

[0133] Focal length of relay lens 23

[0134] The field of view of illumination = the lateral dimensions of the area illuminated in the sample plane.

[0135] Magnification of the lighting path

[0136] Numerical aperture of the illumination beam after multimode optical guide coupling

[0137] For the SIM to function correctly, it is preferable to prevent the 0th and + / -1st order diffraction spots formed in the objective pupil for SIM illumination from becoming too large. Large and incompletely coherent illumination spots in the objective pupil reduce the depth of the z-modulation of the resulting interference pattern. Figure 11 In the diagram, the numerical aperture of the excitation light beam 11 emitted from the multimode optical guide 20 is schematically represented as an angle. As shown. Assuming a refractive index of 1, the numerical aperture is mathematically expressed as...

[0138] = .

[0139] Can be via (i.e., the numerical aperture of the excitation light 11 coupled into the multimode optical guide 20) can be controlled to achieve a larger... SIM / SMLM+TIRF. As described above, by the coupled excitation light 11 The control allows for the selective excitation of modes in the multimode optical guide 20. This results in the excitation light 11 coupled from the multimode optical guide 20 supporting a smaller amplitude compared to the multimode optical guide 20 itself. Due to the numerical aperture being smaller than 20 nm of the multimode optical guide... The excitation light 11 is coupled in such a way that not all modes supported or possible by the multimode optical guide 20 are excited. The number of excited modes determines the spot size in the beam profile at the output of the multimode optical guide 20. To obtain uniform illumination, a certain minimum number of modes must be excited. Therefore, during coupling, it is not advisable to use... The number of modes supported in the multimode optical guide 20 is chosen to be arbitrarily small. This also depends on the geometry of the multimode optical guide 20, particularly its shape and size. Therefore, the minimum possible... It also depends on the geometry of the multimode optical guide 20.

[0140] Figure 12 This schematically illustrates the angle of the excitation light 11 emitted from the exit end 26 of the multimode optical guide 20. Furthermore, numerical aperture The numerical aperture of the excitation light 11 that will enter the inlet 24 of the multimode optical guide 20 The impact. Figure 12 Angles are shown in the figure. and Assuming a refractive index of 1, the numerical aperture is mathematically given by the sine of each angle, i.e. and Given a light source, i.e., given a beam size and a given initial beam divergence value, The focal length of the lens 51 used before the fiber is used to couple the excitation light 11. To control. Lens 51 is used to change the numerical aperture of the excitation light 11 coupled from the multimode optical guide 20. The lens 51 is part of the device 50. It may be part of a variable focal length optics device (e.g., a zoom optics device), or it may be part of a lens changing device that allows the focal length to be changed as desired. And thus change the angle and And ultimately change In particular, it can reduce This is to reduce the beam diameter given in Equation (1) and to make it easier to satisfy the TIRF condition.

[0141] Figure 13 and Figure 14 The following variation is shown, in which the numerical aperture of the excitation light 11 coupled from the multimode optical guide 20 is changed. The device 50 is realized through a tapered end that is materially locked to the multimode optical guide 20. Figure 13 In this configuration, the diameter increases along the beam direction, resulting in a larger diameter at the exit end 26 compared to other multimode optical guides 20. Accordingly, Reduce. In Figure 14 In this case, the diameter decreases along the beam direction; specifically, at the exit end 26, the diameter of the multimode optical guide 20 is smaller compared to other multimode optical guides 20. Correspondingly, This increases the field size in the sample. Therefore, as shown in Formula 2 above, it also affects the field magnitude. .

[0142] This invention introduces a novel microscope for achieving high-resolution laser wide-field microscopy using flat-top illumination. The key feature is that the exit end of a multimode optical guide (especially a multimode fiber), preferably having a rectangular or polygonal cross-section, is critically imaged onto the sample plane via a relay optics. An intermediate image plane is generated by at least one relay optics. This microscope can be, in particular, a fluorescence wide-field microscope, with additional devices for implementing SMLM and / or SIM methods. Furthermore, Hilo / TIRF illumination can optionally be implemented.

[0143] List of reference numerals

[0144] 1 Sample Area

[0145] 2 samples

[0146] 3 Immersion liquid

[0147] 10. Light sources, such as lasers and LEDs

[0148] 11 Excitation Light

[0149] 12 Beam Shaping Optics

[0150] 13. First lens of beam shaping optics 12

[0151] 14. Second lens of beam shaping optics 12

[0152] 15. Optical axis parallel to the z-axis

[0153] 16. Emitting light, such as fluorescence.

[0154] 20. Multimode optical guides, such as multimode fibers

[0155] 21 Output terminal of multimode optical guide 20

[0156] 22, together with lens 23, constitutes the collimating lens of the relay optical device.

[0157] 23 Lens forming a relay optics device with collimating lens 22

[0158] 24. The entrance or input opening of the multimode optical guide 20.

[0159] 26. Output or exit opening of multimode optical guide 20

[0160] 30 Relay Optical Devices

[0161] 31 The first lens of relay optical device 30

[0162] 32 The first lens of relay optical device 30

[0163] 41. The angle of incidence in the intermediate image plane 72 for adjusting the excitation light 11 onto the sample 2 The device, a pivotable mirror or a pivotable glass plate, TIRF mirror

[0164] 42 Wavefront manipulation devices, such as gratings, SLMs

[0165] 43. The angle of incidence in the pupil plane 81 used to adjust the excitation light 11 onto the sample 2. The device features a laterally movable double prism and a TIRF slider.

[0166] 50 Numerical aperture used to change the beam of excitation light 11 coupled from multimode optical guide 20 device

[0167] 51. Numerical aperture used to change the beam of excitation light 11 coupled into the multimode optical guide 20 Lenses, such as a part of a zoom optics device

[0168] 53. An optical component forming the exit end 26 of the multimode optical guide 20, a tapered element.

[0169] 54. An optical component forming the exit end 26 of the multimode optical guide 20, a tapered element.

[0170] 60 A device for shaking or vibrating the multimode optical guide 20

[0171] 61 Housing of device 60

[0172] 62. Diffusers in lighting paths, such as oscillating plates.

[0173] 64 The actuator of diffuser 62, such as a swing device

[0174] 70 Planes in sample region 1, planes in sample 2

[0175] 71. Intermediate image plane, first intermediate image plane

[0176] 72. Intermediate image plane, second intermediate image plane

[0177] 73. Intermediate image plane, third intermediate image plane

[0178] 80 microscope objective lens, 93° back focal plane, pupil

[0179] 81. Pupil plane

[0180] 86 light spots in pupil 80

[0181] 90 Control units, such as PCs

[0182] 91. Lens tube in illumination optical path

[0183] 92 Main beam splitter

[0184] 93. Microscope objectives and illumination objectives

[0185] 94. Lens in the detection optical path

[0186] 95. Detectors, such as cameras

[0187] 96. Sensing plane of detector 95

[0188] 97. Sensing surface of the detector

[0189] The image of sample 2 is projected onto the illuminated area on the sensing surface 97 of detector 95, corresponding to the cross-section of multimode optical guide 20.

[0190] 100 Microscope according to the present invention

[0191] The image of sample 2 is projected onto the illuminated area on the sensing surface 97 of detector 95, corresponding to the cross-section of multimode optical guide 20.

[0192] 200 Microscope according to the present invention

[0193] 297 Detector 95 sensing surface

[0194] The image of sample 298 is projected onto the illuminated area on the sensing surface 297 of detector 95, corresponding to the cross-section of multimode optical guide 20.

[0195] The diameter of the illumination beam immediately downstream (i.e., after) of lens 22

[0196] The diameter of the illumination beam immediately downstream (i.e., after) the relay lens 23

[0197] The beam diameter of the excitation light 11 in the pupil 80 of the illumination objective lens 93

[0198] The diameter of the multimode optical guide 20 at the exit end 26

[0199] Refractive index of immersion liquid 3

[0200] Refractive index of sample 2

[0201] The focal length of lens 51 before the input opening of multimode optical guide 20

[0202] focal length of lens 22

[0203] Focal length of the illumination objective lens 93

[0204] Lens tube length

[0205] The focal length of the telescope lens 91

[0206] Focal length of relay lens 23

[0207] The field of view of illumination = the lateral dimensions of the area illuminated in the sample plane.

[0208] Magnification of the lighting path

[0209] Numerical aperture of microscope objective lens 93

[0210] Numerical aperture of multimode optical guide 20 (e.g., multimode fiber 20)

[0211] The numerical aperture and numerical output aperture of the excitation light 11 coupled from the multimode optical guide 20.

[0212] Numerical aperture of the excitation light 11 coupled into the multimode optical guide 20, numerical incident aperture

[0213] Right-handed Cartesian coordinate system (x, y, z)

[0214] Aperture angle of the excitation beam coupled into the multimode optical guide 20

[0215] Aperture angle of the excitation beam coupled into the multimode optical guide 20

[0216] Radial extension of the TIRF region in the pupil plane 80

[0217] Inner radius of the TIRF region in the pupil plane 80

[0218] Outer radius of the TIRF region in the pupil plane 80

[0219] Radial width of an annulus satisfying the TIRF condition

[0220] The angle of incidence of excitation light 11 onto sample 2

[0221] The minimum angle for total internal reflection, the critical angle for total internal reflection, is equivalent to the radius within the objective lens pupil at 80°.

[0222] References

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Claims

1. Microscope, having a light source (10) for providing excitation light (11), an illumination light path for guiding the excitation light (11) into a sample region (1), a two-dimensionally spatially resolving detector (95) for detecting emission light (16) emitted from a sample (2) in the sample region (1), a detection light path with a microscope objective (93) for guiding the emission light (16) onto the detector (95), and a control unit (90) for evaluating the emission light (16) detected by the detector (95), characterized in that the illumination light path has at least one relay optics (31, 32; 22, 23) for providing an intermediate image plane (72; 73), the illumination light path has a multimode light guide (20), an output end (21) of the multimode light guide (20) is arranged in or near the intermediate image plane (72; 73), the illumination light path has a variable wavefront manipulation device (42) arranged in or near the intermediate image plane (71).

2. Microscope according to claim 1, characterized in that the multimode light guide (20) has at least one multimode fiber or at least one multimode light guide rod.

3. Microscope according to claim 1 or 2, characterized in that the variable wavefront manipulation device (42) has a spatial light manipulator.

4. Microscope according to claim 3, characterized in that the variable wavefront manipulation device (42) has at least one of the following components: a diffractive component, a laterally moveable transmissive grating, a laterally moveable reflective grating, a refractive component.

5. Microscope according to any one of claims 2 to 4, characterized in that the length of the multimode fiber (20) is more than 2 meters.

6. Microscope according to any one of claims 2 to 5, characterized in that the illumination light path has a device for mechanically manipulating the multimode fiber (20), in particular a device (60) for shaking or vibrating the multimode fiber (20).

7. Microscope according to any one of claims 1 to 6, characterized in that the illumination light path has a diffuser (62) downstream of the light beam of the multimode light guide (20) or light guide rod, in particular.

8. Microscope according to claim 7, characterized in that there is a device for moving the diffuser (62).

9. Microscope according to claim 8, characterized in that the device for moving the diffuser (62) has a rocking device and / or a rotating device.

10. Microscope according to any one of claims 1 to 9, characterized in that the multimode light guide (20) has a circular cross section.

11. Microscope according to any one of claims 1 to 9, characterized in that the cross section of the multimode light guide (20) has a polygonal shape, in particular equilateral, a rectangular shape or a square shape.

12. Microscope according to any one of claims 1 to 9, characterized in that ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The sensing surface of the detector (95) and the cross section of the multimode light guide (20) each have, in particular, a regular polygonal shape, a regular hexagonal shape (198, 97), a rectangular shape (297, 298) or a square shape.

13. The microscope according to any one of claims 1 to 12, characterized in that the detection light path is set up for imaging the illuminated face in the sample region (1) onto the sensing plane (96) of the camera (95) such that the overlap between the sensing region of the camera in the sensing plane (96) and the image of the illuminated face in the sample region (1) is maximized.

14. The microscope according to any one of claims 1 to 13, characterized in that The illumination path has an incident angle for adjusting the excitation light (11) onto the sample (2). ) device.

15. The microscope according to claim 14, characterized in that Used to adjust the incident angle of the excitation light (11) The device has a pivotable mirror (41) or a pivotable plane-parallel glass plate, the mirror or the plane-parallel glass plate being arranged in or near the intermediate image plane (72) of the illumination path.

16. The microscope according to claim 14 or 15, characterized in that Used to adjust the incident angle of the excitation light (11) The device has a means for generating an adjustable lateral beam deflection arranged in or near the pupil plane (81) of the illumination path.

17. The microscope according to any one of claims 1 to 16, characterized in that the control unit (90) is set up for ■ evaluating the measurement data of the detector (95), and / or ■ driving the variable wavefront modulation device (42), and / or ■ Drive control is used to adjust the incident angle of the excitation light (11) onto the sample (2). ) device, ■ carrying out at least one of the following methods • a SIM method, • a SMLM method, • a TIRF method, • a TIRF-SIM method, • a TIRF-SMLM method.

18. The microscope according to any one of claims 1 to 17, characterized in that In the illumination optical path, there is at least one numerical exit aperture for changing the excitation light (11) beam coupled from the multimode optical guide (20). ) device (50).

19. The microscope according to claim 18, characterized in that Used to change the numerical output aperture ( The device (50) has a numerical incident aperture for changing the excitation light (11) beam coupled into the multimode optical guide (20). ) device (50).

20. The microscope according to any one of claims 18 or 19, characterized in that the device (50) for changing the numerical exit aperture has at least one variable optical component (52).

21. The microscope according to claim 20, characterized in that the variable optical component (50) is arranged at the entrance end of the multimode light guide (20) and has, in particular, a variable lens group (51), in particular a zoom optics.

22. The microscope according to any one of claims 19 to 21, characterized in that the device (50) for changing the numerical exit aperture has an optical component which is arranged at the exit end (26) of the multimode light guide (20) and, in particular, is shaped on the output end (26) of the multimode light guide (20).

23. The microscope according to claim 22, characterized in that the optical component is or has a lens.

24. The microscope according to any one of claims 22 or 23, characterized in that the optical component has a conical tip (53, 54) at the output end (26) of the multimode light guide (20).

25. Method for microscopy, in which the following method steps are carried out: - guiding excitation light (11) from a light source (10) via an illumination light path onto a sample (2), - guiding emission light (16) emitted by the sample (2) via a detection light path having a microscope objective (93) onto a two-dimensionally spatially resolving detector (95), and - evaluating the measurement data of the detector (95). detecting the emitted light (16) with the detector (95), characterized in that the intensity distribution of the excitation light (11) over the beam cross section of the excitation light (11) is homogenized by guiding the excitation light (11) provided by the light source (10) in an illumination light path via a multimode light guide (20), wherein the output end (21) of the multimode light guide (20) is arranged in or near the intermediate image plane (72; 73), and in particular for carrying out a SIM method and / or a TIRF method, the excitation light (11) is manipulated with a variable wavefront manipulation device (42) arranged in or near the intermediate image plane (71) in the illumination light path.

26. The method according to claim 25, characterized in that at least a portion of the multimode fiber (20) is moved back and forth in the illumination light path.

27. The method according to claim 25 or 26, characterized in that at least a portion of the multimode fiber (20) is shaken or excited to vibrate in the illumination light path.

28. The method according to claim 26 or 27, characterized in that the frequency of the movement, in particular the vibration frequency, of a portion of the multimode fiber (20) is greater than 500 Hz, preferably greater than 1 kHz.

29. The method according to any one of claims 25 to 28, characterized in that a diffuser (62) in the illumination light path is moved, in particular moved back and forth and / or rotated.

30. The method according to claim 29, characterized in that one or the diffuser (62) is in an oscillating state relative to the output end (21) of the multimode light guide (20).

31. The method according to any one of claims 25 to 30, characterized in that at least one of the following methods is carried out: • a SIM method, • a SMLM method, • a TIRF method, • a TIRF-SIM method, • a TIRF-SMLM method, • a HILO method.

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