Method for locating or tracking emitters in a sample

Abstract

A method for locating or tracking emitters in a sample (2), wherein the sample (2) is illuminated in a measuring step with an intensity distribution of illuminating light (B), wherein the illuminating light (B) induces or modulates light emissions (L) from emitters in the sample (2), wherein the intensity distribution has a local minimum, and wherein the intensity distribution and the sample (2) are moved relative to one another by means of a scanning device (6) in a measuring scanning movement, wherein light emissions (L) of a measuring emitter (M) in the sample (2) induced or modulated by the illuminating light (B) are detected, and wherein a position of the measuring emitter (M) is estimated on the basis of the detected light emissions (L) and positions of the local minimum of the intensity distribution associated with the light emissions (L), characterized in thatthat, depending on a speed and / or an acceleration of the measuring scanning movement, the estimated position of the measuring emitter (M) is corrected on the basis of calibration data and / or a control signal of the scanning device (6) is adjusted on the basis of calibration data, wherein the calibration data comprise localization data of a calibration emitter (K) obtained by means of at least one calibration scanning movement of the scanning device (6).

Description

Technical field of the invention

[0001] The invention relates to a method for locating or tracking emitters in a sample, in particular according to the MINFLUX principle. State of the art

[0002] In contrast to classical imaging techniques of light microscopy, in light microscopy localization methods, the positions of individual emitters (e.g., fluorophores or molecules labeled with fluorophores) are calculated based on detected light emissions. From the positions of a large number of emitters, usually determined one after the other, a localization map can be created that visualizes the distribution of the emitters in the sample. Such a localization map is similar to a wide-field image, in which sample structures are imaged onto a detector, or to a raster image, in which the sample is scanned with focused illumination light and the light emission from the sample is recorded for each raster point. However, depending on the localization precision of the corresponding imaging technique, localization maps can have a resolution that is well below the diffraction limit.

[0003] The term "isolated emitters" here means that at a given time, light-emitting emitters whose emission light is indistinguishable are separated by a distance above the diffraction limit, so that their emission light can be separated. This can be achieved, for example, in the case of permanently light-emitting emitters by a labeling density of the sample below a limit value. However, if the emitters flash statistically, for example, a sufficient separation can be achieved at almost any given time even with a higher labeling density. For this purpose, the sample environment (e.g. buffer, embedding medium) can be chemically configured to result in a desired flashing rate. Smaller separations are also tolerable when different types of emitters are present whose emission light can be optically separated, e.g. due to different emission spectra or emission lifetimes.Finally, in exceptional cases, special evaluation methods (e.g., statistical or time-resolved methods) may also make it possible to jointly localize groups of several closely adjacent emitters. This can determine a specific position for each emitter, or an average position of several emitters can be determined.

[0004] Individual emitters moving within the sample can be tracked by several rapid localizations within the sample (tracking). The corresponding measurement data can be displayed, for example, in the form of a trajectory.

[0005] In the so-called MINFLUX technique, a sample containing isolated emitters (e.g., individual fluorophores, molecules labeled with fluorophores, or light-scattering particles) is illuminated with an intensity distribution of excitation light at illumination positions in a range around a roughly estimated position of an individual emitter. The intensity distribution has a, particularly central, intensity minimum (ideally an intensity zero). For each illumination position, the light emissions (in particular, the number of photons) of the isolated emitter are recorded. A new position estimate of the emitter is then calculated from the light emissions and the associated illumination positions. The intensity distribution can, for example, be a so-called 2D donut, a so-called bottle beam, or a superposition of these light distributions, which are known from the field of STED microscopy.

[0006] Various variants of the MINFLUX technique are described, for example, in the publications F. Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612, KC Gwosch et al. (2020) MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 and R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478 as well as in the patent documents US 9 719 928 B1, US 10 900 901 B2, US 10 908 089 B2 and US 10 962 479 B2.

[0007] Due to the excitation light distribution used with intensity minimum, the MINFLUX technique allows localization precision in the single-digit nanometer range to be achieved with a significantly lower number of emitted photons compared to the so-called PALM / STORM technique.

[0008] Patent publication US 11 255 791 B2 describes, among other things, a variant of the MINFLUX technique in which the sample is illuminated with a combination of an excitation light distribution with a local maximum and a STED (stimulated emission depletion) light distribution with a local minimum. The position of an individual emitter is also calculated from the light emissions recorded for different illumination positions, but the closer the minimum of the STED light distribution (and the maximum of the excitation light distribution superimposed on the minimum) is to the actual emitter position, the more light is emitted.

[0009] A special localization technique using an excitation light distribution with a local maximum and a STED light distribution with a local minimum is described in the publication M. Weber et al. (2021) MINSTED fluorescence localization and nanoscopy, Nat. Photonics 15, 361-366 and the patent application WO 2023 / 006176 A1 and is known as MINSTED. The combination of the excitation light distribution and the STED light distribution is moved along a continuous trajectory around an estimated emitter position, and the trajectory is adjusted based on each detected photon. The excitation and STED intensities can also be increased.

[0010] Application US 2023 / 0008453 A1 describes a variant of the MINFLUX technique, in which an intensity distribution with a local minimum is moved by a scanning device along a closed path within a sample region, whereby an intensity distribution over time is calculated from temporally resolved detected fluorescent light. By fitting a periodic function to the measured intensity distribution, the position of an individual emitter in the sample is estimated.

[0011] US 2020 / 0244515 A1 describes a method for acquiring microscopic images of samples with three-dimensional dimensions, as well as a scanning fluorescence microscope. In this method, a raster image of the sample is acquired by scanning with an excitation light distribution parallel to the optical axis of the objective. Based on an evaluation of the raster image, parameters of an aberration correction function are adjusted in order to correct aberrations in the excitation light distribution using the aberration correction function with a correction device.

[0012] US 2022 / 0011559 A1 discloses a device for detecting movements of a sample relative to an objective, the device comprising imaging optics which images light from reference objects in the sample onto a camera in an image plane, wherein an optical device is arranged in a Fourier plane between the objective and the camera, which optical device filters out low spatial frequencies from the image of the reference objects in order to improve the detection of the movements.

[0013] DE 102021 107 704 B4 discloses a method for the light microscopic examination of a sample marked with photostable reference markers in a light microscope, wherein the examination comprises sequentially illuminating the sample with focused laser light at a plurality of illumination positions, and wherein the examination of the sample is repeatedly interrupted between the illumination at the illumination positions and the position of at least one reference marker is determined according to a MINFLUX method, as well as a light microscope for carrying out the method.

[0014] DE 10 2022 112 065 B3 describes a method for microscopic sample imaging, wherein a first image of a first sample is created using a first super-resolution light microscopy method with a resolution below the diffraction limit, wherein the first sample or a further second sample is subsequently expanded by an expansion factor, and wherein, after expansion, a second image of the expanded sample is created using a second light microscopy method with the same light microscope, wherein structural changes caused by the expansion are analyzed on the basis of a comparison between the first and the second image.

[0015] In MINFLUX and related localization methods, where the intensity distribution of the illuminating light is moved along continuous trajectories, photons emitted by an emitter in the sample during movement along the trajectory can be assigned to the corresponding position of the minimum of the intensity distribution along the trajectory to perform a position estimation. This requires precise knowledge of the current position along the trajectory.

[0016] However, in many types of scanning devices, e.g. galvanometer scanners, significant deviations occur between a position of the illuminating light specified by the controller and its actual position, especially at the high scanning speeds targeted in MINFLUX localization and tracking methods.

[0017] For example, with periodic scanning movements, which can be used to create circles and other closed paths, frequency-dependent attenuation (i.e., the specified scan amplitude is not achieved) and phase shifts (i.e., the actual position of the illumination focus lags behind the specified position) often occur. For example, in galvanometer scanners, both resonances of mechanical components and components of the scanner control feedback loop contribute to such deviations. Furthermore, the inertia of a deflection mirror can cause its movement to lag behind the movement of the rotation axis coupled to the mirror.

[0018] Even with non-periodic movements, e.g., rapid jumps between sample positions, both an error in the distance of the jump and a delay can occur.

[0019] Deviations between the target position and the actual position of the intensity distribution lead to an error in positioning, which generally increases with increasing scanning speed and / or scanning acceleration. In MINFLUX methods, the influence of such positioning errors is particularly significant due to the particularly high photon efficiency and the resulting lower number of detected photons, especially compared to conventional orbital tracking methods, in which the sample is scanned on continuous trajectories with a Gaussian focus of the illumination light.

[0020] Especially in mechanical scanning devices such as galvanometer scanners, a feedforward or closed-loop control is often implemented which adjusts the control signal based on position sensors attached directly to the mirror drives in order to correct position errors.

[0021] However, this type of feedforward control has the particular disadvantage that neither the deviation of the mirror movement from the axis movement nor the actual effect of the position errors in the sample is taken into account. Object of the invention

[0022] Therefore, it is the object of the present invention to improve localization or tracking methods in which an intensity distribution of illumination light with a local minimum is scanned over a sample in such a way that errors in the position determination of emitters in the sample are reduced. Solution

[0023] This object is achieved by the subject matter of the independent claims. Advantageous developments of the invention are specified in the subclaims and are described below. Description of the invention

[0024] A first aspect of the invention relates to a method for locating or tracking emitters in a sample, wherein the sample is illuminated in a measuring step with an intensity distribution of an illuminating light, wherein the illuminating light induces or modulates light emissions from emitters in the sample, wherein the intensity distribution has a local minimum, wherein the intensity distribution and the sample are moved relative to one another by means of a scanning device in a measuring scanning movement, wherein light emissions of a measuring emitter in the sample induced or modulated by the illuminating light are detected, and wherein a position of the measuring emitter is estimated on the basis of the detected light emissions and positions of the local minimum of the intensity distribution assigned to the light emissions.

[0025] According to the invention, the estimated position of the measuring emitter is corrected on the basis of calibration data as a function of a speed and / or an acceleration of the measuring scanning movement, wherein the calibration data comprise localization data of a calibration emitter obtained by means of at least one calibration scanning movement of the scanning device, and / or a control signal of the scanning device is adapted on the basis of calibration data, wherein the calibration data comprise localization data of a calibration emitter obtained by means of at least one calibration scanning movement of the scanning device.

[0026] In particular, the at least one calibration scanning movement has different speeds and / or accelerations, ie in particular one calibration scanning movement can have different speeds and / or accelerations or several calibration scanning movements can each have one speed and / or acceleration, wherein the speeds and / or accelerations are different.

[0027] Because the calibration is carried out according to the invention based on localization data from calibration emitters, the effect of the position errors occurring on errors in the localization of emitters in the sample can advantageously be directly corrected or compensated for by feedforward control for various scanning movements, taking into account the entire optical path between the light source and the sample as well as delays in the electronic control of the scanning device. In particular, deviations in the position of the mirror from the current position of the rotational axis coupled to the mirror, caused by the inertia of a deflection mirror, can be corrected or compensated for, which are not detected and thus not compensated for with conventional feedforward control or control based on position data recorded on the axis.

[0028] A method for localizing emitters within the meaning of this specification is characterized by the fact that the positions of individual emitters are computationally estimated in the localization or tracking step based on detected light emissions. This distinguishes such methods from conventional light microscopy imaging techniques, such as wide-field or scanning microscopy, which optically image a large number of emitters without determining the positions of individual emitters.

[0029] The term "measurement step" is used here only to distinguish it from a calibration step (see below) that may optionally be performed prior to this step. Beyond that, the term "measurement step" is not intended to imply any limitations. In particular, the measurement step may also comprise several sub-steps in which an emitter is located, e.g., iteratively, in the sample. It is also possible for several emitters to be located or tracked consecutively in the measurement step. Likewise, several measurement steps may, of course, be performed consecutively within the scope of the method according to the invention.

[0030] In this application, emitters (in particular measurement emitters and calibration emitters) are understood to be objects which, when illuminated with excitation light, can be regarded as point light sources with regard to the measurements according to the invention. The light emanating from the object acting as a point light source can, for example, be scattered light resulting from elastic scattering such as Rayleigh scattering or inelastic scattering such as Raman scattering, or it can be luminescent light, in particular fluorescent light. An emitter can therefore be, for example, a light-reflecting nanoparticle, a quantum dot, a fluorescent dye molecule (fluorophore), or a molecule or nanoparticle labeled with one or more fluorescent dye molecules.Depending on the size of the molecule and the distance between the fluorophores, a molecule labeled with multiple fluorophores or a nanoparticle labeled with multiple fluorophores can of course also have multiple emitters in the sense of the definition used here.

[0031] As mentioned at the beginning, "isolated" emitters are understood here to be emitters that can be optically separated from one another using light microscopy techniques. This can be achieved by a labeling density of the sample that results in an average distance between the emitters above the diffraction limit. Alternatively, asynchronously blinking emitters can be used by adjusting the sample conditions (in particular the composition of the sample buffer and embedding medium) so that the average distances between the light-emitting emitters are above the diffraction limit at all times. Finally, different emitters that are otherwise optically distinguishable can, under certain circumstances, also be separated if their distance is below the diffraction limit, e.g., based on their characteristic emission spectrum or their emission lifetime.

[0032] The intensity distribution and the sample are moved relative to each other by the scanning device. In this case, an illumination light beam can be displaced relative to the sample while the sample remains stationary; the sample can be moved (e.g., by a scanning device mechanically coupled to a sample holder) while the illumination light beam remains stationary; or both the illumination light beam and the sample can be moved in such a way that a relative movement between them results.

[0033] The light emissions are, in particular, individual photons or groups of photons. This can be, for example, luminescent light, especially fluorescent light, or reflected or scattered light. The light emissions can be detected, for example, with a photodiode (in particular an avalanche photodiode, APD) or a photomultiplier, or with a spatially resolving detector, such as a CCD or CMOS camera or a so-called APD array.

[0034] The illumination light can, in particular, be excitation light, which excites an emitter in the sample to emit light, i.e., induces light emission from the emitter. The light emissions can, in particular, be reflected light, scattered light, or luminescence light (e.g., fluorescent light). Alternatively, the illumination light can also modulate light emissions from the emitter. In this case, the illumination light can, for example, be STED light, which causes emitters to de-excite from an excited state to the ground state, or switching light, which converts emitters from an excitable state to a dark state.

[0035] In the measuring step, the sample is illuminated with an intensity distribution of the illumination light which has a local minimum. In particular, regions of intensity increase border the local minimum in at least one spatial direction. The local minimum is in particular a central minimum of the intensity distribution, i.e. it forms a center of the intensity distribution, whereby the center can in particular be arranged at the geometric focus. In this case, the light distribution can in particular be point-symmetric with respect to the geometric focus. The local minimum can in particular be at least approximately an intensity zero. Such intensity distributions include in particular a so-called donut beam and a so-called bottle beam.The intensity distribution can in particular also have two intensity maxima which are separated from one another by a plane of minimum intensity, in particular a zero plane, whereby the zero plane intersects the geometric focus, and whereby the zero plane can in particular be perpendicular to the focal plane, i.e. parallel to an optical axis of the objective. Such light distributions can be generated, for example, by phase modulation of the illuminating light in or near a pupil of an objective lens (for example using a phase plate or a spatial light modulator) and focusing into the sample using the objective lens. A donut can be generated, for example, by a so-called vortex phase pattern (also phase clock), a bottle beam by an annular phase jump (in particular with the phase difference π) and a light distribution with two maxima separated by a zero plane by a linear phase jump (in particular with the phase difference π).

[0036] The localization or tracking method according to the invention can, in particular, be a so-called MINFLUX method (when the illumination light with which the sample is illuminated in the localization step is excitation light that induces the light emissions of the emitters) or a STED-MINFLUX method (when the illumination light with which the sample is illuminated in the localization step is inhibition light that modulates the light emissions of the emitters). In a MINFLUX method or STED-MINFLUX method, the local minimum of the intensity distribution of the illumination light is placed at illumination positions in a near range of the approximate position of an isolated emitter, determined in particular in the at least one search step, and photons emitted by the emitter are detected for each illumination position. The near range can, in particular, have an extent on the order of the optical diffraction limit.The detected photon counts and the corresponding illumination positions can form input values for a position estimator (e.g., a least mean square estimator or a maximum likelihood estimator), which is then used to determine an updated position estimate for the emitter. This process can be repeated iteratively by placing the intensity distribution of the illumination light at updated illumination positions in a range close to the updated position estimate and, in turn, detecting photons for each position. In particular, a radius or an extension of the trajectory around the previously estimated position can be reduced. Optionally, the light intensity of the illumination light can also be increased. The iteration steps can be continued, for example, until the emitter stops emitting light or until a photon limit or a threshold of the localization precision is reached.In a MINFLUX or STED-MINFLUX tracking method, one or more iterations can be repeated at short intervals to track the trajectory of a moving emitter. Illumination patterns specifically tailored to a tracking method can be used.

[0037] The calibration data describe, at least implicitly, a dependency between the deviation between the target and actual position of the local minimum of the intensity distribution and the speed or acceleration of the scanning movement, which affects the position determination of the calibration emitter. Such a dependency between the deviation between the target and actual position and the speed and / or acceleration can, for example, be represented for each spatial coordinate as a diagram in which the speed or acceleration is plotted on the x-axis and the deviation is plotted on the y-axis. The dependency can be linear or non-linear. In particular, the higher the speed or acceleration, the greater the deviation between the target and actual position.For certain, particularly low, values of speed or acceleration, the deviation may also be zero or almost zero, with the deviation increasing significantly, particularly above a threshold of speed or acceleration.

[0038] Alternatively or in addition to the correction, a feedforward control can be implemented based on the calibration data, which ensures that the control signal is adjusted so that the actually controlled position corresponds as closely as possible to the target value. This feedforward control can, in particular, also be a pure phase feedforward control, i.e., for example, in the case of a circular movement of the intensity distribution, the feedforward control only occurs with respect to a spatial angular coordinate, but not, for example, with respect to the circle radius. If the circle radius does not fall below a lower threshold, such a feedforward control can be extremely advantageous for MINFLUX or STED-MINFLUX localization, especially in an iterative process, since here the estimation of the direction of the deviation between the center of the circle and the actual emitter position is crucial for fast and highly accurate position determination.

[0039] According to one embodiment, the calibration data describe a deviation of the estimated position of the calibration emitter from an actual position of the calibration emitter as a function of the speed and / or acceleration of the calibration scanning movement.

[0040] The deviation does not necessarily have to be determined along all spatial coordinates. For example, in a two-dimensional localization, only the deviation of the angular coordinate from the calibration data can be described, leaving the deviation of the radial coordinate undetermined. In this case, too, as explained above, a useful precontrol or correction can be performed if necessary.

[0041] The actual position of the calibration emitter can, for example, be determined using an independent method or be known in advance. Alternatively, the position of a calibration emitter can be determined by a calibration scanning movement with such a low speed and / or acceleration that there is no appreciable speed / acceleration-related deviation between the estimated position and the actual position of the calibration emitter. For example, during a calibration step for a stationary, bleach-insensitive emitter, e.g., a nanoparticle immobilized on a slide (reflective or labeled with fluorophores), a MINFLUX localization can be performed. In this process, an intensity distribution of the illuminating light with a local minimum is scanned by the scanning device in a relatively slow circular motion across the sample.

[0042] According to a further alternative, the actual position can be obtained, for example, from a very high-precision estimated localization, e.g., a MINFLUX localization, of the calibration emitter with constant scanning speed and / or scanning acceleration. The accuracy of this localization can be so high that the speed- and / or acceleration-dependent error becomes negligible. For example, the MINFLUX localization of the calibration emitter can be performed iteratively, with a scanning movement being performed in several iteration steps, resulting in a circular trajectory of the intensity distribution, and an estimated position being determined based on the detected light emissions. The circle center can, for example,can be placed at the position determined in the previous iteration step and the radius of the circle can be successively reduced, in particular until there is no more modulation of the light emission, so that it can be assumed that the calibration emitter is located in the center of the last circular path traveled.

[0043] In particular, the estimated position of the measuring emitter can be corrected based on the deviation between the measured position and the actual position of the calibration emitter, which is associated with the speed and / or acceleration occurring during the measuring scanning movement, or the control signal of the scanning device can be adjusted based on the deviation between the measured position and the actual position of the calibration emitter, which is associated with the speed and / or acceleration occurring during the measuring scanning movement. The speed and / or acceleration in question can correspond to the speed and / or acceleration of the calibration scanning movement when the calibration data is generated.Alternatively, the corresponding deviation between the actual position and the estimated position for a specific velocity and / or acceleration can be determined from calibration data, where the calibration data was obtained from calibration scan movements with different velocities and / or accelerations. A correction value or correction vector interpolated from the calibration data can then be used to correct the position.

[0044] According to a further embodiment, positions of the intensity distribution of the illumination light (that is to say in particular positions of the local minimum of the intensity distribution) in the sample are corrected on the basis of the calibration data, wherein the positions are assigned to light emissions of the measuring emitter.

[0045] By using the corrected position values of the intensity distribution (i.e., the minimum of the intensity distribution) to estimate the position of the measuring emitter, the estimated position of the measuring emitter can be corrected depending on the speed and / or acceleration of the measuring scanning movement.

[0046] According to a further embodiment, the correction of the estimated position is carried out by adjusting a position estimator for the measuring emitter based on the calibration data.

[0047] For example, at least one correction term for the position estimator can be formed based on the calibration data.

[0048] According to a further embodiment, the scanning device is designed to shift the intensity distribution of the illumination light so that the intensity distribution and the sample are moved relative to each other.

[0049] This may mean, in particular, that the scanning device is a beam scanning device that shifts an illuminating light beam of the illuminating light.

[0050] According to a further embodiment, the measuring scanning movement and the calibration scanning movement each comprise a periodic movement with at least one frequency.

[0051] The frequencies of the measurement scanning movement and the calibration scanning movement can be identical or different. For example, the measurement scanning movement can be a circular movement (particularly in the focal plane) or a back-and-forth movement along a line. The measurement scanning movement can therefore have a single, particularly constant, frequency. A circular movement or a back-and-forth movement can then also be used as a corresponding calibration scanning movement, with the calibration scanning movement being performed at multiple frequencies and the calibration emitter being localized in each case. The center of a circular path of the intensity distribution can, in particular, be offset from the actual position of the emitter so that the detected light emissions depend on the position of the intensity distribution on the circular path.During the calibration scan movement, the frequency can be continuously increased or decreased to record a frequency response of the localization deviation. In this way, a frequency-dependent phase and / or amplitude error can be corrected or compensated for by feedforward control.

[0052] According to a further embodiment, the dependence between a desired position and an actual position of the local minimum on the frequency of the scanning movement exists due to a dependence of a phase of the scanning movement and / or an amplitude of the scanning movement on the frequency.

[0053] Attenuation (amplitude change) and phase shift as a function of frequency can be represented, for example, in a so-called Bode plot. Such a frequency response can be known in advance, particularly for a scanning device. Nevertheless, the method according to the invention offers the possibility of determining the direct effect of attenuation and phase shift on the position estimation. This can be particularly advantageous when conditions of the specific experiment affect the frequency response in a way that is difficult to predict in advance. One example could be the heating of a galvanometer scanner during a scan.

[0054] In particular, the periodic movement can be a sine oscillation or a cosine oscillation with respect to a spatial coordinate.

[0055] The scanning movement can in particular be a combination of periodic movements, which is achieved, for example, by a combination of several scanners, e.g. by an x-scanner and a y-scanner, which shift the intensity distribution of the illuminating light in a focal plane that is perpendicular to an optical axis of an objective through which the illuminating light is focused. With the same frequencies and a phase shift of 90°, this results, for example, in a circular movement. In the case of different frequencies, more complex trajectories can result, e.g. Lissajous figures, epitrochoids or hypotrochoids. Under certain circumstances, the resulting attenuation and phase shift for such combinations of frequencies can also be carried out by linear addition of the attenuations or phase shifts for the individual frequencies, especially when harmonic oscillations are present.

[0056] In the case of a periodic scanning movement, the expected deviation can be estimated relatively well in advance, but the generation of the calibration data enables a direct reference to the respective planned experiment and allows a simple prediction of the effect of the scanning speed or scanning acceleration on the position estimation, which, particularly in the case of galvo scanners, also takes into account deviations between mirror movement and rotation axis movement due to mass inertia.

[0057] According to a further embodiment, the measurement scanning movement comprises a superposition of movements of several different frequencies, wherein the at least one calibration scanning movement has the different frequencies. The fact that the at least one calibration scanning movement has the different frequencies can mean, in particular, that a calibration scanning movement has several frequencies (e.g., in the case of a Lissajous figure, an epitrochoid, or a hypertrochoid) or that several calibration scanning movements are carried out to generate the calibration data, wherein each of the calibration scanning movements has a respective frequency and the frequencies differ from one another (e.g., two circular paths with different frequencies).

[0058] As explained further below, this embodiment advantageously allows for improved position estimation even for more complex orbits than, for example, circular orbits. Examples of such more complex circular orbits are Lissajous figures, epitrochoids, and hypotrochoids.

[0059] According to a further embodiment, the calibration scanning movement is identical to the measurement scanning movement.

[0060] For the embodiment described above, this means that the calibration scanning movement is also characterized by a superposition of vibrations with several frequencies, particularly in each spatial direction.

[0061] According to a further embodiment, the calibration scanning movement and the measuring scanning movement differ from one another, wherein the calibration scanning movement can in particular comprise partial movements of the measuring scanning movement and / or wherein a plurality of calibration scanning movements are assigned to one measuring scanning movement.

[0062] According to a further embodiment, the calibration data are obtained based on at least two calibration scanning movements with different frequencies. The calibration scanning movements can, for example, be circular paths with different frequencies.

[0063] In the above case, for example, the calibration scan movement can be performed separately for the different spatial directions.

[0064] According to a further embodiment, the different frequencies of the measuring scanning movement are assigned to a movement in the same spatial direction. This means, in particular, that the measuring scanning movement has a sum or a difference of sine or cosine oscillations in at least one spatial direction, in particular in at least two spatial directions. If superpositions of sine or cosine oscillations are present in two spatial directions (e.g., x and y), two-dimensional paths in the form of hypotrochoids or epitrochoids, for example, can result.

[0065] According to a further embodiment, the different frequencies of the measuring scanning movement are each assigned to movements in different, in particular orthogonal, spatial directions. This can mean, in particular, that only one oscillation with a single frequency is carried out in each spatial direction, although a different frequency is present in one spatial direction than in the other. In this way, it is possible, for example, for two superimposed movements in the x and y directions to generate a two-dimensional trajectory in the form of a Lissajous curve. The scanning movements in the different spatial directions can be implemented, in particular, using independent scanners, e.g., an x-galvo scanner and a y-galvo scanner. In this case, it is generally necessary to pre-control or regulate the phase offset of the two scanners so that the desired trajectory of the intensity distribution is generated. This control orControl can be performed according to the method according to the invention using the calibration data. Alternatively or additionally, phase precontrol or control can also be carried out, for example, based on data from position sensors or position encoders of the scanning device. Control or control using the method according to the invention can be particularly advantageous if the two scanners heat up to different degrees due to the different scanning frequencies, which can lead to a relative phase deviation, due to which the generated trajectory of the intensity distribution in the sample can differ significantly from the desired trajectory.

[0066] Even in the described case of different frequencies in different spatial directions, the calibration scan movement can be identical to or different from the measurement scan movement. If the calibration scan movement differs from the measurement scan movement, the measurement scan movement can, for example, generate a path in the form of a Lissajous curve, and the calibration scan movement can be a circular path that is traversed at at least two different frequencies, for example, at the two frequencies of the Lissajous curve (in particular, also at other frequencies, e.g., in a continuous frequency sweep).

[0067] According to a further embodiment, the measuring scanning movement comprises a continuous, in particular closed, path of the minimum of the intensity distribution of the illumination light.

[0068] This means that according to this embodiment, unlike some MINFLUX methods known from the prior art, several illumination positions are not controlled in a step-by-step manner and light emissions are detected for each illumination position with a stationary intensity distribution. Rather, the intensity distribution is constantly in motion. During the motion, light emissions are detected and, particularly during the experiment, optionally also after the experiment has ended, are assigned to a position of the minimum of the intensity distribution using sampling / binning.

[0069] A closed orbit can be, for example, a circular orbit, a Lissajous figure, an epitrochoid, or a hypotrochoid. In particular, a center of the orbit can be given by the currently estimated position of an isolated mass emitter.

[0070] In some embodiments, a measuring emitter can be localized with sufficiently high accuracy simply by moving the intensity distribution on a single path and detecting the light emissions. However, it is often necessary to traverse a path multiple times, e.g., to capture a sufficient number of light emissions. Furthermore, in many cases it is necessary to use different paths to localize or track a measuring emitter. For example, in an iterative MINFLUX localization, the center of a circular path can be set to the position estimated in the previous iteration step in each iteration step and, in particular, the radius of the path can be successively reduced. Even when tracking over longer distances, it is particularly necessary to adjust the center of a path to the movement of a measuring emitter.

[0071] If the measuring scanning movement comprises a, in particular closed, continuous path, the at least one calibration scanning movement can also be a, in particular closed, continuous path or comprise at least one such continuous path.

[0072] According to a further embodiment, the scanning movement includes a jump. This means that in this case, a continuous path is not desired, but rather the minimum of the intensity distribution is to be moved discontinuously at the highest possible speed between two points in the sample.

[0073] The term “jump” refers in particular to a trajectory of the intensity distribution in the sample, the decomposition of which into harmonic functions (sine or cosine functions) has relative components (weights of a sum of sine or cosine functions) that change over time during the scanning movement.

[0074] Accordingly, a “continuous trajectory” is understood to mean, in particular, a trajectory whose decomposition into harmonic functions has temporally constant relative components during the scanning movement.

[0075] In contrast to continuous trajectories, the effects of scanning speed or scanning acceleration on the position error in jumps are particularly difficult to predict. In particular, these effects cannot be derived from a known frequency response of the scanner. Therefore, the inventive generation of calibration data is particularly advantageous in this case.

[0076] In particular, the scanning movement may include a sudden transition between two paths.

[0077] A scanning motion with jumps can also occur, especially when tracking emitters. For example, it may be necessary to shift the center of a given path abruptly before a complete, closed path has been traversed.

[0078] To obtain the calibration data, for example, a sample containing a calibration emitter at a known position can be scanned with a variety of different scan patterns containing jumps, while the light emissions of the calibration emitter are recorded. The obtained localization data of the calibration emitter can then be stored, for example, in a lookup table for the respective scan patterns and used for correction or feedforward control when using a corresponding scan pattern in the measurement step.

[0079] According to a further embodiment, the determination of the calibration data or the adaptation of the control of the scanning device based on the calibration data comprises a wavelet transformation. In this case, for example, a trajectory or a scan pattern consisting of several trajectories can be decomposed into a superposition of different basic functions or wavelets. In a calibration step, for example, scan patterns corresponding to the basic functions can then be tested on a calibration emitter and the corresponding locations of the calibration emitter can be determined. For a more complex trajectory containing several basic functions, the resulting deviation between the actual and estimated emitter position can then be determined mathematically on the basis of the calibration data, e.g. by weighted (vector) addition of correction vectors assigned to the individual basic functions (wavelets).

[0080] According to a further embodiment, the method comprises a calibration step, in particular carried out before the measuring step, wherein the calibration data are generated in the calibration step.

[0081] According to one embodiment, in the calibration step, a position of a calibration emitter fixed in a sample is determined several times, wherein the sample is illuminated several times with different speeds and / or accelerations of the calibration scanning movement, in particular on a continuous path, with the intensity distribution of the illumination light, wherein the light emissions of the emitter induced or modulated by the illumination light are detected in each case, and wherein a position of the calibration emitter is estimated in each case on the basis of the detected light emissions and the positions of the minimum of the intensity distribution assigned to the light emissions.

[0082] A simple example of this embodiment is the repeated traversing of a circular path with increasing frequency in the vicinity of a stationary calibration emitter with a known position in the sample and the localization of the calibration emitter based on the detected light emissions. The center of the circular path is offset from the actual emitter position so that the light emission from the calibration emitter is modulated while traversing the circular path. As the frequency increases, a systematic deviation of the determined position from the actual position of the calibration emitter occurs, particularly with respect to an angular coordinate (if the position of the calibration emitter is considered in polar coordinates).

[0083] According to a further embodiment, the calibration data are stored after completion of the calibration step, wherein the correction of the position estimate or the adaptation of the control signal is carried out on the basis of the stored calibration data.

[0084] According to a further embodiment, the calibration step is carried out on a calibration sample which contains calibration emitters which are stationary with respect to the calibration sample, wherein the position of a calibration emitter in the calibration sample is estimated several times in the calibration step for generating the calibration data.

[0085] According to a further embodiment, the calibration step is carried out on the same sample, in particular immediately before the measuring step, wherein the calibration data generated in the calibration step are used in the measuring step to correct the position estimate or to adapt the control signal.

[0086] With such a live calibration, the correction of the position determination or the feedforward control can be adapted particularly well to the conditions of the current experiment.

[0087] According to a further embodiment, several measurement steps are performed consecutively for different measuring emitters, with a calibration step being performed between two measurement steps. For example, a calibration step can be performed before each measurement step, or calibration steps can be performed at specific times during the measurement sequence (e.g., after a predetermined number of measurement steps or at irregular intervals).

[0088] According to another embodiment, the calibration emitter belongs to the same species as the measurement emitter. This has the advantage that the calibration data reflect the measurement conditions particularly well.

[0089] According to a further embodiment, the calibration emitter is stationary in the sample and optically separable from measuring emitters to be located.

[0090] According to a further embodiment, the position of a calibration emitter that is optically separable from the measuring emitter to be located or tracked and stationary in the sample is determined multiple times (in particular on different paths or with different scan patterns and / or with changing speed and / or acceleration). The calibration emitter can, for example, be chemically immobilized on a sample carrier such as a microscope slide or a coverslip and / or embedded in a viscous embedding medium. Because the calibration emitter is optically separable from the emitter to be located or tracked, calibration can be carried out independently. "Optically separable" can in particular mean isolated in the sense of the definition given above. With the described embodiment, a live calibration that is particularly well adapted to the measurement conditions can advantageously be carried out.In this case, too, the calibration emitter can belong to the same species or a different species than the measurement emitter. The use of an emitter in the sample as a calibration emitter does not preclude the subsequent localization of this emitter as a measurement emitter, provided it is of the same emitter species. This can be the case, for example, when localizations of a large number of measurement emitters are performed consecutively to determine a high-resolution localization map.

[0091] Furthermore, in the embodiment described above, it is also possible to first carry out a calibration step with a calibration emitter, in which the calibration emitter is localized with high precision at a constant scanning speed and / or scanning acceleration, for example using a MINFLUX method, in order to obtain the actual position of the calibration emitter and is then re-located with a changed speed and / or acceleration (in particular using a circular path of the intensity distribution whose center is offset from the actual emitter position) in order to obtain the speed- and / or acceleration-dependent position error (i.e. the deviation between the estimated position and the actual position at a given speed and / or acceleration).In particular, if the calibration emitter is an emitter that marks sample structures of interest, it may be useful to use not only the localization data of different measuring emitters obtained in several measuring steps but also the highly accurate localization data of the calibration emitter from the calibration step for the measurement carried out, e.g. to generate a localization map of the sample, thus treating the calibration emitter like a measuring emitter.

[0092] According to a further embodiment, a high-resolution image of structures in the sample is determined based on position estimates of multiple measuring emitters in the sample. For example, a so-called localization map can be created from a plurality of determined positions of individual measuring emitters. This map resembles a conventional light microscopic image, but, particularly when using a MINFLUX method, can have a resolution in the single-digit nanometer range, for example.

[0093] According to a further embodiment, a trajectory of the measuring emitter is determined based on several sequentially obtained position estimates of the same measuring emitter moving within the sample. A trajectory is a path of sequentially measured locations of a moving measuring emitter.

[0094] A second aspect of the invention relates to a light microscope for locating or tracking emitters in a sample, in particular according to a method according to the first aspect.The light microscope has at least the following components: a light source designed to generate illumination light that induces or modulates light emissions from emitters in the sample, a light modulator designed to generate an intensity distribution of the illumination light with a local minimum in the sample, a scanning device designed to move the intensity distribution of the illumination light and the sample relative to one another in a measuring scanning movement, a control unit designed to control the scanning device, a detector designed to detect light emissions of a measuring emitter that are induced or modulated by the illumination light, and a computing unit designed to estimate a position of the measuring emitter based on the detected light emissions and positions of the local minimum of the intensity distribution associated with the light emissions.According to the invention, the computing unit is configured to correct the estimated position of the emitter based on calibration data as a function of a speed and / or an acceleration of the measuring scanning movement of the scanning device. Alternatively or additionally, the control unit is configured to adapt a control signal of the scanning device based on calibration data as a function of a speed and / or an acceleration of the measuring scanning movement of the scanning device. The calibration data comprise localization data of a calibration emitter obtained at different speeds and / or accelerations of at least one calibration scanning movement of the scanning device.

[0095] According to one embodiment of the light microscope, the scanning device comprises at least one galvo scanner, at least one resonant mechanical scanner, at least one MEMS (micro electro mechanical system) scanner or at least one Risley scanner.

[0096] A Risley scanner is a mechanical scanner with so-called Risley prism pairs, whereby the illuminating light beam passes successively through two wedge-shaped prisms that rotate around parallel axes of rotation.

[0097] According to a further embodiment, the scanning device comprises at least a first scanner and a second scanner, wherein the first scanner is designed to displace the illumination light relative to the sample along a first spatial coordinate, wherein the second scanner is designed to displace the illumination light relative to the sample along a second spatial coordinate, wherein the first spatial coordinate and the second spatial coordinate are non-parallel, in particular perpendicular to one another.

[0098] According to a further embodiment, the first spatial coordinate and the second spatial coordinate are perpendicular to an optical axis of an objective through which the sample is illuminated with the illumination light.

[0099] The scanning device can therefore comprise, for example, at least one x-scanner and at least one y-scanner, as is common in galvanometer scanning devices.

[0100] In particular, calibration data for the first scanner and the second scanner can then be generated, which can then be used to correct the corresponding coordinate of the position estimate or to pre-control the corresponding scanner.

[0101] According to a further embodiment, the first scanner and the second scanner are designed to carry out scanning movements at different frequencies, in particular wherein the control unit is designed to adapt control signals of the first scanner and the second scanner on the basis of respective calibration data generated for the first scanner and the second scanner.

[0102] By using different frequencies, more complex paths, such as Lissajous figures, hypotrochoids or epitrochoids, can be realized.

[0103] A third aspect of the invention relates to a computer program comprising instructions which cause the light microscope according to the second aspect to carry out the method according to the first aspect.

[0104] Further, in particular functional, features of the light microscope according to the second aspect and of the computer program according to the third aspect result from the features of the method according to the first aspect.

[0105] Advantageous developments of the invention will become apparent from the patent claims, the description, the drawings, and the accompanying explanations of the drawings. The described advantages of features and / or combinations of features of the invention are merely exemplary and may be effective alternatively or cumulatively.

[0106] With regard to the disclosure content (but not the scope of protection) of the original application documents and the patent, the following applies: Further features can be found in the drawings - in particular the relative arrangements and operative connections shown. The combination of features of different embodiments of the invention or of features of different patent claims is also possible, deviating from the selected references of the patent claims, and is hereby suggested. This also applies to features that are shown in separate drawings or mentioned in their description. These features can also be combined with features of different patent claims. Likewise, features listed in the patent claims can be omitted for further embodiments of the invention, but this does not apply to the independent patent claims of the granted patent.

[0107] The reference signs contained in the patent claims do not represent a limitation of the scope of the subject-matter protected by the patent claims. They serve only the purpose of making the patent claims easier to understand.

[0108] In the following, exemplary embodiments of the invention are described with reference to figures. These do not limit the subject matter of this disclosure or the scope of protection. Short description of the characters Fig. 1 schematically shows an example of a continuous circular path of an intensity distribution of an illuminating light with which a sample can be illuminated in the measuring step according to the invention; Fig. Figure 2 shows schematically an example of a temporal sequence of detected light emissions from an emitter; Fig. 3 schematically shows another example of a continuous trajectory of an intensity distribution of an illuminating light with which a sample can be illuminated in the measuring step according to the invention, in the form of a Lissajous figure; Fig. 4 schematically shows the implementation of a calibration step according to an embodiment of the method according to the invention; Fig. 5 shows an example of a possible dependency between scanning frequency and deviation between actual position and estimated position; Fig. 6 shows an embodiment of a light microscope with which the method according to the invention can be carried out. Description of the characters

[0109] Fig. 1 shows a region of a sample 2 with an isolated measuring emitter M, wherein the actual position of the measuring emitter M is marked with a solid star. An estimated position G resulting from a relatively inaccurate pre-localization is, in contrast, represented by a dashed star. During the measuring step of an exemplary MINFLUX localization or tracking method according to the invention, the sample 2 is illuminated with an intensity distribution of an illuminating light B with a local minimum by an objective 8 focusing the illuminating light B (see Fig. 6). The intensity distribution can, for example, be a so-called donut, which can be obtained by phase modulating the illumination light B with a vortex-shaped phase pattern. The illumination light B can, for example, be excitation light that excites the measuring emitters M in sample 2 to fluoresce.

[0110] The intensity distribution of the illumination light B is determined by deflecting the illumination light beam using a scanning device 6 (see Fig. 6) with its local minimum on a continuous path 20 in sample 2. In the example according to Fig. 1, the orbit 20 is a circular orbit whose center is the estimated position G of the measuring transmitter M.

[0111] Fig. Figure 3 shows another example of a continuous trajectory 20 of the intensity distribution, a Lissajous figure with a frequency ratio of 2:1.

[0112] During the movement of the intensity distribution on the track 20, light emissions L of the measuring emitter M are detected by a detector 10 (see Fig. 6) are detected and recorded in a temporally resolved manner. The light emissions L can be, for example, fluorescence photons.

[0113] Fig. Figure 2 shows a temporal distribution of individual detections of light emissions L in a time window T. Detection times are therefore available for the individual light emissions L.

[0114] For each of these detection times, a corresponding position can be determined on the basis of the known path 20, at which the local minimum of the intensity distribution of the illumination light B was located at the time of the corresponding light emission L.

[0115] The obtained values can be used as input values of a position estimation algorithm, which then estimates the position of the measuring emitter M based on the light emissions L.

[0116] However, a source of error in the position estimation arises from the fact that the scanning device 6 (see Fig. 6), especially at high scanning speeds (e.g., at high frequencies of periodic movement and rapid aperiodic position jumps), the sensor does not perfectly follow the control signal, but rather scans with smaller amplitudes (attenuation) and lags behind the control signal (phase shift). Another source of error can arise, for example, from the inertia of a deflection mirror, which does not perfectly follow the movement of the scanner's rotational axis depending on the rotational frequency.

[0117] Therefore, the position of the intensity distribution at which a light emission L was detected cannot be determined unambiguously for each scanning speed.

[0118] According to the invention, in a calibration step carried out before the measuring step, calibration data are collected, which comprise localization data of a calibration emitter K obtained at different speeds and / or accelerations of at least one calibration scanning movement of the scanning device 6. Based on the calibration data, the estimated position of the measuring emitter M can then be corrected and / or a control unit 7 of the scanning device 6 (see Fig. 6) a feedforward control is implemented based on the calibration data, which takes the deviation into account.

[0119] An example of such a calibration step is shown in Fig. 4. In the calibration step, a calibration scanning movement is performed, in which a circular path 20 of the local minimum of the intensity distribution near the actual position of a stationary calibration emitter K (filled symbol) is traversed at different scanning frequencies, of which at least one is also to be used in the measuring step, the corresponding light emissions L of the calibration emitter K are detected, and the position of the calibration emitter K is estimated. Depending on the scanning frequency, a deviation results between the actual position of the calibration emitter K and the respective estimated positions G1, G2, G3, which are indicated for three exemplary frequencies by the dashed symbols. In the example shown, only the measured angular coordinate depends on the frequency, while the radial coordinate of the emitter remains constant with respect to the center of the path 20.

[0120] The actual position of the calibration emitter K can be determined in the calibration step, for example, by a highly accurate iterative MINFLUX localization.

[0121] The determined relationship between frequency f and deviation A is shown in the diagram of the Fig. 5. In the example shown, the relationship is linear, but non-linear relationships are of course also possible.

[0122] From such results, a correction vector for the position of the intensity distribution in the sample can be determined, for example, for a given frequency. This correction vector corrects the deviation between the target and actual positions of the intensity distribution for the corresponding frequency.

[0123] Using such correction vectors, for each light emission L of the emitter E in the measurement step (see e.g. Fig. 1 and Fig. 3) the corresponding actual position of the intensity distribution is determined and taken into account in a position estimator for the measuring emitter in order to correct the estimated position of the emitter. Alternatively, a correction term for a position estimator can be derived directly from the calibration data. Another possibility is to adjust the control signals of the scanning device 6 based on the calibration data so that the actual position of the intensity distribution corresponds to the target position as closely as possible at all times.

[0124] Fig. 6 shows an embodiment of a light microscope 1 according to the invention.

[0125] The light microscope 1 comprises an illumination optics 4 with a light source 3 (in particular a laser) for generating a light beam of the illumination light B and an objective 8 for focusing the illumination light beam into a sample 2. The illumination optics 4 also has a light modulator 5 for phase modulating the illumination light beam, wherein the phase modulation after focusing the illumination light B into the sample 2 creates an intensity distribution with a local minimum. The light modulator 5 can be, for example, a phase plate or a so-called spatial light modulator (SLM) with controllable pixels. This can be used not only in transmission mode (as in Fig. 6) but can also be used alternatively in reflection or diffraction mode.

[0126] The illumination light B is in particular excitation light, which excites emitters in the sample to fluoresce.

[0127] The illumination light B passes between the light modulator 5 and the objective 8 through a beam splitter 9 and a scanning device 6, e.g. a galvanometer scanning device with at least two scanning mirrors each coupled to a galvo drive, which deflect the illumination light B in two orthogonal directions (x and y directions) perpendicular to an optical axis O of the objective 8.

[0128] The scanning device 6 is designed to measure the intensity distribution of the illumination light B on a continuous path 20 (see Fig. 2 and Fig. 3) in sample 2.

[0129] The light emissions L of individual emitters (e.g. measuring emitter M or calibration emitter K) in the sample 2, excited or modulated by the illumination light B, are bundled by the objective 8, descanned by the scanning device 6 and, due to the wavelength of the emission light, reflected by the beam splitter 9 into a detection beam path.

[0130] A detector 10, e.g. an avalanche photodiode, is arranged in the detection beam path, optionally with a detection pinhole (not shown) in front for confocal detection.

[0131] The detector 10 is designed to detect the light emissions L of individual emitters (measurement emitter M and / or calibration emitter K) in the sample 2 in a time-resolved manner.

[0132] The detector 10 is connected to a computing unit 11 which is designed to assign the detected light emissions L to respective positions of the intensity distribution in the sample 2 on the basis of the detection times and to estimate the position of an emitter (measurement emitter M and / or calibration emitter K) in the sample 2 from the light emissions L and the associated positions of the intensity distribution.

[0133] The computing unit 11 is connected to a control unit 7, which controls the scanning device 6.

[0134] According to the invention, calibration data can be recorded with the light microscope 1, which comprise localization data of a calibration emitter K obtained at different speeds and / or accelerations of at least one calibration scanning movement of the scanning device 6.

[0135] The computing unit 11 may be configured to correct the position data (i.e., the positions of the intensity distribution in the sample 2 at the time of detection of the light emissions and / or the estimated position of the measuring emitter M) on the basis of the calibration data.

[0136] Alternatively or additionally, the control unit 7 can be designed to carry out a pre-control or regulation of the scanning device 6 on the basis of the calibration data. List of reference symbols 1 light microscope 2 Sample 3 Light source 4 Lighting optics 5 Light modulator 6 Scanning device 7 Control unit 8 lens 9 beam splitters 10 Detector 11 Computing unit 20 lanes B Illumination light K Calibration emitter L Light emissions M Messemitter O Optical axis T time window G,G1,G2,G3 Estimated Position

Claims

[1] A method for locating or tracking emitters in a sample (2), wherein the sample (2) is illuminated in a measuring step with an intensity distribution of illuminating light (B), wherein the illuminating light (B) induces or modulates light emissions (L) from emitters in the sample (2), wherein the intensity distribution has a local minimum, and wherein the intensity distribution and the sample (2) are moved relative to one another by means of a scanning device (6) in a measuring scanning movement, wherein light emissions (L) of a measuring emitter (M) in the sample (2) induced or modulated by the illuminating light (B) are detected, and wherein a position of the measuring emitter (M) is estimated on the basis of the detected light emissions (L) and the positions of the local minimum of the intensity distribution associated with the light emissions (L). characterized bythat, depending on a speed and / or an acceleration of the measuring scanning movement, the estimated position of the measuring emitter (M) is corrected on the basis of calibration data and / or a control signal of the scanning device (6) is adapted on the basis of calibration data, wherein the calibration data comprise localization data of a calibration emitter (K) obtained by means of at least one calibration scanning movement of the scanning device (6). [2] Method according to claim 1, characterized by that the calibration data describe a deviation of the estimated position of the calibration emitter (K) from an actual position of the calibration emitter (K) as a function of the speed and / or acceleration of the calibration scanning movement. [3] Method according to claim 1 or 2, characterized bythat positions of the intensity distribution of the illumination light (B) in the sample (2) are corrected on the basis of the calibration data, the positions being assigned to light emissions of the measuring emitter (M). [4] Method according to claim 1 or 2, characterized by that the correction of the estimated position of the measuring emitter (M) is carried out by adjusting a position estimator for the measuring emitter (M). [5] Method according to one of the preceding claims, characterized by that the scanning device (6) is designed to shift the intensity distribution of the illumination light (B) so that the intensity distribution and the sample (2) are moved relative to one another. [6] Method according to one of the preceding claims, characterized by that the measuring scanning movement and the at least one calibration scanning movement each comprise a periodic movement with at least one frequency. [7] Method according to claim 6, characterized bythat the measuring scanning movement has a superposition of movements of several different frequencies, wherein the at least one calibration scanning movement has the different frequencies. [8] Method according to claim 7, characterized by that the different frequencies of the measuring scanning movement are assigned to a movement in the same spatial direction. [9] Method according to claim 7, characterized by that the different frequencies of the measuring scanning movement are each assigned to movements in different, in particular orthogonal, spatial directions. [10] Method according to one of claims 7 to 9, characterized by that the calibration data are obtained based on at least two calibration scan movements with different frequencies. [11] Method according to one of the preceding claims, characterized by that the calibration scan movement and the measurement scan movement are different from each other. [12] Method according to one of claims 1 to 11, characterized by that the measuring scanning movement comprises a continuous, in particular closed, path (20) of the minimum of the intensity distribution of the illumination light (B). [13] Method according to one of claims 1 to 11, characterized by that the measuring scanning movement comprises a jump, in particular wherein the scanning movement comprises a jump-like transition between two closed paths (20). [14] Method according to one of the preceding claims, characterized by that the determination of the calibration data or the adaptation of the control of the scanning device (6) on the basis of the calibration data comprises a wavelet transformation. [15] Method according to one of the preceding claims, characterized by that the method comprises a calibration step, wherein the calibration data are generated in the calibration step. [16] Method according to claim 15, characterized bythat the calibration data are saved after completion of the calibration step, whereby the correction of the position estimate or the adjustment of the control signal is carried out on the basis of the saved calibration data. [17] Method according to claim 15, characterized by that the calibration step is carried out before the measuring step on the same sample (2), wherein the calibration data generated in the calibration step are used in the measuring step to correct the position estimate or to adapt the control signal. [18] Method according to claim 17, characterized by that the calibration emitter (K) is stationary in the sample and optically separable from the measuring emitters (M) to be located. [19] Method according to one of the preceding claims, characterized by that the calibration emitter (K) belongs to the same species as the measuring emitter (M). [20] Method according to one of the preceding claims, characterized bythat a high-resolution image of structures in the sample (2) is determined on the basis of position estimates of several measuring emitters (M) in the sample (2) or that a trajectory of the measuring emitter (M) is determined on the basis of several position estimates obtained one after the other of the same measuring emitter (M) moving in the sample (2). [21] Light microscope (1) for locating or tracking emitters in a sample (2), in particular according to a method according to one of claims 1 to 20, comprising - a light source (3) designed to generate illumination light (B) that induces or modulates light emissions (L) from emitters in the sample (2), - a light modulator (5) designed to generate an intensity distribution of the illumination light (B) with a local minimum in the sample (2), - a scanning device (6) which is designed to move the intensity distribution of the illumination light (B) and the sample (2) relative to each other in a measuring scanning movement, - a control unit (7) designed to control the scanning device (6), - a detector (10) designed to detect light emissions (L) of a measuring emitter (M) induced or modulated by the illuminating light (B), - a computing unit (11) designed to estimate a position of the measuring emitter (M) on the basis of the detected light emissions (L) and the positions of the local minimum of the intensity distribution associated with the light emissions (L), characterized bythat the computing unit (11) is designed to correct the estimated position of the measuring emitter (M) on the basis of calibration data as a function of a speed and / or an acceleration of the measuring scanning movement, and / or that the control unit (7) is designed to adapt a control signal of the scanning device (6) on the basis of calibration data as a function of a speed and / or an acceleration of the measuring scanning movement, wherein the calibration data comprise localization data of a calibration emitter (K) obtained by means of at least one calibration scanning movement of the scanning device (6). [22] Light microscope (1) according to claim 21, characterized by that the scanning device (6) comprises a galvo scanner, a resonant mechanical scanner, a MEMS scanner or a Risley scanner. [23] Light microscope (1) according to claim 21 or 22, characterized bythat the scanning device (6) has at least a first scanner and a second scanner, wherein the first scanner is designed to displace the illumination light (B) relative to the sample (2) along a first spatial coordinate, wherein the second scanner is designed to displace the illumination light (B) relative to the sample (2) along a second spatial coordinate, and wherein the first spatial coordinate and the second spatial coordinate are non-parallel, in particular perpendicular to one another. [24] Light microscope (1) according to claim 23, characterized by that the first spatial coordinate and the second spatial coordinate are perpendicular to an optical axis (O) of the objective (8). [25] Light microscope (1) according to claim 23 or 24, characterized bythat the first scanner and the second scanner are designed to carry out scanning movements at different frequencies, wherein the control unit (7) is designed to adapt control signals of the first scanner and the second scanner on the basis of respective calibration data generated for the first scanner and the second scanner. [26] Computer program comprising instructions which cause the light microscope (1) according to one of claims 21 to 25 to carry out the method according to one of claims 1 to 20.

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