Fluorescence microscope and set of components and modules for upgrading a fluorescence microscope

Abstract

A fluorescence microscope (10) comprises a sample illumination beam path comprising a light source (9) for illumination light, a first wavefront modulator (24) for providing a focused illumination light (8) having a central intensity minimum, a beam splitter (26), and a second adjustable wavefront modulator (34) arranged in a pupil plane (30) of an objective (20). A first detection beam path section comprising the second wavefront modulator (34) and a telescope (11) and ending at the beam splitter (26) coincides with the sample illumination beam path. A separate second detection beam path section comprises a detector (38) for detecting fluorescence from the sample. The telescope (11) images a first pupil (31) formed in the pupil plane (30) into a smaller second pupil (32) and converts a beam of the illumination light (8) collimated in the second pupil (32) into an enlarged beam collimated in the first pupil (31).

Description

TECHNICAL FIELD

[0001] The present invention relates generally to scanning fluorescence microscopy, and more particularly to a confocal laser scanning fluorescence microscope for three-dimensional imaging. The technical field includes fluorescence microscopy for confocal fluorescence microscopy, STED microscopy and MINFLUX microscopy. BACKGROUND

[0002] From US patent application publication US 2015 / 0 338 639 Al a transmissive phase modulation element and a microscope comprising an objective and such a phase modulation element are known. The phase modulation element is arranged perpendicular to an optical axis in a pupil plane which is provided by imaging the back aperture of the objective by means of relay optics, as the back aperture of the objective is usually not accessible. The phase modulation element is located in a light path section belonging to the illumination light path and the detection light path. Thus, both the collimated illumination light and the light emanating from the sample and collected by the objective pass the modulation element which changes the phase of the illumination light to correct for aberrations caused by the sample and also partly corrects for aberrations of the light emanating from the sample. US 2015 / 0 338 639 Al further proposes the use of a deformable mirror, i.e. a phase modulation element working in reflection mode. However, no particular instructions are given as to how to integrate such a mirror in the light path, or even how to preferably integrate a transmissive phase modulation element in the light path.

[0003] Japanese patent application publication JP 2004 341 394 A discloses a laser scanning fluorescence microscope comprising a deformable mirror for correcting aberrations occurring in the imaging structure within the medium, i.e. not at the surface of the medium. The deformable mirror is arranged in a pupil plane in a light path section belonging to the illumination light path and the detection light path. In this common light path section of the detection light path and the illumination or excitation light path, a scanner is arranged in another pupil plane. The two mentioned pupil planes are imaged onto each other by relay optics with the same size, i.e. in a 1 : 1 ratio. The disclosure of JP 2004 341 394 A starts from a prior art system which applies the correction of aberrations only by the deformable mirror. The applicant of JP 2004 341 394 A points out that this prior art system does not allow a sufficient correction, in particular for off-axis focusing positions, and that this problem is solved by combining the deformable mirror with a specially designed objective.

[0004] The laser scanning fluorescence microscope known from the European patent application publication EP 1 372 011 A2 and the US patent US 7 633 053 B2, which are of the same patent family, comprises a laser providing illumination light, a beamsplitter reflecting the illumination light and being transparent for detection light, and a polarization beamsplitter. Depending on the polarization of the illumination light, the polarization beamsplitter directs the illumination light into a first or a second branch of an illumination light path. In each of the two branches, the respective illumination light is directed onto an active wave front modulator by a quarter wave plate and a zoom optics, which is made as an adaptive reflecting element, for example as a mirror with a segmented adjustable mirror surface or as a membrane mirror. The zoom optics serves to adapt the aperture of the respective adaptive element to the aperture of the microscope objective. The respective illumination light reflected by the adaptive reflecting element is redirected to the polarization beamsplitter. By two passages through the respective branch comprising the respective zoom optics and the respective associated quarter wave plate, the polarization of the respective illumination light is rotated by 90°. Thus, the deflected part of the light beam, when incident on the beamsplitter, passes through the beamsplitter without deflection when incident in the opposite direction, while the non-deflected part of the light beam, when incident on the beamsplitter, is deflected by the beamsplitter when incident in the opposite direction. In this way, the two parts of the light beam are coaxially coupled into a further common part of the illumination light path. A lens arranged in the further common part images the plane of the wave front modulator into a plane that is conjugate to the back aperture of the objective. A scanning device is arranged in the conjugate plane. The illumination light path is focused in or on the sample by the scanning optics, the tube lens and the objective. Light emitted from the sample, for example fluorescence, is collected by the microscope objective and, depending on its polarization, takes the opposite path to the illumination light, so that the detection light of both polarizations is coaxially emitted from the polarization beamsplitter in the opposite direction to the incident illumination light. Then, the detection light passes through the beamsplitter and is directed to a confocal detection device. Compared to the previously known coupling of light into the adaptive optical system by the beamsplitter, the arrangement of EP 1 372 011 A2 enables a lossless coupling in. An alternative device comprises only one active wave front modulator in the illumination light path, and another alternative device comprises two active wave front modulators, one of which is arranged only in the illumination light path and the other only in the detection light path.

[0005] The international patent application publication WO 2018 / 042 056 Al and the US patent application publication US 2019 / 0 195 800 Al, which belong to the same patent family, relate to a method of adjusting different elements of a conventional STED microscope. These documents describe in particular the overall design of such a STED microscope. It is pointed out here that active or adaptive optical devices, such as wavefront modulators based on liquid crystal technology, often referred to as SLMs, deformable mirrors or MEMS elements, can be arranged in the light path of the excitation light and / or the fluorescence suppression light and / or the fluorescence and are operated by adjustment means for adjusting the laser scanning microscope. No measures for stabilizing the adjustment of the light paths, such as measures for avoiding the influence of angular errors of the light paths relative to each other, are proposed. Since the STED microscope essentially corresponds to that described in WO 2018 / 042 056 Al, it is not further described at this point. Figure 1 The STED microscope described in WO 2018 / 042 056 Al is shown in the attached

[0006] Lenz, Martin O. et al., "3-D stimulated emission depletion microscopy with programmable aberration correction", J. Biophotonics 7, No. 1-2, 29-36 (2014), discloses a special STED microscope which comprises a reflective active wavefront modulator based on liquid crystal technology arranged in the depletion light path. The active part surface of the wavefront modulator is arranged in the pupil or intersects the pupil at an angle. By means of relay optics comprising a mirror and a quarter-wave plate, this pupil is imaged onto a second active part surface of the wavefront modulator, so that this second active part surface is also arranged in the pupil or intersects the pupil at an angle. Each of the two part surfaces is used to modulate a respective one of the two orthogonal polarization directions of the depletion light. Both pupils are generated by imaging the back aperture of the objective by means of lenses which are specifically provided in the depletion light path.

[0007] The thesis "Intelligent-Illumination STED" by Heine, Another STED microscope is disclosed in 2017 (downloadable at http: / / hdl.handle.net / 11858 / 00-1735-0000-002E-E3B0-E) which comprises two excitation lasers. The means for forming the depletion beam comprises a wavefront modulator based on liquid crystal technology designated as SLM. One half of the SLM is used to form a first component of the depletion beam having a first polarization, the other half of the SLM is used to form a second component of the depletion beam having a second polarization orthogonal to the first polarization. This function is achieved by a compact optical means, wherein the incoming depletion beam impinges on the first half of the SLM, which modulates and reflects the first component and does not modulate but also reflects the second component of the depletion beam. Next, the polarization of both components is rotated by 90°. Afterwards, both components impinge on the second half of the SLM, which now modulates and reflects the component which was not modulated before, while it does not modulate but reflects the component which was modulated. This means containing the SLM has the same function as the means known from Lenz, Martin O. et al. "3-D stimulated emission depletion microscopy with programmable aberration correction", J. Biophotonics 7, No. 1-2, 29-36 (2014) described above, but is more compact. However, it has the disadvantage that in any case the two halves of the SLM cannot be precisely arranged in the pupil plane. In the STED microscope, the two halves of the SLM are close to one pupil plane, at the same distance from this pupil plane. Furthermore, this paper discloses a microscope comprising relay optics between the scan lens of a quad-scanner, which is a special design of a scanner particularly suitable for STED and MINFLUX microscopes, and a dichroic mirror for coupling the depletion light into the common optical path for excitation light, depletion light and fluorescence.

[0008] Three German patents DE 100 63 276 C2 and US 6 914 236 B2 and US 7 009 161 B2, which belong to the same patent family, disclose a STED microscope and relate to a laser scanning STED microscope, which focuses a depletion beam onto a first focal region such that the first focal region overlaps with a second, different focal region of an excitation beam. The STED microscope comprises elements for correcting longitudinal and transversal chromatic aberrations between the excitation wavelength and the depletion wavelength, which occur in particular in the scanning optics and the objective. These elements shall ensure that the overlap between the two beams remains constant during scanning. For this purpose, at least one adaptive optical element, for example a deformable mirror, is used. In these patents, different basic arrangements of adaptive optical elements are listed. For example, it is suggested to use an LCD element as adaptive optical element, i.e. an SLM like in the Heine paper, and to arrange this LCD element in the depletion light path in the Fourier plane to the focal plane, i.e. in the image plane with respect to the back aperture of the objective. This element shall be readjusted during scanning to compensate for position-dependent aberrations caused by the optics.

[0009] The German patent application publication DE 10 2011 055 367 A1 and the US patent US 9 291 562 B2, which belong to the same patent family, describe a method and a device for tracking particles in a sample. The device comprises in particular an excitation laser, a beam shaping device, a beam movement device for moving the excitation focus laterally in the sample plane, a sample movement device for moving the sample axially, an objective and a detection device for detecting fluorescence. The beam shaping device has the effect that the excitation beam is formed with an intensity distribution having a central zero point on the focus. The intensity distribution can for example correspond to a STED ring. This setup essentially corresponds to the setup also used for MINFLUX microscopes.

[0010] ​The MINFLUX method is described by Francisco Balzarotti et al. in “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes” ARXiv: 1611.03401 [physics.optics] (2016), including the supplementary material, and in the paper published in the same authors’ title in Science, Vol. 355, Issue 6325, 606-612, (2017). The MINFLUX microscope is a method to determine the position of a single fluorophore in a sample. For applying this method, it is important that the position of one fluorophore in the sample is determined with high precision by another method than MINFLUX with diffraction limited precision or higher, for example by a known localization microscope method. In MINFLUX, this pre-determined position of the fluorophore is used. An intensity distribution with a central zero point, for example a ring-shaped distribution or a one-dimensional distribution, is used to scan the area around the pre-determined position at several sampling points. At all sampling points, the fluorophore is in close proximity to the intensity minimum. From the measured intensity values of the fluorescence emitted by the fluorophore, the position of the fluorophore can be determined with higher precision than in the first determination. Starting from the position of the fluorophore determined with higher precision, the scanning step can be repeated at smaller distances of the sampling points and centered more precisely with respect to the actual position of the fluorophore. The MINFLUX principle is based on scanning the fluorophore with minimal intensity. The MINFLUX principle can be realized by a ring-shaped intensity distribution, an intensity distribution resulting from a standing wave, and a one-dimensional intensity distribution. MINFLUX can also be used for tracking a fluorescent particle, wherein the time period available for scanning the area around the respective temporary position of the fluorescent particle depends on the speed of the fluorescent particle.

[0011] The setup described in the publication of Balzarotti et al. includes elements mentioned when referring to DE 10 2011 055 367 A1 and US 9 291 562 B2. In the supplementary material of the first-mentioned publication, the electro-optical deflector and the piezoelectric tiltable mirror are referred to as beam movement devices for laterally moving the excitation focus. The disclosed microscope includes a plurality of different optical path portions. Both the illumination light and the detection light are guided in one optical path portion. A telescope arrangement, which is not further described, is arranged in this optical path portion. Further, this optical path portion includes the above-mentioned piezoelectric tiltable mirror in a pupil plane. A usable scanning range (about 20 x 20 pm 2), the scanner type was named (PSH-10 / 2, i.e. a device of piezosystem jena GmbH), and the objective used was named as well (HCX PL APO 100x / 1.40-0.70 Oil CS, Leica Microsystems GmbH). From this information, in combination with a data sheet of the scan angle of the tiltable mirror (+ / - 4 mrad), it can be concluded that the telescope images the pupil located between the objective and the telescope in an enlarged fashion into the pupil plane of the tiltable mirror, and that the collimated light beam entering the telescope through the tiltable mirror is converted into a light beam with a reduced cross section towards the objective behind the telescope. The mention of a vortex wave plate as a beam shaping device, which leads to an intensity distribution at the focus with a central minimum. Balzarotti et al. mention the possibility to determine the position of the fluorophore in three-dimensional space by the MINFLUX method as a direct additional means which can be implemented, for example, by a z-ring. However, no specific instructions to implement this additional means are given.

[0012] In the paper "MINFLUX nanoscopy delivers multicolor nanometer 3D-resolution in (living) cells" by Klaus C. Gwosch et al., published on the Preprint Server bioRxiv in August 2019 (doi: http: / / dx.doi.org / 10.1101 / 734251), a method and the used equipment to actually realize three-dimensional imaging by MINFLUX are described. The setup of the equipment essentially corresponds to the setup described by Balzarotti et al. A white light laser is added and an active element, which will be described further below, is used as beam forming device instead of a vortex plate. Furthermore, it is shown how three-dimensional imaging with two different emission wavelengths of fluorophores, i.e. fluorophores that can be spectrally separated in detection, is carried out simultaneously by MINFLUX. The equipment comprises an excitation laser, an amplitude modulator, an active wavefront modulator and an electro-optical x-y-z movement device, which consists of an electro-optical movement device for movement in the x- and y-direction and a variable focus lens in the illumination light path. The type of wavefront modulator is indicated, so that it can be a modulator based on reflective liquid crystals. The wavefront modulator is used to selectively adjust the wavefront of the excitation light, so that the sample experiences an intensity distribution without a central zero point through the objective, experiences a ring or experiences an intensity distribution with an axially delimited central zero point, the so-called 3D ring. At the dichroic mirror, the activation light path is coupled into the common activation and illumination light path. Both the activation and the illumination light path are coupled into the sample through a tiltable mirror for scanning the sample and the objective. In the opposite direction to the illumination light path, the fluorescence emitted from the sample is guided through the objective and the tiltable mirror and is coupled out of the illumination light path and guided towards the detection device through another dichroic mirror.

[0013] From the published publication JP H11-326860 published in Japan, a laser fluorescence microscope scanned by a movable sample stage and a laser scanning microscope with an active wavefront modulator are known, which are used to correct distortions related to the axial scan position, for example caused by the sample, and / or to adjust the axial focus position in the sample. In the arrangement depicted for illustrating the basic principle, when viewed along the excitation beam path, the detection beam path and the excitation beam path merge into a common beam path at a dichroic mirror. The common beam path is guided through a telescope, which enlarges the beam diameter to the required size, a transmissive active wavefront modulator located in the enlarged beam path. In a further course of the common beam path, an objective is arranged for focusing the illumination light onto or into the sample, wherein no further optical elements are located between the wavefront modulator and the objective. As one embodiment of the invention, a confocal laser microscope with a scanning stage is described, which differs from the above-described setup only in that the scanning stage is described and the detection means are described as confocal detection means. As a further embodiment, a confocal laser scanning fluorescence microscope is depicted, which comprises one wavefront modulator each in the excitation beam path and in the detection beam path. Furthermore, a multiphoton laser scanning fluorescence microscope is depicted. None of these embodiments comprises a common detection and excitation beam path section extending after the scanner and comprising a telescope which is scaled down when viewed in the detection direction.

[0014] Rueckel, M. et al. "Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing", PNAS, Vol. 103, No. 46, 17137-17142 (2006) discloses a two-photon microscope comprising a device for correcting sample-induced distortions. The device comprises a deformable mirror in the illumination beam path. A section of the illumination beam path, which especially comprises the microscope objective, is additionally used for guiding light reflected on a calibration object onto a CCD detector. In the direction towards the CCD detector, a telescope is arranged after the deformable mirror, which telescope reduces the beam diameter, so that the beam diameter on the mirror side of the telescope is larger than the beam diameter on the side of the telescope facing away from the mirror. The CCD detector is arranged on a plane which is conjugate to the back aperture of the objective. On the CCD detector, the reflected light without fluorescence is superimposed with light guided by an interferometer device. From the interference pattern, conclusions are drawn about the sample-induced distortions corrected by means of the deformable mirror.

[0015] The international patent application publication WO 2014 / 029978 Al and the US patent US 9 575302 B2, which belong to the same patent family, disclose a scanning laser fluorescence microscope, which is designed as a STED microscope and comprises elements forming an excitation light beam path, which focus excitation light for exciting fluorescence in a sample in the sample, elements forming a depletion light beam path, which focus depletion light for causing stimulated emission of the excited fluorophores, thereby suppressing fluorescence into the sample coaxially with the excitation light, with the intensity of the depletion light having a local intensity minimum on the optical axis, in particular the depletion light is focused such that a so-called STED ring evolves on the focal plane, elements forming a detection light beam path, which comprise a confocal detection device for confocally detecting sample fluorescence, an objective, which is part of all three light beam paths mentioned before and is configured to both focus light beams directed onto the sample onto the sample and to collect and guide fluorescence into the detection light beam path, a dichroic mirror, which merges the excitation light beam path and the depletion light beam path in their course towards the sample and which couples out fluorescence extending along the detection light beam path from the depletion light beam path, the dichroic mirror beam arrangement being in a light beam path section in which all three light beams are collimated, and a piezo stage, which is translatable in all three spatial dimensions, on which a sample to be imaged can be positioned, the piezo stage being configured for moving the sample to be imaged relative to a focal region of the objective and in turn for allowing scanning of the sample in all three spatial dimensions. This setup is also described by Travis J. Gould et al. "Adaptive optics enables 3D STED microscopy in aberrating specimens"; OPTICS EXPRESS, Vol. 20, No. 19, 20999-21009 (2012).

[0016] Furthermore, the microscope according to WO 2014 / 029 978 Al and US 9 575 302 B2 comprises a wavefront modulator, in particular a reflective wavefront modulator based on liquid crystal technology, in its depletion beam path, hereinafter referred to as SLM (abbreviation of the standard term "spatial light modulator"), which is positioned and configured so that it can modulate the depletion light impinging on it with respect to its phase, so that after passing through further elements of the depletion beam path and being focused by the objective, on the one hand a STED ring is formed in the focus and on the other hand the depletion light is provided with a further modulation, so that distortions caused by the sample, for example distortions caused by a mismatch of the refractive index of the sample and the refractive index of the immersion medium, can be compensated by the focus into the sample, so that a high focus quality can be achieved even in deeper regions of the sample. According to the description in WO 2014 029978 Al and US 9575 302 B2, the SLM is arranged in the pupil, i.e. in a plane that is conjugate to the back aperture of the objective, wherein the reflective surface of the SLM is tilted with respect to the plane of the pupil.

[0017] Furthermore, the microscope according to WO 2014 / 029 978 Al and US 9 575 302 B2 comprises a reflective wavefront modulator in its excitation beam path, which is arranged in the pupil like the aforementioned SLM. In one embodiment, the wavefront modulator is arranged only in the excitation beam path and is made as an SLM; in another embodiment, the wavefront modulator is arranged between the aforementioned dichroic mirror and the objective, so that it is located not only in the excitation beam path, but also in the depletion and detection beam paths. In this case, the wavefront modulator is made as a deformable mirror. In both cases, the wavefront modulator is configured so that it can compensate for distortions as explained above with reference to the depletion beam path. In the latter case, in which there is a deformable mirror in all beam paths, the wavefront modulator compensates for distortions in all beam paths, and the SLM, which is located only in the depletion beam path, is used for beam shaping, i.e. for forming the STED ring and for compensating for distortions in the depletion beam path. Even if this is not explicitly described, it is obvious to the person skilled in the art that this relates to a compensation for the remaining distortions that have not yet been compensated by the deformable mirror. While the embodiment first mentioned here is described in detail in WO 2014 029978 Al and US 9 575 302 B2, the latter-mentioned embodiment is only outlined in general terms with reference to the person skilled in the art.

[0018] The common feature of the above-mentioned prior art scanning laser fluorescence microscopes, which are simple confocal microscopes, STED microscopes or MINFLUX microscopes, is that they have a common beam path section in which the detection beam path and the illumination beam path coincide but are directed in opposite directions. In the STED microscope, there is also a beam path section in which at least two illumination beam paths overlap, one of the at least two illumination beam paths being configured for illuminating the sample with a focus having a central intensity minimum. In the MINFLUX microscope, the illumination beam path configured for illuminating the sample with a focus having a central intensity minimum coincides with the detection beam path in a common beam path section. With reference to the STED microscope, the above-mentioned patent application publications WO 2018 / 042 056 A1 and US 2019 / 0 195 800 A1 address the fundamental problem of adjusting the individual beam paths relative to one another such that they coincide as precisely as possible in the common beam path section, or of adjusting the individual beam paths such that the projections of the excitation focus and the STED focus in the confocal detection plane coincide as precisely as possible and are centered within the confocal pinhole. The patent application publications disclose options for how this adjustment can be performed automatically. In practice, such automatic adjustment is also of great interest because misalignments of the individual beam paths can also occur during operation of the microscope, for example as a result of thermal influences. A dichroic mirror used as a beam coupler or beam splitter tilts over time, which leads to the individual beam paths not extending precisely in one direction on one axis after their merging, but rather being misaligned at an angle relative to one another.

[0019] There is still a need for a scanning laser fluorescence microscope which is configured to be able to illuminate a sample with illumination light whose focus has a central intensity minimum, wherein the influence of an angular misalignment between at least one illumination beam path for the illumination light and the detection beam path on the imaging quality is reduced and the quality of the adjustment of the beam paths relative to one another is enhanced. SUMMARY

[0020] According to the application, the fluorescence microscope comprises:

[0021] i) an illumination beam path for illuminating a sample with illumination light, the illumination beam path comprising an illumination light source providing illumination light, a first wavefront modulator for spatially modulating the illumination light, a beam splitter, a second wavefront modulator for further spatially modulating the illumination light, and an objective, wherein the first wavefront modulator is configured for modulating the illumination light such that the modulated illumination light focused by means of the objective causes the sample to be subjected to an intensity distribution of the illumination light comprising a central intensity minimum, the second wavefront modulator being adjustable, wherein an active area of the second wavefront modulator is arranged in or intersects at an angle with a pupil plane of the objective, wherein a first pupil appears at the position of the active area of the second wavefront modulator,

[0022] ii) a detection beam path for confocally detecting luminescence light emitted from the sample, the detection beam path comprising a first section and a second section, wherein the first section of the detection beam path comprising the second wavefront modulator and ending at the beam splitter coincides with the illumination beam path, wherein in the first section of the detection beam path the direction of the detection beam path is opposite to the direction of the illumination beam path, the second section of the detection beam path starting at the beam splitter and comprising a detection unit for confocally detecting the luminescence light does not coincide with the illumination beam path.

[0023] According to its usual definition, the term pupil can be here the image of the aperture or aperture stop of the back aperture of the objective.

[0024] The fluorescence microscope according to the application is characterized in that the first telescope is arranged in the first section between the second wavefront modulator and the beam splitter such that the first telescope images the first pupil in a second pupil smaller than the first pupil in the direction of the detection beam path and transfers the beam of the illumination light collimated in the second pupil into an enlarged beam of the illumination light collimated in the first pupil.

[0025] The first telescope has the effect that all beam groups are narrowed or contracted together when viewed in the direction of propagation of the fluorescence. In this way, it is achieved that an angular deviation of the beam paths relative to each other only leads to a small positional deviation of their focal points in the sample relative to each other, wherein the focal point of the detection beam path in the sample is to be understood as the region in the sample that is arranged confocally relative to the detection device. In other words, it is achieved that the illumination focal point is imaged on the confocal plane with only a very small positional deviation despite the angular deviation of the beam paths relative to each other. The mode of action of the application will be described further below with reference to Figure 4.

[0026] The magnification factor of the first telescope, which determines the ratio of the first diameter of the first pupil to the second diameter of the second pupil, can be at least 1.2:1, preferably at least 1.5:1 or at least 2:1. In particular, the magnification factor or the ratio of the diameters can be about 2:1, 2.5:1 or 3:1. Generally, the magnification factor or the ratio of the diameters will not exceed 20:1. Generally, it will not exceed 10:1, or even 5:1.

[0027] In the context of the present application, the feature that the second wavefront modulator is arranged such that its effective area lies in the pupil plane or intersects it at an angle means that the active surface is located in the vicinity of the geometric position of the pupil plane. The tolerable distance of the effective area to the pupil plane is related to the Rayleigh length of the focal point in the sample and to the back-projection of these Rayleigh lengths from the region of the back focal plane of the objective to the region of the pupil plane in which the second wavefront modulator is arranged. These numbers are in turn related to the wavelength of the illumination light and the detection light and to the available numerical aperture of the objective. In a typical STED microscope, distances in the range of millimetres to several centimetres can be tolerable. However, it is preferred that this distance should be kept as small as possible.

[0028] In the present specification and the appended claims, the term "beam splitter" refers to an optical element which is suitable both for separating and for combining or coupling different beam paths. Thus, each optical element which is referred to herein as a beam splitter can also be referred to as a beam coupler. Typical embodiments of beam splitters or couplers for use in the present application are in particular beam splitting cubes, semi-transparent mirrors, dichroic mirrors, dichroic reflecting prisms, polarization beam splitters. Generally, any beam splitter used in the present application will have a typical design as commonly used in fluorescence microscopes for combining or separating beam paths.

[0029] Different active systems can be used as the second wavefront modulator. Both transmissive and reflective wavefront modulators based on liquid crystal technology, often referred to as SLMs, are considered. However, the second wavefront modulator is preferably made as a deformable mirror. Here, the effective area can be the surface of a deformable membrane. The effective area can also be made by the reflective surface of individual actuators. Membrane mirrors are well known in the art and are also well available on the market. Deformable mirrors whose surface is made by the reflective surface of individual actuators have the advantage over membrane mirrors that their shape can be adjusted at higher speed. Deformable mirrors have the advantage over SLMs that their action or function is independent of the polarization of the light to be modulated. Generally, the second wavefront modulator is used for correcting aberrations, in particular sample-induced aberrations. Due to its position, the second wavefront modulator has the same effect on all beam paths, and thus, it is particularly suitable for correcting aberrations of the illumination light which forms a focal point with a central intensity minimum in the sample as well.

[0030] The first wavefront modulator can be a passive or non-adjustable element, for example a wave plate. In particular, all passive wavefront modulators used in STED microscopes can also be used in the present application. For this purpose, a simple STED microscope can be upgraded by means of an element group according to the application or a module according to the application.

[0031] Preferably, the first wavefront modulator is also an active, i.e. adjustable, wavefront modulator. This active wavefront modulator can be a wavefront modulator based on liquid crystal technology, which is generally referred to as SLM. The first wavefront modulator can be a transmissive SLM or a reflective SLM. A transmissive SLM can be arranged in a simple manner perpendicular to the beam path. Thereby, the effective active area, i.e. the area of the reference area which is used as a component in wavefront formation, can - and preferably - lie completely in the pupil plane, i.e. is not tilted with respect to the pupil plane. A reflective SLM can generally be adjusted at a higher speed. The high-speed adjustability is an advantage, for example, when the increase in axial and / or lateral spatial resolution caused by the STED beam is to be changed during image acquisition. If the first wavefront modulator is also an active wavefront modulator, it can be used to compensate for the difference in distortion correction between the illumination light which forms a focus with a central minimum on the one hand and the fluorescence or more generally the luminescence and / or the further illumination light on the other hand. Furthermore, in this case the high-speed adjustability is an advantage, since the distortions can be dependent on the position in the sample.

[0032] From the prior art, for example from the publication by Lenz, Martin O. et al. ("3-D stimulated emission depletion microscopy with programmable aberration correction") and From the prior art, for example from the publication by Lenz, Martin O. et al. ("3-D stimulated emission depletion microscopy with programmable aberration correction") and

[0033] In an advantageous further development of the application, the fluorescence microscope according to the application comprises a second telescope in the beam path between the second wavefront modulator and the objective. The second telescope serves to adjust the beam diameter and to adjust the image of the active area of the second wavefront modulator in the back focal plane of the objective to fit the back aperture. Further optical elements can contribute to this imaging and changing of the beam path diameter, such as, for example, the scan lens in a laser scanning fluorescence microscope or more generally lenses belonging to the scanner, and the tube lens. The second telescope is configured such that the active area of the second wavefront modulator is imaged onto the back focal plane of the objective in order to fill the aperture or slightly overfill the aperture, if possible.

[0034] Preferably, the microscope according to the application is made as a laser scanning fluorescence microscope. Here, it has a scanner which is arranged in the beam path between the second wavefront modulator and the tube lens.

[0035] In a preferred further development, the laser scanning fluorescence microscope comprises a second telescope. The second telescope for adjusting the beam diameter to fit the back aperture is preferably arranged between the scanner and the second wavefront modulator.

[0036] The fluorescence microscope according to the application preferably has a first deflection mirror and a second deflection mirror, the first telescope and the active area of the second wavefront modulator being arranged along the beam path between the first deflection mirror and the second deflection mirror. These deflection mirrors are positioned such that even when they are removed from the beam path, the functionality of the fluorescence microscope remains, including the beam shaping for forming a focus of the first illumination light having a central intensity minimum. This has the advantage that in the event of a failure of the second wavefront modulator which is to be used to correct the aberrations, at least the basic functionality of the microscope, i.e. the functionality of a STED microscope, can be restored in a simple manner. Furthermore, this construction has the advantage that the components for correcting the aberrations can be combined in a module.

[0037] It is advantageous to position the second beam splitter or coupler as close as possible to or in the pupil plane. This has the advantage that, for example in the event of a heat-induced tilt occurring over the measurement time, the beam splitter only causes a relative tilt of the light beam and not a lateral displacement. It is therefore advantageous to arrange the beam splitter at a distance of no more than the focal length of the first lens of the telescope, preferably no more than half the focal length of the lens, even more preferably no more than 10% of the focal length of the first lens, relative to the pupil plane adjacent to the first lens.

[0038] According to the application, a component group for a fluorescence microscope having an objective comprises:

[0039] i) a support structure,

[0040] ii) a first telescope comprising and being delimited by a first lens and a second lens,

[0041] iii) a deformable mirror having an active area, which in the built-in state of the component group in a fluorescence microscope is arranged in such a way that the first pupil of the objective is imaged on the second pupil of the objective in a direction away from the objective, the second pupil being located at a distance of the focal length of the first lens relative to the first lens and outside the first telescope, wherein the second pupil is smaller than the first pupil.

[0042] Likewise, the magnification factor of the first telescope, which determines the ratio of the first diameter of the first pupil to the second diameter of the second pupil, can be at least 1.2:1, preferably at least 1.5:1 or at least 2:1. In particular, the magnification factor or the diameter ratio can be about 2:1, 2.5:1 or 3:1. Generally, the magnification factor or the diameter ratio will not exceed 20:1. Generally it will not exceed 10:1, even less than 5:1.

[0043] In an alternative component group, another active element, which acts as a wavefront modulator like the deformable mirror, replaces the deformable mirror.

[0044] In a preferred embodiment, the component group according to the application further comprises a second telescope, which comprises and is delimited by a third and a fourth lens. Preferably, the second telescope is made as a zoom optic.

[0045] In another preferred embodiment, the first telescope is made as a zoom optic.

[0046] Preferably, the component group comprises one or more than one deflection mirror folding the beam path in addition to the wavefront modulator. Preferably, at least one of the deflection mirrors is arranged along the optical path within one of the telescopes. Preferably, the mirror is arranged at a minimum distance from the focal plane formed in the respective telescope in which the mirror is arranged. This is of particular importance when using the component group in a STED microscope, since in the focal point of the STED beam there is a very high intensity, which can damage the mirror located in the focal point. The minimum distance is preferably at least 20%, more preferably at least 30%, even more preferably at least 40% of the shorter one of the focal lengths of the lenses constituting the respective telescope. If the deflection mirror is located within the zoom optic, it must also be considered that the intermediate focus should not be formed at the location of the deflection mirror in any operating state of the zoom optic.

[0047] Preferably, the component group has a first deflection mirror which is constructed and arranged for deflecting a light beam impinging thereon in the direction of or on the optical axis of a first telescope or in the direction of or on the optical axis of a first telescope in the beam path of a fluorescence microscope. Furthermore, the component group preferably comprises a second deflection mirror which is constructed and arranged for deflecting a light beam extending in the direction of or on the optical axis of a second telescope onto the optical axis of the microscope. The two deflection mirrors can be arranged in line and as 90° deflection mirrors. Alternatively, the two deflection mirrors can be arranged such that the axes to which the deflection mirrors deflect the light beam outside the component group are not identical but parallel. In this arrangement, the component group or module can be integrated in a fluorescence microscope to replace the periscope.

[0048] In another preferred embodiment, the component group is constructed so as to be integrable in the beam path of a fluorescence microscope such that the beam path outside the component group is not changed by the component group if the deformable mirror is in the zero position.

[0049] In a preferred embodiment, the support structure of the component group has adjustment means, for example at least a slot and an abutment element. The abutment element is preferably constructed so as to be extendable parallel to the optical axis of the microscope. The microscope support comprises a corresponding further abutment element which is constructed so as to align the component group with the optical axis when the two abutment elements abut against each other. The component group can be aligned here by means of the abutment elements and fixed in a movable manner by means of, for example, a screw which extends through at least one slot and is screwed in slightly. Thereafter, the position of the entire component group along the optical axis can be adjusted so as to maintain this alignment, and thereafter the position of the component group is fixed by means of the screw. If at least one of the abutment elements comprises a suitable fine adjustment element, for example a stop screw, misalignments of the component group with respect to the optical axis of the microscope having an angle different from 0 can be corrected.

[0050] The adjustment means are particularly advantageous if the elements of the component group, including the support structure, are adjusted or aligned with respect to each other. In this case, the component group is a module which can be integrated in the beam path of a fluorescence microscope as a whole and then only has to be oriented or aligned as a whole with respect to the beam path of the microscope.

[0051] Advantageous refinements of the application result from the claims, the description and the drawings.

[0052] The advantages mentioned in the description for the features and combinations of features are merely exemplary and can act alternatively or cumulatively, without necessarily having to be achieved by embodiments according to the application.

[0053] Without thereby changing the subject matter of the attached claims, the following is stated with respect to the original application material and the disclosure of the patent, not the scope of protection: further features can be derived from the drawings, in particular the shown geometries and the relative dimensions and the relative arrangement and effective connection of the plurality of components to one another. Combinations of features of different embodiments of the invention or of features of different claims, deviating from the selected reference relationship of the claims, can also be realized, and from this. This also relates to such features shown in separate drawings or mentioned in the description thereof. These features can also be combined with features of different claims. Furthermore, features listed in the claims for a further embodiment of the invention can be omitted, but this does not apply to the independent claims of the granted patent.

[0054] Features mentioned in the claims and the description are to be understood with respect to their number as meaning that exactly this number is present or a number greater than this number is present, without the adverb "at least" having to be used explicitly. Thus, when, for example, one element is spoken of, it is to be understood that exactly one element, two elements or more elements are present. These features can be supplemented by other features or can be individual features for making up the respective product.

[0055] The reference signs contained in the claims are not limiting on the scope of the subject matter protected by patent rights. They are merely intended to make the claims easier to understand. BRIEF DESCRIPTION OF DRAWINGS

[0056] In the following, the invention is further explained and described with respect to preferred exemplary embodiments shown in the drawings.

[0057] Figure 1 is a schematic diagram of a prior art laser scanning fluorescence microscope.

[0058] Figure 2 is a schematic diagram of a prior art laser scanning fluorescence microscope with means for correcting sample induced aberrations.

[0059] Fig. 3 shows schematically an example of a known active wavefront modulator.

[0060] Figure 4A and 4B The effect of the invention is illustrated by comparing two optical paths.

[0061] Figure 5A and 5B Embodiments of a fluorescence microscope according to the invention are shown, which are designed as a STED microscope and in which two pupils are formed.

[0062] Figure 6A and Figure 6BA further embodiment of the fluorescence microscope according to the application is shown, which is designed as a STED microscope, and in which two pupils are formed in the fluorescence microscope.

[0063] Figure 7A and 7B The basic elements of an embodiment of the fluorescence microscope of the application are shown, which is designed as a MINFLUX microscope, and in which two pupils are formed in the fluorescence microscope.

[0064] Figure 8A and 8B A further embodiment of the fluorescence microscope of the application is shown, which is designed as a STED microscope, and in which two pupils are formed in the fluorescence microscope.

[0065] Figure 9A and 9B A further embodiment of the fluorescence microscope of the application is shown, which is designed as a MINFLUX microscope, and in which two pupils are formed in the fluorescence microscope.

[0066] Figure 10 A component group according to the application on a substrate for upgrading a fluorescence microscope is shown, which comprises an active wavefront modulator in the common light path for fluorescence and illumination light and a scanner.

[0067] Figure 11 A further component group of the application on a substrate for upgrading a fluorescence microscope is shown, which comprises an active wavefront modulator in the common light path for fluorescence and illumination light and an objective lens.

[0068] Figure 12 A further component group of the application for upgrading a fluorescence microscope is shown, which comprises an active wavefront modulator in the common light path for fluorescence and illumination light and a further wavefront modulator for forming an illumination focus, and an objective lens. DETAILED DESCRIPTION

[0069] In Figure 1The fluorescence microscope 10 schematically shown in Fig. 1 is a prior art laser scanning fluorescence microscope 15. The laser scanning fluorescence microscope 15 comprises an excitation light source 16 for an excitation light 6. The excitation light 6 is reflected by a first beam splitter 25 towards a second beam splitter 26. Both beam splitters 25 and 26 can be made as dichroic mirrors. The second beam splitter 26 is used to superimpose the excitation light 6 with a first illumination light 8, which here is a fluorescence suppression light 7 from a fluorescence suppression light source 17. Accordingly, the fluorescence suppression light source 17 is also referred to as a first illumination light source 9 in this document. Both beam splitters 25, 26 are also used to separate a detection light path for the fluorescence 19 from an illumination light path for the excitation light 6 and the fluorescence suppression light 7. The beam splitter 26 separates the detection light path from the common light path for the fluorescence 19, the fluorescence suppression light 7 and the excitation light 6 and the light path for the excitation light. Next, the beam splitter 25 separates the detection light beam from the common light path for the fluorescence 19 and the excitation light 6. A first wavefront modulator 24 is arranged in the light path for the fluorescence suppression light 7 between the fluorescence suppression light source 17 and the beam splitter 26. By means of a scanner 27 comprising a lens and by means of a tube lens 28, the fluorescence suppression light 7 and the excitation light 6 are guided through a focusing objective 20 into a sample space 21. A sample 22 is arranged in the sample space 21. The excitation light 6 and the fluorescence suppression light 7 are focused into the sample 22. The first wavefront modulator 24 modulates the fluorescence suppression light 7 such that a focal point with a central intensity minimum is formed in the sample 22 when the objective 20 focuses the fluorescence suppression light 7. Fluorescence 19 emitted by the sample 22 passes the objective 20, the tube lens 28, the scanner 27 and the beam splitters 26 and 25 via a mirror 29 and through a filter 33, a lens 45 and a pinhole 5 onto a detector 18. The lens 45 focuses the fluorescence 19 onto the aperture of the pinhole 5. The first wavefront modulator 24 is only schematically shown. The first wavefront modulator 24 can be a passive non-adjustable element, e.g. a wave plate, or an active adjustable element, e.g. an SLM 61, 61' or a set of components, in particular comprising one or more reflective SLMs 61 or comprising a reflective SLM 61 with two active local surfaces. The above mentioned paper by Lenz, Martin O. et al. (“3-D stimulated emission depletion microscopy with programmable aberration correction”) and An example of this set of components is described in the above-mentioned paper by Heine "Intelligent-Illumination STED". If the first wavefront modulator 24 is a passive element, it is preferably arranged in the pupil in the collimated beam of fluorescence suppression light, i.e. in the pupil plane 30, so that the first wavefront modulator 24 is imaged onto the back aperture 23 of the objective 20 so that the first wavefront modulator 24 fills the back aperture 23. If the first wavefront modulator 24 is a simple reflective active element, the active element is usually arranged so that its effective area 44 intersects the pupil plane 30 at an angle. If the first wavefront modulator is a set of components, preferably each effective area or partial area is arranged in the pupil plane 30 or intersects the pupil plane 30 at an angle, or a pair of effective areas or partial areas is arranged around the pupil plane 30. In general, it is also possible to arrange the effective areas or partial areas of the first wavefront modulator 24 away from the pupil plane, or even in a section of the optical path in which the fluorescence suppression light is not collimated. Passive elements such as wave plates, including segmented vortex wave plates, other segmented wave plates and combinations of these, in addition to vortex wave plates, are in fact usually arranged in the collimated optical path, but outside the pupil plane, when they are used as a first wavefront modulator 24 for static formation of a focus comprising a central intensity minimum. The arrangement in the collimated optical path, in particular in combination with the arrangement in the pupil plane 30, has the result that the relationship between the wavefront shape at the location of the wavefront modulator 24 and at the location of the back aperture can be easily described. With the arrangement in the pupil plane, the wavefront shape is identical, apart from factors corresponding to the imaging scale. This is particularly advantageous if an active first wavefront modulator 24 is used. If the first wavefront modulator 24, which is only schematically shown here, is a set of components, it also comprises additional optical elements for beam guidance. Further optical elements required to image the back aperture 23 of the objective 20 into one or more pupil planes 30 are not shown in Figure 1 The illustrated laser scanning microscope 15 can be used as a confocal laser scanning microscope without the use of fluorescence suppression light 7. The fluorescence suppression light 7 can be a depletion light. In this case, the laser scanning microscope 15 is used as a STED microscope if the fluorescence suppression light 7 used as depletion light is used.

[0070] Figure 2 is a schematic illustration of a laser fluorescence microscope 10 with a configuration for correcting sample-induced aberrations as essentially known from WO 2014 / 029978 A1 and US 9 575 302 B2. The laser fluorescence microscope 10 is also a STED microscope. According to Figure 2The laser fluorescence microscope 10 comprises an excitation light source 16 for the excitation light 6. The excitation light 6 is reflected by a beam splitter 35 towards a further beam splitter 36. The two beam splitters 35, 36 can be dichroic mirrors. The beam splitter 36 serves to superimpose the excitation light 6 with the fluorescence suppression light 7 from a fluorescence suppression light source 17 and to pass the excitation light on its way to the beam splitter 36 through a first wavefront modulator 24. Here, the first wavefront modulator 24 is an active element, for example an SLM 61, 61' or a set of components, in particular comprising one or more reflective SLMs 61 or comprising an SLM 61 with two active areas. If the wavefront modulator 24 is a simple reflective active element, it is typically arranged such that its active area 44 intersects the pupil plane 30 at an angle. If the first wavefront modulator 24 is a set of components, each active area or partial area is arranged in or intersects the pupil plane 30 at an angle or a pair of active areas or partial areas is arranged around the pupil plane 30. The beam splitter 36 also serves to separate the detection light path of the fluorescence 19 and the light path of the excitation light 6 from the light path of the fluorescence suppression light 7. In the common light path of the fluorescence suppression light 7, the excitation light 6 and the fluorescence 19, a second wavefront modulator 34 is arranged which intersects and is tilted with respect to the pupil plane 30 and which is made as a reflective active wavefront modulator, in particular as a deformable mirror 51. The second wavefront modulator 34 directs the illumination beams of the excitation light 6 and the fluorescence suppression light 7 into the back aperture 23 of the objective 20 which focuses said illumination beams into the sample 22 which is located on a positioning stage 37 in the sample space 21. In the opposite direction, the fluorescence emitted by the sample 22 is guided through the objective 20 onto the second wavefront modulator 34 and from there onto the beam splitter 36 which directs the fluorescence 19 onto a further beam splitter 35. This beam splitter 35 separates the detection light path of the fluorescence 19 from the light path for the excitation light 6 and guides the fluorescence 19 through a filter 33 and a lens 45 which focuses the fluorescence 19 towards the detector 18 onto the pinhole 5. In this arrangement, the first wavefront modulator 24 is first used to modulate the light beam of the fluorescence suppression light 7 such that the objective 20 focuses the fluorescence suppression light 7 into the sample 22 such that the focal point has a central intensity minimum. Here, again, the fluorescence suppression light 7 is also referred to as first illumination light 8 and the fluorescence suppression light source 17 is also referred to as first illumination light source 9. The second wavefront modulator 34 is used to adapt the pre-modulated wavefront of the fluorescence suppression light 7 according to the sample-induced aberration, for example also in relation to the axial position of the focal point, such that the intensity distribution of the fluorescence suppression light at the focal point is as independent as possible of the aberration and the axial position of the focal point, respectively. Thus, the second wavefront modulator 34 is used to compensate the aberration of the fluorescence suppression light 7 so as to form a focal point in the sample which is as aberration-free as possible.At the same time, the second wavefront modulator 34 is also used to modulate the aberration wavefront of the fluorescence 19 emitted from the sample 22 in such a way that the emission focus is imaged as aberration-free as possible onto the pinhole 15 and the excitation light 16 is focused as aberration-free as possible into the sample 22. Since the wavelengths of the excitation light 6, the fluorescence suppression light 7 and the fluorescence 19 are different, it is not possible to perfectly compensate the aberrations of all light beams by means of one second wavefront modulator 34. However, since the wavelengths of the excitation light 6 and the fluorescence 19 only differ by a small amount, it is possible to well simultaneously compensate the aberrations of both light beams concerned. The first wavefront modulator 24, which pre-modulates the wavefront of the fluorescence suppression light 7 in respect of the formation of a focus with a central intensity minimum, is now additionally used to compensate the aberrations of the fluorescence suppression light 7 which remain after the second wavefront modulator 34. Methods for detecting aberrations and control devices for the purpose of observing a sample without aberrations are described, for example, in WO 2014 / 029 978 A1 and US 9 575 302 B2.

[0071] Figures 3A to 3C Different designs and functions of active wavefront modulators 41 are shown for illustration.

[0072] Figure 3A A deformable mirror 51 is shown schematically. The deformable mirror 51 comprises a support structure 42 and an active area 44. The active area 44 is mirror-reflecting and may, for example, be a mirror-reflecting surface of a deformable membrane. Alternatively, it can be made of a plurality of mirror-reflecting surfaces of individual actuators such that the active area 44 is a segmented area. The active area 44 is operatively connected to the support structure 42. The elements of the active area 44 to achieve the deformation are integrated in the support structure 42. In Figure 3A In the middle, the active area 44 is shown as a circular area, as is usually present in a membrane mirror. In general, the active area 44 can also have a further shape, for example a hexagonal or rectangular shape. If a plane wavefront hits such a mirror, the mirror causes different local phase delays depending on its current shape or deformation.

[0073] Figure 3BA wavefront modulator based on liquid crystal technology is schematically shown, which is generally referred to as SLM and serves as a reflective element. In such a wavefront modulator comprising a support structure 42 and an active area 44, this active area 44 is not mirror-like reflective as such, but the incident wavefront and the modulated outgoing wavefront pass there through. On the way from the active area 44 up to the backside 43, the wavefront of a certain polarization is subjected to different local phase retardations depending on the local setting of the liquid crystal embedded between the active area 44 and the backside 43, wherein the wavefront is reflected back to the active area 44 at the backside 43. Such an SLM 61 can be operated such that on the zero position of the SLM 61 the dominant angle of incidence equals the angle of exit is achieved. However, generally, such an SLM 61 can be operated such that all liquid crystals together act as a diffraction grating such that the vast majority of the incident wavefront is reflected in the first diffraction order. Furthermore, the SLM 61 serves to locally differently delay the outgoing wavefront in terms of its phase. Most SLMs are pixelated and have a rectangular active area 44 as shown. Generally, the active area 44 can also have a further shape.

[0074] Figure 3C A wavefront modulator based on liquid crystal technology is schematically shown, which is a transmissive element. In the following, the wavefront modulator is referred to as SLM 61'. This SLM 61' has a support structure 42, an entrance area 40 and a backside 43 serving as an active area 44. The incident wavefront enters through the entrance area 40 and leaves the active area 44 as a modulated wavefront. Most transmissive SLMs 61' are also pixelated and have a rectangular active area 44 as shown. Generally, the active area 44 can also have a further shape.

[0075] Figure 4A and 4B are used to explain the basic principles of the present application. In both optical paths according to Figure 4A and 4B the light beams passing orthogonally through the pupil plane 30 in the left-hand part of Figure 4A and 4B are adapted in terms of their beam diameter by means of two telescopes, respectively, and the light beams are focused by means of a further lens. In a microscope, a confocal lens can be arranged in the focal plane. In both optical paths, the further focusing lens has the same focal length and the beam diameter is the same in both optical paths after passing through the second telescope. In the fluorescence microscope according to the present application, a second wavefront modulator 34 can be arranged in the pupil plane 30. In the optical path according to Figure 4A the light beams first pass through a telescope which does not change the beam diameter. In the optical path according to Figure 4B the light beams first pass through a telescope 11 made of lens 2 and lens 1, which reduces the beam diameter by a factor of two. In the optical paths according to Figure 4A and4B In the two optical paths, the further pupil plane 30' is indicated as a dashed line. In the fluorescence microscope according to the application, the beam splitter 26 is arranged in or close to the pupil plane 30', which separates the illumination light path for the illumination light 8, which forms a focus in the sample with a central intensity minimum, from the detection light path for the fluorescence 19, if seen in the direction of the objective 20 of the microscope. At such a beam splitter 26, an angular error between these optical paths can be introduced. According to the application, the influence of this angular error on the imaging quality is reduced. To illustrate this effect, in Figure 4A and 4B the following light beam is shown in dashed lines: This light beam passes through the pupil plane 30' at an angle of inclination a with respect to the optical axis and has the same diameter as the non-inclined light beam in the pupil 30'. In Figure 4A a light beam with the first large beam diameter passes through the telescope 11, which reduces the beam diameter to half. Then, the light beam is focused, wherein the focus is formed at a position with a lateral distance a from the focus of the non-inclined light beam. In Figure 4B a light beam with a reduced beam diameter compared to the beam diameter in the pupil plane 30 passes through the telescope, which does not further change the beam diameter. Then, the light beam is focused, wherein the focus is located at a position with a lateral distance b from the focus of the non-inclined light beam. The reduction ratio of the distance b with respect to the distance a is the ratio by which the telescope 11 narrows the beam diameter. In particular, here the ratio of the distance a to the distance b is approximately 2:1. Thus, Figure 4B the following effect of the application is shown: If the introduced inclined light beam has a smaller beam diameter due to the telescope 11, the inclination of the two partial optical paths that are imaged to the confocal pinhole is smaller.

[0076] Of course, it is possible to introduce the inclination at another position than the pupil plane 30'. In this case, the respective light beam passes through an inclined and offset pupil plane 30*. However, the position of the focus is only influenced by the inclination. In any case, it is advantageous to arrange the beam splitter 26, which separates the illumination light 8, in the pupil plane 30'. In Figure 4A and Figure 4B In the two optical paths shown in

[0077] Instead of tilting, the beam splitter can introduce offset within a pupil plane. Figure 4A and Figure 4B In this context, the lines defining the beam can be viewed as thin beams that are laterally offset from each other. From Figure 4A As can be seen, the offset corresponding to the large beam diameter in the pupil plane 30' is converted into an offset corresponding to a beam diameter that is reduced by half in the pupil plane 30”, while according to Figure 4B In the optical path, the offset in the pupil plane 30” is equal to the offset in the pupil plane 30'. This means that the concept of the present invention is disadvantageous in avoiding the effects of lateral offset. However, the inventors have found that in practical applications, angular errors mainly occur and should be avoided. If, for practical reasons, the first wavefront modulator 24 is not directly arranged in the pupil plane 30, this is also related to the fact that as the distance from the pupil plane increases, a larger angular error will result in a larger lateral offset on the first wavefront modulator 24. By avoiding angular errors through the present invention, the magnification of the lateral offset in the pupil plane is overcompensated.

[0078] Figure 5A The fluorescence microscope 10 schematically shown is a laser scanning fluorescence microscope 15 according to the present invention. To a large extent, it corresponds to... Figure 1 The laser scanning fluorescence microscope 15 shown is illustrated. Additionally, the fluorescence microscope 10 has a second wavefront modulator 34 in a first optical path segment, through which the excitation light 6, the fluorescence suppression light 7, and the fluorescence 19 all pass. The second wavefront modulator 34 is designed as an active element, i.e., designed as an adjustable or adaptive element. It can be a reflective SLM 61. Preferably, the second wavefront modulator 34 is a deformable mirror 51. The second wavefront modulator 34 has an effective region 44, see [reference needed]. Figures 3A to 3C The effective region 44 is arranged to intersect the pupil plane 30 at a certain angle, forming a first pupil 31 at the location of the effective region 44. This means that imaging of the aperture of the objective lens 20 is present at the location of the effective region 44. Since the effective region 44 of the first wavefront modulator 34 is tilted or deflected relative to the optical axis, this is only approximately applicable. Therefore, in this document, the statement "at the location of the effective region 44 of the second wavefront modulator 34" should be understood as meaning that the imaging of the aperture of the objective lens 20 coincides with the effective region 44 of the second wavefront modulator 34 at least along a line intersecting the optical axis. Preferably, this line is orthogonal to the optical axis. Figure 5AIn the middle, the pupil plane 30 and the back aperture of the objective 20 are indicated by dashed lines. In the area of the second wavefront modulator 34, two intersecting lines are drawn, one of which indicates the position of the pupil plane 30 without taking into account the second wavefront modulator 34, which here is embodied as a mirror-reflecting element, and the other of which indicates the position of the pupil plane 30 after reflection at the active area 44 of the second wavefront modulator 34. Along the propagation direction of the fluorescence 19, i.e. in the direction from the second wavefront modulator 34 towards the detector 18, the laser scanning fluorescence microscope 15 according to the application has a first telescope 11, which here is embodied by the first lens 1 and the second lens 2. According to the application, the first telescope 11 images a first pupil 31 as a second pupil 32 which is smaller than the first pupil 31. In the embodiment according to Figure 5A the application, both the telescope 11 and the second pupil 32 are arranged within a light path section which is delimited by the beam splitter 26 and which comprises the second wavefront modulator 34, in which light path section the detection light path coincides with the illumination light path, i.e. with the light paths of the excitation light 6 and the fluorescence suppression light 7, wherein the direction of the detection light path is opposite to the direction of the illumination light path.

[0079] In the block according to Figure 5B the size of the pupils 31 and 32 is schematically shown. Here, the ratio of the first diameter of the first pupil to the second diameter of the second pupil is approximately 2:1.

[0080] The function of the telescope 11 is that all light beams together are narrowed from the point of view of the propagation direction of the fluorescence 19. In this way it is achieved that an angular deviation of the light paths from one another results only in a small positional deviation of the focal points in the sample 22 relative to one another, wherein the focal point of the detection light path in the sample is to be understood here as the region in the sample which is arranged in focus with the pinhole 5. In order to achieve this smallest possible positional deviation, all light beams must have a small effective diameter when being combined or coupled. This can also be achieved, however, if the telescope 11 is arranged only in the above-mentioned first section of the detection light path or of all light paths and forms the pupil 32 in the second light path section of the detection light path which does not coincide with all illumination light paths, i.e. behind the beam splitter 26 when viewed in the propagation direction of the fluorescence 19.

[0081] In this embodiment of the application shown in Figure 6A the pupil 32' is formed in the light path of the fluorescence suppression light 7, which has the same distance from the beam splitter 26 as the pupil 32 and the same size as the pupil 32. The pupil 32' can be located in the pupil plane 30, the first wavefront modulator 24 being arranged in the pupil plane 30 but this is not mandatory. In Figure 5A the same way as in Figure 6AIn the middle, the formation of the pupil 32' which lies outside the plane of the first wavefront modulator 24 is shown. In order to form the one or more pupil planes 30, additional imaging elements are required which are not shown in Figure 6A In the middle, the formation of the pupil 32' which lies outside the plane of the first wavefront modulator 24 is shown. In order to form the one or more pupil planes 30, additional imaging elements are not shown in

[0082] The effective cross section of the illumination beam is to be understood as the largest cross section which contributes to a further variation of the intensity distribution within the back aperture 23 of the objective 20. The actual beam cross section during the coupling or combination of the light paths has no influence on the positional deviations in the case of an overfilling of the back aperture, i.e. in the case of a beam cross section of the illumination light path in the back aperture which is larger than the cross section of the back aperture. In general, the actual beam cross section, in particular of one of the fluorescence suppression lights 7, is chosen such that the back aperture is just overfilled.

[0083] In addition, instead of the pinhole 5 and the detector 18, in Figure 6A A detection device 38 for confocal detection is shown schematically in the middle. This device can be one of different types of detection devices. It is important that the entrance of the detection device 38 for the photons to be detected is arranged confocally with respect to the fluorescent volume to be imaged. For example, an array detector arranged in the focal plane can be used as the detection device 38. In turn, the region of such an array detector which receives the light to be detected is preferably adapted to the size of the diffraction-limited light distribution to be detected with the array detector, which is somewhat larger depending on the actual application, in order to, for example, make the light-receiving surface 1.2 times the Airy disk diameter of the diffraction-limited light distribution to be detected, or even significantly larger, so that the light-receiving region is 1.2 times or 1.5 times or 2 times or not more than 3 times the Airy disk diameter. Preferably, such an array detector is arranged such that the center of its region with the fully adapted light path lies on the optical axis.

[0084] In the block of Figure 6B The size of the pupils 31 and 32 in an embodiment of the fluorescence microscope according to the application is shown schematically in the block. Figure 6A Here, the ratio of the first diameter of the first pupil to the second diameter of the second pupil is approximately 2.5:1.

[0085] Figure 7A An embodiment of a fluorescence microscope 10 according to the application is shown, which is designed as a MINFLUX microscope 70. This MINFLUX microscope 70 is also a kind of laser scanning fluorescence microscope 15. The basic design corresponds to Figure 6AThe shown STED microscope. In contrast to the STED microscope, the first illumination light source 9 is configured as the excitation light source 16 in the MINFLUX microscope 70. Accordingly, the first illumination light 8 is the excitation light 6. This excitation light 6 is modulated in the first wavefront modulator 24 such that it forms an illumination focal point with a central intensity minimum in the sample 22 after being focused by the objective 20. Also in the fluorescence microscope 10 designed as MINFLUX microscope 70, the beam splitter 26 is used to couple or combine the light paths of the fluorescence 19 and the first illumination light 8.

[0086] In Figure 7B the block of the application according to Figure 7A the size of the pupils 31 and 32 in an embodiment of the fluorescence microscope of the application. Here, the ratio of the first diameter of the first pupil to the second diameter of the second pupil is approximately 2.5:1.

[0087] Figure 8A The fluorescence microscope 10 shown in Figure 5A corresponds largely to the fluorescence microscope shown in Figure 5A contrast to the fluorescence microscope 10 shown in Figure 8AThe illustrated fluorescence microscope 10 includes a second telescope 12 made of two lenses 3 and 4. Typically, telescope 12 may include additional lenses. Lenses 3 and 4 then define the telescope 12. The same applies to the first telescope 11. In this and all other embodiments of the fluorescence microscope 10 according to the invention, the first telescope 11 may also include more lenses than those illustrated. In this case, lenses 1 and 2 illustrated are the lenses defining the telescope 11. According to the invention, in all embodiments of the fluorescence microscope 10, the following must be achieved by means of the first telescope 11: the beams of the first illumination light 8 and fluorescence 19, and, if present, additional illumination light (which may be, for example, excitation light 6), must have the smallest possible cross-section or diameter (observed along the detection direction) at the separation position of fluorescence 19. However, it is advantageous to simultaneously make full use of the effective region 44 of the second wavefront modulator 34. This effective region 44 may be the same size as, or larger than, the pupil at the location of the effective region 44 of the second wavefront modulator 34. To effectively utilize the effective region 44 of the wavefront modulator 34, the beam diameter must be adapted along the optical path from the back aperture 23 to the second wavefront modulator 34. This adaptation is preferably achieved by adapting the beam filling the back aperture 23 of the objective lens such that its diameter at the location of the effective region 44 of the second wavefront modulator 34 is no larger than the effective region 44 of the second wavefront modulator 34. Otherwise, the beam guided via the effective region 44 cannot fill the back aperture 23 of the objective lens 20. Simultaneously, the optical elements are designed such that the back aperture 23 is precisely imaged onto the effective region 44 of the second wavefront modulator 34, thereby forming a pupil at the location of the effective region 44 of the second wavefront modulator 34. The diameter of the pupil corresponds to the beam diameter in the corresponding plane. It should be noted in this regard that... Figure 8A None of the accompanying drawings, including those in the series, are drawn to scale. However, Figures 10 to 12 It is geometrically constructed to some extent. Figure 8A The fluorescence microscope shown is a laser scanning fluorescence microscope 15. In addition to the objective lens 20, this laser scanning microscope 15 typically has a tube lens 28 and a scanner 27. The term "scanner" should be understood to refer to a device that includes at least one lens in addition to a deflecting mirror. The light beam is collimated between the objective lens 20 and the tube lens 28. The light beam is collimated by the tube lens 28 and any part belonging to the scanner 27 but not included in the objective lens 20. Figure 8A The lens system shown converts these collimated beams into collimated beams whose diameter can vary with the parameters of the lens system relative to the diameter in the rear aperture 23. Furthermore, the lens system projects the rear aperture 23 onto... Figure 8AThe pupil (not shown) can be located within the scanner 27 and a deflecting mirror can be placed therein. The second telescope 12 is now used to adapt the beam diameter to the rear aperture 23, or, if viewed from the opposite direction, to adapt the beam diameter in the rear focal plane to the effective region 44 of the second wavefront modulator. This means that the second telescope 12 is used to adapt the beam diameter, determined by the rear aperture 23 of the objective lens 20, the barrel lens 23, and any lenses of the scanner 27, and the relative arrangement of these optical components on the output side of the scanner 27 (the output side when viewed along the detection direction), to the effective region 44 of the second wavefront modulator 34 and to adapt it to be able to... Figure 8A The pupil (not shown) is imaged onto the pupil surface 30, wherein the effective region 44 of the second wavefront modulator 34 is placed in the pupil surface 30.

[0088] exist Figure 8B The invention is schematically illustrated in the box. Figure 8A The dimensions of pupils 31 and 32 in the embodiment of the fluorescence microscope. Here, the ratio of the first diameter of the first pupil to the second diameter of the second pupil is approximately 2:1.

[0089] Figure 9A An additional laser scanning fluorescence microscope 15 according to the invention is shown, which is configured as a MINFLUX microscope 70. The MINFLUX microscope 70 includes a scanner 27', which itself does not include a lens, but only includes a specially arranged deflector. The scanner 27' together with the lens 3 forms a unit corresponding to the embodiment of the scanner 27 shown in other figures. The functional unit consisting of the scanner 27' and the lens 3 is also referred to herein as a quad scanner. The lens 3 associated with the scanner 27' together with the tube lens 28 forms a second telescope 12, which images the back aperture 23 in the pupil plane of the second wavefront modulator 34 and adapts the beam diameter to the effective region 44 of the second wavefront modulator 34. The distance of the tube lens 28 and its distance to the back aperture 23 of the objective lens 20 is almost constant in a given microscope. This also applies to the scanner 27, which, when viewed along the illumination direction, includes a scanning lens located behind the deflector, as is common in the prior art. In such a setup, the requirements for the scanning lens are extremely high because the beam passes through the scanning lens at drastically changing angles and with a lateral distance relative to the optical axis. Figure 9A The advantage of the configuration shown is that all beams pass through all lenses on the optical axis and all beams pass perpendicularly through all lenses 3. This allows the microscope configuration to be adapted by appropriately selecting the focal length of the lenses 3 and correspondingly matching the distance from a specific second wavefront modulator 34. It should be noted that... Figure 9A It was not drawn to scale.

[0090] In Figure 9B The size of the pupils 31 and 32 in an embodiment of the fluorescence microscope according to the application is schematically shown in the block diagram. Here, the ratio of the first diameter of the first pupil to the second diameter of the second pupil is about 2.5:1. Figure 9A

[0091] In Figure 10 A set of components for upgrading a fluorescence microscope is shown in the block diagram. It comprises an active wavefront modulator 41 in the common optical path of fluorescence and illumination light and a scanner 27 comprising lenses not shown. The set of components is depicted as arranged on a substrate 60. Here, the description of the set of components partly refers to its function in the installed state or in the operation of the microscope. In the laser scanning fluorescence microscope 15, the light beam or bundle of light rays is collimated on the entrance side of the scanner 27. The set of components comprises a first deflection mirror 55 and a second deflection mirror 56. The deflection mirrors 55 and 56 are arranged relative to each other such that the point of incidence of the light beam to be deflected lies on one optical axis. In the actually illustrated embodiment, the deflection mirrors are oriented at an angle of 45° relative to the optical axis, respectively, so that the light beam is deflected by 90°. The set of components further comprises a first telescope 11 comprising a first lens 1 and a second lens 2, a deformable mirror 51, a second telescope 12 comprising a third lens 3 and a fourth lens 4, a lens holder 38 in which the lens 2 and the lens 3 are held, and two further deflection mirrors 57. In general, a further active reflective wavefront modulator, for example an SLM 61, can be used instead of the deformable mirror 51. However, the deformable mirror 51 is preferred because its effect is not influenced by the polarization of the light beam to be modulated. For example, the effect of a membrane mirror is completely independent of the polarization, while the effect of a mirror comprising an active area consisting of a plurality of individual actuators can be substantially independent of the polarization, if not completely. On the other hand, the SLM 61, 61' only modulates light of one specific polarization. In the optical path of the microscope, an intermediate focus 50 is formed in the set of components at a distance of the focal length f1 behind the first lens in the illumination direction and at a distance f4 in front of the fourth lens 4. In the illustrated embodiment, the first lens 1 and the fourth lens 4 have the same focal length. The second lens 2 and the third lens 3 also have the same focal length. One of the further deflection mirrors 57 deflects the light beam within the first telescope 11 so that the light beam travels towards the deformable mirror 51 at an angle of incidence δ relative to the surface normal of the deformable mirror 51. At the zero position of the deformable mirror 51, the deformable mirror 51 reflects the light beam so that it leaves the deformable mirror 51 at an equal angle of exit δ and enters the second telescope 12. Within the second telescope 12, the light beam is deflected again so that it is incident on the second deflection mirror 56 as a collimated light beam after passing through the fourth lens 4. ​

[0092] In a practical embodiment, the configuration is symmetrical with respect to the normal of the surface of the deformable mirror 51. The light beam entering the component group as a collimated light beam with a certain diameter also leaves the component group as a collimated light beam with the same diameter. This also means that the component group can be integrated into an existing microscope 10 without further changes being necessary and, if the deformable mirror 51 fails, the function of the microscope can be restored by removing the first and second deflection mirrors 55, 56, wherein, naturally, the function additionally provided by the component group also disappears. If the component group is arranged completely in one plane, the orientation of the focused image with the built-in component group is reversed compared to the original orientation, i.e. the orientation without the component group. This can and must be taken into account when imaging by scanning the sample.

[0093] Preferably, the component group will be built into a fluorescence microscope 10, for example a laser scanning microscope 15, such that the effective area 44 of the deformable mirror 51 lies in or intersects the pupil plane 30. In Figure 10 In a special case, it is assumed that, when viewed in the illumination direction, a pupil plane 130 is freely accessible in front of the scanner 27 in the microscope, which should be upgraded by means of the component group. This situation typically occurs, for example, in the applicant's STED microscope, which uses a so-called four scanner. For other microscopes in which the scan lens is arranged behind the scan mirror when viewed in the illumination direction, this situation can occur if the microscope has relay optics, which are arranged in front of the scanner when viewed in the illumination direction, and if there is a free pupil plane in the light path section in which all light paths have merged. Such relay optics are not shown in Figure 10The configuration of the component group is such that, by providing the appropriate integration, the free pupil plane 130 initially present in the microscope is imaged by the deflection mirrors 56, 57 in the pupil plane 30 and in turn by the first telescope 11 and the second telescope 12, respectively, in the pupil plane 30, which intersects the active area 44 of the deformable mirror at a respective angle. If both telescopes are, as actually illustrated, composed of two lenses, the two pupil planes 30 are located at a distance fi = f4 from the first lens 1 and the fourth lens 4, respectively, and the pupil plane that intersects the active area 44 of the deformable mirror 51 is located at a distance f2 = f3 from the second lens 2 and the third lens 3, respectively. By means of the appropriate integration, this configuration has the advantageous property that further components of the microscope that have been aligned with respect to the original pupil plane 130 before the integration of the component group or module, such as the SLM 61, 61’ for the active formation of the beam of the first illumination light 8, which provides a focus with a central intensity minimum, can also be placed correctly after the integration of the component group or module without having to be changed.

[0094] With regard to the application in a STED microscope, it is important that none of the intermediate foci 50 lies on one of the further deflection mirrors 57, otherwise damage or thermally induced deformations can be caused here due to the high STED light intensity occurring at the time. The foci are therefore preferably located at a distance from the point of incidence on the respective further deflection mirror 57 that is at least 20%, more preferably at least 30%, and even more preferably at least 40% of the respective focal length of the first lens 1 and the fourth lens 4. This applies to all embodiments of the component group of a STED microscope with deflection mirrors within the telescopes and thus also to all fluorescence microscopes 10 designed as STED microscopes according to the application.

[0095] Preferably, the angle δ of the direction in which the light beam travels towards and away from the deformable mirror 51 with respect to the surface normal of the deformable mirror 51 is small, in particular less than 45°, preferably less than 30°, and more preferably less than 20°. In the illustrated embodiment, the angle δ is approximately 18°. A small angle δ is advantageous because the smaller the angle δ, the greater the realizability of the complete placement of the deformable mirror 51 within the pupil plane. The angle δ cannot be brought to 0° because the light beam would then be reflected onto itself. Depending on the available construction space, angles of less than 18° can be realized in different ways.

[0096] For example, the further deflection mirror 57 can be tilted in such a way that the light beam is reflected off the drawing plane, i.e. off a plane defined by the tilt axis of the first deflection mirror 55, i.e. by the axis about which the first deflection mirror 55 is tilted by 45°, and the point of incidence of the light beam on the first deflection mirror 55, for example. In this way, it is possible to avoid that the edge of the second lens 2 or the holder of the second lens 2 obstructs the optical path from the first lens 1 to the further deflection mirror 57. Similar aspects apply to the third lens. The second lens 2 and the third lens 3 can then be shifted closer to each other; correspondingly, the two deflection mirrors 55 and 56 must then be shifted closer to each other as well, and all other distances and angles must be adjusted. In general, the achievable minimum distance through the centers of the second lens 2 and the third lens 3 in combination with the focal lengths f2 = f3 limits the angle δ only to small values. The angle δ can then be less than 10°, for example, or even less than 5°. As long as the tilt axes of the first deflection mirror 55 and the second deflection mirror 56 are orthogonal to the same plane, the focused image with the integrated module is simply inverted with respect to the focused image without the integrated module, which can and must be taken into account when imaging a sample in a scanning manner or tracking a movable object.

[0097] Alternatively, the further deflection mirror 57 can be arranged within a distance of the focal length f1 of the first lens 1 with respect to the first lens 1 and within a distance of the focal length f4 of the fourth lens 4 with respect to the fourth lens 4, respectively, wherein the conditions described above with respect to the distance of the intermediate focus from the lens must also be taken into account. Here, the angle δ is limited because the light beam from the first lens must be reflected in such a way that it does not extend through the building space occupied by the first lens 1 and its lens holder, respectively. In this arrangement, the further deflection mirror 57 and the effective area 44 of the deformable mirror 51 can be arranged on opposite sides of the optical axis route of the microscope without the need for an integrated component group.

[0098] If the focal lengths of the first lens 1 and the second lens 2 differ significantly, a further alternative can be implemented. This also applies to the third lens 3 and the fourth lens 4 in the case of a symmetrical design. Here, the first telescope 11 and the second telescope 12 strongly change the beam diameter, respectively. This makes it possible to arrange the further deflection mirror 57, respectively, away from the first lens 1 and the fourth lens 4, outside the focal lengths f1 and f4 of the first lens 1 and the fourth lens 4, but preferably so close to the intermediate focus 50 and at an angle, respectively, that the second lens 2 and the third lens 3 are closer to the course of the optical axis of the microscope without integrated module in relation to the angle and position of the further deflection mirror 57 than the first lens 1 and the third lens 3. In addition, in this arrangement, the effective area 44 of the further deflection mirror 57 and of the deformable mirror 51 can be located on opposite sides of the course of the optical axis of the microscope without module or when the module has been removed. In this arrangement, the angle δ can be less than 10° or even less than 5°, for example, depending on the exact parameters of the lenses used.

[0099] The component group can be made into a module, for example, arranged on a substrate 60 as shown. On this substrate 60, the optical elements can be prearranged or adjusted relative to one another. The installation of the module in the microscope can then be carried out in a simple manner, i.e. the module is arranged in the beam path of the microscope. For this purpose, in a preferred embodiment, the module comprises limiting or abutment elements at a fixed distance from a virtual axis passing through the points of incidence of the beam on the first deflection mirror 55 and the second deflection mirror 56, or extending through limiting or abutment elements such as straight extending edges or abutment edges 64. Here, matching stops or matching abutment elements must be provided in the microscope 10 at the location of the module to be integrated, which here extend along the optical axis of the microscope 10. The substrate 60 is preferably configured such that it can be displaced along the matching stops or matching abutment elements before being fixed. This can be achieved, for example, by the substrate 60 having one or more strip-shaped holes 66 through which the substrate 60 is fixed in the microscope 10 by means of a threaded element. Methods for correctly adjusting the module are known to the person skilled in the art.

[0100] Alternatively or additionally, all or some of the elements of the module can be adjusted after integration into the microscope. Thereby, for example, the further deflection mirror can be fine-tuned in its holder by means of an adjustment screw.

[0101] Figure 11A further component group for upgrading a fluorescence microscope by means of an active wavefront modulator in the common beam path for fluorescence and illumination light is also depicted in Fig. 6. This component group is also shown arranged on a substrate 60 and in this way forms a module. In contrast to the component group according to Figure 10 This component group is, however, asymmetrical with respect to the normal to the surface of the deformable mirror 51. Even with this asymmetrical design, however, the collimated light bundle which enters the component group on one side will leave the component group on the other side in the form of a collimated beam. This arrangement has the advantageous property in combination with suitable integration that further components of the microscope 10 which were adjusted to the original pupil plane 130 before the integration of this component group or module are still correctly adjusted after the integration of this component group or module. A particular benefit of the practical embodiment can be seen in that the construction space required along the optical axis of the microscope is reduced, so that elements of the beam path of the illumination lamp 8, for example, can be arranged in such a way that the component group according to Figure 10 cannot be integrated without collision. This component group comprises the same elements as the component group according to Figure 10 . In contrast to this component group according to Figure 10 the effective area 44 of the deformable mirror 51 is tilted at a small angle, so that the normal to the effective area 44 is not orthogonal to the original optical axis direction of the microscope. The two further deflection mirrors 57 are correspondingly arranged at different distances with respect to the adjacent lenses 2, 3, in particular so that the pupil plane 130 is closer to the first deflection mirror 55 and correspondingly further away from the second deflection mirror 56. In this way, there is more space on the side of the first deflection mirror 55 along the optical axis for further elements of the microscope 10. This component group can also be designed in a similar way with more construction space on the side of the objective lens 28 or the scanner 27 if the installation situation requires it (see Figure 10 ).

[0102] In another preferred embodiment, the second telescope 12 can also in all settings, in particular also in the setting corresponding to Figure 11The component group of the microscope 10 is made up of a special zoom optics, which has the property that the position of the pupil plane 30 remains constant, independently of its magnification factor. Such a zoom optics is disclosed, for example, in European patent application publication EP 1 617 251 Al and in US patent application publication US 2006 049 343 Al, which are part of the same patent family. According to these patent application publications, the zoom optics can be composed of four lens groups, which can be displaced relative to one another. Sourav Pal and Lakshminarayan Hazra "Stabilization of pupils in a zoom lens with two independent movements", Appl. Opt. 52, 5611-5618 (2013) further discloses a zoom optics with only three lenses, the position of whose entrance and exit pupils is identical at least for a plurality of different magnifications. The design of the telescope 12 as such a zoom optics has the advantage that the size of the image of the effective area 44 of the deformable mirror 51 in the back focal plane can be adapted to the back aperture 23 of different objectives. Vice versa, this means that even if an objective with a smaller back aperture is exchanged, the existing deformable surface of the deformable mirror can be used completely as effective area 44.

[0103] In a further preferred embodiment, in all arrangements, in particular also in the component group of the microscope 10 according to Figure 11 The first telescope 11 can be designed as such a special zoom optics in all arrangements, in particular also in the component group of the microscope 10 according to

[0104] The asymmetric module can also be positioned, adjusted and fixed as explained above with reference to the symmetric component group. The same aspects described with reference to the symmetric component group with regard to the angle of incidence δ on the deformable mirror 51 and with regard to the option of further reducing this angle are also similarly applicable to the asymmetric component group.

[0105] If microscope 10 is an STED microscope, then it is necessary to take into account that any existing additional deflector 57 will not enter the central focus even when using zoom optics as the first telescope 11 or the second telescope 12. However, in the case of using zoom optics, the position of the deflector can be reasonably no longer expressed as a distance relative to the first lens and its focal length.

[0106] exist Figure 12 The image depicts a component assembly or module including a first wavefront modulator 24. The beam path, via two telescopes 11 and 12 and through a deformable mirror 51, is configured such that, when viewed in the detection direction, the pupil surface 30 behind the first lens 1 of the first telescope 11 is located behind the first deflecting mirror 55. In this pupil surface 30, a second beam splitter 26 is arranged, which separates the detection beam path from the first illumination light 8. By means of a pair of relay lenses 62, 63, this pupil surface is imaged onto another pupil surface 30, in which the effective region 44 of the first wavefront modulator 24 is arranged. The first wavefront modulator 24 is shown here only as an exemplary transmissive SLM 61', and the first illumination light 8 is also shown here only as an exemplary excitation light 6 provided by an excitation source 16. The pair of relay lenses 62, 63 and the first wavefront modulator are all connected to the first wavefront modulator 6. Figure 10 and 11 The known optical elements are arranged together on a common substrate 60. The arrangement of the second beam splitter 26 in the pupil plane 30 has the advantage that the tilt of the beam splitter 26 only causes the beam of the first illumination light 8 to tilt in the other pupil plane, without causing deflection. Since the beam diameter is very small at the location of the beam splitter 26 according to the invention, the tilt has minimal impact on image quality. In summary, maximum insensitivity to, for example, thermally induced misalignment is achieved. However, the arrangement of the second beam splitter 26 in the pupil plane is not mandatory, that is, it is not mandatory even in embodiments having an integrated first wavefront modulator 24 as a module. The effects of the invention can be achieved even if the distance from the pupil plane 30 is not taken into account when positioning the beam splitter 26. Arranging the beam splitter 26 within or adjacent to the pupil plane produces additional advantages. Imaging the pupil plane 30 onto the effective area 44 of the first wavefront modulator is non-mandatory by a pair of relay lenses 62, 63, but can also be achieved by means of another telescope. Even if the effective region 44 of the first wavefront modulator 24 is arranged here, or even if a passive element is arranged here instead, the effective region of this passive element should be utilized effectively. This means that the effective regions 44 of the first wavefront modulator 24 and the second wavefront modulator 34 should be imaged onto each other in the most suitable manner possible. More importantly, the effective region of the first wavefront modulator 24 should also be imaged onto the rear aperture 23 of the objective lens 20 in a suitable manner.

[0107] List of reference signs

[0108] 1 first lens

[0109] 2 second lens

[0110] 3 third lens

[0111] 4 fourth lens

[0112] 5 pinhole aperture

[0113] 6 excitation light

[0114] 7 fluorescence suppression light

[0115] 8 first illumination light

[0116] 9 first illumination light source

[0117] 10 fluorescence microscope

[0118] 11 first telescope

[0119] 12 second telescope

[0120] 15 laser scanning fluorescence microscope

[0121] 16 excitation light source

[0122] 17 fluorescence suppression light source

[0123] 18 detector

[0124] 19 fluorescence

[0125] 20 objective

[0126] 21 sample space

[0127] 22 sample

[0128] 23 back aperture

[0129] 24 first wavefront modulator

[0130] 25 (first) beam splitter

[0131] 26 (second) beam splitter

[0132] 27, 27' scanner

[0133] 28 tube lens

[0134] 29 mirror

[0135] 30, 30', 30" pupil plane

[0136] 31 first pupil

[0137] 32, 32' second pupil

[0138] 33 filter

[0139] 34 second wavefront modulator

[0140] 35 beam splitter

[0141] 36 beam splitter

[0142] 37 positioning stage (sample station)

[0143] 38 detection unit

[0144] 40 entrance plane

[0145] 41 active wavefront modulator

[0146] 42 support structure

[0147] 43 back side

[0148] 44 active area

[0149] 45 lens

[0150] 50 intermediate focus

[0151] 51 deformable mirror

[0152] 55 first deflection mirror

[0153] 56 second deflection mirror

[0154] 57 further deflection mirror

[0155] 58 lens holder

[0156] 60 substrate

[0157] 61, 61' SLM

[0158] 62 relay lens

[0159] 63 relay lens

[0160] 64 abutment edge

[0161] 66 strip aperture

[0162] 70 MINFLUX microscope

[0163] 130 pupil plane

Claims

1. A fluorescence microscope (10), comprising: - Illumination optical path, which is used to illuminate the sample using illumination light (8), the illumination optical path includes: - Illumination source (9), which provides illumination light (8), - First wavefront modulator (24), which is used to spatially modulate illumination light (8), - Beam splitter (26), - A second wavefront modulator (34), which is used to further spatially modulate the illumination light (8), and -Objective lens (20), -In this configuration, the first wavefront modulator (24) is configured to modulate the illumination light (8). So that the modulated illumination light (8) focused by the objective lens (20) causes the sample (22) to experience the intensity distribution of the illumination light (8), including the minimum central intensity value. -The second wavefront modulator (34) is adjustable, wherein the effective area (44) of the second wavefront modulator (34) is arranged in the pupil plane (30) of the objective lens (20) or intersects the pupil plane (30) of the objective lens (20) at an angle, wherein the first pupil (31) appears at the position of the effective area (44) of the second wavefront modulator (34); - The detection optical path, used for confocal detection of fluorescence emitted from the sample, includes a first segment and a second segment. -The first segment of the detection optical path includes a second wavefront modulator (34) and terminates at a beam splitter (26). The first segment of the detection optical path coincides with the illumination optical path, wherein the direction of the detection optical path is opposite to the direction of the illumination optical path in the first segment of the detection optical path, and -The second section of the detection optical path begins at the beam splitter (26) and includes a detection unit (38) for confocal fluorescence detection. The second section of the detection optical path does not coincide with the illumination optical path. - First telescope (11), The first telescope (11) is connected to the second wavefront modulator (34) and beam splitter (26). Arranged between the first section, such that the first telescope (11): - Image the first pupil (31) onto the second pupil (32) along the direction of the detection optical path. The second pupil (32) is smaller than the first pupil (31), and - The collimated illumination light (8) beam in the second pupil (32) is transformed into a collimated and enlarged illumination light (8) beam in the first pupil (31); The distance between the beam splitter (26) and the second pupil (32) adjacent to the first lens (1) of the first telescope (11) is not greater than the focal length of the first lens (1) of the first telescope (11).

2. The fluorescence microscope (10) according to claim 1, characterized in that, The ratio of the first diameter of the first pupil (31) to the second diameter of the second pupil (32) is at least 1.2:

1.

3. The fluorescence microscope (10) according to claim 1, characterized in that, The second wavefront modulator (34) is a deformable mirror (51) including a deformable membrane, the surface of which is the effective region (44).

4. The fluorescence microscope (10) according to any one of claims 1 to 3, characterized in that, The first wavefront modulator (24) is: - Non-adjustable passive optical components; or - Adjustable active wavefront modulator (41).

5. The fluorescence microscope (10) according to any one of claims 1 to 3, characterized in that, The second telescope (12) is arranged in the first section between the second wavefront modulator (34) and the objective lens (20).

6. The fluorescence microscope (10) according to claim 1, characterized in that, The scanner (27) is arranged between the second wavefront modulator (34) and the telescope lens (28) in the first section, and the telescope lens (28) is arranged between the second wavefront modulator (34) and the objective lens (20).

7. The fluorescence microscope (10) according to claim 6, characterized in that, The second telescope (12) is arranged in the first section between the second wavefront modulator (34) and the objective lens (20), and the second telescope (12) is arranged between the second wavefront modulator (34) and the scanner (27) so that the beam diameter of the illumination light is adapted to the back aperture (23) of the objective lens (20).

8. The fluorescence microscope (10) according to any one of claims 1-3, 6, and 7, characterized in that, The effective area (44) of the first telescope (11) and the second wavefront modulator (34) is arranged in the first section between the first deflector (55) and the second deflector (56), wherein the first deflector (55) and the second deflector (56) are arranged in the illumination optical path and the detection optical path, such that after the first deflector (55) and the second deflector (56) are removed from the illumination optical path and the detection optical path, the fluorescence microscope (10) is still able to perform operations including the intensity distribution including the minimum central intensity value of the illumination light (8) on the sample (22).

9. The fluorescence microscope (10) according to any one of claims 1-3, 6, and 7, characterized in that, The distance between the beam splitter (26) and the second pupil (32) adjacent to the first lens (1) of the first telescope (11) is no greater than half the focal length of the first lens (1) of the first telescope (11).

Images