WO2025016752A1 - Method, light microscope and computer program for locating or tracking individual emitters in a sample - Google Patents

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

METHOD, LIGHT MICROSCOPE AND COMPUTER PROGRAM FOR LOCALIZING OR TRACKING INDIVIDUAL EMITERS IN A SAMPLE

Technical field of the invention

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

State of the art

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 illumination 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.

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.

The term “M INFLUX” is 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 by first pre-localizing a single fluorophore by scanning with a first Gaussian excitation light distribution and then placing a second, donut-shaped excitation light distribution at points that form a symmetrical pattern of illumination positions around the position of the fluorophore 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.

Further variants and embodiments of a 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. The sample is illuminated in several iteration steps at illumination positions with the minimum of a donut-shaped excitation light distribution, whereby the illumination positions form a symmetrical illumination pattern centered around the position of the fluorophore estimated in the previous step, and whereby the illumination positions are placed more closely around the currently estimated position of the fluorophore in each iteration step. This allows a very high position accuracy to be achieved in just a few steps.

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)”.

The light that induces or modulates the light emission of the particles can also be, for example, 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 that are based on superimposing 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 values of the fluorescence intensity at different positions of the STED intensity distribution.

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 taken 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 taken with the pinhole aperture opened to different widths in the detection beam path, wherein a position of the pinhole aperture is adjusted on the basis of the determined offset.

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 in order to correct aberrations of the STED light beam and optionally of 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 with 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 method.

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 in order to determine the position of the reference marker using the MINFLUX method. The sample is then tracked starting from the determined position in order to compensate for a drift of the sample relative to the objective of the light microscope or a drift of objects within the sample, depending on the type of reference marker used.

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

However, localization microscopy methods such as MINFLUX differ fundamentally from these methods in their image generation principle. There is no optical imaging of the sample, but an image of several individual emitters or a trajectory of a moving emitter (in the case of a tracking experiment) is mathematically composed from many individual localizations. Misalignments in the beam path and aberrations therefore have very different effects than in imaging microscopy, whereby the specific effect depends largely on the localization principle and the specific implementation of the localization experiment.

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

Known methods in which localization errors in localization microscopy are subsequently corrected by data processing are, however, relatively difficult to integrate into live measurements or extend the measurement time and therefore have a negative impact on user-friendliness. task of the invention

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

Solution

This object is solved 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.

Description of the Invention

A first aspect of the invention relates to a method for localizing 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 order given: illuminating the sample with illumination light (in particular with an illumination light beam of illumination light), wherein an intensity distribution of the illumination light is formed in the sample, wherein the intensity distribution has a local intensity minimum, and wherein the illumination 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 adjusted 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 adjusted 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 adjusted relative to one another.

By adjusting the wavefront or the beam paths based on the light emissions of a single emitter, specific effects of misalignments and aberrations (caused by components in the beam path of the light microscope or by the refractive index distribution in the sample) on the localization accuracy in localization microscopy can advantageously be compensated or corrected. In contrast to known methods from the state of the art, this is not done by subsequent processing of the position data but by adjusting the beam path, which is particularly useful in live Interactions with the user, iterative localization procedures and tracking procedures have a positive effect on user-friendliness.

In particular, the adjustment of the wavefront or the adjustment of the beam paths can be carried out in such a way that the geometric focus of the illumination light does not shift relative to the sample due to this adjustment. The adjustment of the beam paths according to the invention does not, of course, mean pure scanning of the illumination light over the sample or pure tracking of the sample, e.g. as part of a drift correction.

The wavefront can be adjusted, for example, by modulating the phase and/or amplitude of the illumination light.

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.

The beam path of the illumination light beam and the beam path of the detection light beam can be adjusted relative to one another by shifting the illumination light beam while the detection light beam remains unchanged, by shifting the detection light beam while the illumination light beam remains 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 one another.

The term emitter refers to the unit whose position in the sample is to be determined using the localization method. The light emission can therefore occur directly through the emitter itself (particularly if the emitter is a fluorescent dye molecule) or indirectly through markers coupled to the emitter (e.g. fluorescent dye molecules covalently or non-covalently bound to a protein). Light emission can not only mean the active emission of light by the emitter or the markers in the sense of luminescence, but also light emission caused by (Raman/Rayleigh/Mie) scattering. In concrete terms, 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.

Individual emitters are those emitters that can be separated or resolved using optical means. This can mean in particular that the emitters are at a spatial distance from one another that is above the optical diffraction limit. In this sense, emitters are also individual if they can be registered one after the other, for example by registering light emanating from a first emitter at a time. at which a neighboring emitter does not emit light because it is (in the case of fluorophores) in a dark state. In this way, emitters that are at a distance below the diffraction limit but flash asynchronously can also be resolved using a light microscope. Finally, it is also possible to resolve emitters that are at a distance below the diffraction limit but emit light of different wavelengths using a light microscope by spectrally separating the emitted light or to excite two emitters with different excitation spectra at different wavelengths in order to optically separate the emitters. Finally, emitters that have 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”.

The sample is illuminated with the illumination light, particularly in the vicinity of a suspected position of the individual emitter. The illumination light can in particular be excitation light, which excites the emitter(s) to fluoresce or is scattered by the emitter(s), thus inducing light emission. Alternatively, the illumination light can also modulate, in particular inhibit, the light emission. Examples of this are STED light, which extinguishes 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. Illumination light that modulates light emission is used in particular in combination with excitation light.

To detect the light emission of the emitter, either a point detector (such as an Avalanche photodiode, 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 position data of the emitter are determined from the light emission, whereby the position can be determined in particular on the basis of light emissions registered at several illumination positions of the illumination light. The registered light emissions of the emitter can be individual photons registered one after the other (e.g. with an APD). However, the light emissions can also be registered as groups of several photons (as is sometimes the case with so-called hybrid detectors).

The emitter can be localized in particular by a method according to the MINFLUX principle, ie the emitter is illuminated at several illumination positions arranged around the previously determined position of the emitter with an intensity distribution of excitation light having a local intensity minimum, the position of the emitter being estimated from the light emissions assigned to the illumination positions. The illumination light is therefore excitation light. When localizing according to the MINFLUX principle, the Less light emissions can be expected the closer the intensity minimum of the illumination light (excitation light) is to the actual position of the emitter, which makes the method particularly photon efficient.

Alternatively, the emitter can also be localized in particular according to a STED-MINFLUX principle, i.e. the emitter is illuminated at several illumination positions arranged around the previously determined position of the emitter with a superposition of an intensity distribution of excitation light having a local intensity maximum with an intensity distribution of prevention light (e.g. STED light) having a local intensity minimum, whereby the position of the emitter is estimated from the light emissions assigned to the illumination positions. In this case, the illumination light is the prevention light. When localizing according to the STED-MINFLUX principle, the closer the intensity minimum of the illumination light (prevention light) is to the actual position of the emitter, the more light emissions occur.

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 only covers 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.

According to a further embodiment of the method, the illumination positions are not arranged at discrete points around the emitter, but 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 carried out in a similar way to continuous scanning in scanning microscopy, e.g. by defining corresponding time intervals (so-called dwell times) and assigning the light emissions to an average illumination position during the corresponding time interval. Alternatively, the assignment of light emission to illumination position can also be carried out by conversely registering the current illumination position as soon as a photon of the light emission is detected.

A sequence of lighting positions can be run through either once or multiple times. Alternatively, a variable sequence of lighting positions is also possible. It is particularly advantageous to recalculate the lighting positions based on a localization of the emitter, especially iteratively, and to arrange them successively closer to the (actual) position of the emitter. To determine the position data of the emitter, the light emissions of the emitter are detected at the illumination positions, and the position of the emitter is finally determined from the light emissions detected at the illumination positions. For this purpose, position estimators are used in particular, such as those known from the state of the art for the MINFLUX method (see e.g. “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 indicate a position in one, two or three spatial directions.

The local intensity minimum can be point-shaped, linear or formed as a surface. Ideally, it is an intensity zero, zero line or zero surface. The local minimum can also be a global minimum of the intensity, in particular 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.

The intensity distribution can specifically be a 2D donut or a 3D donut (also known as a bottle beam). Such intensity distributions can be generated, for example, by phase modulation of the illumination light with a phase plate or a so-called spatial light modulator (SLM). The person skilled in the art is familiar with corresponding methods from the state of the art for STED and MINFLUX microscopy.

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

According to one embodiment, the adaptation of the wavefront or the beam paths is carried out on the basis of the position data of the individual emitter, wherein the position data is determined on the basis of the detected light emissions. In particular, the adaptation of the wavefront or the beam paths can be carried out on the basis of a comparison of different position data of the same emitter. For example, the position data of an emitter obtained from several localization steps can be analyzed in order to detect 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 localized with comparatively higher accuracy in earlier localization steps.

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 determination of the position of an emitter or several consecutive determinations of the position of different emitters.

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, include 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.

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 in a moving manner for the last n emitters and used to adapt the wavefront or the beam paths.

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 for different axial positions of the intensity distribution are recorded, and the adjustment of the wavefront or the beam paths is carried out on the basis of the light emissions recorded for the various 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 various 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. This type of evaluation can be used to correct localization errors that depend on the axial coordinate (z-coordinate) (ie in particular systematic effects of the z-position on the position estimator).

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.

According to a further embodiment, the illumination light is excitation light that stimulates 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 in particular. This has the advantage that a highly precise localization of the emitter can be carried out with particularly few photons emitted by the emitter.

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. In particular, a control unit of the light microscope can therefore automatically pass on 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 on the basis of the control signal. Alternatively or additionally, for example, the same control unit or another control unit can automatically pass on a corresponding control signal to a wavefront modulator, which modulates the wavefront of the illumination light on the basis of the control signal. This type of automatic adjustment can also be implemented 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 for adjusting the beam path can in particular be the same emitters that are used for 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 carried out. In particular, the refractive index of the sample is identical during adjustment and measurement.

According to a further 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 by localization microscopy.

According to a further embodiment, the emitter is a molecule labeled with a fluorophore. The link between the fluorophore and 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, position information about the target structure can be collected indirectly by localizing the emitter (here antibody or nanobody with fluorescent dye). Labeling with only one fluorophore has the advantage that the light emissions tend to come from a smaller area of the sample, and thus more meaningful information about the target structure can be obtained.

According to a further embodiment, the emitter is a molecule labeled with several fluorophores. This can be, for example, an antibody covalently coupled to several fluorophores, which in turn binds to a structure of interest in the sample. Coupling to several fluorophores has the advantage that more photons are emitted overall and thus a higher positional accuracy can be achieved during localization. In addition, irreversible bleaching only occurs after a longer period of time, which allows position determination to be carried out over a longer period of time.

According to a further embodiment, a close area of the sample in which the emitter to be localized or tracked is located according to a presumed position determined in advance is illuminated with the intensity distribution of the illumination light with the local minimum. This close area is typically derived from an estimated position of a previous localization step. The first localization step can be carried out, eg in a MINFLUX method, with an independent localization technique of lower accuracy, which is also referred to as pre-localization. According to a further embodiment, by adjusting 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), a deviation between a desired intensity distribution of the illumination light and an actual intensity distribution of the illumination light in the sample is corrected. 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.

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 a skewed axis tilted to the optical axis due to aberrations. Such light distributions are used, for example, in 3D MINFLUX localization, and in some cases both for axial and lateral localization. A skewed 3D donut leads to a systematic deviation in the position estimation in lateral localization, depending on the current focal plane. Such deviations can be corrected, for example, by recentering the illumination light beam on the pupil of the objective.

According to a further embodiment, a phase pattern is adjusted which is displayed on a wavefront modulator, 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 which 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 which 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 by 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. By using 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, a 3D donut-shaped intensity distribution can be generated under ideal conditions. 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 due to 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.

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). Due to certain optical aberrations, for example, the intensity distribution of the illumination light in the sample can deviate from a desired intensity distribution, e.g. 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.

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 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 compared to the position determined in the second localization step estimated position of the same emitter, wherein on the basis of 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 each other and/or the beam path of the illuminating light beam and the beam path of the further illuminating light beam are adjusted relative to each other.

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.

In particular, the position of the emitter can be determined in the second localization step with an accuracy that is better than a threshold value of the accuracy. In particular, the accuracy in the second localization step can reach an optimum or approach an optimum. In the latter cases in particular, the position of the emitter determined in the second localization step can be regarded as 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 adjustment of the wavefront or the beam paths can be carried out.

The first localization step can in particular be a pre-localization step that is not carried out according to the MINFLUX principle, but with an independent imaging or localization technique in order to obtain an initial position estimate for an individual emitter, on the basis of which a MINFLUX localization is then carried out. In this pre-localization step or in an upstream step, the emitter can in particular also be found, i.e. the sample is searched for individual emitters by registering light emissions, which are then pre-localized and finally localized with high accuracy.

According to a further embodiment, the first localization step is carried out according to a stochastic localization method. This includes in particular STORM (stochastic optical reconstruction microscopy) and PALM (photoactivated localization m/croscopy) microscopy as well as SOFI (superresolution optical fluctuation imaging) microscopy and PAINT (points accumulation for imaging in nanoscale topography) microscopy. The emitters are in a non-fluorescent dark state, and individual emitters are spontaneously converted from this dark state to a fluorescent state by photoactivation or by chemical reaction, or there is an equilibrium between the non-fluorescent dark state and the fluorescent state that can be influenced by setting 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 simultaneously stimulated to emit light. In this case, the light emission is preferably detected using a spatially resolving or imaging detector, i.e. a detector that has a large number 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, a large number 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. A balance between an emitting state and a dark state is also typically set in MINFLUX microscopy in order to be able to localize emitters that are spatially densely arranged one after the other in the sample.

According to a further embodiment, the first localization step is carried out by scanning the sample with the illumination light. This means that in particular the first localization step is carried out by means of laser scanning, which can optionally be carried out, for example, as conventional confocal laser scanning or also as STED laser scanning. To do this, 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. 2D or 3D donut) and an image is reconstructed from the light emissions detected at each scan point, in which the individual emitters can in turn be localized. For image acquisition in STED mode, the excitation light can be superimposed with a distribution of de-excitation light having a local intensity minimum, which improves the resolution beyond the optical diffraction limit and enables more precise localization of the emitter in the first localization step.

In the second localization step, the emitter can be localized in particular by a method according to the MINFLUX principle, i.e. the emitter is illuminated at several illumination positions arranged around the position of the emitter determined in the first localization step with an intensity distribution of illumination light having 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 take place between the first and second localization steps; the only decisive factor for the embodiment described above is the presence of a first and a second localization step.

Like the illumination light used in the first localization step, the illumination light used in the second localization step can also induce or modulate the light emission of the emitter, in particular inhibit it. The illumination light used in the second The illumination light used in the localization step can be identical to the illumination light used in the first localization step, but this is not absolutely necessary. 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 localization of an emitter from a confocal image in the first localization step and localization of 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 with an intensity minimum. Such switching can be implemented using a programmable phase modulator (spatial light modulator, SLM), among other things.

In practice, there is often the problem 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) be location-dependent, i.e. dependent on the position of the emitter in the field of view. A systematic deviation is only to be understood as the fact that, when one and the same emitter is localized repeatedly, the same deviations occur between the first and second localizations within the scope of the measurement accuracy. Regardless of this, the systematic deviations can be subject to (slow) changes, for example as a result of drift effects.

According to a further embodiment, the sample is also illuminated in the first localization step with an intensity distribution of the illumination light with a local intensity minimum. According to a further embodiment, the intensity minimum in the first localization step is arranged at illumination positions around a previously estimated position of the individual emitter. For example, the first localization step can already be carried out according to a MINFLUX method (according to a MINFLUX principle), whereby the first and second localization steps can be steps of an iterative MINFLUX method, for example, in which the position of the emitter is determined in each iteration and the illumination positions are adjusted on the basis of the last position determination, in particular arranged closer 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 in successive iterations of the MINFLUX method is not obvious, a systematic offset is regularly observed in practice, which is probably 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, whereby 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 recorded 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 as high as that of conventional image recording.

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 - the localization of the emitter in the first localization step by means of scanning (in particular laser scanning) or according to 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 recognized, for example, if several emitting emitters are located within a diffraction-limited area and cannot be individually localized.

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 several detector elements (in particular pixels) of the spatially resolving detector.

According to a further embodiment, the systematic deviation is caused by optical imaging errors. Systematic deviations between the positions of the emitter determined in the first localization step and in the second localization step often occur as a result of optical imaging errors and in particular when the optical beam path used for determining the position in the first localization step differs from the optical beam path used for determining the position in the second localization step or the first and second localization steps are carried out using optical means that differ from one another in at least one optical element.

According to a further embodiment, 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. As soon as the beam paths differ, it becomes necessary to carry out an initial adjustment of the beam paths to one another and, if necessary, to keep them constant over a longer period of time. In addition, the imaging properties of the two beam paths are usually different, so that due to different imaging errors, in particular spherical aberrations, transverse chromatic aberrations, coma and astigmatism, the images produced by the two beam paths are no longer congruent over 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.

However, a systematic deviation in the position determination of the emitter in the first localization step and in the second localization step can also occur 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 case of the donut-shaped light distribution) and thus from the center of the light distribution used in the first localization step, as a result of which the localizations systematically deviate 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 light emission of the emitter on the intensity of the illumination light at the location of the emitter. This case can occur, for example, if the first localization step is also carried out with a MINFLUX method, but with illumination points further apart and/or lower intensity of the illumination light.

According to a further embodiment, the adaptation of the wavefront and/or 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) 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.

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.

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

The initial pre-localization step in MINFLUX microscopy is partly carried out using pinhole orbit scanning. When the intensity distribution of the illumination light is stationary relative to the sample (in particular an intensity distribution with a local minimum such as a 2D or 3D donut), the projection of the pinhole into the sample is moved on a circular path, the light emissions from the sample are detected and a rough position estimate for the emitter is made based on the detected light emissions, on the basis of which the subsequent MINFLUX localization steps are then carried out. The rotation of the pinhole projection can be enabled, 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).

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

In the event that 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 that is carried out using a pinhole orbit scan method. This means that the projection of the pinhole into the sample is moved on a circular path, in particular using beam scanners that are independent of one another, wherein 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, wherein light emissions from the sample are detected, and wherein a position of an individual emitter in the sample is estimated on the basis of 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 one another on the basis of the detected light emissions of the individual emitter in such a way that a local 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 marked with fluorophores) in the sample can be excited in order to carry out multi-color MINFLUX localization. In this case, for example, the different illumination light beams can be shifted parallel to one another in such a way that the corresponding intensity distributions formed by the different illumination 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 with the illumination light beam and a second position of the emitter determined with the further illumination light beam can be determined and minimized in particular by adjusting the relative beam path. Illumination beams of different wavelengths can lead to the emission of light emissions, in particular even with the same emitter, eg in the case of fluorescence excitation, when the excitation spectrum of the emitter includes both wavelengths.

A second aspect of the invention relates to a light microscope for localizing 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 designed 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 designed to form an intensity distribution of the illumination light with a local intensity minimum in the sample, at least one detector designed to detect light emissions of the individual emitter, a computing unit designed to determine position data of the individual emitter on the basis of the detected light emissions, wherein the light microscope has a control unit designed to adapt a wavefront of the illumination light on the basis of the detected light emissions of the individual emitter and/or to determine 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 emitters relative to each other and/or to adjust a beam path of the illumination light beam and a beam path of another illumination light beam relative to each other. According to a further embodiment, the control unit is designed to control the wavefront modulator or another wavefront modulator on the basis of the detected light emissions of the individual emitter such that the wavefront of the illumination light is adjusted.

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 change the phase distribution in particular, so that the intensity distribution in the sample is changed, in particular so that the actual intensity distribution corresponds to a desired intensity distribution.

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.

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.

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 be coupled directly 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 a beam deflection element, e.g. a mirror, to which the actuator is coupled, wherein the beam deflection element adjusts the beam position of the detection light beam relative to the pinhole. Of course, several actuators can also be provided, each of which is coupled to an optical component, for example, in order to adjust the beam position of the detection light beam in several spatial directions. According to a further embodiment, the detector has a plurality of detector elements, in particular those that can be read out individually. 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 AVC photodiodes.

The light source in particular comprises one or more lasers.

According to a further embodiment, the light microscope has a

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

intensity distribution of the illumination light in the sample.

The beam positioning device is designed in particular to position the intensity distribution of the illumination light at illumination positions in the sample within a field of view of interest. The beam positioning device is preferably designed such that the intensity distribution can be repositioned within 10 ps, in particular within 5 ps and further in particular within 1 ps, at least between illumination positions that are less than 500 nm apart, in particular less than 250 nm and further in particular less than 100 nm apart. For this purpose, the beam positioning device can in particular have 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) that covers a larger positioning range.

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

Advantageous developments of the invention emerge from the patent claims, the description and the drawings and the associated explanations of the drawings. The described advantages of features and/or combinations of features of the invention are merely examples and can be used alternatively or cumulatively.

The following applies to the disclosure content (but not the scope of protection) of the original application documents and the patent: 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 different from the selected References to the patent claims are possible and are hereby suggested. This also applies to features that are shown in separate drawings or are mentioned in their description. These features can also be combined with features of different patent claims. Likewise, features listed in the patent claims can be omitted for further embodiments of the invention, but this does not apply to the independent patent claims of the granted patent.

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

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

Short description of the characters

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

Fig. 2 shows MINFLUX localization data of an emitter;

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

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

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

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

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

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

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;

Fig. 10 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 a shifted phase pattern;

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

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

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;

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;

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;

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;

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;

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

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

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

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 illumination light beam B of illumination light, in particular excitation light. The illumination 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 illumination light in order to generate an intensity distribution of the illumination light with a local intensity minimum, e.g. a 2D donut or a 3D donut (bottle beam), at the focus in the sample 2.

The phase-modulated light beam is then reflected at 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.

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.

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 also be designed as separate processors.

The processor 10 is designed to receive data from the detector 5, which represent 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 etcher. In particular, in order 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 also connected to the beam positioning devices 6, 7 in order to control them.

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 so that a wavefront of the illumination light is adjusted in order to adjust the intensity distribution of the illumination light so to change it so that it approaches a desired intensity distribution or controls the actuator 8 so that the detection light beam D is centered on the pinhole 16. The latter is carried out in particular in such a way 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.

To generate the localization data shown in Fig. 2, the position of a fluorescent nanoparticle (emitter) was determined several times by shifting a 2D donut-shaped intensity distribution of excitation light with electro-optical deflectors to six illumination positions 20 distributed symmetrically on a circle 19 with a diameter of L=300 nm and recording the light emissions for each illumination position 20. 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 scans the excitation light beam and descans 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.

When measuring 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.

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.

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 symmetrically distributed, but rather maxima of the intensity form diagonally to the x and y axes.

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

This implies that localizations of emitters in the presence of astigmatism aberrations of the excitation PSF show systematic deviations, especially when there is no perfect confocal capture of the emission light, but the center of the projection of the pinhole into the sample is shifted relative to the actual position of the emitter in the sample. Such deviations occur regularly in MINFLUX methods, since the true position of the emitter is only known with great uncertainty at the beginning of the method.

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

Fig. 5 and Fig. 6 show xy precision maps of a 2D MINFLUX localization experiment in which Alexa Fluor 647 fluorescence 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 method. Iteration 0 was a pre-localization using pinhole orbit scanning. The projection of the pinhole into the sample was moved on a circular path with a stationary intensity distribution in the sample by controlling two scanning devices, both of which scan 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 method with the pattern shown in Fig. 2 of six illumination positions 20 distributed symmetrically on a circle 19, with the iterations being assigned different diameters L of the circle 19, 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 in order to obtain further highly precise localizations of an emitter, in particular until the bleaching or transition to a dark state of the respective emitter. The photon-weighted mean of the positions of the respective emitter determined in 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 different emitters were determined and used to generate the precision maps.

A regular 2D donut (Fig. 5) or a 2D donut with additional astigmatism aberration (Fig. 6, Zernike coefficient +0.2) was used as excitation light distribution. It is clear from the data that in the presence of astigmatism aberration there is a systematic deviation of the estimated position, which is expressed in an elliptical distribution of the localizations (Fig. 6). In contrast, without aberrations (Fig. 5) there is a symmetrical circular distribution of the localizations. To better visualize the elliptical or circular distributions, the data in the plots are overlaid with a black elliptical or circular ring, from which the respective ellipticity can also be determined, particularly when the data is adjusted accordingly.

Such deviations can lead to the localizations converging more slowly to the true position and pose the risk that emitters fall out of the capture range of the localization and thus cannot be successfully localized. In addition, with a large systematic deviation, the emitters can often be illuminated with unnecessarily high light intensities, which, depending on the type of sample, can lead to problems with photobleaching or phototoxicity. This generally applies to all systematic deviations in the position determination described below.

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.

Fig. 9 and Fig. 10 show an effect of shifting a phase pattern used to phase modulate an excitation light beam relative to the excitation light beam in a 2D MINFLUX localization of 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 using 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, i.e. a 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.

Fig. 10 shows corresponding deviations when the phase pattern is shifted by 10 pixels on a controllable light modulator (spatial 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.

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.

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

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

From the data it is evident 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.

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

Fig. 13 and Fig. 14 show systematic deviations (average values of the deviations of the localizations of several emitters from the respective actual position) 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 botf/e-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.

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 the respective actual position) 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 and in Fig. 13 and Fig. 14. With the deformed intensity distribution, a strong positive deviation of the axial coordinate (z) results for the 0th iteration (pre-localization with pinhole orbit scanning).

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

Fig. 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 Fig. 5 and Fig. 6 and overlaid with elliptical and circular rings, respectively.

The left column contains precision maps with a correctly adjusted 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.

Fig. 18 to Fig. 20 show the corresponding intensity distributions 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 the corresponding intensity distribution with a shift of 5 pixels and Fig. 20 the corresponding intensity distribution with a shift of 10 pixels.

The distorted excitation light distribution results in an asymmetric, especially in the xz-section elliptical, distribution of the localizations. In the xz-section in particular, A significant broadening of the distribution can be observed, which is probably due to the degeneration of the zero point of the intensity distribution (see Fig. 19 and Fig. 20).

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.

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.

list of reference symbols

1 light microscope

2 samples

3 light source

4 wavefront modulator

5 detector

6 Beam positioning device, in particular electro-optical modulator

7 Beam positioning device, in particular galvanometer scanner

8 actuators

9 lens

10 processor

10a storage

11 mirrors

12 tube lens

13 Dichroic beam splitter

14 emission filters

15 lens

16 pinhole aperture

17 computing units

18 control unit

19th district

20 lighting positions

B Illumination light beam

D detection light beam

L diameter

Claims

patent claims 1. Method for localizing or tracking individual emitters in a sample (2) by means of a light microscope (1), comprising a. illuminating the sample (2) with illumination light, 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), b. detecting light emissions of 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 adjusted 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 adjusted 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 adjusted 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 stimulates 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 wave front 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. Method according to claim 9, characterized in that a phase pattern displayed on a wavefront modulator (4) arranged in a beam path of the illumination 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 the wavefront of the illumination light is adjusted on the basis of the determined deviation and/or the beam path of the illumination 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 illumination light beam (B) and the beam path of the further illumination 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 in the first localization step is arranged 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 one another 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 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. 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 illumination light beam (B) and the further illumination light beam have different wavelengths, wherein the beam path of the illumination light beam (B) and the beam path of the further illumination light beam are adapted relative to one another such that a local deviation between the illumination light beam (B) and the further illumination light beam is corrected. 20. Light microscope (1) for localizing 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 of a single emitter in the sample (2), - a wavefront modulator (4) which is 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) which is 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) which is designed to adapt a wavefront of the illumination light on the basis of the detected light emissions of the individual emitter and/or to adapt 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 relative to one another and/or to adapt a beam path of the illumination light beam (B) and a beam path of a further illumination 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 illumination light beam (B), the further illumination 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 illumination 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 illumination light beam (B) and the beam path of the further illumination 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 detector elements, in particular individually readable detector elements. 27. 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.