DE102019120960A1 - Method for calibrating a microscope, microscope arrangement and use - Google Patents

Abstract

The invention relates to a method for calibrating a microscope, wherein a sample in the form of a solid body with at least one optical center is used for calibration. For example, calibrations with regard to a collection efficiency, to a distance, to a polarization or to a vector electric field of an excitation light beam can thus be carried out. The sample used has proven to be stable and easy to handle.

Description

The invention relates to a method for calibrating a microscope, an associated microscope arrangement and an associated use.

Microscopes, particularly high resolution microscopes, are used for a variety of applications. For example, they can be used for biological, microbiological and material science issues. By means of various super-resolution techniques, resolutions can now be achieved which are considerably better than the resolution that is limited due to diffraction.

In order to be able to correctly interpret results obtained using a microscope, it is advantageous if a reference is available for various properties of a microscope. Known references, for example for a measure of length, are based on the fact that fluorophores are deliberately introduced into DNA molecules. However, such samples have proven to be less durable and are more suitable for sizes of several hundred nanometers.

It is therefore an object of the invention to provide a method for calibrating a microscope which, compared to known solutions, is implemented as an alternative or better. It is further objects of the invention to provide an associated microscope arrangement and an associated use.

This is achieved according to the invention by a method, a microscope arrangement and a use according to the respective main claims. Advantageous configurations can be found in the respective subclaims, for example. The content of the claims is made part of the description by express reference.

The invention relates to a method for calibrating a microscope.

• - einen Probenhalter zur Aufnahme einer Probe,
• - eine Anregungsoptik zum Richten eines Anregungslichtstrahls auf die Probe,
• - eine Abfrageoptik zum Aufnehmen von durch die Probe ausgesendetem Licht,
• - einen Detektor zum Erfassen von durch die Abfrageoptik aufgenommenem Licht.

The microscope has the following:

• - a sample holder for holding a sample,
• - excitation optics for directing an excitation light beam onto the sample,
• - an interrogation optics for receiving light emitted by the sample,
• a detector for detecting light picked up by the interrogation optics.

• - Einsetzen eines Festkörpers als Probe in den Probenhalter, wobei der Festkörper zumindest ein erstes optisches Zentrum aufweist, welches nach Anregung Licht aussendet,
• - Richten eines Anregungslichtstrahls auf die Probe, so dass das erste optische Zentrum angeregt wird,
• - Erfassen des von der Probe ausgesendeten Lichts mittels der Abfrageoptik und des Detektors,
• - Kalibrieren des Mikroskops basierend auf dem vom Detektor erfassten Licht.

The procedure consists of the following steps:

• - Insertion of a solid body as a sample in the sample holder, the solid body having at least a first optical center which emits light after excitation,
• - Directing an excitation light beam onto the sample so that the first optical center is excited,
• - Detection of the light emitted by the sample by means of the interrogation optics and the detector,
• - Calibrate the microscope based on the light captured by the detector.

In the method according to the invention, a solid body with an optical center is used. It has been shown that such solid bodies with an optical center are considerably more resistant and have a longer service life than samples known from the prior art. Accordingly, a calibration can be carried out much more reliably and different comparisons can be made, for example between different times and / or between different microscopes. In addition, comparisons can be made between different microscope parts such as objectives or different evaluation methods or algorithms. Exemplary statements are described in more detail below.

The method is particularly suitable for use with a microscope as indicated.

• - Einsetzen eines Festkörpers als Probe in einen Probenhalter eines Mikroskops, wobei der Festkörper zumindest ein erstes optisches Zentrum aufweist, welches nach Anregung Licht aussendet,
• - Richten eines Anregungslichtstrahls mittels einer Anregungsoptik des Mikroskops auf die Probe, so dass das erste optische Zentrum angeregt wird,
• - Erfassen des von der Probe ausgesendeten Lichts mittels einer Abfrageoptik des Mikroskops und eines Detektors des Mikroskops,
• - Kalibrieren des Mikroskops basierend auf dem vom Detektor erfassten Licht.

An alternative formulation of the procedure can be as follows:

• - Insertion of a solid body as a sample in a sample holder of a microscope, wherein the solid body has at least one first optical center which emits light after excitation,
• - Directing an excitation light beam by means of an excitation optics of the microscope onto the sample, so that the first optical center is excited,
• - Detection of the light emitted by the sample by means of interrogation optics of the microscope and a detector of the microscope,
• - Calibrate the microscope based on the light captured by the detector.

The solid body can be a continuous or one-piece solid body. However, it can also be a composite solid, for example. For example, several NanoDiamonds can be put together to form a solid. Particular advantages result in the case of a crystalline, in particular monocrystalline solid, but it is also possible, for example, to use polycrystalline, glass-like or composite solid bodies. Such solids can be produced, for example, by spin coating, for example by spin coating nanodiamonds in a polymer such as polymethyl methacrylate (PMMA). Nanodiamonds can also be unassembled in fixed positions in a carrier material. Crystals have particular advantages in terms of fixed geometry and known orientations based on surfaces. However, terms such as lattice or lattice space can also be reasonably used in amorphous materials.

It should also be understood that the emission of light after excitation can be understood to mean, for example, an emission, the optical center being excited in a known manner by means of an excitation light, that is to say changes into a more energetic quantum mechanical state. From here it can return to a ground state via one or more decay paths and emit a photon in the process. However, the emission of light can also be a matter of scattering, which is understood here as emission after excitation. In other words, an excitation light beam is directed onto the optical center and the light is scattered so that it can be detected at another location. This is also understood here as the emission of light after being stimulated.

The sample holder can, for example, be designed to hold the sample in a defined and / or reproducible position. This can be monitored, for example, by means of the method, which will be discussed in more detail below.

The excitation optics can, for example, be designed to direct a laser beam onto the sample. For this purpose, for example, a laser can be provided as part of the excitation optics. This can be manipulated by optical components such as mirrors, lenses, beam splitters and other components as required for the respective purpose. For example, the excitation light beam can be focused on the sample and / or on the optical center. However, a non-focused light beam can also be used.

The detector can be, for example, an analog detector such as a photo plate. It can also be a digital detector. For example, a CCD detector, a camera, in particular a sensitive camera, a CCD camera, or a CMOS camera or a film, in particular a photographic film or an optical film, can be used. The detector can, for example, be designed in such a way that it can detect at only one location, for example with a photodiode, avalanche photodiode (APD), or a photomultiplier. However, it can also be designed to be movable, for example, so that a larger area can be imaged by means of a detector that basically only detects at one location. It can also be provided that the detector is formed from a plurality of individual detectors at different locations. This can also cover a larger area. Alternatively, the sample, the beam and / or an objective can also be moved relative to the detector, for example.

According to one embodiment, the sample has at least one second optical center. This enables further functionalities relating to the calibration of the microscope, which will be discussed in greater detail below.

The first optical center and the second optical center can, for example, have a spacing of at least one grid position or of at least 1 nm or of at least 3 nm from one another. Such distances as a minimum distance have proven to be advantageous for typical applications and can be measured as well as used for the purpose of a reference.

The first optical center and the second optical center are preferably at a distance of at most 100 nm or at most 50 nm or at most 20 nm from one another. Such distances as the upper limit have proven particularly advantageous because up to such distances in typical constellations, coupling of the optical centers is possible, which can be used for different functionalities such as independent distance determination or emitter-selective control.

According to the respective embodiments, spin-active optical centers, magnetically couplable optical centers or otherwise couplable optical centers can be used as optical centers. This can be used in particular to couple these when at least two optical centers are present and thus to be able to fall back on quantum mechanical effects, for example to determine their distance or to address them selectively. Even in the case of a single optical center, such an embodiment, for example by using a spin-active optical center, can for example achieve an optical transition dipole moment of the optical center, which can be used for calibration tasks. This will continue detailed below. If the optical center is embedded in a crystal, there are particular advantages with regard to fixed lattice spacing, fixed crystal axes, fixed geometry and crystal axes that can be measured on the crystal surfaces.

Diamond or silicon carbide (SiC) in particular can be used as the solid. Such solid bodies have proven to be particularly durable and suitable for the purposes pursued here. It should be understood, however, that other solid bodies can also be used.

For example, nitrogen vacancies can be used as optical centers. In particular, nitrogen vacancies in diamond can be used. These have proven to be advantageous for typical calibration tasks, such as those which are described in more detail below, and are otherwise well known with regard to their physical behavior. It should be mentioned, however, that other types of optical centers, for example other types of defects or other designs of optical centers, can also be used.

The excitation light beam can for example have a wavelength corresponding to an excitation maximum of the optical centers. The wavelength can, for example, be in the optical spectrum, in the UV spectrum or in the IR spectrum. A particularly effective excitation can thereby be achieved. However, it is also possible to use an excitation light with a broad spectrum or a continuous spectrum or with a wavelength which lies outside an excitation maximum of the optical centers, in which case, for example, the characteristics of the excitation optics or other behavior of the optical centers can be taken into account .

The microscope can be, for example, a confocal microscope, a near-field microscope, a scattering microscope or a wide-field microscope. The method described herein has proven to be particularly advantageous for such types of microscopes. All other types of microscopes or any other optical device that meets the requirements described at the outset can also be used to carry out the method.

According to one embodiment, the calibration is a calibration of a distance. The sample preferably has at least one second optical center. This second optical center can, for example, have a distance from the first optical center which lies within the lower and / or upper limits already indicated above as advantageous.

• P. Neumann, R. Kolesov, B. Naydenov, J. Beck, F. Rempp, M. Steiner, V. Jacques, G. Balasubramanian, M. L. Markham, D. J. Twitchen, S. Pezzagna, J. Meijer, J. Twamley, F. Jelezko, J. Wrachtrup, „Quantum register based on coupled electron spins in a room-temperature solid“, Nature Physics, vol. 6, no. 4, pp. 249-253, Apr. 2010 . [Online]; http://www.nature.com/articles/nphys1536
• H. S. Knowles, D. M. Kara, M. Atatüre, „Demonstration of a coherent electronic spin cluster in diamond“, Physical Review Letters, vol. 117, no. 10, Sep. 2016 . [Online]; https-//doi.org/10.1103/physrevlett.117.100802
• F. Dolde, M. W. Doherty, J. Michl, I. Jakobi, B. Naydenov, S. Pezzagna, J. Meijer, P. Neumann, F. Jelezko, N. B. Manson, J. Wrachtrup, „Nanoscale Detection of a Single Fundamental Charge in Ambient Conditions Using the NV Center in Diamond“, Physical Review Letters, vol. 112, no. 9, p. 097603, März 2014 . [Online]; https://link.aps.org/doi/10.1103/PhysRevLett. 1 12.097603

The method can preferably include a step of determining a distance between the first optical center and the second optical center by means of an electromagnetic coupling measurement. Such an electromagnetic coupling measurement is known in principle, for example from the following publications:

• P. Neumann, R. Kolesov, B. . Naydenov, J. Beck, F. Rempp, M. Steiner, V. Jacques, G. Balasubramanian, ML Markham, DJ Twitchen, S. Pezzagna, J. Meijer, J. Twamley, F. Jelezko, J. Wrachtrup, “Quantum register based on coupled electron spins in a room-temperature solid,” Nature Physics, vol. 6, no. 4, pp. 249-253, Apr. 2010 . [On-line]; http://www.nature.com/articles/nphys1536
• HS Knowles, DM Kara, M. Atatüre, “Demonstration of a coherent electronic spin cluster in diamond”, Physical Review Letters, vol. 117, no.10, Sep. 2016 . [On-line]; https - // doi.org/10.1103/physrevlett.117.100802
• F. Dolde, MW Doherty, J. Michl, I. Jakobi, B. Naydenov, S. Pezzagna, J. Meijer, P. Neumann, F. Jelezko, NB Manson, J. Wrachtrup, “Nanoscale Detection of a Single Fundamental Charge in Ambient Conditions Using the NV Center in Diamond, "Physical Review Letters, vol. 112, no.9, p. 097603, March 2014 . [On-line]; https://link.aps.org/doi/10.1103/PhysRevLett. 1 12.097603

• - Anlegen eines Magnetfelds an die Probe,
• - Anregen der beiden optischen Zentren mittels eines Anregungslichtstrahls,
• - Anlegen eines ersten elektromagnetischen Wechselfelds bevorzugt im Mikrowellenspektrum, wobei beobachtet wird, dass jedes der beiden optischen Zentren bei beispielsweise jeweils zwei Frequenzen des ersten elektromagnetischen Wechselfelds das magnetische Moment ändert, was sich beispielsweise bei einer Stickstofffehlstelle in Diamant dadurch äußern kann, dass es dunkel wird,
• - Anlegen des ersten elektromagnetischen Wechselfelds mit einer dieser Frequenzen, so dass eines der beiden optischen Zentren dunkel wird, wobei das erste elektromagnetische Wechselfeld ein zeitlich begrenzter Puls ist, so dass das magnetische Moment des ersten optischen Zentrums in einer Überlagerung des hellen und dunklen Zustandes endet und eine Präzession um das angelegte Magnetfeld startet (Larmor-Präzession),
• - Anlegen eines zweiten elektromagnetischen Wechselfeldes mit einer Frequenz, so dass das andere optische Zentrum dunkel wird, wobei das elektromagnetische Wechselfeld ein zeitlich begrenzter Puls ist, so dass das magnetische Moment des zweiten optischen Zentrums verändert wird, was im Falle einer Wechselwirkung der beiden optischen Zentren zu einer Änderung der Präzessionsfrequenz des ersten optischen Zentrum führt,
• - Anlegen des ersten elektromagnetischen Wechselfelds mit derselben Frequenz und Pulsdauer, so dass das erste optische Zentrum die Präzession beendet und entsprechend der Phase/des Fortschritts in der Präzession entweder dunkel oder hell wird,
• - Variieren der Zeitpunkte, zu denen die elektromagnetischen Pulse angelegt werden, so dass der Verlauf der Präzession und somit die Änderung der Präzessionsfrequenz beobachtet werden kann,
• - Rückrechnen aus der Änderung der Präzessionsfrequenz auf den Abstand zwischen den beiden optischen Zentren.

Roughly summarized, such a coupling measurement or distance measurement can take place, for example, as follows:

• - applying a magnetic field to the sample,
• - excitation of the two optical centers by means of an excitation light beam,
• - Applying a first alternating electromagnetic field, preferably in the microwave spectrum, whereby it is observed that each of the two optical centers changes the magnetic moment at, for example, two frequencies of the first alternating electromagnetic field, which can be expressed, for example, in the case of a nitrogen vacancy in diamond, in that it becomes dark ,
• - Applying the first alternating electromagnetic field with one of these frequencies so that one of the two optical centers becomes dark, the first alternating electromagnetic field being a time-limited pulse, so that the magnetic moment of the first optical center ends in a superposition of the light and dark state and a precession starts around the applied magnetic field (Larmor precession),
• - Applying a second electromagnetic alternating field with a frequency so that the other optical center becomes dark, the electromagnetic alternating field being a time-limited pulse so that the magnetic moment of the second optical center is changed, which occurs in the event of an interaction between the two optical centers leads to a change in the precession frequency of the first optical center,
• - Applying the first alternating electromagnetic field with the same frequency and pulse duration, so that the first optical center ends the precession and becomes either dark or light according to the phase / progress in the precession,
• - Varying the times at which the electromagnetic pulses are applied, so that the course of the precession and thus the change in the precession frequency can be observed,
• - Calculating back from the change in the precession frequency to the distance between the two optical centers.

The procedure can also be referred to as a Ramsey sequence and can easily be extended to an extended pulse sequence, for example as a Hahn echo sequence.

It has been shown that the distance between two optical centers can be determined very precisely by means of such an electromagnetic coupling measurement, so that a resolution with regard to individual grid positions is possible. A timing of the pulses can determine when the respective field is present and when not.

A timing of pulses that is reduced to a standard measure is preferably used in the coupling measurement. This can be the aforementioned first and / or second alternating electromagnetic fields, for example. A return to a standard measure means that the timing or the frequency of this electromagnetic wave is calibrated to the SI unit second. For this purpose, an atomic clock or another correspondingly precise clock, for example from a satellite navigation signal, can be used. This can in particular be calibrated to the corresponding SI standard dimension. For example, such an atomic clock can be made available by a standardization organization.

By tracing the timing in a coupling measurement back to a standard dimension, a sample can be obtained in which two optical centers are present at a distance which is calibrated to a standard dimension or to an SI standard dimension. A sample is thus provided which can serve as a reference in the nanometer range and is SI-calibrated.

It should be understood that the tracing back to a standard dimension or the provision of a correspondingly calibrated sample, such a sample per se and an associated method as well as the use of such a sample for the purposes mentioned herein can represent a respective independent aspect of the invention.

A distance determined by means of a detector is preferably calibrated. This can mean, for example, that a distance is determined by means of the detector, which distance is specified, for example, in pixels of the detector, in a distance traveled by the detector or another unit of measurement used by the detector. For example, lens or beam scanners or wide-field cameras can also be used. The distance between the two optical centers in the sample is typically known, for example by means of the coupling measurement already described above. This makes it possible to calibrate a dimension or coordinate system used in the detector to the actual distance. This allows a very precise calibration of the detector, the microscopic structure and / or the evaluation method used, so that one can be sure to measure exact distances, especially in the nanometer range.

The distance can be determined for example by means of emitter-selective activation of the optical centers and / or by means of super resolution methods, correlation evaluation or cross-correlation evaluation of signals recorded by the detector based on the detected light.

This can mean, for example, that, as already described above, by applying an electromagnetic wave from the microwave spectrum, the optical centers, which can be referred to as emitters, are selectively activated or deactivated and corresponding light is detected by the detector. Signals generated based on this can be evaluated, for example by fitting a respective Gaussian function to a signal and using the maximum of the Gaussian function as the position of the respective optical center in the coordinate system of the detector. A cross-correlation evaluation can also be carried out accordingly. Corresponding techniques are known, for example, as super resolution methods or super resolution microscopy, which generally enable a resolution which is better by considering certain areas in the microscopic structure, for example in the detector or detector system is than the resolution of the microscope, which is limited due to the diffraction limit.

According to one embodiment, a plurality of distances between the first optical center and the second optical center are determined vectorially at different times. This can mean in particular that not only a length, but also the orientation of the distance is determined in a three-dimensional coordinate system. A rotation, for example, can then be determined from the vectorial distances.

According to one embodiment, a plurality of distances between the first optical center and the second optical center are determined at different times in each case by means of the light detected by the detector. The distance can be determined in three dimensions or in two dimensions, in particular transversely to the direction of propagation of the excitation light beam and / or an optical axis of the microscope. This can mean in particular that not only a length, but also the orientation of the distance is determined in a three-dimensional coordinate system or as a projection in a two-dimensional coordinate system. A rotation of the sample can then be determined, for example, from the three-dimensional vectorial distances or from the two-dimensional projections of the distances. With knowledge of the three-dimensional vectorial distance from the magnetic distance determination, for example as described above, a more precise statement about this rotation can be determined, for example.

• Travis J. Gould, Daniel Burke, Joerg Bewersdorf, Martin J. Booth, „Adaptive optics enables 3D STED microscopy in aberrating specimens“, Optics Express 20, 19, 20998-21009 (2012)
• B. Huang, W. Wang, M. Bates, X. Zhuang, „Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy“, Science 319, 810-813 (2008)

Exemplary procedures are described in the following publications:

• Travis J. Gould, Daniel Burke, Joerg Bewersdorf, Martin J. Booth, "Adaptive optics enables 3D STED microscopy in aberrating specimens", Optics Express 20, 19, 20998-21009 (2012)
• B. Huang, W. Wang, M. Bates, X. Zhuang, "Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy", Science 319, 810-813 (2008)

With such a design, for example, rotations of the sample due to long-term changes in the sample holder or other components of the microscope can be determined. This enables long-term monitoring of the microscope. In particular, the same sample can always be used.

According to one embodiment, the calibration is a measurement of the excitation light beam. The first optical center preferably has at least one optical transition dipole moment. This is preferably immutable.

By means of such a method, the excitation light beam can be measured very precisely, so that data such as intensity, electric field vector or polarization of the excitation light beam can be recorded with a high spatial resolution. As a result, for example, results obtained by means of the microscope can be interpreted much more precisely and better. It should be mentioned that the term polarization of the excitation light beam can be understood to mean, in particular, a state before it passes through an optical system located in front of the sample, wherein such a polarization can be set in a targeted manner. For the interaction with an optical center, the vector electric field at the location of the optical center is fundamentally relevant, which can depend on the polarization, but also on other factors.

The first optical center can, for example, be moved relative to the excitation light beam, which can be done, for example, by moving the sample holder or using appropriate mechanisms in the sample holder. In particular, changes in the emitted light can be determined.

Such changes can be, for example, changes in intensity or changes in color. These can be detected, for example, by means of the microscope's detector. This makes it possible to determine how the light emitted by the sample changes when the optical center is at different points relative to the excitation light beam. It should be mentioned that this is not possible using samples known from the prior art, which are based, for example, on DNA molecules, since these can, for example, rotate, wobble or fade.

The sample can be moved, for example, in a plane transverse to the excitation light beam. However, it can also take place differently, for example, alternatively or additionally, a movement can take place parallel to the excitation light beam. Planes can also be defined which are perpendicular to the excitation light beam and in which the method takes place. In principle, the movements described can be superimposed as desired, depending on the application.

According to one embodiment, the excitation light beam is polarized. The polarization can be, for example, linear, circular, trivial or highly complex, trivial polarization being understood to be a spatially invariable polarization and a highly complex polarization is understood to mean that it is different in different places.

During the calibration, the polarization can be rotated relative to the sample and the sample can be left locally unchanged relative to the excitation light beam. Polarization-dependent changes in the emitted light can be determined. This makes it possible to determine the reaction of the sample or the optical center to changes in polarization. As a result, it can be determined much more precisely than in previous versions how a sample behaves in the event of changes, in particular when the polarization is rotated, and the excitation light beam can be characterized very precisely, since its polarization or electrical field vectors can be measured in this way.

The measurement is three-dimensional and vectorial. For example, the already mentioned, preferably invariant, optical transition dipole moment of the optical center can be used for this purpose. For example, the optical transition dipole moment can be designed in such a way that maximum emission occurs when the optical transition dipole moment is parallel to the electric field of the excitation light beam, and that minimal emission or even no emission occurs when the optical transition dipole moment is transverse to the electric field of the excitation light beam . This can be used, for example, in that the sample and thus also the optical transition dipole moment is rotated relative to the excitation light beam, with the sample or optical center being able to remain in the same location, for example. In this way, for example, the excitation light beam can also be measured. It should be understood that a change in an intensity can be measured, for example, starting from a maximum intensity, for example with an optical transition dipole moment lying parallel to the electric field of the excitation light beam, but also starting from a non-existent emission, for example when the electric field of the Excitation light beam lying optical transition dipole moment can be measured. In the latter case, detection can be simpler in certain situations, since a change can be determined from a zero signal.

It should be understood that any optical field can be characterized using the method described herein. For example, it can be a plane wave, a focus or a focused wave, evanescent fields or any mode such as a TEM00 mode. In particular, the method just described can be used to characterize the polarization or a vector electric field.

The excitation light beam can be polarized, for example as already described above. During the calibration, the polarization can be left unchanged relative to the sample and the sample can be left locally unchanged relative to the excitation light beam, the emitted light being determined at different times. Changes in the emitted light can then be determined between the points in time. Such a procedure enables the microscope to be monitored at different times, with the same sample preferably always being able to be used. Due to the high stability of the sample, which is formed by a solid body, comparisons between different points in time, even those that are longer apart, are possible. This enables the stability of the microscope to be continuously monitored over a longer period of time.

The excitation light beam can be polarized, for example as already described above. During the calibration, the polarization can be rotated and the first optical center can be moved relative to the excitation light beam in order to determine polarization-dependent intensity maxima, whereby the measured intensity is meant here. In particular, an associated intensity maximum or an associated intensity distribution can be determined for each applied polarization. The measured intensity typically depends on the scalar product between the electric field at the location of the optical center and the optical transition dipole moment of the optical center. The electric field can be distorted, for example, by astigmatism in an excitation optics. This makes it possible to determine at which points the sample shows a particularly high emission of light, whereby the polarization or the electric field of the excitation light beam can also be calibrated. In other words, from the procedure just described, it is known at which points the effect of the excitation light beam on a specific sample or on a specific optical center is particularly high or even lower.

According to one embodiment, the calibration is a calibration of an absolute intensity or a collection efficiency of the microscope. In this way it can be recorded, for example, how many photons are detected by a microscope and / or which proportion of photons which are emitted by a sample is actually detected by the microscope or its detector. This allows the optical system of the microscope to be characterized.

The excitation light beam can, for example, have an intensity at which the first optical center emits at a rate corresponding to the inverse of the lifetime of its excited state. The intensity can also be so high that the first optical center emits at the maximum photon emission rate.

Such an embodiment can ensure that the number of photons emitted, for example emitted, is known in a certain period of time, for example within one second, and the proportion of photons emitted by the interrogation optics can thus easily be calculated back using the photons detected by the detector reaches the detector and is detected by it.

A rate corresponding to the inverse of the lifetime of the excited state can be achieved in particular if the first optical center is a purely two-level system or corresponds as closely as possible to an idealized two-level system, which can mean, for example, that no competing Disintegration paths are available. In this case, the system is typically excited from a ground state, remains in the excited state for the life of the excited state, returns to the ground state by emitting a photon and is immediately brought back into the excited state due to the sufficient intensity of the excitation light beam . A maximum photon emission rate is more general and includes not only the case just described of a correspondingly excited two-level system, but also takes into account the case that competing decay processes are present. For example, from an excited state, not only can relaxation directly into the ground state be possible, but there can also be an intermediate state which is energetically located between the ground state and the excited state and in which a decay from the excited state can take place. This typically takes place without photon emission or at least with photon emission in a different wavelength. The intermediate state can, for example, have a different, for example significantly longer, lifetime than the excited state. Such states can also be referred to as dark states. They can arise, for example, from trapping, caused for example by defects, contamination or other features. This can lead to the fact that the photon emission rate can be lower, for example also significantly lower, than the inverse of the lifetime of the excited state, even with high excitation. Nevertheless, the corresponding lifetimes of the states are known in typical optical centers, so that the photon emission rate can be calculated, or it is known from other measurements. The excitation of an optical center described here can also be referred to as saturation.

The excitation light beam can, for example, have the same intensity when the method is carried out multiple times at different times and / or with different microscopes and / or with different parts of microscopes and / or with different configurations and / or with different evaluation methods, but with the same sample. This enables a comparison of the detected photons per unit of time at different times and / or between different microscopes. Such comparisons are possible with the samples described herein in the form of solids, since these remain constant and unchanged over long periods of time and show constant behavior. Due to their robustness, corresponding samples can also be transported uncooled over long distances without any other great effort and damage, for example, in order to compare microscopes at different locations with one another.

A defined excitation power is understood to mean, for example, a predetermined power of the excitation light beam or a predetermined intensity of the excitation light beam, which can be so high, for example, that, as already described above, a maximum photon emission rate and / or a photon emission rate corresponding to the inverse of the lifetime of the excited state is achieved However, a defined excitation power which is smaller can also be used. Because of the otherwise unchanged and defined conditions, such a defined excitation power at different times and / or with different microscopes typically leads to the same emission of light, changes in such emission of light also being due to changes in a microscope between the times and / or Differences between different microscopes can be inferred.

With the measures described here, in particular, the detection or a behavior of the detector and / or an interrogation optics can also be calibrated. They are applicable accordingly. A sample can for example also be rotated or moved relative to the detector.

The collection efficiency of the microscope can be calculated, for example, as the quotient of photons captured in a time interval divided by photons emitted in the time interval. This enables the collection efficiency to be calculated simply and conveniently. It indicates how many of the emitted photons are actually recorded by the detector.

The method can for example be carried out by means of a plurality of microscopes using the same sample. Results when calibrating the microscope can be compared with one another. This enables a comparison of different microscopes, which can also be located at different locations, for example. Comparisons between different parts of microscopes or evaluation processes are also possible. The fact that the samples described herein in the form of solids can also be transported over longer distances and longer periods of time is advantageously used without the need for special expenditure such as cooling. This is not possible with samples known from the prior art. It should be understood that a corresponding comparison between microscopes can represent an independent aspect of the invention. The method can be used in all of the embodiments and variants described herein.

• - einen Probenhalter zur Aufnahme einer Probe,
• - eine Anregungsoptik zum Richten eines Anregungslichtstrahls auf die Probe,
• - eine Abfrageoptik zum Aufnehmen von durch die Probe ausgesendetem Licht,
• - einen Detektor zum Erfassen von durch die Abfrageoptik aufgenommenem Licht,
• - eine elektronische Steuerungsvorrichtung, welche dazu konfiguriert ist, ein Verfahren wie hierin beschrieben auszuführen.

The invention also relates to a microscope arrangement. The microscope arrangement has a microscope. The microscope has the following:

• - a sample holder for holding a sample,
• - excitation optics for directing an excitation light beam onto the sample,
• - an interrogation optics for receiving light emitted by the sample,
• - A detector for detecting light picked up by the interrogation optics,
• an electronic control device which is configured to carry out a method as described herein.

The microscope arrangement also has a sample in the form of a solid, the solid having at least one first optical center which emits light after being excited.

The advantages described above with reference to the method according to the invention in all of its variants can be achieved by means of the microscope arrangement according to the invention. With regard to the method, all embodiments and variants described herein can be used. It should be understood that the microscope arrangement is a combination of a microscope including its control device with a sample in the form of a solid body. These can for example be arranged spatially adjacent to one another. In particular, the sample can also be inserted into the sample holder.

The control device can for example be a computer or another programmable device which can be located adjacent to the other components of the microscope or also at a location remote therefrom. It should be understood that in the case of a microscope, data acquisition and data evaluation can in principle also take place spatially separated from one another, so that, for example, the implementation of the method can also be outsourced to a data center. It should also be understood that the control device is typically configured to carry out the method only to the extent that this is technically possible. For example, steps such as inserting the sample and / or certain adjustment tasks can be carried out otherwise, for example manually.

The invention further relates to a non-volatile computer-readable storage medium on which a program code is stored, which, when executed, a processor carries out a method described herein. With regard to the method, all embodiments and variants described herein can be used.

The invention also relates to a use of a sample for calibrating a microscope. The sample is a solid body with at least one first optical center which emits light after being excited. By means of such a use, the advantages already described above with reference to the method according to the invention can be achieved. With regard to all variants of the use, the variants described with reference to the method can be used.

• - einen Probenhalter zur Aufnahme einer Probe,
• - eine Anregungsoptik zum Richten eines Anregungslichtstrahls auf die Probe,
• - eine Abfrageoptik zum Aufnehmen von durch die Probe ausgesendetem Licht,
• - einen Detektor zum Erfassen von durch die Abfrageoptik aufgenommenem Licht.

In particular, the microscope can have the following when used:

• - a sample holder for holding a sample,
• - excitation optics for directing an excitation light beam onto the sample,
• - an interrogation optics for receiving light emitted by the sample,
• a detector for detecting light picked up by the interrogation optics.

The sample can for example have only one optical center or a first and a second optical center.

The first optical center and the second optical center can be at a distance of have at least one lattice site or at least 1 nm or at least 3 nm from one another. The first optical center and the second optical center can also be at a distance of at most 100 nm or at most 50 nm or at most 20 nm from one another.

Spin-active optical centers, magnetically couplable optical centers or otherwise couplable optical centers can be used as optical centers.

Diamond or silicon carbide, for example, can be used as the solid. However, other types of solids can also be used. In particular, nitrogen vacancies can be used as optical centers, it also being possible to use other types of optical centers.

Otherwise, it should also be understood that all of the designs and variants described with reference to the method with regard to the use can be used.

The calibration can in particular take place according to the method. With regard to the method, all embodiments and variants described herein can be used.

The calibration can in particular also take place by means of a microscope arrangement described herein. In this regard, too, all of the embodiments and variants described herein can be used.

The invention also relates to a sample in the form of a solid body with at least one optical center which emits light after being excited. The sample is provided and / or configured for use in a method according to the invention, in a microscope arrangement according to the invention or in a use according to the invention. With regard to the method, the microscope arrangement and the use, all of the embodiments and variants described herein can be used.

In general, by means of the measures described here, for example the method, the use and the microscope arrangement, a plurality of tasks can be carried out which were not possible with embodiments previously known from the prior art.

1. 1. Kalibrierung des genauen Abstands zweier Fluorophore, beispielsweise für die Konfokalmikroskopie oder für Konfokalmikroskope,
2. 2. Verwendung zweier Fluorophore als rückführbares Standardmaß (Normal) für Nanometerabstände,
3. 3. Kalibrierung der genauen Verteilung der Lichtintensität und der Polarisation bzw. elektrischem Feld am Ort des Fokus,
4. 4. Kalibrierung der absoluten Intensität und der Sammlungseffizienz des mikroskopischen Aufbaus,
5. 5. Vergleich von Mikroskopen unter Verwendung der gleichen Probe.

These are for example:

1. 1. Calibration of the exact distance between two fluorophores, for example for confocal microscopy or for confocal microscopes,
2. 2. Use of two fluorophores as a traceable standard measure (normal) for nanometer distances,
3. 3. Calibration of the exact distribution of the light intensity and the polarization or electric field at the location of the focus,
4. 4. Calibration of the absolute intensity and the collection efficiency of the microscopic structure,
5. 5. Comparison of microscopes using the same sample.

It should be understood that this list is only to be understood as an example and in no way restrictive. The points mentioned can, however, be understood as independent aspects of the invention.

In the prior art, for example, certain standards are known for investigations into the quantum efficiency of emitters. For example, DNA origami samples are known for colocalization. For example, these can contain two or more fluorophores that can be imaged. However, such samples have not proven to be long-lived, which excludes their use as a reference for different times and / or for microscopes or microscope parts located at different locations.

It has also been shown that in the case of DNA molecules which contain two or more fluorophores, fluorophores can be bleached, whereby these lose their fluorescent properties. This can be considered to be a major problem with such samples. If this happens to a molecule, it can no longer be used for calibration. The samples are temperature-sensitive and often have to be cooled, which causes considerable effort. Even if stored properly, the molecules can only be used for a few months. It is also known that the distance between the fluorophores in DNA origami can only be specified to within a few nanometers for a molecule. Measurements with it do not allow any statement about the polarization or the vectorial composition of the applied light field. As a result of the bleaching of the fluorophores, a comparison of different microscopes on the same emitter pair is only possible to a limited extent or not possible. For use as a standard measure, distance measurements on molecules would have to be carried out with a calibrated microscope, for which calibrated micrometers would have to be used. However, these are nowhere near as accurate as the time reference described above.

It has been shown that, especially when using techniques for measuring below the refraction limit (super resolution), an exact determination of the field in the focus can be important. It is also an advantage if microscopes are comparable and accurate. The colocalization can be of particular importance because drift or other changes in position, for example reloading the sample, do not change the precision of the measurement.

The sample described herein can, for example, be based on fluorescent or luminescent defect centers in solids, such as nitrogen vacancies in diamond. These do not bleach and can be located using alternative electron spin resonance (ESR) methods. It is also possible to determine the number of emitters using their photon statistics. Since the distance between two emitters in a solid body is constant over long periods of time due to the low diffusion probability of such defects, such a sample enables a long-lasting sample for microscope calibration.

In particular, fluorescent defect centers in solids can be used for the examination of microscopes. For example, nitrogen vacancies in diamond can be used, the fluorescence properties of which have been very well investigated and can correspond to those of biological samples relevant in microscopy. In the case of defect centers, the localization of the individual emitters can also be determined independently of optical microscopy using electron spin resonance methods, for example by applying a gradient field or, in the case of colocalization, using magnetic dipolar coupling or electrical field measurements between two emitters. This can be used, for example, to cover distances of approx. 3 nm to 100 nm.

• I. Jakobi, S. A. Momenzadeh, F. F. De Oliveira, J. Michl, F. Ziem, M. Schreck, P. Neumann, A. Denisenko, J. Wrachtrup, „Efficient creation of dipolar coupled nitrogen-vacancy spin qubits in diamond“, Journal of Physics: Conference Series, vol. 752, no. 1, 2016

Fluorescent defect centers or other optical centers in solids are usually photostable, i.e. they do not bleach or diffuse. This means that the samples are stable and usable for a long time. Since the emitters are in a solid body, handling, storage and transportation of the samples are easy. They are also temperature resistant. With regard to the colocalization, it is also advantageous that the distance is precisely fixed to the size of the fluorophore and no longer changes. That means: Once the relative distance of an emitter pair has been determined, the sample can be used repeatedly to adjust and characterize microscopes. Since the distance is precisely defined, no statistics have to be carried out on an ensemble of emitter pairs. By tracing the distance determination back to the SI system of units, emitter pairs can be used as standard nanometer measurements (normal). In the case of magnetic dipolar coupling, the distance is determined, for example, by measuring the time of the electron spin resonance. The optical dipolar transition moments are also fixed and do not rotate. This enables not only the measurement of the intensity of the focus, but also the vectorial composition of the electric field. If the fluorophores are embedded in a crystal lattice, as is the case with nitrogen vacancies in diamond, the optical transition dipole moments can be derived from the crystal axes. Methods for the production of solid samples with individual fluorophores and fluorophore pairs are known, for example in the case of nitrogen vacancies by ion implantation through nanoscopic masks. Such methods are described, for example, in the following publication:

• I. Jakobi, SA Momenzadeh, FF De Oliveira, J. Michl, F. Ziem, M. Schreck, P. Neumann, A. Denisenko, J. Wrachtrup, "Efficient creation of dipolar coupled nitrogen-vacancy spin qubits in diamond", Journal of Physics: Conference Series, vol. 752, no.1, 2016

• M. Haruyama, S. Onoda, T. Higuchi, W. Kada, A. Chiba, Y. Hirano, T. Teraji, R. Igarashi, S. Kawai, H. Kawarada, Y. Ishii, R. Fukuda, T. Tanii, J. Isoya, T. Ohshima, O. Hanaizumi, „Triple nitrogen-vacancy centre fabrication by C5N4Hn ion implantation“, Nature Communications, vol. 10, no. 1, p. 2664, 2019 . [Online]; http://www.nature.com/articles/s41467-019-10529-x

A version without masks is described, for example, in the following publication:

• M. Haruyama, S. Onoda, T. Higuchi, W. Kada, A. Chiba, Y. Hirano, T. Teraji, R. Igarashi, S. Kawai, H. Kawarada, Y. Ishii, R. Fukuda, T. Tanii, J. Isoya, T. Ohshima, O. Hanaizumi, "Triple nitrogen-vacancy center fabrication by C5N4Hn ion implantation", Nature Communications, vol. 10, no. 1, p. 2664, 2019 . [On-line]; http://www.nature.com/articles/s41467-019-10529-x

The scattering, depth and concentration of the fluorophores can be tailored. For example, corresponding optical centers can be located at a uniform depth of approximately 30 nm with a deviation of, for example, +/- 1 nm.

• 1: eine Mikroskopanordnung,
• 2: Details einer Probe,
• 3: Details eines optischen Zentrums,
• 4: einen Verlauf eines Anregungslichtstrahls,
• 5: einen zu 4 korrespondierenden Verlauf eines elektrischen Feldes des Anregungsl ichtstrah ls,
• 6: Verläufe von Anregungseffizienzen,
• 7: eine Probe,
• 8: eine Probe und deren Abbildung,
• 9: eine Probe zur Vermessung des Anregungslichtstrahls,
• 10: eine Intensitätsverteilung in einer Ebene,
• 11: Schwerpunkte von Intensitäten in Abhängigkeit von einer Polarisation,
• 12: polarisationsabhängige Intensitätsverteilungen,
• 13: polarisationsabhängige Intensitätsverteilungen mit Astigmatismus,
• 14: eine Legende für die vorhergehenden 10 bis 13.

The person skilled in the art will find further features and advantages in the exemplary embodiments described below with reference to the accompanying drawings. Show:

• 1 : a microscope assembly,
• 2 : Details of a sample,
• 3 : Details of an optical center,
• 4th : a course of an excitation light beam,
• 5 : one too 4th Corresponding course of an electric field of the excitation light beam,
• 6 : Curves of excitation efficiencies,
• 7th : a sample,
• 8th : a sample and its image,
• 9 : a sample for measuring the excitation light beam,
• 10 : an intensity distribution in a plane,
• 11 : Centers of intensities depending on a polarization,
• 12 : polarization-dependent intensity distributions,
• 13 : polarization-dependent intensity distributions with astigmatism,
• 14th : a legend for the previous ones 10 to 13 .

1 shows schematically a microscope arrangement 1 according to an embodiment of the invention. The microscope arrangement 1 exhibits a microscope 5 as well as a sample 100 on. These components will be discussed in more detail below.

The microscope 5 has excitation optics 10 as well as a query optics 30th on. The excitation optics 10 in the present case has a light source 12 in the form of a laser, a mirror 14th and corrective optics shown purely schematically 16 on. It should be understood that the described components of the excitation optics are only mentioned here as examples and that completely different components can also be used. The excitation optics 10 generates an excitation light beam 20th which in a manner to be described on the sample 100 can be directed.

The query optics 30th is designed to take from the sample 100 emitted light 40 absorb and process. The query optics indicate this 30th for example two lenses 32 , 36 as well as a diaphragm arranged in between 34 on. The aperture 34 can for example be used in confocal microscopy to select a visible area.

The microscope 5 has a detector 60 on. The query optics are aimed at this 30th the emitted light 40 .

The microscope 5 further comprises an electronic control device 70 which is configured to carry out a method according to the invention. This will be discussed in more detail below. The electronic control device 70 is as shown with the detector 60 connected and can thus receive signals that the detector 60 based on detected light 40 generated.

The microscope 5 has another optic in the form of a beam splitter plate 50 on which optically the excitation optics 10 and the query optics 30th separates from each other. The excitation light beam 20th is from the beam splitter plate 50 towards the sample 100 to headed. Between the beam splitter plate 50 and the sample 100 there is still a purely schematically illustrated sample optics 80 . This ensures that the excitation light beam 20th as desired to the test 100 hits, the excitation light beam 20th in the present case is focused.

The microscope 5 also has a sample holder 90 on in which the sample 100 is stored. The sample 100 can thus in a defined manner from the excitation light beam 20th be illuminated. It should be mentioned that, for example, a sample scanner, a beam scanner or an objective scanner can be used as an embodiment.

Light emerges from the sample 100 off, for example due to emission or due to scattering of the excitation light beam 20th This is how the already mentioned emitted light is created 40 , which is also from the sample optics 80 added and through the beam splitter plate 50 into the query optics 30th is directed. There it is attached to the detector as already described 60 fed. However, it should be mentioned that, in principle, completely different optical paths can also be used for excitation and query.

At the rehearsal 100 In the present case, it is a sample in the form of a solid body in which at least one optical center is present. Such an optical center can be based on the excitation light beam 20th emitted light 40 produce. This can be done by scattering or by exciting a system with at least two levels into an excited state and subsequent relaxation with emission of photons into a ground state. Possibilities that result from this are described in more detail below.

2 shows a typical interior of a sample 100 in the present case with two optical centers, namely a first optical center 110 and a second optical center 120 . Such optical centers 110 , 120 can for example be implemented as nitrogen vacancies in diamond as a solid. A distance d of such optical centers 110 , 120 is, for example, between 3 nm and 20 nm, in which the effects already described above and below are particularly evident can be observed and used advantageously. However, other distances are also possible.

The first optical center 110 has a first orientation 115 on. The second optical center 120 exhibits a second orientation 125 on. With these orientations 115 , 125 it is a respective main axis of symmetry which is relevant for the magnetic coupling. It is known that nitrogen vacancies in diamond have four possible orientations relative to the crystal lattice, each of which has an angle of 54.7 ° to a (001) surface. Among other things by the shown orientations 115 , 125 creates a magnetic coupling between the two optical centers 110 , 120 defines which by in 2 field lines shown is shown schematically.

As already mentioned above, the magnetic coupling between two optical centers 110 , 120 can be used to determine their distance independently of optical measurements. In other words, one of the in 1 illustrated microscope 5 independent procedure available to determine the distance d between the two optical centers 110 , 120 to be measured very precisely.

In particular, the method already described above can be used for this purpose. A spatial calibration of the detector 60 is not required for this.

The timing already mentioned above can, as described in the references already cited above, be assigned to intervals. This timing can be measured very precisely and can in particular also be traced back to a standard measure of the SI system of units, namely the second. For example, an atomic clock that is appropriately calibrated can be used for this purpose. In this way, not only the distance d, which in 2 is shown, can be determined very precisely, but this can also be traced back to an SI standard. It can also be used here that the distance d can basically only have discrete values which are predetermined by the grid.

3 shows the first optical center 110 in a spatial orientation with a specified coordinate system and its axes x, y and z. The respective projections onto the (x, y) plane, the (y, z) plane and the (x, z) plane are also shown. The orientation 115 is also shown.

As with arrows, which are in a plane perpendicular to the orientation 115 is indicated, has the first optical center 110 , which in the present case is in the form of a nitrogen vacancy, has two optical transition dipole moments 117 , 118 on which in a plane transverse to the orientation 115 lie. The two optical transition dipole moments 117 , 118 are to be understood as preferential orientations in which an excitation of the first optical center 110 is particularly efficient with a parallel electric field of an excitation light. Incidentally, this applies equally to the two optical transition dipole moments 117 , 118 spanned plane, a rotation of the electric field in this plane has no effect. If, however, the electric field of the excitation light is different from that caused by the optical transition dipole moments 117 , 118 If the spanned plane is twisted out, the result is that the excitation is less efficient.

The phenomenon just described can be used to measure an excitation light beam 20th be used. This is in 4th as well as the associated 5 and 6 shown in more detail, with only components in one plane being shown here for the sake of simplicity. The z-axis lies in the direction of the propagation direction of the excitation light beam 20th . 4th shows the excitation light beam 20th especially before and after exiting the sample optics 80 , where the excitation light beam 20th on a focal plane 25th is focused. Due to the focusing, the electric field of the excitation light beam changes 20th , this in front of the sample optics 80 is oriented transversely to the direction of propagation, as indicated by the unfilled arrows below, but then changes and only in the focal plane 25th is again perpendicular to the direction of propagation. In one level 27 behind the focal plane 25th is the electric field of the excitation light beam 20th depends on the location across the direction of propagation. This is in 5 shown in more detail, with the arrows not filled in the electric field of the excitation light beam 20th show depending on the location.

In 4th and in 5 two points are drawn in, at which, for example, optical centers can be located. These can basically come from the excitation light beam 20th be stimulated. The positions are shown by vertical dashed lines. They are also represented by the circles shown. It should be mentioned that providing two optical centers is equivalent to shifting one optical center between two locations.

Corresponding optical transition dipole moments of the two optical centers are in 5 shown with filled arrows. As can be seen, the two optical transition dipole moments are aligned in the same way. The electric field of the excitation light beam 20th is however clearly different in the two locations, whereby in the left position the electric field of the Excitation light beam 20th is almost perpendicular to the optical transition dipole moment, whereas the electric field of the excitation light beam 20th occupies only a small angle to the optical transition dipole moment in the right position.

This leads to the in 6 shown curve of the excitation efficiency. If the already described phenomenon of polarization dependence or field dependence of the excitation did not exist, an excitation efficiency would have that in 6 The course shown in dashed lines, which is caused by the intensity distribution of the excitation light beam 20th is determined. Due to the phenomenon of field dependency just described, however, the excitation efficiency has the curve shown in solid lines, whereby due to the better alignment of the field, the right optical center is excited much more strongly than the left optical center, despite the same distance from a solid vertical center line. This is also due to the larger illuminated dot in 6 shown.

The phenomenon just described of the field dependence of the excitation can be used to generate an excitation light beam 20th to measure. A corresponding sample can be used for this purpose, for example 100 with such an optical center 110 in the sample holder 90 can be used and targeted. In this way, changes in intensity can be determined, with a scalar product between the optical transition dipole moment of the optical center and the electric field of the excitation light beam 20th The higher the brightness detected at a certain location, the greater it is. It is also possible to change the polarization of the excitation light beam 20th to actively rotate in order to detect corresponding changes in brightness.

7th shows an exemplary sample 100 in the form of a solid 105 , in which the already mentioned optical centers 110 , 120 at a distance d from each other. There are typical electric fields E. and / or magnetic fields B. drawn, which are exemplified here by the sample 100 go through it and with the two optical centers 110 , 120 can interact.

8th shows the already in 7th shown sample 100 and its effect on the detector 60 , with the optics in between 30th , 50 , 80 are only shown purely schematically. The two optical centers 110 , 120 thanks to the optics in between 30th , 50 , 80 on the detector 60 as the first point 62 and second point 64 mapped which on the detector 60 have a distance d 'from each other. This distance d 'can easily be determined and initially relates to the coordinate system of the detector 60 . For example, the distance d 'can be a number of between the two points 62 , 64 lying pixels or by a distance by which the detector 60 proceeded as a whole.

Since the distance d between the two optical centers 110 , 120 in the sample, as already mentioned above, very precisely independent of a spatial calibration of the detector 60 can be measured, it is now possible to determine the distance d 'in the detector 60 to be calibrated because it can be assumed that the coordinate system of the detector 60 measured distance d 'is the distance d in the sample 100 corresponds. The sample 100 can thus be used as a reference for the measured distance. If another sample is subsequently used in which an unknown distance is to be measured, this calibration can be used to determine the distance in the other sample. It has been found to be particularly advantageous that the sample 100 with the solid 105 is very photostable, remains stable over long periods of time and is easy to handle. It does not require any special storage and can also be transported over long distances without changing its properties. This enables a comparison of different microscopes 5 , which are in different places, and also enables a control of a single microscope, for example 5 which at different times with the same sample 100 can be calibrated. There may be deviations over time, for example due to changes in the components of the microscope 5 can occur. For example, a respective calibration by means of the sample can also be performed on other samples before measurements to be carried out 100 be carried out so that a respective distance in the other sample can be determined very precisely. For example, it can also be used to detect rotations, which are due to long-term changes in the sample holder, for example 90 may occur.

In 9 is shown separately as a sample 100 for measuring the excitation light beam 20th or can be used to determine a collection efficiency. The excitation light beam 20th is done by the optics already mentioned 10 , 50 , 80 , what a 9 are only shown schematically on the sample 100 focused and thus already has one in the 4th , 5 and 6 depicted location-dependent course of its electric field. The electric field is shown schematically by means of arrows 22nd shown.

At the in 9 shown embodiment with only one optical center 110 or even with a rehearsal 100 with multiple optical centers it is possible to increase the collection efficiency of the microscope 5 to calibrate. An excitation light beam can be used for this purpose 20th used with a defined intensity which thus also lead to a defined emission of the optical center 110 leads. In particular, it can be an excitation light beam 20th can be used with an intensity so great that the first optical center 110 emitted at its maximum photon emission rate. This is for typical optical centers 110 known as nitrogen vacancies in diamond. Using the detector 60 can then be determined how many photons at the detector 60 actually arrive. The collection efficiency of the microscope can then be calculated from a quotient of the photons actually arriving in a certain time interval divided by the number of photons emitted in this time interval 5 be determined. This can be, for example, a few percent, but also below or above.

That in the rehearsal 100 contained optical center 110 exhibits an optical transition dipole moment already described 117 The more parallel the electric field of the excitation light beam, the more efficient the excitation 20th and the optical transition dipole moment 117 are. This enables the excitation light beam to be measured 20th , for example by moving the sample 100 in the sample holder 90 .

Such a spatial method can, for example, allow an in 10 intensity distribution shown can be determined. The excitation light beam is polarized at 90 ° 20th used, which corresponds to a polarization parallel to the y-axis. The optical center is located in a plane 200 nm behind a focal plane of the excitation light beam.

A focus of the measured brightness is on the x-axis, but about 66 nm above the y-axis. The already in 4th drawn central line in the direction of the direction of propagation of the excitation light beam 20th used. The surrounding lines indicate at which points the measured intensity, which is indicative of the excitation strength, drops by ten percentage points. The immediately surrounding line thus shows an intensity of 90% of the maximum intensity or the intensity at the center of gravity, the line labeled “0.8” shows an intensity of 80% of the maximum intensity or the intensity at the center of gravity, and so on. 10 thus shows that a maximum or a focus of the excitation efficiency occurs outside an actual zero point at which the highest excitation efficiency would be expected without taking into account the polarization dependence or field dependence. This phenomenon can be used to generate the excitation light beam 20th as already mentioned to be measured.

11 shows how the in 10 The focus of the excitation shown depends on an output polarization of the excitation light beam 20th behaves, a plane 200 nm behind a focal plane of the excitation light beam is also used here. Please refer to the in 14th illustrated legend, which one applies to all 10 to 13 applies. With a polarization of 0 °, which corresponds to a polarization parallel to the x-axis, the excitation maximum is very close to the zero point, that is to say at an intersection of the y-axis and the x-axis. With a polarization of + 45 °, the excitation maximum is around (+20 nm, +18 nm). With a polarization of -45 °, the maximum excitation is around (-20 nm, + 18 nm). The solid line shows the elliptical course, the excitation maximum with continuous rotation of the polarization. The excitation maximum, which, for example, by moving the sample 100 in the sample holder 90 can be determined, is thus dependent on the polarization of the excitation light beam 20th . This can be used to measure the excitation light beam 20th than calibration of the microscope 5 be used.

12 shows a three-dimensional dependence of the intensity maxima, with the direction of propagation of the excitation light beam 20th is shown as the z coordinate and the zero point in the z direction in the focal plane 25th which is already in 4th is shown. It is also a level E. plotted which of the 10 and 11 (x, y) -plane shown corresponds.

In 12 respective curves of excitation maxima for different polarizations are shown. Regarding the polarizations, be on 14th referenced. For example, it can be seen that in the case of a polarization of 0 °, the excitation maximum does not change with the z coordinate. For other polarizations, however, the excitation maximum changes with the z coordinate along respective straight lines or at least along lines that are straight lines in the first order. This behavior can be used to measure the excitation light beam 20th be used.

13 shows the course of corresponding excitation maxima for the case that in the excitation optics 10 astigmatism is present. A first focus level is an example here FE1 and a second plane of focus FE2 shown. With these two levels of focus FE1 , FE2 the excitation maxima are no longer as a function of the polarization, for example in 11 shown running along an ellipse, but along a respective straight line. These straight lines can be measured, and an intersection of these straight lines can indicate an actual sample position. This can be used to measure the excitation light beam 20th and in particular for the detection of any astigmatisms in the excitation optics 10 or in other optics of the microscope 5 be used. This also enables calibration of the microscope 5 possible.

Overall, it has been shown that the use of samples 100 in the form of a solid 105 with optical centers 110 , 120 different calibrations of a microscope 5 enabled, such a calibration in comparison to calibrations with known calibration samples such as DNA molecules brings with it considerable advantages. In particular here are the longevity of the sample 100 , their easy handling and storage, high resistance to fading, easy portability and a long-term stable distance between optical centers 110 , 120 to call.

Mentioned steps of the method according to the invention can be carried out in the order given. However, they can also be carried out in a different order if this is technically reasonable. The method according to the invention can, in one of its embodiments, for example with a specific combination of steps, be carried out in such a way that no further steps are carried out. In principle, however, further steps can also be carried out, including those which are not mentioned.

It should be pointed out that features can be described in combination in the claims and in the description, for example to facilitate understanding, although they can also be used separately from one another. The person skilled in the art recognizes that such features can also be combined with other features or feature combinations independently of one another.

References back in subclaims can identify preferred combinations of the respective features, but do not exclude other combinations of features.

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.

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Claims (28)

Procedure for calibrating a microscope (5), - The microscope (5) having the following: - a sample holder (90) for receiving a sample (100), - excitation optics (10) for directing an excitation light beam (20) onto the sample (100), - an interrogation optics (30) for receiving light (40) emitted by the sample (100), - A detector (60) for detecting light picked up by the interrogation optics (30), - wherein the method comprises the following steps: - Insertion of a solid body (105) as a sample (100) in the sample holder (90), the solid body (105) having at least one first optical center (110) which emits light after excitation, - Directing an excitation light beam (20) onto the sample (100) so that the first optical center (110) is excited, - Detecting the light (40) emitted by the sample (100) by means of the interrogation optics (30) and the detector (60), - Calibrating the microscope (5) based on the light detected by the detector (60). Procedure according to Claim 1 - wherein the sample (100) has at least one second optical center (120). Procedure according to Claim 2 - wherein the first optical center (110) and the second optical center (120) are at a distance (d) of at least one grid position or of at least 1 nm or of at least 3 nm from one another; and / or - wherein the first optical center (110) and the second optical center (120) have a distance (d) of at most 100 nm or of at most 50 nm or of at most 20 nm from one another. Method according to one of the preceding claims, - The optical centers (110, 120) used are spin-active optical centers, magnetically couplable optical centers or otherwise couplable optical centers. Method according to one of the preceding claims, - Diamond or silicon carbide being used as the solid body (105). Method according to one of the preceding claims, - With nitrogen vacancies being used as optical centers (110, 120). Method according to one of the preceding claims, - The microscope (5) being a confocal microscope, a near-field microscope, a scattering microscope or a wide-field microscope. Method according to one of the preceding claims, - wherein the calibration is a calibration of a distance (d, d '), and - wherein the sample (100) has at least one second optical center (120). Procedure according to Claim 8 - which includes a step of determining a distance (d) between the first optical center (110) and the second optical center (120) by means of an electromagnetic coupling measurement. Procedure according to Claim 9 - With the coupling measurement using a timing of pulses traced back to a standard measure. Method according to one of the Claims 8 to 10 - wherein a distance (d ') determined by means of the detector (60) is calibrated. Procedure according to Claim 11 - The distance (d, d ') being determined by means of emitter-selective activation of the optical centers (110, 120) and / or by means of super resolution methods, correlation evaluation or cross-correlation evaluation of signals recorded by the detector (60) based on the detected light. Method according to one of the Claims 8 to 12 - wherein a plurality of distances (d, d ') between the first optical center (110) and the second optical center (120) are determined vectorially at different times, and - where from the vectorial distances (d, d') a rotation is determined. Method according to one of the Claims 1 to 7th - wherein the calibration is a measurement of the excitation light beam (20), and - wherein the first optical center (110) has at least one optical transition dipole moment (117, 118). Procedure according to Claim 14 - wherein the first optical center (110) is moved relative to the excitation light beam (20) and changes in the emitted light (40) are determined. Method according to one of the Claims 14 or 15th , - wherein the excitation light beam (20) is polarized, and - during the calibration, the polarization is rotated relative to the sample (100) and the sample (100) is left locally unchanged relative to the excitation light beam (20) in order to avoid polarization-dependent changes in the emitted light (40) to determine. Method according to one of the Claims 14 to 16 - the excitation light beam (20) being polarized, - the polarization relative to the sample (100) being left unchanged during the calibration and the sample (100) being left locally unchanged relative to the excitation light beam (20), the emitted light (40 ) is determined at different times, and - changes in the emitted light (40) between the times are determined. Method according to one of the Claims 14 to 17th - wherein the excitation light beam (20) is polarized, and - wherein the polarization is rotated during calibration and the first optical center (110) is moved relative to the excitation light beam (20) in order to determine polarization-dependent intensity maxima. Method according to one of the Claims 1 to 7th - wherein the calibration is a calibration of an absolute intensity or a collection efficiency of the microscope (5). Procedure according to Claim 19 - wherein the excitation light beam (20) has an intensity with which the first optical center (110) emits at a rate corresponding to the inverse of the lifetime of its excited state and / or emits at the maximum photon emission rate. Method according to one of the Claims 19 or 20th - The excitation light beam (20) when the method is carried out several times at different times and / or with different microscopes (5) and / or with different parts of microscopes (5) and / or with different configurations and / or with different evaluation methods, however with the same sample (100), each has the same intensity. Procedure according to Claim 21 - The collection efficiency of the microscope (5) being calculated as the quotient of the photons captured in a time interval divided by the photons emitted in the time interval. Method according to one of the preceding claims, - which is carried out by means of a plurality of microscopes (5) using the same sample (100), - The results of the respective calibration of the microscopes (5) are compared with one another. Microscope arrangement (1), which has the following: - having a microscope (5) - a sample holder (90) for receiving a sample (100), - excitation optics (10) for directing an excitation light beam (20) onto the sample (100), - an interrogation optics (30) for receiving light (40) emitted by the sample (100), - A detector (60) for detecting light picked up by the interrogation optics (30), - An electronic control device (70) which is configured to carry out a method according to one of the preceding claims, and - A sample (100) in the form of a solid body (105), the solid body (105) having at least one first optical center (110) which emits light after being excited. Microscope assembly (1) Claim 24 - wherein the sample (100) is inserted into the sample holder (90). Using a sample (100) to calibrate a microscope (5), - The sample (100) being a solid body (105) with at least one first optical center (110) which emits light after being excited. Use after Claim 26 - the microscope (5) having the following: - a sample holder (90) for holding a sample (100), - excitation optics (10) for directing an excitation light beam (20) onto the sample (100), - interrogation optics (30) ) for receiving light (40) emitted by the sample (100), - a detector (60) for detecting light received by the interrogation optics (30); and / or - wherein the sample (100) has at least one second optical center (120); and / or - wherein the first optical center (110) and the second optical center (120) are at a distance (d) of at least one grid position or of at least 1 nm or of at least 3 nm from one another; and / or - wherein the first optical center (110) and the second optical center (120) have a distance (d) of at most 100 nm or of at most 50 nm or of at most 20 nm from one another; and / or - wherein the optical centers (110, 120) are spin-active optical centers, magnetically coupled optical centers Centers or optical centers that can be coupled in some other way are used; and / or - diamond or silicon carbide being used as the solid body (105); and / or - nitrogen vacancies being used as optical centers (110, 120). Use after one of the Claims 26 or 27 , - the calibration according to one of the Claims 1 to 23 he follows; and / or - which by means of a microscope arrangement (1) according to one of the Claims 24 or 25th he follows.

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