DE102019119213A1 - Devices and methods for microscopy for three-dimensional super resolution - Google Patents

Abstract

One object of the invention is to improve devices and methods for microscopy and to facilitate their handling. For this purpose, in a method, an image of a test object is reconstructed in super-resolution based on fluorescence from a plurality of NV centers which the test object has. A spatial area with the test object is irradiated with an excitation light for the NV centers and with microwave radiation, the frequency of which corresponds to the frequency at which resonant microwave absorption occurs. Spatial sections of the spatial area are scanned with a resolution beyond an Abbe limit, with each spatial segment of the spatial segment generating the microwave radiation for irradiation in such a way that it has a microwave field course, disappears locally in the respective spatial segment and a respective spatial extent of the disappearance of the microwave radiation in the respective spatial segment at least is limited in one dimension to an extent which is smaller than the Abbe limit. In addition, when scanning for each spatial segment of the spatial segments, a light in the respective spatial segment is optically detected at least in a spectral range which corresponds to the emission light of the NV centers. Finally, the image of the test object is reconstructed based on the light captured in the respective space sections.

Description

The invention is in the field of microscopy and optical position determination and relates in particular to a method for super-resolution microscopy, a method for determining a position of an NV center, a method for microscopy based on a large number of mesoscopic solid-state elements, a microscope and a device for Determination of a position of an NV center of a solid-state mesoscopic element.

In the microscopy of biological materials, conventional fluorescent substances or fluorophores are usually used for staining - for example, molecules that optically fluoresce when excited. With a specific staining process, certain parts of the biological material, such as certain areas or organelles of biological cells, can be provided with the fluorescent substance - the fluorescent substance binds to such certain parts. Those parts to which the fluorescent substance is bound are then highlighted or identified in relation to other parts of the biological material when appropriately excited due to the fluorescence. By staining - and thus in particular highlighting in color - certain parts of the biological material can also be used to determine the respective positions of these certain parts based on a detection of a light that corresponds to the staining or to track them over time.

In addition to conventional fluorescent substances such as fluorescent molecules when excited, mesoscopic solid-state elements with at least one color center could also be used, for example, for coloring.

Nanodiamonds (or, more generally, mesoscopic solid-state elements) with nitrogen-vacancy centers as color centers have a high brightness when optically stimulated. H. especially high light emission and photostability, d. H. especially a slight fading. In addition, an emission light emitted by such a nitrogen defect center is dependent on a magnetic field effective there and on further influencing factors such as microwave radiation. In addition, nanodiamonds are biocompatible.

Fluorescence or phosphorescence (or general luminescence, henceforth briefly referred to as “fluorescence” and correspondingly fluorescent substance or fluorophore) from nitrogen vacancy centers or more generally from NV centers - for example in applications such as the determination of a magnetic field or in quantum cryptography or for quantum computer systems - depending on the magnetic field effective there and any other influencing variables. For example, such a nitrogen vacancy center could serve as an emitter in a solid-state-based single photon source at room temperature. For example, due to the controllable fluorescence, a high achievable sensitivity and / or a large achievable range over which the intensity of the emission light depends on the magnetic field and the microwave radiation, such nitrogen vacancy centers are also used for various commercial fields such as for the investigation of electrical Circuits or for an optically integrated biosensor based on an optically detected (microwave) resonance of a nitrogen vacancy center. Such an optically integrated biosensor is described in the patent US 8,193,808 B2 described.

Techniques have also been developed for this purpose in order to determine a position and any properties of a nitrogen vacancy center in a diamond with super resolution. These are usually adaptations of known techniques for super-resolution microscopy such as STED (STimulated Emission Depletion), which require a high dose of light, or techniques for diamonds in general, i. H. in particular for macroscopic diamonds, which can be aligned in a controlled manner, the (relative) position of nitrogen vacancy centers in such a diamond is determined based on the fact that the light emitted by the nitrogen vacancy center is not only dependent on the strength and / or frequency of the magnetic field or the microwave radiation, but also on the relative orientation of the nitrogen vacancy center to the magnetic field profile of the magnetic field.

While nanodiamonds with a nitrogen void center - due to their bio-compatibility and fluorescent properties such as high brightness and high photostability - could be used as a supplement or a replacement for conventional fluorophores or fluorescent substances in microscopy, especially of biological material, for example for staining Such nanodiamonds usually and especially in a biological material do not align in a controlled manner. Rather, such nanodiamonds can usually rotate in a biological material and / or change their orientation.

There is a need to improve fluorescence-based devices and methods for microscopy and position determination - such as in the microscopy of a biological material stained by means of a fluorescent substance - and in particular to increase and / or increase the resolution thereof - for example the optical resolution in microscopy To enable or replace fluorescent substances so that biocompatibility, reliability and / or efficiency can be increased.

The invention fulfills this need in each case by a method for super-resolution microscopy of a test object, by a method for determining a position of a NV center of a mesoscopic solid-state element within a spatial region, by a method for microscopy based on a plurality of mesoscopic solid-state elements, by a Microscope and a device for determining a position of a NV center of a mesoscopic solid-state element within a spatial area, each according to the teaching of one of the main claims. Advantageous embodiments, developments and variants of the present invention are in particular the subject of the subclaims.

A first aspect of the invention relates to a method for super-resolution microscopy of a test object. The test object has a multiplicity of mesoscopic solid-state elements, each of which has at least one NV center. In addition, the test object is arranged within a spatial area. The method comprises irradiating the spatial region with an excitation light for the NV centers of the plurality of mesoscopic solid-state elements. The method further comprises irradiating the spatial region with microwave radiation, the frequency of which corresponds to the frequency at which resonant microwave absorption occurs without a local magnetic field at the NV centers. The method furthermore includes a scanning of spatial sections of the spatial area with a resolution beyond an Abbe limit for an emission light which can be emitted by the NV centers - in particular when optically excited by the excitation light. When scanning, a magnetic field is generated over the spatial area with a magnetic field curve for each spatial segment of the spatial segment, so that the magnetic field disappears locally in the respective spatial segment and a respective spatial extent of the disappearance of the magnetic field in the respective spatial segment is limited at least in one dimension to an extent that is smaller than the Abbe limit is. As an alternative to generating the magnetic field or in combination with it, when scanning each space of the space, the microwave radiation for irradiation is generated in such a way that it has a microwave field course, disappears locally in the respective space and a respective spatial extent of the disappearance of the microwave radiation in the respective space in at least one dimension is limited to an extent which is smaller than the Abbe limit. In addition, when scanning for each spatial segment of the spatial segments, a light in the respective spatial segment is optically detected at least in a spectral range which corresponds to the emission light. Finally, the method also has a reconstruction of an image of the test object based on the space sections and the light captured in the respective space sections, wherein in some variants the reconstruction with respect to the space sections at least on a known form and a known arrangement of the space sections - at least relative to one another - based.

In the sense of the disclosure, “super-resolution microscopy” is to be understood as at least a microscopy with a resolution beyond an Abbe limit of a light used for microscopy. The resolution - for example when using light around the visible spectrum - can be higher than 500 nm, i.e. about 300 nm, 150 nm, 100 nm or 50 nm. In three-dimensional microscopy, the resolution for a resolution in a focal plane of the microscope and a resolution with respect to an axis through the focal plane - for example, for a three-dimensional image which is composed of several two-dimensional images from several focal planes - differ. Accordingly, “super-resolution” is to be understood as a spatial precision or accuracy with such a resolution. For super-resolution microscopy, see for example "Super-resolution microscopy" at https://en.wikipedia.org/wiki/Super-resolution_microscopy (for example, version dated June 28, 2019 at https://en.wikipedia.org/w/ index.php?title=Super-resolution_ microscopy&oldid=903884823).

In the sense of the disclosure, an “NV center” is to be understood as at least one color center, the color center being optically excitable by means of an excitation light as a function of a magnetic field effective there, and emission light being emitted from the excited color center. Such a color center can be a defect in a matrix structure, in particular in a (possibly crystalline) solid. An intensity of the emission light can also be dependent on a resonant microwave absorption, the resonant microwave absorption being dependent on the magnetic field at the color center. Such a NV center can also be a nitrogen vacancy center in a diamond lattice, for example a so-called [NV] ^- center, which is the subject of current research. For such a [NV] ^- center, a model is currently based on a multi-electron system, which is a 3-level system with a triplet ground state and an excited triplet state and at least one energetically between the ground state and the excited state Intermediate state - in particular a singlet state - (or approximately two intermediate states according to Doherty, Marcus W .; Manson, Neil B .; Delaney, Paul; Jeletsko, Fedor; Wrachtrup, Jörg; Hollenberg, Lloyd CL (2013-07-01). "The nitrogen-vacancy color center in diamond". Physics Reports. The nitrogen-vacancy color center in diamond. 528 (1): 1-45) is described. In such a [NV] ^- center, an electron spin resonance can also be excited between several energetically different states in the triplet ground state, which differ energetically due to a spin interaction and a magnetic field possibly acting at the NV center. Microwave radiation of a suitable frequency can be used to excite the electron spin resonance, so that the electron system is raised from an energetically lower state of the triplet ground state to an energetically higher state of the triplet ground state by means of energy from the microwave radiation.

In the sense of the disclosure, a “mesoscopic solid-state element” is to be understood as at least an object made of a solid-state material which has a spatial extent - that is, for example, a maximum diameter - of less than 1 μm. The spatial extent can also be greater than approximately 1 nm. Such a mesoscopic solid-state element can be a matrix structure made up of atoms or molecules, for example a crystalline solid. In the case of an NV center of a mesoscopic solid-state element, the mesoscopic solid-state element has, for example, at least this NV center or forms it. The NV center can be a defect in a matrix structure of the mesoscopic solid-state element. The mesoscopic solid-state element can also have further NV centers. Such a mesoscopic solid-state element can be or comprise a nanodiamond, for example. Such a nanodiamond can have a nitrogen vacancy center as the NV center, that is to say for example an [NV] ^- center or an [NV] ⁰ center. Such a nanodiamond can also have an ST1 or “Stuttgart 1” color center as the NV center. A mesoscopic diamond matrix can also (more generally) be such a mesoscopic solid-state element and have a color center in the diamond matrix as the NV center. Such a mesoscopic solid-state element can also be made from 4H-SiC and have a solid-state matrix, in particular a crystal lattice, made from 4H-SiC. Such a mesoscopic solid-state element made of 4H-SiC can be a color center as a NV center such as a so-called "VcVsi DiVacancy" or a so-called "NV Nitrogene Vacancy" or a so-called "hexagonal lattice site silicon vacancy (V _Si )" (see for example NATURE COMMUNICATIONS | (2019) 10: 1954 | https://doi.org/10.1038/s41467-019-09873 | High-fidelity spin and optical control of single silicon-vacancy centers in silicon carbide), especially in the crystal lattice.

A second aspect of the invention relates to a method for determining a position of a NV center of a mesoscopic solid-state element within a spatial region. The method includes irradiating the spatial region with an excitation light for the NV center. The method further comprises irradiating the spatial region with microwave radiation, the frequency of which corresponds to the frequency at which resonant microwave absorption occurs without a local magnetic field at the NV center. The method furthermore comprises a scanning of spatial sections of the spatial area, with each spatial segment of the spatial areas either generating a magnetic field over the spatial area with a magnetic field curve so that the magnetic field disappears locally in the respective spatial segment, or the microwave radiation for irradiation is generated in such a way that it has one Has microwave field progression and disappears locally in the respective spatial section, or both a magnetic field and microwave radiation are generated in such a way that at least the magnetic field or the microwave radiation disappears locally in the respective spatial section and possibly the other either also disappears or does not disappear in the respective local spatial section, and where For each space segment of the space segments, a light is optically detected in the respective space segment at least in a spectral range which corresponds to an emission light emitted by the NV center. The method also includes determining the position of the NV center based on the detected light in the respective space sections.

One advantage of scanning and the magnetic field or the disappearing microwave radiation that disappears in the respective space segment can be that it allows the intensity of the emission light of the NV center to be changed when it is in the respective space segment, whereby a distinction is made between this during scanning respective space section is made possible by the remaining space sections and thus an optical resolution and / or an accuracy in determining the position can be increased. An advantage of differentiating the respective section on the basis of a change in the intensity of the emission light due to the disappearance of the magnetic field or the microwave radiation can be in particular that the disappearance is independent of an orientation in space and thus local to the NV center - regardless of its spatial orientation Orientation - the magnetic field or the microwave radiation disappears, whereas the change in the intensity of the emission light due to a finite magnetic field or a finite microwave radiation - i.e. in particular not disappearing - depends on the spatial orientation of the NV center with respect to the magnetic field or the microwave radiation can depend. When differentiating between the respective spatial section based on the disappearance of the magnetic field / microwave radiation, a spatial orientation of the NV center does not have to be known, which can be advantageous for a biological material or for other materials in which the spatial orientation of the NV center is is not determined or can change due to rotation with respect to the material - such as with liquids. The same applies to the large number of mesoscopic solid-state elements with the NV centers, whereby the independence of the disappearance of the magnetic field or the microwave radiation from the spatial orientation for each NV center of the NV centers synergistically simplifies the super-resolution microscopy, since none the respective orientation of the NV centers must be determined. Thus, in some embodiments, the multiplicity of mesoscopic solid-state elements has at least 100, at least 1000 or at least 12000 mesoscopic solid-state elements and a corresponding multiplicity of NV centers - that is, at least 100, at least 1000 or at least 12000. In this way, the method can advantageously be made more efficient - even if a determination of the respective orientation of the NV centers were possible at all - since, for example, no determination of the respective orientation is required.

An advantage of the mesoscopic solid-state element, which has the NV center - that is to say, for example, as a defect in a solid matrix of the mesoscopic solid-state element such as a diamond matrix - can in particular be that the NV center has high photostability and / or high brightness - d. H. In particular, the emission light has a high intensity at a certain intensity of the excitation light - and / or the mesoscopic solid-state element shields the NV center from external influences - for example more strongly than with conventional fluorophores such as individual fluorescent molecules - which increases the efficiency and / or reliability - for example when staining by means of such mesoscopic solid-state elements - can be increased and their application simplified. Such mesoscopic solid-state elements also usually interact less with a - for example biological - material and thus have an increased (bio) compatibility, which advantageously reduces (unwanted) influencing of the material being examined and thus simplifies the application. Increased (bio) compatibility can also enable longer measurement times and thus, for example, increased accuracy and / or reliability. Thus, when examining a biological material, for example, its properties can be examined without (significantly) changing them due to the coloring by means of a fluorescent substance - that is to say in the present case a large number of such NV centers. It can also be used to improve other applications - that is, in particular not for a biological material - with certain (partial) parts of a product such as a chemical substance or a workpiece being able to be marked using such NV centers and this marking due to the high brightness and / or photostability can be used reliably to determine the position of these markings.

The possible advantages, embodiments or variants of the first aspect of the invention also apply accordingly to the method for determining a position of an NV center.

A third aspect of the invention relates to methods for microscopy based on a plurality of mesoscopic solid-state elements, each of the solid-state elements having at least one NV center. The method includes arranging a test object within a spatial region, the test object having the plurality of mesoscopic solid-state elements - that is to say is colored by means of the mesoscopic solid-state elements, for example. In addition, the method includes performing the method according to the first aspect of the invention, the respective NV centers of the mesoscopic solid-state elements being distinguished from one another by means of the scanning of the spatial sections and thus being resolved by imaging.

The possible advantages, embodiments or variants of the preceding aspects of the invention also apply accordingly to the method for microscopy based on a large number of mesoscopic solid-state elements.

A fourth aspect of the invention relates to a microscope. The microscope has a spatial area for the arrangement of a test object - such as a biological material or a workpiece. The microscope also has a light source for irradiating the spatial area with an excitation light, by means of which, when a test object with a plurality of NV centers each formed in a mesoscopic solid-state element is arranged in the spatial area, one or more of the NV centers of the plurality NV centers are optically stimulable. The microscope also has a microwave antenna arrangement for irradiating the spatial area with microwave radiation, the frequency of which corresponds to the frequency at which resonant microwave absorption of the NV centers occurs without a local magnetic field. The microscope furthermore has a control device which is set up to scan spatial sections of the spatial area and thereby to select one spatial section of the spatial sections and for the respectively selected one To control a magnetic field device in such a way that it generates a magnetic field with a magnetic field profile over the spatial area, through which the magnetic field disappears locally in the selected spatial segment, or to control the microwave antenna arrangement in such a way that it irradiates the spatial area with the microwave radiation in such a way that the microwave radiation has a microwave field course and disappears locally in the selected space section. The microscope also has a sensor device which is set up to detect an emission light emitted by the NV centers at least in the spatial section selected during the scanning. In addition, the microscope has an evaluation device which is set up to determine a — in particular two-dimensional or three-dimensional — image of the test object based on the emission light which is emitted by the NV centers in the respective spatial sections.

The possible advantages, embodiments or variants of the preceding aspects of the invention also apply accordingly to the microscope. The microscope can for example be set up to carry out a method according to the first or third aspect of the invention.

In some embodiments, the microscope furthermore has the magnetic field device for generating the magnetic field, an intensity of the emission light emitted by the NV centers being dependent on the resonant microwave absorption and the magnetic field at the respective NV center. Alternatively, in some embodiments the microscope does not have a magnetic field device, an intensity of the emission light emitted by the NV centers being dependent at least on the resonant microwave absorption, but not necessarily on the (local) magnetic field at the respective NV center. In such embodiments for NV centers whose intensity of the emission light does not depend on a magnetic field and / or - in some variants - without a magnetic field device, the control device is set up to control the microwave antenna arrangement, for example in order to address a particular section of space during scanning, or the control device is not set up to control a magnetic field device. Some embodiments also have a shielding device for shielding an (external) magnetic field or for shielding (external) microwave radiation.

A fifth aspect of the invention relates to a device for determining a position of an NV center of a mesoscopic solid-state element within a spatial region, the device being set up to carry out a method according to the first, second or third aspect of the invention.

The possible advantages, embodiments or variants of the preceding aspects of the invention also apply accordingly to the device for determining a position of an NV center. In some variants, the microscope can also be set up to carry out a method according to the second aspect, and thus form some variants of the device according to the fifth aspect of the invention, wherein not necessarily a plurality of NV centers are detected but also a single NV center based detected on a change in its intensity of the emission light and its position can be determined.

Further advantages, features and possible applications emerge from the following detailed description of exemplary embodiments and / or from the figures.

The invention is explained in more detail below with reference to the figures on the basis of advantageous exemplary embodiments. The same elements or components of the exemplary embodiments are essentially identified by the same reference symbols, unless otherwise described or if the context does not indicate otherwise.

• 1 ein Modell eines [NV]^--Zentrums;
• 2 ein Energiediagramm für ein NV-Zentrum;
• 3 ein Mikroskop nach einer Ausführungsform;
• 4 ein Raumbereich mit Raster nach einer Ausführungsform;
• 5 eine Abhängigkeit einer Fluoreszenz eines [NV]^--Zentrums von einer Frequenz einer Mikrowellenstrahlung nach einer Ausführungsform;
• 6 ein Flussdiagramm eines Verfahrens zur superauflösenden Mikroskopie in drei Dimensionen basierend auf einer Vielzahl an Nanodiamanten nach einer Ausführungsform; und
• 7 ein Flussdiagramm eines Verfahrens zur zweidimensionalen Bestimmung einer Position eines NV-Zentrums nach einer Ausführungsform.

To this end show, partly schematically:

• 1 a model of a [NV] ^- center;
• 2 an energy diagram for an NV center;
• 3 a microscope according to one embodiment;
• 4th a spatial area with a grid according to one embodiment;
• 5 a dependence of a fluorescence of a [NV] ^- center on a frequency of microwave radiation according to one embodiment;
• 6th a flowchart of a method for super-resolution microscopy in three dimensions based on a plurality of nanodiamonds according to an embodiment; and
• 7th a flowchart of a method for two-dimensional determination of a position of an NV center according to an embodiment.

The figures are schematic representations of various embodiments and / or exemplary embodiments of the present invention. Elements and / or components shown in the figures are not necessarily shown true to scale. Rather, the various elements and / or components shown in the figures are such reproduced so that their function and / or their purpose can be understood by a person skilled in the art.

Connections and couplings shown in the figures between the functional units and elements can also be implemented as indirect connections or couplings. In particular, data connections can be wired or wireless, that is to say in particular as radio connections. Certain connections, for example electrical connections, for example for energy supply, can also not be shown for the sake of clarity. Furthermore, optical connections - for example between optical elements - which can be represented in particular as a straight light beam, can also be implemented in some variants by means of a light guide and / or optical elements such as mirrors for deflecting light beams, such connections for the sake of clarity are not necessarily shown.

1 illustrates an atomic structure of an NV center such as a nitrogen vacancy center schematically with a ball-and-stick model of a [NV] ^- center ( 140 ) without surrounding diamond lattice. There are three carbon atoms 146 arranged in three places of the diamond lattice, while in one of these three carbon atoms 146 (immediate / nearest neighbor) neighboring lattice space a defect 144 (Vacancy: V) exists - this lattice site is therefore not occupied - as well as a nitrogen atom instead of a carbon atom for a lattice site adjacent to it (immediate / nearest neighbor) 142 (Nitrogen: N) is arranged. In addition, in 1 a vector for an external magnetic field 80 as well as an axis of the NV center 148 - with respect to which a spin of a multi-electron system of the NV center is defined - is shown. It goes without saying that in addition to the external magnetic field 80 a magnetic field is also effective on electrons of the multi-electron system due to the magnetic moments of the atomic nuclei or these magnetic fields overlap, whereby - unless reference is made separately to these additional magnetic moments - in the sense of the invention under the "magnetic field effective there" that magnetic field is assigned is to understand which occurs there, i.e. at the respective NV center due to the external magnetic field.

In 2 is an energy diagram 40 for an NV center such as a [NV] ^- center according to current modeling (cf. Rogers, LJ; Armstrong, S .; Sellars, MJ; Manson, NB (2008). "Infrared emission of the NV center in diamond: Zeeman and uniaxial stress studies". New Journal of Physics. 10 (10): 103024 .) (cf. for example Doherty, Marcus W .; Manson, Neil B .; Delaney, Paul; Jeletsko, Fedor; Wrachtrup, Jörg; Hollenberg, Lloyd CL (2013-07-01). "The nitrogen-vacancy color center in diamond". Physics Reports. The nitrogen-vacancy color center in diamond. 528 (1): 1-45 .) shown. The multi-electron system of the NV center has a triplet ground state | g>, an excited triplet state | e> and two intermediate states | ze> and | zg>, which are energetically between the ground state | g> and the excited state | e> , on. Two of the electrons of the multi-electron system can be aligned parallel or antiparallel with regard to their spin in the triplet ground state, so that the multi-electron system has a spin +1 (m _s = +1) or -1 (m _s = -1) or a spin 0 (m _s = 0). Due to their spin interaction, the electrons have a higher energy level with spin +1 and -1 than with an anti-parallel alignment with spin 0. In addition, - in 2 not shown - the energy levels for m _s = + 1 and m _s = -1 can differ from one another due to an interaction with the magnetic moments of the atomic nuclei (see hyperfine structure), with this splitting, i.e. a difference between the energy levels for m _s = + 1 and m _s = -1 is usually much smaller than an energy difference between the energy levels for m _s = + 1 / m _s = -1 compared to the energy level for m _s = 0.

Starting from 2 emission light can be emitted by an NV center 140 corresponding 1 and / or with regard to energy levels accordingly 2 and a dependency of an intensity of the emission light on a resonant microwave absorption and a possible magnetic field at the NV center as follows. Using excitation light 46 with sufficient energy per photon, i.e. a green laser with a wavelength less than about 532 nm - such as a wavelength of 515 nm as shown - the [NV] ^- center can be converted from the ground state | g> (initially into vibronic Bands and then from there) optically excite the excited state | e>, whereby the spin of the multi-electron system is retained, i.e. when the ground state | g> with m _s = + 1 is excited, correspondingly as an excited state | e> with m _s = +1 is reached. The NV center can then emit a photon, i.e. emission light 56 and thus fluorescence, return to the corresponding state of the triplet ground state - that is, from the excited state le> with m _s = + 1 to the ground state | g> with m _s = + 1 -; at a [NV] ^- center, for example, a photon with 637 nm, i.e. red emission light, can be produced 56 are emitted. This transition is also called a radiating transition or optical transition, with the (emission / fluorescence) light emitted here usually being detected optically.

In addition to this radiant transition, a further transition via the intermediate states | ze> and | zg> is also possible, for example at Transition from | zg> to | ze> a photon with a larger wavelength, i.e. a photon with 1042 nm is emitted at an [NV] ^- center. In other models, only one intermediate state is assumed, so that no corresponding photon is emitted. In these transitions there is no emission of photons or at least one emission 58 of photons with a different, especially with a longer wavelength take place, and these transitions are also called non-radiating transitions. In these non-radiating transitions, the spin of the multi-electron system is not necessarily retained, the rate or the probability of a transition from an excited state with m _s = + 1 or m _s = -1 of the excited triplet state | e> to State with m _s = 0 of the triplet ground state is greater than the rate or the probability of a transition from the excited state | e> with m _s = 0 to one of the ground states with m _s = + 1, 0 or -1. The further transition via the intermediate states | ze> and | zg> competes with the radiant transition. Thus, if an NV center has a spin m _s = 0, it emits more emission light than with a spin m _s = + 1 or m _s = -1, since there is a transition at m _s = + 1 or m _s = -1 takes place more frequently over the intermediate states. In addition, in the case of an NV center, repeated excitation can increase the population probability for the ground state and / or for the excited state with m _s = 0, since over the further transition the ground state | g> with m _s = 0 (and then after possibly renewed excitation the excited state | e> with m _s = 0) is reached - this is also called spin polarization.

By certain measures - such as radiation with a certain energy (in particular per radiation quantum), which corresponds to an energy difference between | g> with m _s = 0 and Ig> with m _s = ± 1 or an energy difference between | e> with m _s = 0 and | e> with m _s = ± 1 corresponds - the occupation probability for the ground state and / or the excited state can be increased with m _s = + 1 or -1 for an NV center. At an [NV] ^- center (without an external magnetic field), microwave radiation 48 a transition from the ground state | g> with m _s = 0 to one of the ground states with m _s = ± 1 can be resonantly excited at a frequency of about 2870 MHz, i.e. an electron spin resonance can be achieved - i.e. in the sense of the disclosure in particular a resonant absorption of the ( Microwave) radiation through the NV center with transition from Ig> with m _s = 0 to | g> with m _s = + 1 or -1. In a broader sense, electron spin resonance can also be understood as such an excitation by varying the (external) magnetic field and measuring the magnetic field-dependent absorption of the microwave radiation (cf. e.g. https://de.wikipedia.org/wiki/Elektronenspinresonanz).

When an external magnetic field is applied, the energy levels of the ground state with m _s = + 1 and the ground state shift at an NV center, where the intensity of the emission light depends on the (local) magnetic field - such as the [NV] ^- center with m _s = -1 (the same applies to the states of the excited triplet state | e> with m _s = ± 1). Thus, for the transition from Ig> with m _s = 0 to | g> with m _s = -1, a different frequency of the microwave radiation is required than for the transition from Ig> with m _s = 0 to | g> with m _s = + 1.

When a [NV] ^- center is irradiated (initially without an external magnetic field) with microwave radiation with a frequency of about 2870 MHz, the probability for the states with m _s = ± 1 is increased, which causes the fluorescence, i.e. the emitted emission light 56 decreases. By an external magnetic field 80 , which at the NV Center 140 acts, the frequency required for the electron spin resonance is shifted, as a result of which the probability for the states with m _s = ± 1 is less or no longer increased and thus the fluorescence does not decrease or increase again.

The change in fluorescence - i.e. in particular the change in the intensity of the emission light - is therefore dependent on the specific measure to increase the occupation probability for states with m _s = ± 1 - i.e. about the frequency and field strength of the irradiated microwave radiation - as well as from the (external) magnetic field effective at the NV center. Thus, the intensity of the emission light can be changed by means of a specific measure to increase the occupation probability, such as microwave radiation, and this specific measure and thus a change in intensity can be counteracted if there is a corresponding dependence on the magnetic field. For example, a corresponding microwave radiation at an [NV] ^- center causes a decrease in the intensity of the emission light compared to the emission light without microwave radiation (with the same excitation light), with a magnetic field acting at a [NV] ^- center shifting and shifting the frequency required for resonant microwave absorption thus prevents a decrease in the intensity - or causes an increase in the intensity of the emission light compared to the emission light with microwave radiation and the same excitation light.

3 shows schematically a microscope 300 according to one embodiment of the present invention.

In one embodiment, the microscope 300 a room area 320 for the arrangement of a test object, this spatial area lying in a focal plane of the microscope. In some Further training is the spatial area through a slide 321 of the microscope 300 defined, the slide 321 and the microscope are set up, one from the slide 321 carried test object in a focal plane of the microscope 300 to arrange. Correspondingly, the microscope can in some further developments 300 be set up to receive a slide, which is not necessarily part of the microscope, in such a way that a test object lies in the focal plane.

In 3 is also a biological material, particularly a biological tissue, as a test object 200 shown which in the room area 320 on the slide 321 arranged, but not necessarily part of the microscope 300 is. The biological tissue 200 exhibits a multitude of biological cells 222 , 224 as well as a variety of nanodiamonds 240 as a plurality of mesoscopic solid-state elements, each of the nanodiamonds 240 at least one nitrogen vacancy center in each case 140 as NV center. As shown, an area can be a 224 of biological cells by means of several nanodiamonds 240 be stained. In addition - in some variants - the nanodiamonds 240 have a functionalized surface, so that they fit into the area of this biological cell 224 tie. Correspondingly, mesoscopic solid-state elements can - in some variants - have a functionalized surface which enables binding to other areas of a biological cell or to a specific material or a solution in a specific fluid. Also - as shown - another area of the test object can include some of the nanodiamonds 240 exhibit.

In one embodiment, the microscope 300 still a light source 340 for irradiating the room area with an excitation light 46 by means of which one or more of the nitrogen vacancy centers 140 the multitude of nitrogen vacancy centers 140 are optically stimulable. In some variants, the light source has 340 a laser for green light, so in particular with a wavelength less than 532 nm, such as with a wavelength of 515 nm. So is in 3 an optical excitation of the nitrogen vacancy center 140 shown, which is in a room section 324 of the room area 320 whereas the nitrogen vacancy center is located 140 in the room section 326 of the room area 320 not from the excitation light 46 is irradiated and therefore not optically stimulated. An advantage of irradiating only those spatial sections which are selected during the scanning - and possibly some surrounding spatial sections - can in particular be that a radiation dose can be reduced by the excitation light.

The microscope 300 further comprises a microwave antenna arrangement 380 for controllable irradiation of the room area 320 with a microwave radiation, the frequency of which corresponds to the frequency at which, without a local magnetic field, resonant microwave absorption of the nitrogen vacancy centers 140 occurs. In some variants for nanodiamonds with a [NV] ^- center as the nitrogen vacancy center is the microwave antenna array 380 set up to emit microwave radiation between 2.5 GHz and 3.5 GHz and / or at a frequency of about 2870 MHz, the respective frequency can be regulated depending on a decrease in intensity.

The microscope 300 furthermore has a control device 360 on that is set up all room sections 324 , 326 of the room area 320 raster and thereby a space section of the space sections - roughly as shown space section 324 - select as well as the microwave antenna arrangement for the selected room section 380 to control in such a way that these the spatial area 320 irradiated with the microwave radiation in such a way that the microwave radiation has a microwave field profile and locally disappears in the respectively selected spatial section. Here are the control device 360 and the microwave antenna assembly 380 set with respect to a field strength microwave radiation such that in the respective space section in which microwave radiation disappears, the intensity of the emission light of the NV centers there corresponds at least substantially to the intensity without microwave radiation - i.e. in particular at least 50%, 80% or 90% - and that in the other sections of the room, in which the field strength of the microwave radiation is finite, the intensity of the emission light is reduced compared to the intensity with vanishing microwave radiation - i.e. in particular the intensity of the emission light with non-vanishing microwave radiation at most 95%, 90%, 70% or 10% compared to the intensity of the emission light with vanishing microwave radiation.

The microscope 300 furthermore has a sensor device 350 on that is set up a light 56 , 305 , in particular to detect in a spectral range which corresponds to the emission light 56 corresponds, at least in the space section selected during the scanning - i.e. roughly as shown in the space section 324 - capture.

In some variants the sensor device is 350 furnished part of the room sections 324 , 326 with the section of space selected when scanning - for example the section of space as shown 324 - To be captured by imaging, and has an image sensor for this purpose. In some variants of it the image sensor is sensitive to red light, in particular with a wavelength of 637 nm. Alternatively or additionally, the sensor device 350 in some variants a photodiode for detecting light 56 , 503 from the respectively selected space section, in particular with a high sensitivity for a wavelength of 637 nm.

In some advanced training courses, the microscope 300 a beam splitter device 330 on that is set up excitation light 46 from the light source 340 into a beam path of the microscope 300 and from one of the NV centers 140 Emission light emitted into the beam path 56 and / or additional light 305 from the spatial section selected during the scanning to a beam path exit of the microscope in which the sensor device 350 is arranged to lead. In some variants the beam splitter is device 330 designed as a dichroic mirror.

In some advanced training courses, the microscope 300 optical elements 316 - approximately comprehensive an objective 310 - In the beam path, which is set up to guide the excitation light 46 to the room area 320 or to certain sections of the room 324 , 326 thereof and / or set up to guide light 56 , 305 from the room area 320 or from certain sections of the room 324 , 326 of which to a beam exit of the microscope 300 , in which the sensor device 350 is arranged.

The microscope 300 furthermore has an evaluation device 390 on that is established, the positions of the nitrogen vacancy centers 140 based on the emission light 56 to determine which in the respective room sections 324 , 326 emitted from the nitrogen vacancy centers.

In 4th is a space 320 , which by means of a grid 322 in several room sections 324 , 326 is divided, according to one embodiment of the present invention, in particular in combination with the microscope with respect to 3 or with the procedure regarding 6th , illustrated schematically.

In one embodiment, the space sections are 324 , 326 lines through the space 320 which are particularly in the focal plane of the microscope 300 lie and move in one main direction through the room area 320 extend, while these have a smaller extent in other spatial directions, which can in particular be dependent on the desired spatial resolution - i.e. have a spatial extent in other directions that is smaller than 1 µm, 300 nm, 100 nm or 5 nm. In this case, lines in some variants are spatial extensions each around a straight line lying in the focal plane. Also point the lines 324 , 326 - in some variants, such as in 4th shown - a common central point 328 and are relative to the space area 320 , in particular in the focal plane, each rotated by a certain step angle, with the step angle in each case being constant with respect to a preceding line in some variants. Accordingly, the straight lines intersect at the central point 328 and are each rotated to each other by a certain step angle. While the step angles are exemplarily illustrated as 20 ° steps, in some variants - depending on the desired spatial resolution - they are selected to be smaller, such as 1 ° steps, 0.1 ° steps, 0.01 ° steps or 0.003 ° -steps This advantageously allows the room area 320 in room sections 324 , 326 subdivide.

Correspondingly, in an exemplary embodiment for three-dimensional microscopy, the spatial sections are 324 , 326 each area through the room area 320 which extends in a first and a second spatial direction through the spatial area 320 extend, while these have a smaller extent in third spatial directions, which can in particular be dependent on the desired spatial resolution - i.e. have a spatial extent in the third spatial direction that is smaller than 1 µm, 300 nm, 100 nm or 5 nm. In some variants, surfaces are spatial extensions each around a plane intersecting a focal plane. In addition, the surfaces 324 , 326 - in some variants - a common central point 328 and are relative to the space area 320 , in particular in the focal plane as well as to the focal plane, each rotated by a specific first step angle and a specific second step angle, wherein in some variants the first step angle and / or the second step angle is constant to a previous surface. In some variants, the areas can be calculated by specifying a first angle for a first group of areas, the areas of the first group of areas being rotated relative to one another by the determined second step angle, and also iteratively increasing the first angle per iteration by the first step angle and iterative determination of areas of further groups of areas which each have the respective first angle relative to the spatial area and are each rotated to one another by the determined second step angle. Accordingly, the levels intersect at the central point 328 and are rotated with respect to one another by the first and second step angles.

Furthermore, in one exemplary embodiment, the surfaces can be rotated to one another only by a step angle and intersect in a straight line which passes through a common central point in a focal plane goes. Several focal planes can be scanned for three-dimensional microscopy.

In some further training courses in which the spatial area 320 by means of a grid 322 based on a central point 328 - as regarding 4th described - is subdivided, is the microwave antenna assembly 380 set up to controllably generate the microwave radiation in such a way that it is in a selected one of the space sections 324 , 326 disappears and has a finite field strength in the rest of the space. In addition, the sensor device 350 set up, when scanning the selected room sections 324 , 326 at least one-dimensionally resolved, ie the light 56 , 305 to capture it. Finally, there is the evaluation device 390 set up to carry out an (possibly three-dimensional) inverse Radon transformation and thus an image of the spatial area 320 to reconstruct from the recorded space sections.

In some developments, the microwave antenna arrangement is 380 set up to controllably generate the microwave radiation in such a way that it is in a selected one of the space sections 324 , 326 disappears and has a finite field strength in the remaining space sections, the microwave radiation having a sigmoidal field curve and / or a fat curve with a gradient in the selected one of the space sections which is so steep there that the microwave radiation disappears only in this selected space section, while the field strength in the remaining space sections is so large that the emission of the emission light is reduced by at least a predetermined value.

In some developments, the microwave antenna arrangement is 380 set up to controllably generate the microwave radiation in such a way that it is in a selected one of the space sections 324 , 326 disappears and has a finite field strength in the remaining space sections, with microwave radiation of different frequencies being superimposed for the disappearance in the selected space section or in the selected space sections in such a way that it leads to interference and, accordingly, a destructive overlay, i.e. in particular extinction in the selected space section (s) Room sections comes. In this advantageous way, large local gradients and thus, in particular, precise disappearance in the selected space sections can be achieved and / or precise control - for example a shift for the space section to be selected along an axis through the space area - can be made possible.

In 5 is a dependence of a fluorescence of a [NV] ^- center on a frequency of a microwave radiation, by means of which according to one embodiment of the invention in particular in combination with the microscope with respect to 3 or with the procedure regarding 6th or 7th The intensity of the emission light is changed in certain - ie in particular selected - spatial sections of a spatial region.

In one embodiment, the fluorescence is in arbitrary units (arb. Units, au) - ie in particular an intensity of the emission light of the [NV] ^- center - on the ordinate of 5 plotted and the frequency of the microwave radiation on the abscissa. Starting from a fluorescence of approx 300 au the fluorescence at a frequency of about 2870 MHz to about 265 au from. At this 2870 MHz, the resonant microwave absorption by the [NV] ^- center is maximum and thus the fluorescence - without an external magnetic field - is reduced to a maximum, which can correspond to a reduction to 88%. Depending on the NV centers used in each case, the frequency of the (maximum) resonant microwave absorption can be different and selected as a predetermined frequency or - for example within a predetermined frequency range - set by a control system, for example as part of the control device.

6th shows a flow diagram of a method 400 for super-resolution microscopy, especially in three dimensions, based on a large number of nanodiamonds, the method 400 in some variants a procedure 430 includes; the proceedings 400 , 430 depending on an embodiment of the invention.

In one embodiment, the method 400 the procedural steps 420 , 460 , 462 , 434 , 468 , 438 , 465 and 490 as well as the procedural condition 410 on. The procedure 400 begins at the start of the procedure 402 and ends at the end of the procedure 404 .

In some variants, a microscope according to the invention, in particular a with respect to 3 described microscope can be used.

In the process step 420 a test object is arranged within a three-dimensional spatial area, the test object having the plurality of nanodiamonds as a plurality of mesoscopic solid-state elements, the nanodiamonds each having a [NV] ^- center.

In the process step 460 the room area is based on a grid like that around in terms of 4th is described, scanned. This is done in process step 460 the procedural steps 462 , 434 , 468 , 438 and 465 executed iteratively until according to the procedural requirements 410 all space sections of the space area or at least a certain selection of these space sections have been scanned.

In the process step 462 a first or, in the case of further iterations, a corresponding further spatial section is selected based on the grid from the spatial area. The grid is selected in such a way that the individual room sections are at a distance from other neighboring room sections that corresponds to a resolution beyond the Abbe limit, or adjoin or overlap one another and that the individual room sections have a spatial extent that at least in one dimension Resolution beyond the Abbe limit. With the grid according to 4th the step angle is selected so small that the distance is even smaller than the Abbe limit even in areas further away from the central point. Correspondingly, the grid is related to 4th For three-dimensional microscopy, the first step angle and the second step angle are chosen to be so small that the distance is even smaller than the Abbe limit even in areas of the spatial area further away from the central point and all lines or surfaces intersect at a common central point. In this advantageous way, the three-dimensional spatial area can be subdivided into spatial sections with super-resolution. In some variants, the control device leads 360 of 3 the procedural step 462 out.

In the process step 434 the respectively selected spatial section is irradiated as part of the spatial area with an excitation light for the [NV] ^- center, i.e. with a light with a wavelength in the green range such as 515 nm. In some variants, the excitation light is generated by means of the light source 340 of 3 generated and possibly by means of optical elements 316 to the room area or to the respectively selected room section.

In the process step 468 a microwave radiation is generated in such a way that it has a microwave course and disappears locally in the selected space section and has a frequency which corresponds to the frequency at which resonant microwave absorption occurs without a local magnetic field at the [NV] ^- center, i.e. around 2870 MHz. In some variants, the control device generates 360 a corresponding signal for the microwave radiation.

In the process step 438 the area of the room is irradiated with this microwave radiation. In some variants, the room area is created by means of the microwave antenna arrangement 380 of 3 , in particular based on a corresponding signal from the control device 360 , irradiated, in particular the microwave antenna arrangement 380 in cooperation with the control device 360 is set up to generate and emit the microwave radiation in such a way that the disappearance of the microwave radiation is limited - at least essentially - to the respectively selected space section, whereby a resolution beyond the Abbe limit can particularly advantageously be achieved.

In the process step 465 a light is optically detected in the respectively selected spatial section at least in a spectral range which corresponds to an emission light emitted by the [NV] ^- center, i.e. in a spectral range between 570 nm and 700 nm, with a sensitivity for the detection of around 637 nm is maximized. In some variants, the light is generated by means of the sensor device 350 of 3 captured by imaging.

With procedural condition 410 it is checked whether a sufficient number of space sections - depending on the desired resolution, for example - or also all space sections of the space area have been scanned, and if this is the case - symbolized by <y> - the method 400 at process step 490 continued. Otherwise - symbolized by <n> - the procedure 400 at process step 462 for a further space segment, that is to say in particular for a space segment that has not yet been scanned.

In the process step 490 a three-dimensional inverse Radon transformation is carried out for the detected light from all scanned space sections and thus an image of the spatial area - i.e. in particular the [NV] ^- centers and the test object colored with these NV centers - super-resolution, ie in particular with a resolution beyond of the Abbe limit, reconstructed.

As shown, the procedure 400 the procedure 430 include, the method 430 the procedural steps 460 , 462 , 434 , 468 , 438 , 465 , 490 and 494 as well as the procedural condition 410 includes. In the additional process step 494 the positions of the nanodiamonds or [NV] ^- centers are determined from the reconstructed image by means of pattern recognition.

In some variations of the procedure 400 is iterated not over the first step angle and the second step angle, but over several focal planes, a first focal plane and then correspondingly further focal planes of the focal planes being selected and a two-dimensional image of the test object for each of the focal planes by scanning accordingly 460 is determined with (only) a step angle at which the spatial sections lie in the respective focal plane, and the three-dimensional image is assembled from these two-dimensional images.

7th shows a flow diagram of a method 430 for two-dimensional determination of a position of an NV center according to an embodiment of the invention.

In one exemplary embodiment, the method corresponds at least substantially and unless otherwise described to the method 430 in terms of 6th according to the procedure 430 at the start of the procedure 402 begins and at the end of the procedure 404 ends. The method can also be used for a large number of NV centers and thus a large number of positions can be determined, for example for super-resolution microscopy.

In one embodiment, the method is based 430 on a change in the intensity of the emission light by means of a magnetic field.

This is done in process step 438 the entire spatial area, which lies in a focal plane, is irradiated with microwave radiation with a suitable frequency and field strength such that the intensity of the emission light of the NV center or several NV centers in different spatial sections of the spatial area decreases.

In the process step 460 the spatial sections, which also lie in the focal plane, are scanned two-dimensionally - that is, for (only) one step angle, the process steps 462 , 466 and 465 be carried out and, if procedural condition 410 is fulfilled, the process then at process step 470 continued.

In the process step 466 a magnetic field - in some variants, for example by means of an anti-Helmholtz coil or an arrangement with several such coils - is generated with a magnetic field curve in such a way that the magnetic field disappears locally in the selected spatial section. Thus, the intensity increases in the remaining room sections, while the intensity in the selected room section - due to the non-detuned frequency for the resonant microwave absorption - remains reduced.

In the process step 465 the spatial area is recorded in an imaging manner, that is to say in particular the light from the spatial area for an area in the spectrum in which the emission light is located is recorded in an imaging manner.

In the process step 470 a reference image is recorded when irradiated with microwave radiation but without a magnetic field - magnetic fields can in particular also be shielded by a magnetic field shielding device - i.e. the (reduced) intensity of the emission light for the NV center or for the NV centers is recorded using an image.

In the process step 472 a change in this intensity - in particular due to a non-disappearance of the magnetic field - is determined based on the reference image.

In the process step 494 the position of the NV center is determined based on the change in intensity, with this in method step 496 the position of the NV center is determined by means of pattern recognition (directly - that is to say in particular without prior reconstruction of an image of the spatial area) based on the change in the intensity in the spatial sections.

While some exemplary embodiments have been described in relation to one or more [NV] ^- centers, those skilled in the art can also adapt these for other NV centers. For example, in some modifications with a so-called "hexagonal lattice site silicon vacancy (V _Si )" as excitation light, a light with a wavelength of at most 861 nm, i.e. an excitation light with a wavelength of 730 nm, is generated with a laser diode and emitted as light the spatial area or from the respectively selected spatial section, in particular the emission light for a wavelength at least in the range between 875 nm and 890 nm and generated as microwave radiation with a frequency in the range of 4.5 MHz.

With the above, the following statements also result and / or are exemplified with the above.

Some of the embodiments advantageously enable three-dimensional super-resolution microscopy. One advantage of super-resolution microscopy based on the large number of mesoscopic solid-state elements can in particular be that the mesoscopic solid-state elements influence a test object such as a biological cell less than other fluorescent substances - for example in combination with the high required light doses -, which improves microscopy and / or can be simplified and / or made more reliable. Another advantage can be that the fluorescence of the NV centers of the mesoscopic solid-state elements can be changed over all three spatial dimensions by means of the microwave radiation and / or by means of the magnetic field - with corresponding field profiles in three dimensions, whereby, in particular, each of the spatial sections can be specifically addressed - i.e. in particular scanned - whereby three-dimensional imaging - i.e. in particular a three-dimensional reconstruction of the test object or its image or the mesoscopic solid-state elements arranged on the test object or exhibited by the test object - can be achieved.

For three-dimensional super-resolution microscopy, in some embodiments, the spatial area extends in three dimensions, the spatial area with a resolution beyond an Abbe limit for an emission light that emanates from the NV center or from the NV centers upon optical excitation by the Excitation light can be emitted, is scanned.

In some embodiments in which the spatial area extends in three dimensions, the spatial sections are each lines or areas through the spatial area, the light being detected at least one-dimensionally resolved along the respective line or for the respective area.

In some embodiments for three-dimensional, in particular super-resolution, microscopy, in which the spatial sections are each lines or surfaces, the lines or surfaces have a common central point and are each rotated relative to the spatial area by a certain first step angle and a certain second step angle . Furthermore, the image is determined as a three-dimensional, in particular super-resolution, image by means of a three-dimensional, inverse Radon transformation. In some variants, the light is also recorded at least two-dimensionally resolved over the respective surface or two-dimensionally resolved over a (common) projection plane - for example an image plane - for the surfaces.

In some embodiments for three-dimensional, in particular super-resolution, microscopy, in which the spatial segments are each lines, the spatial area is divided into several focal planes of a microscope for capturing the light in the spatial segments. The focal planes are each offset by a certain increment relative to the spatial area. In addition, for each focal plane of the focal planes, a subset of the lines lies in the respective focal plane, the respective lines each having a respective common central point and each being rotated relative to the spatial area by a certain step angle in the respective focal plane around the respective central point. In addition, the microscope is set up to detect the light resolved at least two-dimensionally for each focal plane and for the respective lines lying in the respective focal plane. In addition, a two-dimensional image for a part of the test object in the respective focal plane is determined for each focal plane by means of an inverse Radon transformation and the image of the test object is determined as a three-dimensional image based on the two-dimensional images.

In some embodiments in which the spatial area extends in three dimensions, the spatial area is in the form of a cuboid or envelops a cuboid, the cuboid having a base area of at least 1 μm by 1 μm, at least 3 μm by 10 μm, at least 20 μm 100 µm or at least 200 µm by 200 µm and a height of at least 1 µm, at least 10 µm or at least 150 µm. In some variants of this, in which the spatial area is subdivided into several focal planes, the focal planes each extend over the base area and are arranged along the height - in particular depending on the desired resolution. For example, a cuboid spatial area with a base area of 3 µm by 4 µm and a height of 10 µm can be divided into 200 focal planes, each with an area of at least 3 µm by 4 µm, which in the direction of the height of the cuboid each have a distance of 50 nm to each other.

In some embodiments, in particular for super-resolution microscopy or to determine a position of an NV center with a high resolution, in particular a super-resolution, the spatial area is provided with a resolution beyond an Abbe limit for the emission light - i. H. especially super resolution - scanned. Correspondingly, in some such embodiments, a respective spatial extent of the disappearance of the magnetic field or of the microwave radiation is limited at least in one dimension to an extent which is smaller than the Abbe limit. In this advantageous way, a super-resolution resolution, that is to say in particular imaging with a resolution beyond the Abbe limit, can be achieved or a position of the NV center can accordingly be determined with an accuracy beyond the Abbe limit.

In some embodiments, in particular for two-dimensional microscopy or for two-dimensional determination of a position of a NV center, the spatial area is a focal plane of a microscope for capturing the light in the spatial area and in particular the light of the spatial sections of the spatial area. In some variants, the spatial sections are each lines in the focal plane, the microscope being set up to detect the light resolved at least one-dimensionally along the respective line or two-dimensionally in the focal plane.

In some embodiments in which the space segments are lines, the lines have a common central point and are each rotated by a certain step angle relative to the space region or to a preceding / adjacent line. In addition, for imaging, for example for two-dimensional microscopy, in some such embodiments the image is determined as a two-dimensional image of the test object by means of an inverse Radon transformation. For a determination of a position of an NV center, in some such embodiments, the position of the NV center or NV centers is used by means of an inverse Radon transformation and - in some variants thereof - by means of one based on a result of the inverse Radon transformation Pattern recognition determined. Alternatively, in some such embodiments, a pattern recognition is carried out (directly) based on the detected light in the respective space sections for the determination of the position.

In some embodiments, the solid-state mesoscopic element comprises or consists of a nanodiamond, the nanodiamond having a nitrogen vacancy center as the NV center. Correspondingly, in some embodiments the mesoscopic solid-state elements of the plurality of mesoscopic solid-state elements each comprise or consist of a nanodiamond, the respective nanodiamond each having a nitrogen vacancy center as the respective NV center.

In some embodiments, a change in the light that is dependent on the magnetic field or the microwave radiation is determined in the respective space sections - that is to say in particular the respectively selected space section. In this case, the image is reconstructed based on the respective change in the detected light or the position of the NV center or the positions of the NV centers are determined.

In some embodiments, in order to determine the change in the detected light, a reference image of the light in the spatial region is recorded without microwave radiation or without a magnetic field, and the respective change with respect to this reference image is determined.

In some embodiments, the light in the respective spatial section is recorded using an image - for example two-dimensional or three-dimensional.

In some embodiments, only that section of space is irradiated with the excitation light from which the light is detected.

In some embodiments, the microwave radiation has a gradient over the spatial area as the microwave field curve, so that the microwave radiation in all spatial sections, except when scanning the respective spatial section, differs from at least essentially zero in such a way that the microwave radiation stimulates the resonant microwave absorption and an intensity of the emission light of the NV center or the NV centers reduced. For example, such a gradient can be used to generate a microwave field course which disappears along a straight line - for example in a focal plane of a microscope - and / or such a gradient can be rotated two-dimensionally or three-dimensionally within the spatial region.

In some embodiments, the magnetic field has a gradient over the spatial area as the magnetic field profile, so that the magnetic field in all spatial sections, except when scanning the respective spatial section, differs from at least essentially zero in such a way that the magnetic field has the frequency at which the resonant microwave absorption in NV -Zentrum or occurs at the NV centers, shifts in relation to the frequency of the microwave radiation and accordingly reduces an excitation of the resonant microwave absorption. Correspondingly, magnetic field profiles can be generated in this advantageous manner so that the magnetic field disappears along a straight line.

In some embodiments, the magnetic field is generated with an anti-Helmholtz coil, the disappearance of the magnetic field in the respective space section being controlled by means of electrical currents through at least one coil and a further coil of the anti-Helmholtz coil.

In some embodiments, the test object is or has a biological cell - for example a biological tissue with cells - wherein a large number of mesoscopic solid-state elements are applied or introduced into the biological cell or on the biological cell - that is, the biological cell is stained, for example becomes.

In some embodiments, the mesoscopic solid-state element has a functionalized surface.

In some embodiments, the light, that is to say in particular the emission light, is averaged over a predetermined period of time. In this advantageous way - also in the case of Rabi oscillations or other rapid fluctuations in fluorescence - the evaluation, that is to say in particular the reconstruction of the image, can be simplified and / or made more resource-efficient.

, wobei Ω die Rabi Frequenz, $\overrightarrow{x}$

der Ort und y die Rate ist, mit welcher das Anregungslicht die Spinpolarisation bewirkt.In some embodiments, the light, that is to say in particular the emission light, is recorded with a time resolution which enables a resolution of Rabi oscillations which are caused by the microwave radiation. For example, the fluorescence signal can be proportional to $$\frac{{\left[\Omega \left(\overrightarrow{x}\right)\right]}^2/\mathrm{4th}}{\frac{{\left[\Omega \left(\overrightarrow{x}\right)\right]}^2}{2}+\gamma }$$

, where Ω is the Rabi frequency, $\overrightarrow{x}$

is the location and y is the rate at which the excitation light causes spin polarization.

The resolution can be increased further in this advantageous way.

In order to further increase the resolution, in some embodiments a pulsed excitation light, a pulsed microwave radiation and / or a pulsed magnetic field is generated.

In order to further increase the resolution and / or to make the implementation of the method faster and / or to enable a larger spatial area to be captured, in some embodiments the spatial area is represented by means of a complex microwave field or a complex magnetic field, which in particular has several spatial sections at the same time which the respective field disappears, scanned.

While exemplary embodiments, possible applications and application examples have been described in detail, in particular with reference to the figures, it should be pointed out that a large number of modifications are possible. It should also be pointed out that the exemplary designs and applications are merely examples that are not intended to limit the scope of protection, the application and the structure in any way. Rather, the preceding description provides the person skilled in the art with guidelines for the implementation and / or application of at least one exemplary embodiment, with various modifications, in particular alternative or additional features and / or modifications of the function and / or arrangements of the described components, as desired by the person skilled in the art can be made without deviating from the subject matter specified in the attached claims and its legal equivalents and / or leaving their scope of protection.

QUOTES INCLUDED IN THE DESCRIPTION

This list of the documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent literature cited

• US 8193808 B2 [0005]

Non-patent literature cited

• Doherty, Marcus W .; Manson, Neil B .; Delaney, Paul; Jeletsko, Fedor; Wrachtrup, Jörg; Hollenberg, Lloyd C. L. (2013-07-01). "The nitrogen-vacancy color center in diamond". Physics Reports. The nitrogen-vacancy color center in diamond. 528 (1): 1-45) [0012]
• Rogers, L. J .; Armstrong, S .; Sellars, M. J .; Manson, N.B. (2008). "Infrared emission of the NV center in diamond: Zeeman and uniaxial stress studies". New Journal of Physics. 10 (10): 103024 [0031]
• Doherty, Marcus W .; Manson, Neil B .; Delaney, Paul; Jeletsko, Fedor; Wrachtrup, Jörg; Hollenberg, Lloyd C. L. (2013-07-01). "The nitrogen-vacancy color center in diamond". Physics Reports. The nitrogen-vacancy color center in diamond. 528 (1): 1-45 [0031]

Claims (19)

Method (400) for super-resolution microscopy of a test object (200), wherein the test object has a plurality of mesoscopic solid-state elements (240), each of which has at least one NV center (140), and is arranged within a spatial region (320), wherein the method (400) comprises: - (434) irradiating the spatial region (320) with an excitation light (46) for the NV centers (140) of the plurality of mesoscopic solid-state elements (240); - (438) irradiating the spatial region with microwave radiation (48), the frequency of which corresponds to that frequency at which resonant microwave absorption occurs without a local magnetic field at the NV centers (140); - (460) Scanning of space sections (324, 326) of the space area (320) with a resolution beyond an Abbe limit for an emission light (56) which can be emitted by the NV centers (140), with each space section of the space sections: - A magnetic field over the spatial area is generated (466) with a magnetic field profile, so that the magnetic field (80) locally disappears in the respective spatial section (324) and a respective spatial extent of the disappearance of the magnetic field in the respective spatial section is limited to at least one dimension Expansion that is smaller than the Abbe limit, or the microwave radiation (48) for irradiation is generated (468) in such a way that it has a microwave field course, disappears locally in the respective spatial section (324) and a respective spatial expansion of the disappearance of the microwave radiation (48 ) in the case of the respective space section (324), at least one dimension is limited to an extent which is smaller than the Abbe limit; and - (465) a light (56, 305) in the respective spatial section (324) is optically detected at least in a spectral range which corresponds to the emission light (56); - (490) Reconstructing an image of the test object (200) based on the space sections (324, 326) and the light (56, 305) detected in the respective space sections (324, 326). Method (400) for three-dimensional super-resolution microscopy according to Claim 1 , wherein the spatial area (320) extends in three dimensions and the spatial sections are each lines (324, 326) or areas through the spatial area (320), the light (56,305) resolved at least one-dimensionally along the respective line or for the respective Area is recorded (465). Method (400) according to Claim 2 wherein: the lines or areas have a common central point (328) and are each rotated relative to the spatial region (320) by a specific first step angle and a specific second step angle; and (490) the image is determined as a three-dimensional image by means of a three-dimensional, inverse Radon transformation. Method (400) according to Claim 2 wherein: the spatial region (320) is divided into a plurality of focal planes of a microscope (300) for detecting the light in the spatial sections; the focal planes are each offset by a certain increment relative to the spatial area; for each focal plane of the focal planes a subset of the lines (324, 326) lies in the respective focal plane and the respective lines each have a respective common central point (328) and in each case relative to the spatial area (320) by a certain step angle in the respective focal plane around the respective central point (328) are rotated; the microscope (300) is set up to detect the light resolved at least two-dimensionally for each focal plane and for the respective lines (324, 326) lying in the respective focal plane; and for each focal plane a two-dimensional image for a part of the test object (200) in the respective focal plane is determined by means of an inverse Radon transformation and the image of the test object (200) is determined (490) as a three-dimensional image based on the two-dimensional images. Method (430) for determining a position of an NV center (140) of a mesoscopic solid-state element (240) within a spatial region (320), the method comprising: - (434) irradiating the spatial area with an excitation light (46) for the NV center; - (438) irradiating the spatial region with microwave radiation (48), the frequency of which corresponds to that frequency at which resonant microwave absorption occurs without a local magnetic field at the NV center; - (460) Scanning of space sections (324, 326) of the space area (320), with each space section of the space sections: - A magnetic field is generated over the spatial area (466) with a magnetic field curve, so that the magnetic field (80) disappears locally in the respective spatial section (424), or the microwave radiation (48) for irradiation is generated (468) in such a way that it has a microwave field curve and disappears locally in the respective space section (324); and - (465) a light (56, 305) in the respective spatial section (324) is optically detected at least in a spectral range which corresponds to an emission light (56) emitted by the NV center (140); - (494) determining the position of the NV center (140) based on the detected light (305) in the respective space sections (324, 326). Method (430) according to Claim 5 , whereby the spatial area is scanned with a resolution beyond an Abbe limit for the emission light and a respective spatial extent of the disappearance of the magnetic field or the microwave radiation in the respective spatial section is limited at least in one dimension to an extent which is smaller than the Abbe limit is. Method (400, 430) according to Claim 1 , 5 or 6th wherein the spatial area is a focal plane of a microscope (300) for capturing the light in the spatial sections and the spatial sections are each lines (324, 326) in the focal plane, the microscope being set up to resolve the light at least one-dimensionally along the respective line or two-dimensionally to capture in the focal plane. Method (400, 430) according to Claim 7 wherein: the lines have a common central point (328) and are each rotated relative to the spatial region (320) by a certain step angle; and (490) the image is determined as a two-dimensional image of the test object (200) by means of an inverse Radon transformation or (494) the position of the NV center (140) is determined by means of an inverse Radon transformation and pattern recognition (496) . The method (400, 430) according to any one of the preceding claims, wherein: the mesoscopic solid-state element (240) comprises or consists of a nanodiamond or the plurality of mesoscopic solid-state elements (240) each comprise or each consist of a nanodiamond; and the respective nanodiamond in each case has a nitrogen vacancy center as the respective NV center (140). The method (400, 430) according to one of the preceding claims, further comprising: - (472) determining a change in the light in the respective space sections (324) which is dependent on the magnetic field or the microwave radiation; wherein the image is reconstructed (490) or the position of the NV center is determined (494) based on the respective change in the detected light. Method (400, 430) according to Claim 10 , wherein a reference image of the light in the spatial region without microwave radiation or without a magnetic field is recorded (470) and the respective change with respect to this reference image is determined. The method (400, 430) according to any one of the preceding claims, wherein the light in the respective space section is captured by imaging. Method (400, 430) according to one of the preceding claims, wherein in each case only that space section (324) is irradiated with the excitation light from which the light is detected. The method (400, 430) according to any one of the preceding claims, wherein: the microwave radiation as the microwave field profile has a gradient over the spatial area (320), so that the microwave radiation in all spatial sections (326), except when scanning the respective spatial section (324), differs from at least essentially zero in such a way that the microwave radiation excites the resonant microwave absorption and an intensity of the emission light of the NV center (140) or the NV centers (140) is reduced; or the magnetic field as the magnetic field profile has a gradient over the spatial area (320), so that the magnetic field in all spatial segments (326) except when scanning the respective spatial segment (324) differs from at least essentially zero in such a way that the magnetic field has that frequency at which the resonant microwave absorption occurs at the NV center (140) or at the NV centers (140), shifts it with respect to the frequency of the microwave radiation and accordingly reduces an excitation of the resonant microwave absorption. Method (400, 430) according to one of the preceding claims, wherein the magnetic field is generated with an anti-Helmholtz coil and the disappearance of the magnetic field in the respective space section (324) by means of electrical currents through at least one coil and another coil of the anti-Helmholtz Coil is controlled. Method (400) for microscopy based on a multiplicity of mesoscopic solid-state elements (240), each of the solid-state elements (240) each having at least one NV center (140), the method comprising: - (420) arranging a test object (200) within a spatial region (320), the test object having the plurality of mesoscopic solid-state elements (240); and - Carrying out the method (400, 430) according to one of the preceding claims, the respective NV centers (140) of the mesoscopic solid-state elements (240) being distinguished from one another by means of the scanning (460) of the spatial sections and thus resolved by imaging. Method (400) according to Claim 16 , wherein the test object (200) has a biological cell (222, 224) and the method further comprises: - (424) applying to and / or introducing the plurality of mesoscopic solid-state elements (240) into the biological cell. A microscope (300) comprising: a space area (320) for the arrangement of a test object (200); a light source (340) for irradiating the spatial area with an excitation light (46), by means of which, when a test object (200) with a plurality of NV centers (140) each formed in a mesoscopic solid-state element (240) is in the spatial area (320) is arranged, one or more of the NV centers (140) of the plurality of NV centers are optically excitable; a microwave antenna arrangement (380) for irradiating the spatial region (320) with microwave radiation (48), the frequency of which corresponds to that frequency at which resonant microwave absorption of the NV centers (140) occurs without a local magnetic field (80); a control device (360) which is set up to scan space sections (324, 326) of the space area (320) and select a space section (324) of the space sections and to control a magnetic field device for the respectively selected space section in such a way that it has a magnetic field a magnetic field profile is generated over the spatial area (324), through which the magnetic field disappears locally in the respectively selected spatial section, or to control the microwave antenna arrangement (380) such that it irradiates the spatial area (324) with the microwave radiation in such a way that the microwave radiation has a microwave field profile and disappears locally in the respectively selected space section (324); a sensor device (350) which is set up to detect an emission light (56) emitted by the NV centers at least in the spatial section (324) selected during the scanning; and an evaluation device (390) which is set up to determine a two- or three-dimensional image of the test object (200) based on the emission light which is emitted in the respective spatial sections (324, 326) by the NV centers. Device (300) for determining a position of a NV center (140) of a mesoscopic solid-state element (240) within a spatial region, in particular a microscope (300) according to FIG Claim 18 , wherein the device is set up, a method (400, 430) according to one of Claims 1 to 17th execute.

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