Method, light microscope and computer program for locating or tracking individual emitters in a sample

Abstract

The invention relates to a method for locating or tracking individual emitters in a sample (2) by means of a light microscope (1), comprising illuminating the sample (2) with illumination light (B), wherein an intensity distribution of the illumination light (B) with a local intensity minimum is formed in the sample (2), and wherein the illumination light induces or modulates light emissions of an individual emitter in the sample (2); detecting light emissions of the individual emitter and determining position data of the individual emitter on the basis of the detected light emissions, wherein on the basis of the detected light emissions a wavefront of the illumination light is adjusted and / or a beam path of an illumination light beam (B) of the illumination light and a beam path of a detection light beam (D) of the light emissions are adjusted relative to one another and / or a beam path of the illumination light beam (B) and a beam path of a further illumination light beam are adjusted relative to one another. The invention also relates to a light microscope (1) and to a computer program for carrying out the method.

Description

[0001] Method, light microscope and computer program for locating or tracking individual emitters in a sample

[0002] Technical field of the invention

[0003] The invention relates to a method, a light microscope and a computer program for locating or tracking individual emitters in a sample, in particular according to the MINFLUX principle.

[0004] State of the art

[0005] The term "MINFLUX microscopy" or "MINFLUX method" refers to a family of localization and tracking methods for individual light-emitting emitters in which a light distribution of illuminating light that induces or modulates light emissions from the emitter is generated at the focus in the sample, wherein the light distribution has a local minimum in at least one spatial direction, and in which the position of an individual emitter is determined by detecting light emissions from the emitter.

[0006] The patent application DE 10 2013 114 860 A1 describes in particular a localization method in which the sample is scanned at grid points with the local minimum of an excitation light distribution in order to localize individual fluorophores.

[0007] The term "MINFLUX" was used for the first time in the publication "F. Balzarotti et al., "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes", Science 355 (6325), 606-612 (2017)". There, the MINFLUX principle described above is implemented in concrete terms. A single fluorophore is first pre-localized by scanning with a first Gaussian excitation light distribution and then a second, donut-shaped excitation light distribution is placed at points that form a symmetric pattern of illumination positions around the fluorophore position estimated in the pre-localization. From the photon numbers registered for the individual illumination positions, the position of the fluorophore is then determined to within a few nanometers using a maximum likelihood estimator.

[0008] Further variants and embodiments of MINFLUX localization are described in the patent applications DE 10 2016 119 262 A1, DE 10 2016 119 263 A1 and DE 10 2016 119 264 A1. The publication "KC Gwosch et al., "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells", Nat. Methods, 17 (2), 217-224 (2020)" describes iterative 2D and SDMI NFLUX localization methods. In several iterative steps, the sample is illuminated at illumination positions with the minimum of a donut-shaped excitation light distribution. The illumination positions form a symmetric illumination pattern centered around the fluorophore position estimated in the previous step. In each iterative step, the illumination positions are placed more closely around the currently estimated fluorophore position. This allows for very high positioning accuracy to be achieved in just a few steps.

[0009] Another iterative MINFLUX localization and tracking method using a modified position estimator and based on a commercial microscope setup is described in “R. Schmidt et al., “MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope”, Nat. Commun. 12 (1), 1478 (2021)”.

[0010] The light that induces or modulates the light emission of the particles can also be STED (stimulated emission cfep / et / on) light, for example. Patent applications DE 10 2017 104 736 A1 and EP 3 372 989 A1 describe MINFLUX-like methods based on the superposition of an excitation light distribution with a local maximum with a STED light distribution with a local minimum. The sample is scanned by shifting the STED distribution with the STED minimum, and the position of the fluorophore is determined from the measured fluorescence intensity values ​​at different positions of the STED intensity distribution.

[0011] US 2019 / 0195800 A1 describes an adjustment method for a laser scanning microscope in which, in particular, an offset between raster images of a sample acquired with different STED intensities is determined, and based on the offset, a STED light distribution at the focus is adjusted relative to an excitation light distribution, or in which an offset is determined between raster images acquired with a pinhole aperture opened to different widths in the detection beam path, wherein a position of the pinhole aperture is adjusted based on the determined offset.

[0012] US 2015 / 0226950 A1 describes a STED microscopy method and a corresponding microscope with which a phase pattern displayed on a spatial light modulator is adjusted using an image quality metric to correct aberrations of the STED light beam and, optionally, the excitation light beam. The metric is based on an image sharpness parameter and an image brightness parameter. In the multi-color MINFLUX method according to WO 2022 / 112155 A1, individual emitters are localized using light of different wavelengths, and a difference between the obtained localizations is determined, which can then be used, for example, for co-registration of the localizations in image post-processing following the measurement process.

[0013] WO 2022 / 200549 A1 describes a method for highly accurate drift compensation using MINFLUX localization of a reference marker (e.g., a fluorescent or light-reflecting nanoparticle). A light microscopic measurement is interrupted to determine the position of the reference marker using the MINFLUX method. The sample is then tracked from the determined position to compensate for any drift of the sample relative to the objective of the light microscope or any drift of objects within the sample, depending on the type of reference marker used.

[0014] The effects of misalignments in the beam path of a light microscope and aberrations on image quality in conventional imaging microscopy (including laser scanning microscopy and related super-resolution techniques such as STED and RESOLFT microscopy) and methods for their correction or compensation are known from the state of the art (see above).

[0015] However, localization microscopy techniques such as MINFLUX differ fundamentally from these methods in their image generation principle. Instead of optical imaging of the sample, an image of several individual emitters or the trajectory of a moving emitter (in the case of a tracking experiment) is mathematically combined from many individual localizations. Misalignments in the beam path and aberrations therefore have completely different effects than in imaging microscopy, with the specific impact depending largely on the localization principle and the specific implementation of the localization experiment.

[0016] Correction or compensation methods known from the state of the art for imaging microscopy are therefore not directly applicable to localization microscopy (e.g., MINFLUX methods).

[0017] Known methods, in which localization errors in localization microscopy are subsequently corrected through data processing, are relatively difficult to integrate into live measurements or extend the measurement time, thus negatively impacting user-friendliness. Object of the invention

[0018] Based on the above-described disadvantages of the prior art, the object of the present invention is to improve localization microscopy methods in such a way that the localization quality with regard to misalignments in the beam path and aberrations can be improved in a user-friendly manner.

[0019] Solution

[0020] This object is achieved by the subject matter of independent claims 1, 20 and 27. Advantageous embodiments of the invention are specified in subclaims 2 to 19 and 21 to 26 and are described below.

[0021] Description of the invention

[0022] A first aspect of the invention relates to a method for locating or tracking individual emitters in a sample by means of a light microscope, wherein the method comprises the following steps, which do not necessarily have to be carried out one after the other in the specified order: illuminating the sample with illuminating light (in particular with an illuminating light beam of illuminating light), wherein an intensity distribution of the illuminating light is formed in the sample, wherein the intensity distribution has a local intensity minimum, and wherein the illuminating light induces or modulates light emissions of an individual emitter in the sample, detecting light emissions of the individual emitter (in particular a detection light beam of the light emissions) and determining position data of the individual emitter on the basis of the detected light emissions.According to the invention, on the basis of the detected light emissions of the individual emitter, a wavefront of the illumination light is adapted and / or a beam path of an illumination light beam of the illumination light and a beam path of a detection light beam of the light emissions of the individual emitter are adapted relative to one another and / or a beam path of an illumination light beam of the illumination light and a beam path of another illumination light beam are adapted relative to one another.

[0023] By adjusting the wavefront or beam paths based on the light emissions of a single emitter, specific effects of misalignments and aberrations (caused by components in the light microscope's beam path or by the refractive index distribution in the sample) on the localization accuracy in localization microscopy can be advantageously compensated or corrected. In contrast to known prior art methods, this is not achieved by subsequent processing of the position data but by adjusting the beam path, which has a positive effect on user-friendliness, especially during live interactions with the user, iterative localization methods, and tracking procedures.

[0024] In particular, the wavefront adjustment or the beam path adjustment can be performed in such a way that the geometric focus of the illumination light does not shift relative to the sample as a result of this adjustment. The adjustment of the beam paths according to the invention, of course, does not mean simply scanning the illumination light across the sample or simply tracking the sample, for example, as part of a drift correction.

[0025] The wavefront can be adjusted, for example, by modulating the phase and / or amplitude of the illuminating light.

[0026] The detection light beam can be formed in particular by bundling the light emissions of the emitter with an objective, in particular by means of the same objective that focuses the illumination light into the sample.

[0027] The beam paths of the illumination light beam and the beam paths of the detection light beam can be adjusted relative to each other by shifting the illumination light beam while leaving the detection light beam unchanged, by shifting the detection light beam while leaving the illumination light beam unchanged, or by shifting both light beams in different ways. The same applies to adjusting the beam paths of the illumination light and the additional illumination light relative to each other.

[0028] The term "emitter" refers to the unit whose position in the sample is to be determined using the localization method. Accordingly, light emission can occur directly from the emitter itself (especially if the emitter is a fluorescent dye molecule) or indirectly from markers coupled to the emitter (e.g., fluorescent dye molecules covalently or non-covalently bound to a protein). Light emission can refer not only to the active emission of light by the emitter or markers in the sense of luminescence, but also to light emission caused by (Raman / Rayleigh / Mie) scattering. Specifically, the emitter or the markers coupled to the emitter can be molecules of a fluorescent dye, fluorescent nanoparticles (e.g., quantum dots), or light-scattering nanoparticles such as gold nanoparticles or gold nanorods.

[0029] Single emitters are defined here as emitters that can be separated or resolved using optical means. This can mean, in particular, that the emitters are separated by a spatial distance that lies above the optical diffraction limit. However, emitters are also considered single in this sense if they can be recorded one after the other, for example, by recording light emanating from a first emitter at a time when a neighboring emitter is not emitting light because it is (in the case of fluorophores) in a dark state. In this way, even emitters that are separated by a distance below the diffraction limit but blink asynchronously can be resolved using light microscopy.Finally, it is also possible to resolve emitters that are separated below the diffraction limit but emit light at different wavelengths using a light microscope by spectrally separating the emitted light, or to excite two emitters with different excitation spectra at different wavelengths to optically separate the emitters. Finally, emitters with different emission lifetimes can be differentiated from one another by measuring the lifetime (e.g., by time-resolved single-photon counting) and thus detected separately. All of these embodiments fall under the term "individual emitters."

[0030] The sample is illuminated with illuminating light, particularly in the vicinity of a suspected position of the individual emitter. The illuminating light can be excitation light, which excites the emitter(s) to fluoresce or is scattered by the emitter(s), thus inducing light emission. Alternatively, the illuminating light can also modulate, particularly inhibit, the light emission. Examples of this are STED light, which quenches the excited state of fluorophores through stimulated emission, or switching light, which can, for example, convert fluorophores from a fluorescent state to a dark state, such as a triplet state. Illuminating light that modulates light emission is used particularly in combination with excitation light.

[0031] To detect the emitter's light emission, either a point detector (such as an APD, a photomultiplier, or a hybrid detector) or a spatially resolving area detector (e.g., a camera or an APD array) can be used. The emitter's position data is determined from the light emission, whereby the position determination can be carried out, in particular, based on light emissions recorded at multiple illumination positions. The recorded light emissions from the emitter can be individual photons recorded one after the other (e.g., in the case of an APD). However, the light emissions can also be recorded as groups of multiple photons (as is sometimes the case with so-called hybrid detectors).

[0032] The emitter can be localized, in particular, using a method based on the MINFLUX principle. This means that the emitter is illuminated at several illumination positions arranged around the predetermined position of the emitter with an intensity distribution of excitation light exhibiting a local intensity minimum. The emitter position is estimated from the light emissions assigned to the illumination positions. The illumination light is therefore excitation light. With localization based on the MINFLUX principle, the closer the intensity minimum of the illumination light (excitation light) is to the actual position of the emitter, the fewer light emissions can be expected, which makes the method particularly photon-efficient.

[0033] Alternatively, the emitter can also be localized, in particular, according to a STED-MINFLUX principle. This means that the emitter is illuminated at several illumination positions arranged around the predetermined position of the emitter, with an intensity distribution of excitation light exhibiting a local intensity maximum superimposed on an intensity distribution of inhibition light (e.g., STED light) exhibiting a local intensity minimum. The position of the emitter is estimated from the light emissions assigned to the illumination positions. In this case, the illumination light is the inhibition light. When localizing according to the STED-MINFLUX principle, the closer the intensity minimum of the illumination light (inhibition light) is to the actual position of the emitter, the more light emissions occur.

[0034] According to one embodiment of the method, the illumination positions are arranged at discrete positions around the emitter. These positions can be freely selected, whereby the number of illumination positions can be reduced to a minimum. Alternatively, the positions can also be arranged regularly, i.e., on a grid, whereby the grid covers only a close range of, in particular, at most 1 pm, further in particular at most 500 nm, and even further in particular at most 100 nm, around the emitter.

[0035] According to a further embodiment of the method, the illumination positions are not arranged at discrete points around the emitter, but rather the illumination occurs continuously along an illumination trajectory that encloses the emitter. The assignment of the detected light emissions to an illumination position can be performed in a similar manner to continuous scanning in scanning microscopy, e.g., by defining corresponding time intervals (so-called dwell times) and assigning the light emissions to a mean illumination position during the corresponding time interval. Alternatively, the assignment of light emission to illumination position can also be performed by conversely registering the current illumination position as soon as a photon of the light emission is detected.

[0036] A sequence of illumination positions can be run through either once or multiple times. Alternatively, a variable sequence of illumination positions is also possible. It is particularly advantageous to recalculate the illumination positions based on a localization of the emitter, especially iteratively, and to arrange them successively more closely around the (actual) position of the emitter. To determine the emitter position data, the emitter's light emissions are detected at the illumination positions, and the emitter's position is ultimately determined from the light emissions detected at the illumination positions. For this purpose, position estimators are used, such as those known from the state of the art for the MINFLUX method (see, for example, "F. Balzarotti et al., "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes", Science 355 (6325), 606-612 (2017)".The position data can specify a position in one, two or three spatial directions.

[0037] The local intensity minimum can be point-shaped, linear, or surface-shaped. Ideally, it is an intensity zero, zero line, or zero surface. The local minimum can also be a global minimum of the intensity, especially if the intensity at the local minimum is zero. Otherwise, however, it is also possible that the intensity distribution has further local minima whose intensity is lower than the intensity of the local minimum. In particular, the intensity minimum can be a central minimum that forms a geometric center of a symmetrical light distribution. This center can be located, in particular, at the geometric focus of the light microscope. Global intensity maxima can border the intensity minimum, in particular in at least one spatial direction, in particular on two opposite sides of the intensity minimum.

[0038] The intensity distribution can be a 2D donut or a 3D donut (also known as a bottle beam). Such intensity distributions can be generated, for example, by phase modulating the illumination light with a phase plate or a spatial light modulator (SLM). Those skilled in the art are familiar with corresponding methods from the state of the art for STED and MINFLUX microscopy.

[0039] The positioning of the intensity distribution of the illumination light in the sample can be achieved by beam displacement (e.g. electro-optical or by means of a galvanometric scanner), sample displacement (e.g. by means of a sample holder movable with a piezoelectric actuator) or by controlling certain point light sources such as fiber ends of an optical fiber.

[0040] According to one embodiment, the adjustment of the wavefront or the beam paths is performed based on the position data of the individual emitter, wherein the position data is determined based on the detected light emissions. In particular, the adjustment of the wavefront or the beam paths can be performed based on a comparison of different position data of the same emitter. For example, the position data of an emitter obtained from multiple localization steps can be analyzed to determine a systematic deviation between the ultimately estimated position of the emitter (according to the last localization step for the emitter in question) and an estimated position from an earlier localization step of lower accuracy. Alternatively, a systematic deviation of the localizations caused by misalignments or aberrations can also be determined by an independent method.The determined deviation can be used to compensate or correct the misalignments or aberrations, so that the next emitter to be localized can be located with comparatively higher accuracy in earlier localization steps.

[0041] According to a further embodiment, the adaptation of the wavefront or the beam paths is carried out during a measurement sequence, wherein the measurement sequence comprises several steps in which the sample is illuminated with the illumination light (i.e. with the intensity distribution of the illumination light with the local intensity minimum) and the light emissions of the individual emitter or individual emitters are recorded. This means that the adaptation is not carried out in an adjustment step before a measurement, but rather "live" during a measurement. This has the particular advantage that the adaptation is particularly well adapted to the conditions prevailing during the measurement. A measurement sequence can comprise a position determination of one emitter or several successive position determinations of different emitters.

[0042] According to a further embodiment, position data of the individual emitter are determined multiple times and / or position data of multiple emitters in the sample are determined, wherein the adaptation of the wavefront or the beam paths is carried out on the basis of a statistical analysis of the position data. The statistical analysis can, for example, comprise determining a scatter (e.g., a variance or a standard deviation in at least one spatial direction). In this case, the adaptation of the wavefront or the adaptation of the beam paths can then, for example, be carried out in such a way that the scatter is reduced, in particular minimized. Minimization of the scatter can, for example, be achieved by iteratively carrying out the statistical evaluation and the adaptation of the wavefront or the beam paths. In this way, the quality of the localization can be improved.

[0043] The statistical analysis can also involve averaging, particularly a moving average, between parameters obtained from the position data of several individual emitters. For example, a systematic deviation between an earlier step of a localization and a later step of a localization can be determined for the last n emitters on a moving average basis and used to adjust the wavefront or beam paths.

[0044] According to a further embodiment, an axial position of the intensity distribution of the illumination light is adjusted, wherein light emissions of the individual emitter are detected for different axial positions of the intensity distribution, and wherein the adjustment of the wavefront or the beam paths is carried out on the basis of the light emissions detected for the different axial positions. The term "axial position" refers in particular to the position of the local minimum of the intensity distribution on the optical axis of the objective of the light microscope. For the different axial positions, position data of the individual emitter can optionally be determined from the light emissions. This can in particular be two-dimensional position data or three-dimensional position data. By this type of evaluation, localization errors dependent on the axial coordinate (z-coordinate) (iein particular systematic effects of the z-position on the position estimator).

[0045] The adaptation of the wavefront or the beam paths can in particular also be carried out on the basis of the light emissions detected successively by several emitters, whereby for each of the emitters, light emissions detected for different axial positions of the intensity distribution of the illumination light are used.

[0046] According to a further embodiment, the illumination light is excitation light, which excites the emitter in the sample to emit light. In this case, the sample is illuminated with an intensity distribution of excitation light with a local minimum, in particular a central intensity zero. The method can therefore be a MINFLUX method. This has the advantage that highly precise localization of the emitter can be achieved with a particularly small number of photons emitted by the emitter.

[0047] According to a further embodiment, the adjustment of the wavefront of the illumination light and / or the adjustment of the beam paths (i.e., the beam path of the illumination light beam and the beam path of the detection light beam relative to one another and / or the beam path of the illumination light beam and the beam path of the further illumination light beam relative to one another) takes place automatically. Thus, in particular, a control unit of the light microscope can automatically transmit a control signal calculated on the basis of the registered light emissions of an individual emitter, for example, to a mechanical actuator, which adjusts an optical component in the beam path of the illumination light beam or the detection light beam based on the control signal. Alternatively or additionally, for example,The same control unit or another control unit can automatically transmit a corresponding control signal to a wavefront modulator, which modulates the wavefront of the illumination light based on the control signal. This type of automatic adjustment can also be implemented, in particular, in the form of a control loop. According to a further embodiment, the emitter is a fluorophore or a molecule labeled with one or more fluorophores. The emitters used to adjust the beam path can, in particular, be the same emitters used for the microscopic examination of sample structures. In contrast to a separate adjustment step, for example with reference particles such as light-scattering or fluorescent nanoparticles, this has the advantage that the adjustment is carried out under exactly the same conditions under which the localization experiment is conducted.In particular, the refractive index of the sample is identical during adjustment and measurement.

[0048] According to another embodiment, the emitter is a fluorophore, i.e., a self-fluorescing molecule. This can be, for example, an organic fluorophore or a fluorescent protein. The position of such individual molecules can be determined with high accuracy using localization microscopy.

[0049] According to a further embodiment, the emitter is a molecule labeled with a fluorophore. The linkage of the fluorophore to the molecule can be covalent or non-covalent. The molecule labeled with the fluorophore can be, for example, an antibody or a nanobody that is covalently coupled to a fluorescent dye and binds to a target structure to be examined in the sample. In this case, positional information about the target structure can be indirectly collected by localizing the emitter (here, an antibody or nanobody with a fluorescent dye). Labeling with only one fluorophore has the advantage that the light emissions tend to originate from a smaller area of ​​the sample, and thus more meaningful information about the target structure can be obtained.

[0050] According to another embodiment, the emitter is a molecule labeled with multiple fluorophores. This can be, for example, an antibody covalently coupled to multiple fluorophores, which in turn binds to a structure of interest in the sample. Coupling to multiple fluorophores has the advantage that more photons are emitted overall, thus allowing greater positioning accuracy. Furthermore, irreversible bleaching only occurs after a longer period of time, allowing for positional determination over a longer period.

[0051] According to a further embodiment, a near area of ​​the sample, in which the emitter to be localized or tracked is located according to a predetermined assumed position, is illuminated with the intensity distribution of the illumination light with the local minimum. This near area is typically derived from an estimated position of a previous localization step. The first localization step can, e.g. in a MINFLUX method, be carried out with an independent localization technique of lower accuracy, which is also referred to as pre-localization. According to a further embodiment, by adapting the wavefront and / or the beam paths (ie, the beam path of the illumination light beam and the beam path of the detection light beam relative to each other and / or the beam path of the illumination light beam and the beam path of the further illumination light beam relative to each other) corrects a deviation between a desired intensity distribution of the illumination light and an actual intensity distribution of the illumination light in the sample. Such a deviation can be caused, for example, by aberrations. For example, the minimum of an intensity distribution of the illumination light can shift, or the intensity distribution can become asymmetrical.

[0052] According to a further embodiment, the actual intensity distribution in the sample is skewed relative to an optical axis of the light microscope. For example, the axial intensity maxima of a so-called bottle beam (also referred to as a 3D donut in this application), which ideally lie on the optical axis of the objective, can lie on an oblique axis tilted to the optical axis due to aberrations. Such light distributions are used, for example, in 3D MINFLUX localization, sometimes for both axial and lateral localization. A skewed 3D donut leads to a systematic deviation in the position estimation during lateral localization, depending on the current focal plane. Such deviations can be corrected, for example, by recentering the illumination light beam onto the pupil of the objective.

[0053] According to a further embodiment, a phase pattern displayed on a wavefront modulator is adjusted, wherein the wavefront modulator is located in a beam path of the illumination light, wherein the phase pattern is adjusted to correct the deviation between the desired intensity distribution and the actual intensity distribution. Such a wavefront modulator can in particular be located in a pupil plane that is conjugated to a rear aperture of the objective of the light microscope by imaging via an optical relay, i.e. is a Fourier plane with respect to a focal plane that intersects the geometric focus of the objective in the sample. If the illumination light is modulated in this plane with a suitable phase pattern, an intensity distribution with a local minimum at the geometric focus is created at the focus due to destructive interference.For example, a 2D donut-shaped intensity distribution can be generated by a vortex-shaped phase pattern with a phase angle increasing circularly from 0 to 2K or a multiple thereof. A ring-shaped phase pattern with a phase jump of the phase difference K running along a circle, in which the inner partial area (inner circle) and the outer partial area (ring) of the pattern have equal areas, can, under ideal conditions, generate a 3D donut-shaped intensity distribution. According to a further embodiment, the phase pattern comprises a phase jump running along a circle, wherein a phase difference of the phase jump is adjusted in order to correct the deviation between the desired intensity distribution and the actual intensity distribution. In this way, for example,an axial deviation of the position determination can be corrected, which can arise from defocus aberrations, for example due to different refractive indices between a calibration sample with which the beam path of the light microscope was adjusted and a sample to be examined.

[0054] According to a further embodiment, an aberration correction is carried out by adjusting the wavefront of the illumination light and / or by adjusting the beam paths (i.e., the beam path of the illumination light beam and the beam path of the detection light beam relative to one another and / or the beam path of the illumination light beam and the beam path of the further illumination light beam relative to one another). Certain optical aberrations can, for example, cause the intensity distribution of the illumination light in the sample to deviate from a desired intensity distribution; for example, the local minimum of the intensity distribution can shift. These deviations or their effects on a position estimator can be compensated or corrected by the method according to the invention.

[0055] According to a further embodiment, the method comprises a first localization step and a second localization step, wherein in the first localization step, the sample is illuminated with the illumination light, first light emissions of the individual emitter are detected, and first position data of the individual emitter are determined based on the first light emissions, and wherein in the second localization step, the intensity minimum of the intensity distribution of the illumination light is arranged at illumination positions, wherein the illumination positions are arranged around a position of the individual emitter estimated on the basis of the first position data, second light emissions of the individual emitter are detected for the respective illumination positions, and second position data of the individual emitter are determined based on the second light emissions and the associated illumination positions,wherein, based on the second position data, the estimated position of the individual emitter is determined with greater accuracy than based on the first position data, wherein a systematic deviation between the estimated position of the emitter determined in the first localization step and the estimated position of the same emitter determined in the second localization step is determined, wherein, based on the determined deviation, the wavefront of the illumination light is adjusted and / or the beam path of the illumination light beam and the beam path of the detection light beam are adjusted relative to one another and / or the beam path of the illumination light beam and the beam path of the further illumination light beam are adjusted relative to one another.

[0056] The first localization step and the second localization step do not have to follow one another directly, but one or more further localization steps can of course be carried out between the first localization step and the second localization step.

[0057] In particular, the position of the emitter can be determined in the second localization step with an accuracy that is better than a threshold accuracy. Furthermore, in particular, the accuracy in the second localization step can reach or approach an optimum. Particularly in the latter cases, the position of the emitter determined in the second localization step can be considered the actual emitter position (in the sense of a so-called "ground truth"), on the basis of which the systematic deviation is determined. The better the accuracy of this determined position, the better the systematic deviation can be determined, and thus the adaptation of the wavefront or the beam paths can be performed.

[0058] The first localization step can, in particular, be a pre-localization step that is not performed according to the MINFLUX principle, but rather with an independent imaging or localization technique to obtain an initial position estimate for a single emitter, on the basis of which a MINFLUX localization is then performed. During this pre-localization step or in a preceding step, the emitter can also be located, i.e., the sample is scanned for individual emitters by registering light emissions, which are then pre-localized and finally localized with high accuracy.

[0059] According to a further embodiment, the first localization step is carried out using a stochastic localization method. These include, in particular, STORM (stochastic optical reconstruction microscopy) and PALM (photoactivated localization microscopy) microscopy, as well as SOFI (superresolution optical fluctuation imaging) microscopy and PAINT (points accumulation for imaging in nanoscale topography) microscopy. In this case, the emitters are in a non-fluorescent dark state, and individual emitters are converted from this dark state to a fluorescent state spontaneously, through photoactivation, or through a chemical reaction. An equilibrium exists between the non-fluorescent dark state and the fluorescent state, which can be influenced by adjusting the experimental parameters.To localize these activated emitters, the field of view is illuminated in particular homogeneously with the illumination light so that all activated emitters in the field of view are stimulated to emit light simultaneously. In this case, the light emission is preferably detected using a spatially resolving or imaging detector, i.e. a detector which has a plurality of individually readable detector elements, in particular with a camera. The position of the individual, i.e. optically separable emitters, can thus be determined by determining the center of gravity or adapting a model function in the image. In the event that the first localization step is carried out using a stochastic localization method, in particular a plurality of emitters are imaged in parallel. The position of one of these emitters can then serve as the basis for the illumination positions in the second localization step.An equilibrium between an emitting state and a dark state is also typically established in MINFLUX microscopy in order to be able to locate emitters that are spatially densely arranged in the sample one after the other.

[0060] According to a further embodiment, the first localization step is performed by scanning the sample with the illumination light. This means, in particular, the first localization step is performed using laser scanning, which can be performed either as conventional confocal laser scanning or as STED laser scanning. For this purpose, the sample is scanned point by point with focused excitation light or with an intensity distribution of excitation light with a local minimum (e.g., a 2D or 3D donut). An image is reconstructed from the light emissions detected at each scan point, in which image the individual emitters can be localized.For image acquisition in STED mode, the excitation light can be superimposed with a distribution of de-excitation light exhibiting a local intensity minimum, thereby improving the resolution beyond the optical diffraction limit and enabling more precise localization of the emitter in the first localization step.

[0061] In the second localization step, the emitter can be localized, in particular, using a method based on the MINFLUX principle, i.e., the emitter is illuminated at multiple illumination positions arranged around the emitter position determined in the first localization step with an intensity distribution of illumination light exhibiting a local intensity minimum in at least one spatial direction. The second localization step does not necessarily have to follow immediately after the first localization step; optionally, further localization steps can be performed between the first and second localization steps. For the embodiment described above, only the presence of a first and a second localization step is decisive.

[0062] Like the illumination light used in the first localization step, the illumination light used in the second localization step can also induce or modulate, in particular inhibit, the light emission of the emitter. The illumination light used in the second localization step can be identical to the illumination light used in the first localization step, but this is not mandatory. For example, the wavelength of the illumination light can be identical in both localization steps, but different intensity distributions of the light can be switched between. For localizing an emitter from a confocal image in the first localization step and localizing the emitter according to a MINFLUX principle in the second localization step, it is necessary, for example, to switch from a Gaussian mode to a donut-shaped mode having an intensity minimum.Such switching can be achieved, among other things, with a programmable phase modulator (spatial light modulator, SLM).

[0063] In practice, the problem regularly arises that a systematic deviation occurs between the position determination of an emitter in the first localization step and in the second localization step. This systematic deviation does not have to be homogeneous across the field of view, but can (and in most applications is) location-dependent, i.e., depends on the position of the emitter in the field of view. A systematic deviation is therefore only understood to mean that, during repeated localizations of one and the same emitter, identical deviations occur between the first and second localizations within the limits of the measurement accuracy. Nevertheless, the systematic deviations can be subject to (slow) changes, for example as a result of drift effects.

[0064] According to a further embodiment, the sample is also illuminated in the first localization step with an intensity distribution of the illumination light having a local intensity minimum. According to a further embodiment, the intensity minimum is arranged in the first localization step at illumination positions around a previously estimated position of the individual emitter. In this case, the first localization step can, for example, already be carried out according to a MINFLUX method (according to a MINFLUX principle), wherein the first and second localization steps can, for example, be steps of an iterative MINFLUX method in which the position of the emitter is determined in each iteration and the illumination positions are adjusted based on the last position determination, in particular are arranged more closely around the emitter. At the same time, the intensity of the illumination light can also be increased in each iteration.Although a systematic deviation in the localization of an emitter is not obvious in successive iterations of the MINFLUX method, a systematic offset is regularly observed in practice. This offset is presumably due to a not perfectly rotationally symmetric intensity distribution of the illumination light, which can be corrected in particular by adapting the wavefront or the beam paths according to the invention. In a further embodiment of the method, the sample is illuminated with structured illumination light in the first localization step, wherein the position and / or orientation of the structured illumination light relative to the sample is gradually shifted or rotated, an image of the sample is acquired with an imaging detector for each shift or orientation, and the individual images are combined to produce a higher-resolution image.This procedure is known from structured illumination microscopy (SIM) and also provides a resolution that is up to twice that of conventional image acquisition.

[0065] According to a further embodiment, the emitter is imaged in the first localization step onto a spatially resolving detector, in particular onto a camera or a detector array. Although in some of the previously described embodiments—locating the emitter in the first localization step by scanning (in particular laser scanning) or using a MINFLUX method—the detection of the light emission can be carried out using a point detector (APD, photomultiplier), the use of a spatially resolving detector, in particular a camera or a detector array, is often advantageous in these embodiments as well. By analyzing the spatially resolved image information, it can be detected, for example, if multiple emitting emitters are located within a diffraction-limited area and cannot be individually localized.

[0066] The spatially resolving detector can, in particular, resolve a diffraction image of the individual emitter in a detection plane, wherein the detection light beam is imaged, in particular, by a suitable optics onto a plurality of detector elements (in particular pixels) of the spatially resolving detector.

[0067] According to a further embodiment, the systematic deviation is caused by optical aberrations. Systematic deviations between the emitter positions determined in the first localization step and in the second localization step often occur as a result of optical aberrations, particularly when the optical beam path used for position determination in the first localization step differs from the optical beam path used for position determination in the second localization step, or when the first and second localization steps are performed using optical means that differ from one another in at least one optical element.

[0068] According to a further embodiment, the first localization step and the second localization step are carried out using optical means that differ from one another in at least one optical element. As soon as the beam paths differ, it becomes necessary to initially adjust the beam paths to one another and, if necessary, to keep them constant over an extended period. Furthermore, the imaging properties of the two beam paths are generally different, so that due to different imaging errors, in particular spherical aberrations, lateral chromatic aberrations, coma, and astigmatism, the images produced by the two beam paths are no longer congruent across the entire image area. The method according to the invention advantageously allows these deviations to be corrected, in particular during the measurement or between measurements.

[0069] However, a systematic deviation in the position determination of the emitter in the first localization step and in the second localization step can occur even if the same optical beam path with the same optical elements but different intensity distributions is used in both steps. For example, the actual center of gravity of an intensity distribution of the illumination light (e.g., a donut-shaped light distribution) in the second localization step can deviate from the nominal center of gravity (the central zero point in the donut-shaped light distribution) and thus from the center of the light distribution used in the first localization step, causing the localizations to deviate systematically from one another. An intensity-dependent deviation can also occur if the illumination positions include positions at which the emitter is illuminated with light intensities that lead to saturation of the excitation, i.e.There is no longer a linear dependence of the emitter's light emission on the intensity of the illumination light at the emitter's location. This can occur, for example, if the first localization step is also performed using a MINFLUX method, but with illumination points further apart and / or lower illumination light intensity.

[0070] According to a further embodiment, the adaptation of the wavefront and / or the beam paths (ie, the beam path of the illumination light beam and the beam path of the detection light beam relative to one another and / or the beam path of the illumination light beam and the beam path of the further illumination light beam relative to one another) is carried out such that the systematic deviation between the position of the emitter estimated in the first localization step compared to the position of the same emitter estimated in the second localization step is reduced, in particular minimized.

[0071] According to a further embodiment, a position of a pinhole arranged in a beam path of the detection light beam is adjusted such that the systematic deviation between the position of the emitter estimated in the first localization step compared to the position of the same emitter estimated in the second localization step is reduced.

[0072] Such a pinhole is known from confocal microscopes, but is also used in MINFLUX microscopes, in particular to reduce background fluorescence from planes above and below the focal plane.

[0073] The initial pre-localization step in MINFLUX microscopy is partially performed using pinhole orbit scanning. With a stationary intensity distribution of the illumination light relative to the sample (especially an intensity distribution with a local minimum such as a 2D or 3D donut), the pinhole projection is moved in a circular path within the sample, the light emissions from the sample are detected, and a rough position estimate for the emitter is made based on the detected light emissions. This estimate then forms the basis for the subsequent MINFLUX localization steps. The rotation of the pinhole projection can be achieved, for example, by two independent beam scanners, one of which affects both the position of the illumination light in the sample and the position of the detection light (e.g.,a galvanometer scanner in a de-scanned configuration), while the other beam scanner only affects the position of the illumination light beam (e.g. electro-optical deflectors arranged in the illumination beam path but not in the detection beam path).

[0074] If the detection light beam is not centered on the pinhole, a systematic error in the rough position estimation results during pre-localization by pinhole orbit scanning. Although MINFLUX localization determines the emitter position with very high precision despite the misalignment in the detection beam path, too many emitter photons are required because the initial positioning of the illumination pattern of illumination positions of the intensity distribution is faulty due to the lack of centering on the pinhole. Therefore, the above-described embodiment of the method according to the invention, in which the position of the pinhole is corrected relative to the detection light beam, improves the photon efficiency during MINFLUX localization.

[0075] If the position of the pinhole arranged in a beam path of the detection light beam is adjusted, the first localization step can, in particular, be a pre-localization step performed using a pinhole orbit scan method. This means that the projection of the pinhole into the sample is moved along a circular path, in particular using independent beam scanners, while the position of the illumination light beam, in particular of the intensity distribution of the illumination light with the local minimum, remains constant relative to the sample. Light emissions from the sample are detected, and the position of an individual emitter in the sample is estimated based on the light emissions.According to a further embodiment, the illumination light beam and the further illumination light beam have different wavelengths, wherein the beam path of the illumination light beam and the beam path of the further illumination light beam are adjusted relative to each other based on the detected light emissions of the individual emitters in such a way that a spatial deviation between the illumination light beam and the further illumination light beam is corrected. With the illumination light beam and the further illumination light beam, for example, different emitters (e.g. different fluorophores or molecules labeled with fluorophores) in the sample can be excited in order to perform multi-color MINFLUX localization.the different illuminating light beams are shifted parallel to one another such that the corresponding intensity distributions formed by the different illuminating light beams (in particular one after the other) in the sample are congruent. In this embodiment, for example, a systematic deviation between a first position of the emitter determined by the illuminating light beam and a second position of the emitter determined by the further illuminating light beam can be determined and minimized, in particular by adjusting the relative beam path. Illuminating beams of different wavelengths can lead to the emission of light, in particular even from the same emitter, e.g. in the case of fluorescence excitation, when the excitation spectrum of the emitter comprises both wavelengths.

[0076] A second aspect of the invention relates to a light microscope for locating or tracking individual emitters in a sample, in particular according to a method according to the first aspect, wherein the light microscope comprises at least the following components: a light source configured to illuminate the sample with illumination light, wherein the illumination light induces or modulates light emissions of an individual emitter in the sample, a wavefront modulator configured to form an intensity distribution of the illumination light in the sample with a local intensity minimum, at least one detector configured to detect light emissions of the individual emitter, a computing unit configured to determine position data of the individual emitter based on the detected light emissions, wherein the light microscope has a control unit configured toBased on the detected light emissions of the individual emitter, to adapt a wavefront of the illumination light and / or to adapt a beam path of an illumination light beam of the illumination light and a beam path of a detection light beam of the light emissions of the individual emitter relative to one another, and / or to adapt a beam path of the illumination light beam and a beam path of another illumination light beam relative to one another. According to a further embodiment, the control unit is designed to control the wavefront modulator or another wavefront modulator based on the detected light emissions of the individual emitter such that the wavefront of the illumination light is adapted.

[0077] According to a further embodiment, the wavefront modulator is a phase modulator, in particular a controllable spatial light modulator (SLM) connected to the control unit. By phase modulating the illumination light, intensity distributions with a local minimum (e.g., 2D or 3D donuts) can be generated in the sample. The control unit can then, in particular, change the phase distribution, so that the intensity distribution in the sample is changed, in particular such that the actual intensity distribution corresponds to a desired intensity distribution.

[0078] According to a further embodiment, the further wavefront modulator is a deformable mirror, wherein the control unit is designed to control the deformable mirror on the basis of the detected light emissions of the individual emitter such that the wavefront of the illumination light is adapted, wherein in particular an aberration correction is carried out by adapting the wavefront.

[0079] According to a further embodiment, the light microscope has an actuator which is designed to displace an optical component arranged in a beam path of the illumination light beam, the further illumination light beam and / or the detection light beam, wherein the control unit is designed to control the actuator on the basis of the detected light emissions of the individual emitter such that the beam path of the illumination light beam and the beam path of the detection light beam are adapted relative to one another and / or the beam path of the illumination light beam and the beam path of the further illumination light beam are adapted relative to one another.

[0080] According to a further embodiment, the light microscope has a pinhole arranged in a beam path of the detection light beam, wherein the actuator is designed to adjust the beam position of the detection light beam relative to the pinhole. For this purpose, the actuator can, for example, be directly coupled to the pinhole and adjust its position in the beam path, i.e. the pinhole can be the optical component. Alternatively, the optical component can be, for example, a beam deflecting element, e.g. a mirror, to which the actuator is coupled, wherein the beam deflecting element adjusts the beam position of the detection light beam relative to the pinhole. Of course, multiple actuators can also be provided, each of which is, for example, coupled to an optical component in order to adjust the beam position of the detection light beam in multiple spatial directions.According to a further embodiment, the detector has a plurality of detector elements, in particular individually readable ones. This can be, for example, a CCD or CMOS camera. In particular, the detector elements are designed to register individual photons. The detector can be, for example, a so-called APD array, i.e., a two-dimensional arrangement of avalanche photodiodes.

[0081] The light source comprises in particular one or more lasers.

[0082] According to a further embodiment, the light microscope has a

[0083] Beam positioning device, e.g. a galvanometric scanner, an acousto-optic deflector or an electro-optical deflector, which is designed to

[0084] Intensity distribution of the illumination light in the sample.

[0085] The beam positioning device is particularly designed to position the intensity distribution of the illumination light at illumination positions in the sample within a field of view of interest. Preferably, the beam positioning device is designed such that the intensity distribution can be repositioned within 10 ps, ​​in particular within 5 ps, and furthermore in particular within 1 ps, at least between illumination positions that are spaced apart by less than 500 nm, in particular less than 250 nm, and furthermore in particular less than 100 nm. For this purpose, the beam positioning device can in particular comprise an electro-optical deflector (EOD) or an acousto-optical deflector (AOD). Optionally, the beam positioning device can be designed as a combination of a fast positioning device (e.g.comprising EODs or AODs) with a slower positioning device (e.g. a galvo scanner) covering a larger positioning range.

[0086] A third aspect of the invention relates to a computer program comprising instructions that cause the light microscope according to the second aspect to carry out the method according to the first aspect.

[0087] 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.

[0088] 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; however, this does not apply to the independent patent claims of the granted patent.

[0089] 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 solely to make the patent claims easier to understand.

[0090] In the following, exemplary embodiments of the invention are described with reference to a figure. This does not limit the subject matter of this disclosure or the scope of protection.

[0091] Short description of the characters

[0092] Fig. 1 shows an embodiment of a light microscope according to the invention;

[0093] Fig. 2 shows MINFLUX localization data of an emitter;

[0094] Fig. 3 shows MINFLUX localization data of an emitter with negative astigmatism aberration;

[0095] Fig. 4 shows MINFLUX localization data of an emitter with positive astigmatism aberration;

[0096] Fig. 5 shows precision maps of different iteration steps of a MINFLUX localization of an emitter;

[0097] Fig. 6 shows precision maps of different iteration steps of a MINFLUX localization of an emitter with astigmatism aberration;

[0098] Fig. 7 shows a 2D donut-shaped intensity distribution of illumination light;

[0099] Fig. 8 shows a 2D donut-shaped intensity distribution of illumination light with astigmatism aberration;

[0100] Fig. 9 shows a systematic deviation between different iteration steps of a MINFLUX localization with a 2D donut-shaped intensity distribution of illumination light generated by phase modulation with an optimally adjusted phase pattern;

[0101] Fig. 10 shows a systematic deviation between different iteration steps of a

[0102] MINFLUX localization with a 2D donut-shaped intensity distribution of illumination light generated by phase modulation with a shifted phase pattern;

[0103] Fig. 11 shows a precision map of a pre-localization using pinhole orbit scanning with an optimally adjusted pinhole;

[0104] Fig. 12 shows a precision map of a pre-localization using pinhole orbit scanning with a shifted pinhole;

[0105] Fig. 13 shows a systematic deviation of different iteration steps of an SDMI NFLUX localization with a botf / e-beam-shaped intensity distribution of illumination light;

[0106] Fig. 14 shows a systematic deviation of different iteration steps of a 3D MINFLUX localization with a botf / e-beam-shaped intensity distribution of illumination light with coma aberration;

[0107] Fig. 15 shows a systematic deviation of different iteration steps of another 3D MINFLUX localization with a botf / e-beam-shaped intensity distribution of illumination light;

[0108] Fig. 16 shows a systematic deviation of different iteration steps of a 3D MINFLUX localization with a botf / e-beam-shaped intensity distribution of illumination light with a deviating phase jump;

[0109] Fig. 17 shows precision maps of a final iteration of a 3D-MINFLUX localization with a botf / e-beam-shaped intensity distribution of illumination light generated by phase modulation with phase patterns with different shifts relative to an optimally adjusted phase pattern, in xy-, xz-, and yz-sections;

[0110] Fig. 18 shows a boft / e-beam-shaped intensity distribution of illumination light;

[0111] Fig. 19 shows a botf / e-beam-shaped intensity distribution of illumination light with a shift of the phase pattern by 5 pixels;

[0112] Fig. 20 shows a botf / e-beam-shaped intensity distribution of illumination light with a phase pattern shift of 10 pixels. Description of the figures

[0113] Fig. 1 shows an embodiment of a light microscope 1 according to the invention for localizing individual emitters E in a sample 2. The light microscope 1 has a light source 3, e.g. a laser, for generating an illuminating light beam B of illuminating light, in particular excitation light. The illuminating light beam B passes through a beam positioning device 6, e.g. in the form of one or more electro-optical deflectors, and is reflected by a mirror 11 onto a wavefront modulator 4, which modulates the phase of the illuminating light in order to generate an intensity distribution of the illuminating light with a local intensity minimum, e.g. a 2D donut or a 3D donut (bottle beam), at the focus in the sample 2.

[0114] The phase-modulated light beam is then reflected by a dichroic beam splitter 13 and passes via a further beam positioning device 7, in particular a galvanometric scanner, and a tube lens 12 to an objective 9, which focuses the illumination light into the sample 2.

[0115] The light emitted by emitters E in the sample 2 (in particular fluorescent light) is transmitted by the dichroic beam splitter 13 and passes via an emission filter 14, a lens 15 and a confocal pinhole 16 to a detector 5, which detects light emissions from the emitters E.

[0116] The light microscope 1 further comprises a processor 10 with a memory 10a, which is designed as a combined computing unit 17 and control unit 18. Of course, the computing unit 17 and the control unit 18 can alternatively be designed as separate processors.

[0117] The processor 10 is configured to receive data from the detector 5, which represents the light emissions detected by the detector 5. In its function as a computing unit 17, the processor 10 determines the position of an individual emitter in the sample 2 from the data, e.g., using a maximum likelihood estimator. In particular, to implement an iterative MINFLUX method, in which the intensity distribution of the illumination light is arranged at illumination positions around a previously estimated position of an individual emitter, the processor 10, in its function as a control unit 18, is further connected to the beam positioning devices 6, 7 in order to control them.

[0118] In its function as a control unit 18, the processor 10 uses the data representing the light emissions to determine a systematic deviation in the localization and, in particular, controls the wavefront modulator 4 such that a wavefront of the illumination light is adjusted to change the intensity distribution of the illumination light so that it approximates a desired intensity distribution or controls the actuator 8 such that the detection light beam D is centered on the pinhole 16. The latter is carried out, in particular, such that a systematic deviation between a first localization step and a second localization step (with increased accuracy compared to the first localization step) is reduced.

[0119] To generate the localization data shown in Fig. 2, the position of a fluorescent nanoparticle (emitter) was determined multiple times by shifting a 2D donut-shaped intensity distribution of excitation light using electro-optical deflectors to six illumination positions 20 distributed symmetrically on a circle 19 with a diameter of L=300 nm, and the light emissions for each illumination position 20 were recorded. From the light emissions and the associated illumination positions 20, the position of the nanoparticle was estimated using a position estimator based on a vector sum. The multiple position determination was repeated at the nine positions of a 3x3 grid, with the excitation light beam being shifted to a position on the 3x3 grid relative to the position of the fluorescent nanoparticle using a galvanometric scanner that scanned the excitation light beam and descanned the detection light.At the central position of the 3x3 grid, the light emissions of the nanoparticle were detected confocally; at the other positions, the center of the projection of the detection pinhole into the sample was slightly shifted relative to the actual position of the emitter.

[0120] In the measurement according to Fig. 2 with a 2D donut without significant aberrations, the distribution around the points of the 3x3 grid was reproduced in the measurement data as expected.

[0121] To record the localization data shown in Fig. 3 and Fig. 4, an astigmatism aberration was imposed on the intensity distribution of the excitation light in the sample by appropriately controlling a wavefront modulator, namely a negative astigmatism with a coefficient of the corresponding Zernike polynomial of -0.07 for the data in Fig. 3 and a positive astigmatism with a coefficient of the corresponding Zernike polynomial of +0.07 for the data in Fig. 4.

[0122] Fig. 7 shows an aberration-free 2D donut, while Fig. 8 shows an example of a 2D donut with astigmatism aberration (Zernike polynomial Z 2 , coefficient -0.07). Fig. 7 and Fig. 8 are cross-sectional views in the focal plane (xy plane). It can be seen that the light intensity around the central minimum with astigmatism aberration is no longer distributed symmetrically, but rather, intensity maxima form diagonally to the x- and y-axes.

[0123] The data in Fig. 3 and Fig. 4 show that the Cartesian 3x3 grid, at whose positions the observation field was centered for the localization measurements, is no longer correctly reproduced in the presence of an astigmatism aberration, but is distorted in different ways depending on the sign of the Zernike coefficient.

[0124] This implies that emitter locations exhibit systematic deviations in the presence of astigmatism aberrations of the excitation PSF, especially when the emission light is not captured perfectly confocally, but rather the center of the pinhole projection into the sample is shifted relative to the actual position of the emitter in the sample. Such deviations regularly occur in MINFLUX methods, since the true position of the emitter is known only with great uncertainty at the beginning of the method.

[0125] According to the invention, systematic localization errors in non-confocal detection can be reduced by correcting aberrations of the excitation light with a wavefront modulator.

[0126] Fig. 5 and Fig. 6 show xy precision maps of a 2D MINFLUX localization experiment in which Alexa Fluor 647 fluorescent emitters bound to the nuclear pore protein Nup96 were localized in U2OS cells. The cells were suspended in GLOX buffer. From left to right, distributions of localizations of a large number of emitters around the respective actual position of the emitter (origin of the coordinate system) are shown for different iterations of the MINFLUX procedure. Iteration 0 was a pre-localization using pinhole orbit scanning. The projection of the pinhole into the sample was moved in a circular orbit with a stationary intensity distribution in the sample by controlling two scanning devices, both of which scanned the excitation light, but only one descanned the emission light, and the light emissions from the sample were recorded. This was followed by an iterative MINFLUX procedure with the method shown in Fig.2 shows a pattern of six illumination positions 20 distributed symmetrically on a circle 19, with different diameters L of the circle 19 being assigned to the iterations, namely 1: 290 nm, 2: 150 nm, 3: 75 nm, 4: 40 nm. Following this sequence (so-called capture iterations), certain iteration steps (e.g., iterations 3 and 4) were repeated several times to obtain further highly precise localizations of an emitter, in particular until bleaching or transition to a dark state of the respective emitter. The photon-weighted mean of the positions of the respective emitter determined during iteration 4 was assumed to be the actual position of the corresponding emitter. On this basis, the deviations of the localizations of the capture iterations (0 to 4) from the actual position for the various emitters were determined and used to generate the precision maps.

[0127] A regular 2D donut (Fig. 5) or a 2D donut with additional astigmatism aberration (Fig. 6, Zernike coefficient +0.2) was used as the excitation light distribution. The data show that in the presence of astigmatism aberration, a systematic deviation of the estimated position results, which manifests itself in an elliptical distribution of the locations (Fig. 6). In contrast, without aberrations (Fig. 5), a symmetrical circular distribution of the locations results. To better visualize the elliptical and circular distributions, the data in the plots are overlaid with a black elliptical or circular ring, respectively. From this, the respective ellipticity can also be determined, especially with an appropriate fit to the data.

[0128] Such deviations can lead to slower convergence of the localizations to the true position and pose the risk of emitters falling outside the capture range of the localization and thus not being successfully localized. Furthermore, with a large systematic deviation, the emitters can often be illuminated with unnecessarily high light intensities, which, depending on the sample type, can lead to problems with photobleaching or phototoxicity. This generally applies to all systematic deviations in the position determination described below.

[0129] According to the invention, for example, an astigmatism aberration of an intensity distribution of the illumination light can be corrected with a wavefront modulator in order to reduce a systematic deviation, such as the deviations shown in Fig. 2 to Fig. 6.

[0130] Figures 9 and 10 show the effect of shifting a phase pattern used to phase modulate an excitation light beam relative to the excitation light beam during a 2D MINFLUX localization using Alexa Fluor 647 fluorescence emitters bound to the nuclear pore protein Nup96 in U2OS cells. A vortex phase pattern was used to generate a 2D donut-shaped intensity distribution in the sample. Fig. 9 shows the deviations (average values ​​of the deviations of the localizations of several emitters from the respective actual position) of iterations 0 (pre-localization by pinhole orbit scanning), 1 (MINFLUX localization with L=290 nm), 2 (MINFLUX localization with L=150 nm), 3 (MINFLUX localization with L=75 nm) and 4 (MINFLUX localization with L=40 nm) relative to the actual position of the emitter with a vortex phase pattern optimally adjusted to the excitation light beam, iea concentric arrangement of the beam and the phase pattern. The photon-weighted average of the positions of the respective emitter determined in additional repetitions of iteration 4 was assumed to be its actual position.

[0131] Fig. 10 shows corresponding deviations when shifting the phase pattern by 10 pixels on a controllable light modulator (SLM) compared to the optimally adjusted pattern. Such a shift leads to a distorted intensity distribution in which the center of gravity of the intensity distribution is shifted laterally. Fig. 10 shows a clear systematic deviation of the localization in iterations 0 and 1.

[0132] According to the invention, for example, misalignments of the phase pattern against the illumination light beam can be corrected by adjusting the phase pattern on the SLM in order to reduce the systematic deviation.

[0133] Fig. 11 and Fig. 12 show precision maps of the 0th iteration (pre-localization with pinhole orbit scanning) of a 2D MINFLUX localization with a 2D donut-shaped intensity distribution, which localized Alexa Fluor 647 fluorescent emitters bound to the nuclear pore protein Nup96 in U2OS cells. The precision maps were generated analogously to the data shown in Fig. 5 and Fig. 6 and overlaid with elliptical and circular rings, respectively (see above).

[0134] The data in Fig. 11 were acquired with a confocal pinhole in the detection beam path. The data in Fig. 12 were measured with a pinhole shifted by ten steps.

[0135] The data show that a misalignment of the pinhole leads to a systematic shift of the center of the distribution of the localizations from the zero position in the xy plane.

[0136] According to the invention, the position of the pinhole can be adjusted to reduce such systematic displacement.

[0137] Fig. 13 and Fig. 14 show systematic deviations (mean values ​​of the deviations of the localizations of several emitters from their respective actual positions) between different iteration steps of a 3D-MINFLUX localization of Alexa Fluor 647 fluorescence emitters bound to the nuclear pore protein Nup96 in U2OS cells with a non-aberrated bottle-beam-shaped excitation light intensity distribution (Fig. 13) and a bottle-beam-shaped excitation light intensity distribution with additional coma aberration (Fig. 14) with a Zernike coefficient of +0.2. The iteration steps were the same as for the data shown in Fig. 9 and Fig. 10. In particular, during the 0th iteration (pre-localization, pinhole orbit scanning) and the 1st iteration (MINFLUX localization with L=290 nm), systematic deviations of one of the lateral coordinates (here y, depending on the coma aberration in question) occur.

[0138] Coma aberrations can be corrected according to the invention by shaping the wavefront of the illumination light with a wavefront modulator (e.g., deformable mirror or SLM) to reduce the demonstrated systematic deviations. Fig. 15 and Fig. 16 show systematic deviations (average values ​​of the deviations of the localizations of several emitters from their respective actual positions) of different iteration steps of a 3D-MINFLUX localization of Alexa Fluor 647 fluorescence emitters bound to the nuclear pore protein Nup96 in U2OS cells, whereby a regular symmetric bottle-beam-shaped excitation light intensity distribution was generated by phase modulation with an annular phase pattern with a phase jump of 2K (Fig. 15) or a deformed, asymmetric bottle-beam-shaped excitation light intensity distribution was generated by phase modulation with an annular phase pattern with a phase jump of 2.2K (Fig. 16).The iteration steps were the same as for the data shown in Fig. 9 and Fig. 10 as well as in Fig. 13 and Fig. 14. With the deformed intensity distribution, a strong positive deviation of the axial coordinate (z) is observed for the 0th iteration (pre-localization with pinhole orbit scanning).

[0139] According to the invention, for example, a light modulator can be controlled to adjust the amount of phase shift of an annular phase pattern and thus to shape the intensity distribution of the illumination light in order to reduce systematic deviations in localization.

[0140] Figure 17 shows precision maps of the final iteration of a 3D MINFLUX localization of Alexa Fluor 647 fluorescence emitters bound to the nuclear pore protein Nup96 in U2OS cells with a bottle-beam-shaped excitation light intensity distribution in the xy-section (top), xz-section (middle), and yz-section (bottom). The precision maps were generated as above for the data shown in Figures 5 and 6 and overlaid with elliptical and circular rings, respectively.

[0141] The left column contains precision maps with a correctly aligned phase pattern to generate the bottle-bea-shaped intensity distribution. For the data in the second column, the phase pattern was shifted by 5 pixels on a controllable spatial light modulator, and for the data in the third column, by 10 pixels to distort the intensity distribution.

[0142] Fig. 18 to Fig. 20 show the corresponding intensity distributions, each in a sectional view parallel to the optical axis. Fig. 18 shows the bottle-beam-shaped intensity distribution without shifting the phase pattern, Fig. 19 shows the corresponding intensity distribution with a shift of 5 pixels, and Fig. 20 shows the corresponding intensity distribution with a shift of 10 pixels.

[0143] The distorted excitation light distribution results in an asymmetric, elliptical, distribution of the localizations, particularly in the xz-section. A significant broadening of the distribution is also observed, particularly in the xz-section, which is presumably due to the degeneration of the zero point of the intensity distribution (see Fig. 19 and Fig. 20).

[0144] From Fig. 18 to Fig. 20 it can also be seen that the intensity distribution distorted by the offset of the phase pattern is tilted relative to the optical axis and in the case of a strong shift (Fig. 20) the zero point of the intensity distribution is degenerated by the distortion.

[0145] According to the invention, for example, a light modulator can be controlled to shift a phase pattern for modulating the illumination light beam and thus reduce systematic deviations caused by distortion of the intensity distribution.

[0146] List of reference symbols

[0147] 1 light microscope

[0148] 2 Sample

[0149] 3 Light source

[0150] 4 Wavefront modulator

[0151] 5 Detector

[0152] 6 Beam positioning device, in particular electro-optical modulator

[0153] 7 Beam positioning device, in particular galvanometer scanner

[0154] 8 Actuator

[0155] 9 Lens

[0156] 10 processor

[0157] 10a storage

[0158] 11 mirrors

[0159] 12 tube lens

[0160] 13 Dichroic beam splitter

[0161] 14 emission filters

[0162] 15 lens

[0163] 16 pinhole

[0164] 17 Computing unit

[0165] 18 Control unit

[0166] 19 Circle

[0167] 20 lighting positions

[0168] B Illumination light beam

[0169] D Detection light beam

[0170] L diameter

Claims

Patent claims 1. A method for locating or tracking individual emitters in a sample (2) using a light microscope (1), comprising a. illuminating the sample (2) with illuminating light, wherein an intensity distribution of the illuminating light (B) with a local intensity minimum is formed in the sample (2), and wherein the illuminating light induces or modulates light emissions from an individual emitter in the sample (2), b. detecting light emissions from the individual emitter, and c.determining position data of the individual emitter on the basis of the detected light emissions, characterized in that a wavefront of the illumination light is adapted on the basis of the detected light emissions of the individual emitter and / or a beam path of an illumination light beam (B) of the illumination light and a beam path of a detection light beam (D) of the light emissions are adapted relative to one another and / or a beam path of an illumination light beam (B) of the illumination light and a beam path of a further illumination light beam are adapted relative to one another.

2. Method according to claim 1, characterized in that the adaptation of the wavefront or the beam paths is carried out on the basis of the position data of the individual emitter.

3. Method according to claim 1 or 2, characterized in that the adaptation of the wavefront or the beam paths is carried out during a measuring sequence, wherein the measuring sequence comprises several steps in which the sample is illuminated with the illumination light and the light emissions of the emitter or of individual emitters are detected.

4. Method according to one of the preceding claims, characterized in that position data of the individual emitter are determined multiple times and / or that position data of several emitters in the sample (2) are determined, wherein the adaptation of the wavefront or the beam paths is carried out on the basis of a statistical analysis of the position data.

5. Method according to one of the preceding claims, characterized in that an axial position of the intensity distribution of the illumination light is adjusted, wherein light emissions of the individual emitter are detected for different axial positions of the intensity distribution, and wherein the adjustment of the wavefront or the beam paths is carried out on the basis of the light emissions detected for the different axial positions.

6. Method according to one of the preceding claims, characterized in that the illumination light (B) is excitation light which excites the emitter in the sample (2) to emit the light emissions.

7. Method according to one of the preceding claims, characterized in that the adaptation of the wavefront and / or the beam paths takes place automatically.

8. Method according to one of the preceding claims, characterized in that the emitter is a fluorophore or a molecule labelled with one or more fluorophores.

9. Method according to one of the preceding claims, characterized in that by adjusting the wavefront and / or the beam paths, a deviation between a desired intensity distribution of the illumination light and an actual intensity distribution of the illumination light in the sample (2) is corrected.

10. The method according to claim 9, characterized in that a phase pattern displayed on a wavefront modulator (4) arranged in a beam path of the illuminating light is adapted in order to correct the deviation between the desired intensity distribution and the actual intensity distribution.

11. Method according to one of the preceding claims, characterized in that an aberration correction is carried out by adjusting the wavefront and / or the beam paths.

12. Method according to one of the preceding claims, characterized in that the method comprises a first localization step and a second localization step, wherein in the first localization step the sample (2) is illuminated with the illumination light, first light emissions of the individual emitter are detected and first position data of the individual emitter are determined on the basis of the first light emissions, and wherein in the second localization step the intensity minimum of the intensity distribution of the illumination light is arranged at illumination positions, wherein the Illumination positions are arranged around a position of the individual emitter estimated on the basis of the first position data, second light emissions of the individual emitter are detected for the respective illumination positions, and second position data of the individual emitter are determined on the basis of the second light emissions and the associated illumination positions, wherein the estimated position of the individual emitter is determined with greater accuracy on the basis of the second position data than on the basis of the first position data, wherein a systematic deviation between the estimated position of the emitter determined in the first localization step and the estimated position of the same emitter determined in the second localization step is determined,wherein, on the basis of the determined deviation, the wavefront of the illuminating light is adjusted and / or the beam path of the illuminating light beam (B) and the beam path of the detection light beam (D) are adjusted relative to one another and / or the beam path of the illuminating light beam (B) and the beam path of the further illuminating light beam are adjusted relative to one another.

13. The method according to claim 12, characterized in that the sample (2) is also illuminated in the first localization step with an intensity distribution of the illumination light with a local intensity minimum, in particular wherein the intensity minimum is arranged in the first localization step at illumination positions around a previously estimated position of the individual emitter.

14. The method according to claim 12 or 13, characterized in that the emitter is imaged in the first localization step onto a spatially resolving detector, in particular onto a camera or a detector array.

15. Method according to one of claims 12 to 14, characterized in that the systematic deviation is caused by optical imaging errors.

16. Method according to one of claims 12 to 15, characterized in that the first localization step and the second localization step are carried out with optical means which differ from each other in at least one optical element.

17. Method according to one of claims 12 to 16, characterized in that the adaptation of the wavefront and / or the beam paths is carried out in such a way that the systematic deviation between the position of the emitter estimated in the first localization step compared to the position of the same emitter estimated in the second localization step is reduced.

18. The method according to claim 17, characterized in that a position of a pinhole diaphragm (16) arranged in a beam path of the detection light beam (D) is adjusted such that the systematic deviation between the position of the emitter estimated in the first localization step compared to the position of the same emitter estimated in the second localization step is reduced.

19. Method according to one of the preceding claims, characterized in that the illuminating light beam (B) and the further illuminating light beam have different wavelengths, wherein the beam path of the illuminating light beam (B) and the beam path of the further illuminating light beam are adapted relative to one another such that a local deviation between the illuminating light beam (B) and the further illuminating light beam is corrected.

20. Light microscope (1) for locating or tracking individual emitters in a sample (2), in particular according to a method according to one of claims 1 to 19, comprising - a light source (3) designed to illuminate the sample (2) with illumination light, wherein the illumination light induces or modulates light emissions from a single emitter in the sample (2), - a wavefront modulator (4) designed to form an intensity distribution of the illumination light with a local intensity minimum in the sample (2), - a detector (5) designed to detect light emissions from the individual emitter, - a computing unit (10, 17) designed to determine position data of the individual emitter on the basis of the detected light emissions, characterized in that the light microscope (1) has a control unit (10, 18) designed to adapt a wavefront of the illuminating light on the basis of the detected light emissions of the individual emitter and / or to adapt a beam path of an illuminating light beam (B) of the illuminating light and a beam path of a detection light beam (D) of the light emissions relative to one another and / or to adapt a beam path of the illuminating light beam (B) and a beam path of a further illuminating light beam relative to one another.

21. Light microscope (1) according to claim 20, characterized in that the control unit (10, 18) is designed to control the wavefront modulator (4) or a further wavefront modulator on the basis of the detected light emissions of the individual emitter such that the wavefront of the illumination light (B) is adapted.

22. Light microscope (1) according to one of claims 21, characterized in that the further wavefront modulator is a deformable mirror.

23. Light microscope (1) according to one of claims 20 to 22, characterized in that the light microscope (1) has an actuator (8) which is designed to displace an optical component arranged in a beam path of the illuminating light beam (B), the further illuminating light beam and / or the detection light beam (D), wherein the control unit (10, 18) is designed to control the actuator (8) on the basis of the detected light emissions of the individual emitter such that the beam path of the illuminating light beam (B) and the beam path of the detection light beam (D) are adapted relative to one another and / or the beam path of the illuminating light beam (B) and the beam path of the further illuminating light beam are adapted relative to one another.

24. Light microscope (1) according to claim 23, characterized in that the light microscope (1) has a pinhole diaphragm (16) arranged in a beam path of the detection light beam (D), wherein the actuator (8) is designed to adjust the beam position of the detection light beam (D) relative to the pinhole diaphragm (16).

25. Light microscope (1) according to one of claims 20 to 24, characterized in that the light microscope (1) has a beam positioning device (6, 7) which is designed to position the intensity distribution of the illumination light (B) in the sample (2).

26. Light microscope (1) according to one of claims 20 to 25, characterized in that the detector (5) has a plurality of, in particular individually readable, detector elements.

27. A computer program comprising instructions which cause the light microscope (1) according to one of claims 20 to 26 to carry out the method according to one of claims 1 to 19.

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