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

Abstract

The invention relates to a method for localizing or tracking emitters in a sample, wherein the sample is illuminated with an intensity distribution of an illuminating light having a local minimum, wherein the illuminating light excites emitters in the sample to emit photons or modulates the emission of photons by emitters in the sample, wherein detection events are registered with a detector, wherein the detection events each indicate one photon emitted by an emitter in the sample or several photons emitted by an emitter in the sample, wherein weights are assigned to the detection events on the basis of an analysis of a signal pulse shape of the respective detection event, and wherein a position of the emitter in the sample is estimated on the basis of the weighted detection events.

Description

Technical Field of the InventionThe invention relates to a method for locating or tracking emitters in a sample, in particular a MINFLUX method, to a device having a computing unit for carrying out the method, to an optical microscope, in particular a MINFLUX microscope, for locating or tracking emitters in a sample, and to a computer program which causes the device and / or the optical microscope to carry out the method according to the invention.Prior ArtIn light microscopic localization methods, in contrast to classic imaging methods of light microscopy, positions of individual emitters (e.g. fluorophores or molecules labeled with fluorophores) are calculated on the basis of detected light emissions, wherein a localization map can be created from the usually successively determined positions of a multiplicity of emitters, which localization map visualizes the distribution of the emitters in the sample. Such a localization map resembles an image of the sample that can have a resolution significantly below the diffraction limit, i.e. is "super-resolved".The term "individually" is used here to mean that, at a specific point in time, light-emitting emitters whose emission light cannot be distinguished have a distance above the diffraction limit, so that their emission light can be separated. This can be achieved, for example, in the case of permanently light-emitting emitters by a marking density of the sample below a limit value. If the emitters blink asynchronously, for example, a sufficient distance can be achieved at any given point in time even at a higher marking density. For this purpose, in particular the sample environment (e.g. buffer, embedding medium) can be chemically configured such that a desired flashing rate results. Smaller distances can also be tolerable if different types of emitters are present, the emission light of which is optically separable, for example by different emission spectra or emission lifetimes. Finally, in exceptional cases, it may also be possible by means of special evaluation methods (e.g. statistical or time-resolved methods) to locate groups of a plurality of closely adjacent emitters jointly. In this case, a respective position can be determined for each emitter or an average position of a plurality of emitters can be determined.Individual emitters moving in the sample can be tracked by a plurality of rapidly successive localizations in the sample (tracking). The corresponding measurement data can be represented, for example, in the form of a trajectory.In the so-called MINFLUX technique, a sample comprising individual emitters (e.g. individual fluorophores or molecules labeled with fluorophores or light-scattering particles) is illuminated at illumination positions in a region around a roughly estimated position of an individual emitter with an intensity distribution of excitation light, wherein the intensity distribution has an, in particular central, intensity minimum (ideally an intensity zero point). For each illumination position, the light emissions (in particular photon numbers) of the individual emitter are detected. A new position estimate of the emitter is then calculated from the light emissions and the associated illumination positions. The intensity distribution can be, for example, a so-called 2D-doughnut, a so-called bottom beam, or a superposition of these light distributions, which are known from the field of STED microscopy.Various variants of the MINFLUX technique are described, for example, in the publications F. Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612, K. C. Gwosch et al. (2020) MINFLUX nanoscopy detectors 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 and Schmidt, R. et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478 and in the patents U.S. Pat. No. 9,719,928 B1, U.S. Pat. No. 10,900,901 B2, U.S. Pat. No. 10,908,089 B2 and U.S. Pat. No. 10,962,479 B2.Due to the excitation light distribution used with intensity minimum, localization precisions in the single nanometer range can be achieved with the MINFLUX technique with a significantly smaller number of emitted photons compared to the so-called PALM / STORE technique. This is mainly explained in that the emitter is illuminated with less excitation light the closer the minimum of the excitation light distribution is to the actual emitter position.The patent publication U.S. Pat. No. 11,255,791 B2 describes, inter alia, a variant of the MINFLUX technique in which the sample is illuminated with a combination of an excitation light distribution with a local maximum and a STED (stimulated emission depletion) light distribution with a local minimum. The position of a single emitter is also calculated from the light emissions detected for different illumination positions, but the closer the minimum of the STED light distribution (and the maximum of the excitation light distribution superimposed with the minimum) is to the actual emitter position, the more light is emitted. Therefore, there is no advantage here of the particularly high photon efficiency.A specific localization technique using a local maximum excitation light distribution and a local minimum STED light distribution is described in the publication M. Weber et al. (2021) MINSTED fluorescence localization and spectroscopy, Nat. Photonics 15, 361-366 and patent application WO 2023 / 006176 A1 and known under the designation MINTTED. In this case, the combination of the excitation light distribution and the STED light distribution is moved on a continuous path around an estimated emitter position, and the path is adjusted on the basis of the detected photon. In this case, the excitation and STED intensity can additionally be increased.International patent publication WO 2021 / 214312 A1 describes a method for conventional imaging light microscopy in which light emissions, e.g. fluorescence emissions, are detected from a sample with a detector which is capable of registering individual photons. Depending on the temporal sequence of the photons that strike the detector, multiphoton events may occur, i.e., the shortly consecutive photons cannot be resolved as single signal pulses, but generate a single broader signal pulse of the detector. WO 2021 / 214312 A1 relates to an evaluation method which, while determining the width of the signal pulse shape, counts such multiphoton events in order to improve the dynamic range of the detector and thus the image quality and an optional fluorescence lifetime analysis.In localization and tracking methods for individual emitters, such as MINFLUX methods, according to the prior art, the light emissions of the emitters are usually detected using avalanche photodiodes. These detectors have a dead time, i.e. after detection of a single photon, the detector or the electronics coupled to the detector requires a certain time until the next photon can be detected. During this dead time, the detector is "blind" for further arriving photons.However, it would be desirable to be able to use all arriving photons for the position estimation of an individual emitter in order to localize the emitter with as few photons as possible as accurately and as quickly as possible.Object of the InventionThe object of this is to provide a method for locating or tracking emitters in a sample which is improved with regard to its photon efficiency, its position accuracy and its measurement time.SolutionThis object is achieved by the subject matter of the independent claims. Advantageous refinements of the invention are specified in the dependent claims and are described below.DESCRIPTION OF THE INVENTIONA first aspect of the invention relates to a method for locating or tracking emitters in a sample, in particular a MINFLUX method, wherein the sample is illuminated with an intensity distribution of an illumination light with a local minimum, wherein the illumination light excites emitters in the sample to emit photons or modulates the emission of photons by emitters in the sample.In the method, detection events are registered with a detector, wherein the detection events each indicate a photon emitted by an emitter in the sample or a plurality of photons emitted by an emitter in the sample, wherein weights are assigned to the detection events on the basis of an analysis of a signal pulse shape of the respective detection event, and wherein a position of the emitter in the sample is estimated on the basis of the weighted detection events.A method for locating emitters in the sense of the present specification is characterized in that the positions of individual emitters are estimated arithmetically on the basis of detected light emissions. This distinguishes such methods from classic light microscopic imaging methods, such as far-field or scanning microscopy, in which an optical imaging of a plurality of emitters takes place without knowing the positions of the individual emitters.In the context of the present specification, a method of tracking emitters is a method that tracks the position of a moving single emitter over time.These include, for example, so-called single molecule tracking methods. In this case, in particular a trajectory of the emitter is recorded, i.e. a track which connects locations of the emitter that follow one another in time.Emitters in this application are understood to mean objects which, when illuminated with excitation light, can be viewed as point light sources with a view to the measurements according to the invention. The light emanating from the object acting as a point light source can be, for example, scattered light resulting from elastic scattering such as, for example, Rayleigh scattering or inelastic scattering such as, for example, Raman scattering, or it can be luminescent light, in particular fluorescent light. An emitter can thus be, for example, a light-reflecting nanoparticle, a quantum dot, a fluorescent dye molecule (fluorophore) or a molecule or nanoparticle labeled with one or more fluorescent dye molecules. Depending on the size of the molecule and the distance between the fluorophores, a molecule labeled with a plurality of fluorophores or a nanoparticle labeled with a plurality of fluorophores can of course also have a plurality of emitters in the sense of the definition used here.In the context of the present specification, "individual" emitters are understood to mean emitters which can be optically separated by methods of light microscopy. This can be achieved by a labeling density of the sample, which results in an average distance of the emitters above the diffraction limit. Alternatively, asynchronously flashing emitters can also be used if the sample conditions (in particular the composition of the sample buffer and embedding medium) are set such that the mean distances of the light-emitting emitters in each case are above the diffraction limit at any point in time. Finally, different emitters that can be optically distinguished in another way can under certain circumstances also be separated if they have a distance below the diffraction limit, for example on the basis of their characteristic emission spectrum or their emission lifetime. Optionally, a spatially limited region of the sample can be illuminated with activation light in order to transfer emitters in this region from an inactive state, in which the emitters do not emit light when irradiated with excitation light, into an active state, in which the emitters emit light when irradiated with excitation light.The illumination light can be, in particular, excitation light which excites an emitter in the sample to emit light, i.e. induces a light emission of the emitter. The light emissions can in this case be in particular reflected light, scattered light or luminescent light (e.g. fluorescent light). Alternatively, the illumination light can also modulate light emissions of the emitter. In this case, the illumination light can be, for example, STED light which causes emitters to be de-excited from an excited state to the ground state, or switching light which transfers emitters from an actively emitting state to a dark state.The sample is illuminated with an intensity distribution of the illumination light, which forms an intensity distribution with a local minimum in the sample. Intensity increase regions adjoin the local minimum in at least one spatial direction. The local minimum can be, in particular, a central minimum of the intensity distribution, i.e. form a center of the intensity distribution, wherein the center can be arranged, in particular, at the geometric focus. In this case, it can therefore be a light distribution which is point-symmetric with respect to the geometric focus. The local minimum can be, in particular, at least approximately an intensity zero. Such intensity distributions include, in particular, a so-called doughnut and a so-called bottom beam. Such light distributions can be generated, for example, by phase modulation of the illumination light (for example using a phase plate or a spatial light modulator) and focusing into the sample by means of an objective lens.The localization or tracking method according to the invention is in particular a so-called MINFLUX method (if the illumination light with which the sample is illuminated in the localization step is excitation light that induces the light emissions of the emitters) or STED MINFLUX method (if the illumination light with which the sample is illuminated in the localization step is prevention light that modulates the light emissions of the emitters). In a MINFLUX method or a STED MINFLUX method, the local minimum of the intensity distribution of the illumination light is placed at illumination positions in a vicinity of the approximate position of a single emitter, and photons emitted from the emitter are detected for each illumination position. The short range can in particular have an extension of the order of magnitude of the optical diffraction limit. The detected photon numbers and the associated illumination positions can form input values of a position estimator (e.g. a least mean square estimator or a maximum likelihood estimator), with which a position estimate for the emitter is then determined. This process can be repeated iteratively by placing the intensity distribution of the illumination light at updated illumination positions in a vicinity of the updated position estimate and again detecting photons for each position. In this case, in particular a radius of an illumination pattern formed by the illumination positions can be reduced by the previously estimated position. In this case, the light intensity of the illumination light can optionally be additionally increased. The iteration steps can be continued, for example, until the emitter ceases to emit light or until a photon limit or a threshold value of the localization precision is reached. In a MINFLUX or STED MINFLUX (tracking) tracking method, in particular, one iteration or several iterations can be repeated at short time intervals in order to track the path of a moving emitter. In this case, in particular illumination patterns specifically matched to a tracking method can be used.In the context of the present specification, a detection event is understood to mean the impingement of one or more photons on the detector, which leads to a single signal pulse of the detector. This signal pulse can have different signal pulse shapes, in particular different pulse widths and / or pulse areas, depending on the number of photons belonging to the detection event.The weight assigned to a detection event may be dependent in particular on how many photons are assigned to the detection event. For example, a detection event corresponding to the detection of two photons may be assigned twice the weight as a detection event indicating the detection of a single photon. The weights can be in particular whole, rational and / or real numbers, which correspond in particular to the ratios of photons which are indicated by the detection events.Due to the weighted detection events, the photons taken into account in the position estimator advantageously correspond better to the actual number of photons arriving on the detector than in the case of methods according to the prior art. As a result, the position estimation is more accurate and possibly faster (in particular in the case of iterative MINFLUX methods), since an approximation to the actual position of the emitter tends to take place in earlier iterations.Due to the possibility of using the method according to the invention other detectors (e.g. hybrid photodetectors) for single photon counting in localization microscopy instead of avalanche photodiodes, which detectors have essentially no dead time, the photon efficiency (already high in the case of MINFLUX methods) can advantageously also be increased even further.According to one embodiment, the position of the emitter in the sample is estimated on the basis of the weighted detection events and positions of the minimum of the intensity distribution assigned to the detection events. In this case, the intensity distribution of the illumination light can be displaced in a region by a previously estimated position of an individual emitter, for example using a beam scanner (for example using electro-optical deflectors or galvo scanners). Embodiments known from the prior art are, for example, the stepwise displacement of the intensity distribution on an illumination pattern from three to six positions which lie on a circle around the position estimated in advance by means of electro-optical deflectors (see, for example, Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612, K. C. Gwosch et al. (2020) MINFLUX nanoscopy detectors 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 and Schmidt, R. et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478) or a continuous circular movement of the intensity distribution about the position estimated in advance (see, for example, US 2023 / 0008453 A1). Value pairs of position vectors of the illumination positions and corresponding numbers of photons measured at the illumination positions can then be used in the position estimator. When traversing a continuous path, instead of the position vectors of the previously known illumination positions, the position vectors of positions on the path can be used at which the local minimum of the intensity distribution has just been found in a specific detection event. This position can be derived, for example, from control data of the beam scanner or from a measurement by means of a position sensor. Alternatively, during the travel of the continuous path, as described for example in US 2023 / 0008453 A1, a temporal modulation of the light emission signal can be analysed in order to estimate the position of the emitter.According to a further embodiment, the position of the emitter in the sample is estimated on the basis of the weighted detection events and shapes and / or alignments of the intensity distribution assigned to the detection events. When illuminating the sample with the illumination light, instead of a displacement of the intensity distribution (see above) or in addition thereto, the shape and / or orientation of the intensity distribution can be adapted such that an emitter in the sample to be localized or tracked is exposed to different intensities of the illumination light depending on its actual position in the sample (which is known only imprecisely or not at all). Specifically, an intensity distribution with two opposite intensity maxima, which are separated by a two-dimensionally formed intensity minimum intersecting the geometric focus (in particular a zero area of the intensity), can be rotated about the geometric focus, in particular without displacing the geometric focus with respect to the sample, in order to expose the emitter to different illumination intensities depending on its position. Such intensity distributions can be achieved, for example, by phase modulating the illumination light beam with a phase pattern which has a linear phase jump. The switching of the intensity distribution for changing the shape and / or alignment can be realized, for example, with a controllable light modulator (SLM) or electro-optical elements. For each shape and / or orientation of the intensity distribution, the light emissions of the emitter are detected. On this basis, the position of the emitter in the sample is estimated. A corresponding method is described, for example, in U.S. Pat. No. 2023 / 0236401 A1.According to a further embodiment, the position of the emitter in the sample is estimated with a maximum likelihood estimator or with an LMS (least mean square) estimator. Corresponding estimation methods are described, for example, in Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612 (Maximum Likelihood Estimator) and R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478 and EP 3 951 470 A1 (LMS estimator).According to a further embodiment, the illumination light is excitation light that excites the emitters in the sample to emit photons. According to this embodiment, the method according to the invention is thus a MINFLUX method in which the sample is illuminated with an intensity distribution of excitation light with a local minimum (e.g. an excitation light doughnut or an excitation light bottom beam). This has the advantage, in particular, of a particularly high photon efficiency.According to a further embodiment, the detector is a hybrid photodetector. A hybrid photodetector is a combination of a photomultiplier and an avalanche photodiode. The photon striking the detector first generates a photoelectron on a photocathode. The photoelectron is accelerated in vacuum and then impinges on an avalanche photodiode where it is duplicated. Such detectors have the advantage with regard to the method according to the invention that, in contrast to detectors based on avalanche photodiodes (without photomultipliers), they do not have any significant dead time. They can therefore improve the photon efficiency of a tracking method. On the other hand, hybrid photodetectors have the problem that photons which are rapidly successive are not resolved in time, but rather can result in a single signal pulse. By analyzing the signal pulse shape according to the invention and weighting the detection events for position estimation, hybrid photodetectors can nevertheless advantageously be used for a localization or tracking method of emitters, in particular a MINFLUX method.According to a further embodiment, the detector comprises at least one photomultiplier, in particular a microchannel plate photomultiplier (MCP-PMT).According to a further embodiment, the weights are determined on the basis of a width of the signal pulse shape and / or an area of the signal pulse shape. In this case, a greater width and / or area of the signal pulse shape can indicate a greater number of photons on which the corresponding detection event is based. In particular, the width and / or area is determined automatically by a data analysis algorithm. In this case, artificial intelligence, for example a trained neural network, can optionally also be used.According to a further embodiment, the analysis of the signal pulse shape determines a number of photons detected by the detector, which indicates the respective detection event. This can be done, for example, by analyzing the width and / or area of the signal pulse shape. The number of photons is generally at least 1, but can also be zero. This may be the case if the signal pulse shape does not indicate a detection event, but is due to interference signals, for example.According to a further embodiment, the analysis of the signal pulse shape and the assignment of the weights to the detection events are carried out depending on whether an expected photon rate exceeds a predefined limit value. At low photon rates, the probability of detection events being caused by a plurality of photons overlapping on the time axis may be so low that the analysis of the signal pulse shape and the assignment of weights do not significantly improve the result of the localization. That is, almost all weights determined would be identical (e.g., equal to 1 or equal to the inverse of the total photon number). In this case, a reduction in the computing time can be achieved by selectively omitting the evaluation according to the invention.In particular in the case of iterative MINFLUX methods, the photon emission rate frequently gradually decreases during the passage through the iterations. This is because the actual position of the emitter to be localized or tracked is known with ever higher accuracy. Therefore, the positions of the local minimum of the intensity distribution of the illumination light or the shape and / or orientation of the intensity distribution of the illumination light can be selected in the later iterations such that they are always closer to the actual position of the emitter. This successively results in a lower excitation and a lower photon rate. In this method, the limit value for carrying out the analysis of the signal pulse shape and the assignment of the weights can thus be reached in particular.The expected photon emission rate can be determined by measurement, for example it can be determined continuously from the signals of the detector by determining the number of detected emissions in a predetermined time interval and dividing it by the time interval. This determination can be carried out, for example, at periodic time intervals. If the same reference time interval is always used, a limit value of the expected number of photons can of course also be used as a criterion instead of the expected photon emission rate in order to determine whether the method according to the invention is used under the current measurement conditions.Alternatively, the expected photon emission rate (or number of photons) can also be estimated, e.g. on the basis of current measurement parameters, such as e.g. illumination intensity or repetition rate of an illumination laser (in the case of a pulsed excitation), and optionally on the basis of the type of the sample examined and the type of emitters contained in the sample. For example, for certain steps or iterations of a MINFLUX method, the expected photon emission rate may be known and stored in advance. In this case, the steps can have, for example, different extents (e.g. diameters) of an illumination pattern, different laser powers (and thus total intensities of the illumination light) or different shapes or alignments of the intensity distribution of the illumination light.According to a further embodiment, the position of the emitter is estimated multiple times one after the other in respective iteration steps, in particular with increasing position accuracy, wherein the analysis of the signal pulse shape and the assignment of the weights to the detection events is carried out in at least one first iteration step, and wherein the position estimation is carried out in at least one second iteration step, which is carried out after the at least one first iteration step, on the basis of the unweighted detection events.According to a further embodiment, the method has a prelocalization step, wherein the sample is illuminated with illumination light in the prelocalization step and detection events induced or modulated by the illumination light are detected with the detector, wherein weights are assigned to the detection events detected in the prelocalization step on the basis of the analysis of the signal pulse shape of the detection events, wherein a rough position determination of the emitter to be localized or tracked is carried out on the basis of the weighted detection events, and wherein subsequently the intensity distribution of the illumination light is placed in the sample on the basis of the rough position determination.In this way, the photon efficiency or position accuracy of the rough position determination can be improved.The analysis of the signal pulse shape and the assignment of the weights on the basis of the analysis can in particular also be carried out only for the prelocalization step and not in the subsequent localization / tracking steps (in particular MINFLUX steps). This is advantageous because a higher photon rate tends to occur in a prelocalization step in which the position of the emitter in the sample is not yet known than in the subsequent steps (in particular in a MINFLUX method), so that detection events which are attributable to a plurality of photons increasingly occur in this step.In the prelocalization step, the same intensity distribution of the illumination light can be used as in the subsequent steps of the method, but a different intensity distribution, in particular a regular Gaussian focus or a homogeneous illumination of an image field (wide field illumination) can also be used. The illumination light therefore does not have to have an intensity distribution with a local minimum in the prelocalization step.Prelocalization can be performed by various methods. The prior art discloses, for example, the recording of a wide field image, the scanning of the sample with a Gaussian excitation focus and the so-called pinhole orbit scanning (see WO 2022 / 029280 A1). If the corresponding image recording is carried out with detectors which can capture single photons and in which different numbers of photons generate different signal pulse shapes, the method according to the invention can be used in all these prelocalization methods.A second aspect of the invention relates to a device for locating or tracking emitters in a sample, in particular according to the first aspect of the invention, having a computing unit which is designed to analyze signal pulse shapes of detection events registered with a detector. The detection events each indicate photons emitted from an emitter in a sample illuminated with an intensity distribution of illumination light with a local minimum, or a plurality of photons emitted in a sample illuminated with an intensity distribution of illumination light with a local minimum. The illumination light excites the emitter to emit the photons or modulates the emission of photons by the emitter. The arithmetic unit is designed to assign weights to the detection events on the basis of the analysis of the signal pulse shape of the respective detection event and to estimate a position of the emitter in the sample on the basis of the weighted detection events.A third aspect of the invention relates to an optical microscope, in particular a MINFLUX microscope, for locating or tracking emitters in a sample, having an illumination optical unit which is designed to illuminate a sample with an intensity distribution of illumination light with a local minimum, wherein the illumination light excites emitters in the sample to emit photons or modulates the emission of photons by emitters in the sample, and a detector, in particular a hybrid photodetector which is designed to register detection events, wherein the detection events each indicate a photon emitted by an emitter in the sample or a plurality of photons emitted by an emitter in the sample. The light microscope further comprises a device for locating or tracking emitters in a sample according to the second aspect described above.A fourth aspect of the invention relates to a computer program having program code which causes the device for locating or tracking emitters according to the second aspect and / or the light microscope according to the third aspect to carry out the method according to the first aspect.Further features of the second to fourth aspects of the invention are derived analogously from the features of the first aspect described further above.Advantageous further developments of the invention are evident from the patent claims, the description and the drawings and the associated explanations regarding the drawings. The described advantages of features and / or combinations of features of the invention are merely exemplary and can be applied alternatively or cumulatively.With regard to the disclosure content (but not the scope of protection) of the original application documents and of the patent, the following applies: Further features can be gathered from the drawings-in particular the relative arrangements and operative connections illustrated. The combination of features of different embodiments of the invention or of features of different patent claims is likewise possible deviating from the selected relations of the patent claims and is hereby excited. This also relates to features which are illustrated in separate drawings or are mentioned in the description thereof. These features can also be combined with features of different patent claims. Features listed in the patent claims can likewise be omitted for further embodiments of the invention, but this does not apply to the independent patent claims of the issued patent.The reference numerals included in the claims do not represent a limitation on the scope of the subject matter protected by the claims. They are intended merely to make the claims more readily understood.Exemplary embodiments of the invention are described below with reference to figures. These do not limit the subject matter of this disclosure and the scope of protection.Brief Description of the FiguresFIG. 1 schematically shows an exemplary embodiment of the method according to the invention; FIG. 2 shows an optical microscope according to the invention according to an exemplary embodiment.DESCRIPTION OF THE FIGURESFIG. 1 schematically shows an exemplary embodiment of the method according to the invention, in particular of a MINFLUX method, for locating or tracking emitters E in a sample 2. the sample 2 is illuminated in a near region of a position, estimated roughly in advance, of a singulated emitter E with an intensity distribution of illumination light (in particular excitation light, which excites the emitters in the sample 2 to give light emissions, in particular fluorescence), wherein the intensity distribution has a local minimum. The intensity distribution can be, for example, a so-called doughnut or a so-called bottom beam.The minimum of the intensity distribution is shifted in particular one after the other at positions 21 a, 21 bin the sample 2 which form an illumination pattern 20 around the position estimated in advance. In FIG. 1, the actual position of the emitter E is marked within the illumination pattern 20. The illumination pattern 20 comprises three positions 21a,21b,21c distributed symmetrically on a circle, wherein the center of the circle is located at the position of the emitter E estimated beforehand (roughly in the first step).The intensity distribution can be shifted, for example, stepwise successively to the three positions 21 a, 21 b, 21 c, for example using fast beam scanners such as electro-optical or acousto-optical deflectors. Between such jumps, the intensity distribution can remain stationary, in particular for a certain dwell time at the corresponding position 21 a, 21 b, 21 c. Alternatively, it is also possible to move the intensity distribution on a continuous path around the position of the emitter estimated in advance, wherein beam scanners such as galvo scanners or resonant scanners are also possible. In the latter case, for example, the individual detection events 22 a, 22 b, 22 bmay have a time stamp and the associated position of the intensity distribution on the path may be determined on the basis of the time stamp, for example on the basis of control data of the beam scanner or measurement data of a position sensor which may measure a current position of optical elements (for example mirrors) of the beam scanner, for example. Alternatively, a time-modulated emission signal can also be analyzed in order to estimate the position of the emitter, for example by Fourier analysis or in the manner described in US 2023 / 0008453 A1.For each position 21 a, 21 b, photons of the singulated emitter E are detected by a detector 10 (see FIG. 2 ). These trigger detection events 22 a, 22 b, 22 con the detector 10, wherein the detection events 22 a, 22 b, 22 cmay be caused by the incidence of one or more photons on the active surface of the detector 10.FIG. 1 also schematically shows a time series of three signal pulses of the detector 10, which indicate respective detection events 22 a, 22 b, 22 c. The three detection events 22 a, 22 b, 22 ccorresponding to the three exemplary positions 21 a, 21 b, 21 cof the illumination pattern 20. The signal pulse of the detection event 22 ais, for example, wider than the signal pulses of the detection events 22 band 22 c, since the detection event 22 ais attributable to the incidence of two photons overlapping in time, while the detection events 22 band 22 care each attributable only to the incidence of a photon. This is to be explained in that the actual position of the emitter E is farther from the position 21 aof the local minimum of the intensity distribution than from the positions 21 band 21 c.In real localization experiments, a plurality of photons tend to be detected for each position of the intensity distribution, typically about one hundred to a few thousand photons. The simple example illustrated here, in which only a single detection event with one or two photons is present for each position, is intended only to aid in understanding the method according to the invention, but is in no way limiting to the scope of protection of the claims. The principle according to the invention can of course also be applied to a substantially larger number of detection events. For individual detection events, however, the number of photons is often one or two in real localization experiments, wherein higher values (e.g. in the range of 3-10) are also possible.According to the invention, weights w are assigned to the detection events 22 a, 22 b, 22 cbased on an analysis of the signal pulse shape 23 a, 23 b, 23 c. In the present example, the weight 2 is assigned to the detection event 22 abecause of the greater width and / or the greater area of the signal pulse shape 23 a, while the weight 1 is assigned to the detection events 22 band 22 c. This corresponds here to the numbers of photons which have caused the detection events 22 a, 22 b, 22 c, which is advantageous for the position estimation. However, it is also possible that the weights are not identical to the photon numbers. In particular, however, the weights are in a ratio to the photon numbers determined by a function. Another possibility is for the present example (FIG. 1 ) the weight 0.5 for the detection event 22 aand the weight 0.25 for the detection events 22 band 22 c, respectively. These values are obtained by dividing the photon numbers (2, 1, 1) by the total number of photons (4) detected for all three positions 21a, 21b, 21c.Based on the LMS estimator for a MINFLUX method described in EP 3 951 470 A1, an example of an uncalibrated position estimator according to the invention using weighted detection events is the vector sumIn this case, the position estimator, d j denotes the detection event with index j, the position vector of the minimum of the intensity distribution of the illumination light with index j at the corresponding position 21a,21b,21c at which the minimum was at the time of the detection event 22 a, 22 b, 22 c, and w j denotes the corresponding weight with index j which was assigned to the detection event 22 a, 22 b, 22 cin accordance with the invention on the basis of the analysis of the signal pulse shape 23 a, 23 b, 23 c.This has the result that instead of the inaccurate detection events 22 a, 22 b, 22 c, values are taken into account in the estimator itself, which better reflect the actually detected photon numbers. This improves the position estimation and the utilization of the photons.The estimator described above can in particular be calibrated, e.g. as in EP 3 951 470 A1, using a scaling constant or a calibration polynomial.The above-described principle of taking account of weighted detection events in the position estimator instead of the single photons can of course also be applied to other position estimation methods, for example to the maximum likelihood estimator described in F. Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612, the analysis of the light emission modulation described in US 2023 / 0008453 A1 or the method described in WO 2023 / 006176 A1.Of course, the weighting of the detection events can also be applied to a localization or tracking method in which the intensity distribution of the illumination light is not displaced in the sample using a beam scanner, but rather in which the intensity distribution is changed, so that different light emissions result for different actual positions of the emitter. In this case, for example, a light distribution with a planar minimum and two opposing regions of high light intensity separated by the planar minimum can be rotated about the geometric focus, such that the regions of high light intensity lie in the focal plane along different directions. Such a method is described, for example, in US 2023 / 0236401 A1.FIG. 2 shows an exemplary embodiment of an optical microscope 1 according to the invention, in particular a MINFLUX microscope, which is set up to carry out the method according to the invention and has an apparatus 100 according to the invention for locating or tracking emitters E in a sample 2. The light microscope 1 has a light source 3, in particular a laser, which generates illumination light B. The illumination light B is phase- or amplitude-modulated by a light modulator 5 and focused by an objective 8 into a sample 2, so that an intensity distribution with a local minimum, e.g. a doughnut or a bottom beam or a superposition of a doughnut and a bottom beam, is formed in the sample 2. In the case of phase modulation of the illumination light beam, the light modulator 5 can stand, for example, in a pupil plane conjugate to the back aperture of the objective 8 or close to such a plane. The light modulator 5 can be, for example, a phase plate or a phase filter with a permanently impressed phase pattern. Alternatively, the light modulator 5 can also be a spatial light modulator with pixels whose optical properties can be controlled via electrical or optical signals. As shown in FIG. 2, an active surface of the light modulator 5 can be crossed by the illumination light beam, i.e. transmitted, or alternatively reflected or refracted by the active surface (in the case of a spatial light modulator, e.g. by superposition of a phase pattern with a blazed grating). The light modulator may optionally present a hologram on its active surface.Between the light modulator 5 and the objective 8, a beam splitter 9 for separating the illumination light from light emissions from emitters originating from the sample and a scanning device 6, for example a Galvo scanner, are provided, which scans the illumination light beam over the sample 2. In the example shown in FIG. 2, the scanning device 6 is arranged between the beam splitter 9 and the objective 8, i.e. the light emissions originating from the sample 2 pass through the scanning device 6 and the emission light is scanned out of it. Alternatively, the scanning device 6 can also be arranged between the light source 3 and the beam splitter 9 (so-called non-decanned arrangement). In addition to the scanning device 6 shown, the light microscope 1 can optionally have a further scanning device which can be arranged, for example, between the light source 3 and the light modulator 5 or between the light modulator 5 and the beam splitter 9. This additional scanning device can be, for example, a fast scanning device based on electro-optical deflectors or acousto-optical deflectors. Such scanning devices can be advantageously used in MINFLUX methods in order to rapidly shift the intensity distribution of the illumination light B with respect to the sample 2, wherein a high scanning speed is particularly important when tracking an individual emitter E moving in the sample 2. By means of the scanning device 6, which can then be configured in particular as a galvo scanner, a slower coarse positioning of the illumination light B in the sample 2 over a large image field can be achieved. In addition, the scanning device 6 can be used, in particular in a prelocalization step, for the rough position determination of the emitter E. However, if only one scanning device 6, e.g. a gallo scanner, is provided as shown in FIG. 2, this can also be used in particular for MINFLUX localization, e.g. by traversing a continuous path (e.g. a circular path in the sample 2.In particular when the method according to the invention is designed as a STED-MINFLUX method, the light microscope 1 can have, in addition to the configuration shown in FIG. 2, a further light source, the light of which (in particular excitation light, the illumination light being STED light) is combined with the illumination light B in a known manner.In particular for a 3D localization or tracking method, an axial scanning device, e.g. a deformable mirror or an, in particular electro-optical, variable focus lens, can furthermore be provided.In the sample 2, light emissions of individual emitters E are induced or modulated by the illumination light B. These light emissions are focused by the objective 8, scanned by the scanning device 6 and subsequently reflected by the beam splitter 9 into a detection beam path in which a detector 10, in particular a hybrid photodetector, is arranged, which detects detection events 22 a, 22 b, 22 cwhich are caused by the incidence of individual photons or in each case of a plurality of photons on the detector 10.A pinhole may optionally be arranged in front of the detector 10 in order to detect the light emissions confocally (not shown). Furthermore, the emission light in the detection beam path can be split (e.g. spectrally) by optical elements such as mirrors or prisms, and a plurality of detectors 10 (in particular with corresponding spectral sensitivity) can be provided.The detector 10 shown in FIG. 2 is coupled to a computing unit 11 (in particular an FPGA, ASIC or microcontroller or a conventional computer) of a device 100 according to the invention for locating or tracking emitters E in a sample 2. The arithmetic unit 11 is designed to perform an analysis of signal pulse shapes 23 a, 23 b, 23 cof the detector 10, to assign weights w to the detection events 22 a, 22 b, 22 cbased on the analysis, and to estimate the position of the emitter E in the sample 2 based on the weighted detection events 22 a, 22 b, 22 cand also based on the associated positions of the intensity distribution of the illumination light B in the sample 2 or the shapes or alignments of the intensity distribution.The analysis of the signal pulse shapes 23a,23b,23c the assignment of the weights w and the position estimation can, but need not necessarily, be carried out using the same processor, i.e. the computing unit 11 can also be a system of processors connected to one another (and possibly, for example in the case of cloud computing locally distributed).The light microscope 1 furthermore has a control unit 7, which is connected to the arithmetic unit 11 or forms a unit therewith, and is configured to control at least one scanning device of the light microscope 1 (e.g. the scanning device 6 or the above-described further scanning device, which can have e.g. electro-optical or acousto-optical deflectors) on the basis of the position estimated by the arithmetic unit 11. In this way, for example, an iterative MINFLUX method can be implemented, in which an illumination pattern 20 of positions 21 a, 21 b, 21 cis arranged in an iteration step around a position of the emitter E estimated in a preceding iteration step. Alternatively or additionally, the control unit 7 can also control e.g. the light modulator 5, the light source 3 and / or additional switching elements in order to change the orientation and / or shape of the intensity distribution of the illumination light B.From the successively estimated positions of a plurality of emitters E, a high-resolution image of the sample 2 can be calculated. Alternatively, a trajectory of the emitter E with high spatial and temporal resolution can be determined from the successively estimated positions of the same emitter E moving in the sample 2.List of reference characters1 Light microscope 2 Sample 3 Light source 4 Illumination optical system 5 Light modulator 6 Scanning device 7 Control unit 8 Objective 9 Beam splitter 10 Detector 11 Computing unit 20 Illumination pattern 21 a, 21 b, 21 cPosition 22 a, 22 b, 22 c Detektions event 23 a, 23 b, 23 cSignal pulse shape 100 Device for locating or tracking emitters B Illumination light E Emitter O Optical axis w WeightReferences included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedU.S. Pat. No. 9,719,928 B1

[0006] U.S. Pat. No. 10,900,901 B2

[0006] U.S. Pat. No. 10,908,089 B2

[0006] U.S. Pat. No. 10,962,479 B2

[0006] U.S. Pat. No. 11,255,791 B2

[0008] WO 2023 / 006176 A1 [0009, 0066]WO 2021 / 214312 A1

[0010] US 2023 / 0008453 A1 [0029, 0057, 0066]US 2023 / 0236401 A1 [0030, 0067]EP 3 951 470 A1 [0031, 0062, 0065]WO 2022 / 029280 A1

[0046] Cited Non-Patent LiteratureBalzarotti, F., et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325) 606-612, Gwach, K.C. et al. (2020) MINFLUX nanoscopy detectors 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 and Schmidt, R. et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478

[0006] Deflectors (see, e.g., Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612, K.C. Gwosch et al. (2020) MINFLUX nanoscopy detectors 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 and Schmidt, R. et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478

[0029] Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612 (Maximum Likelihood Estimator) and R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478

[0031] Balzarotti, F. et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612

[0066]

Claims

Method for locating or tracking emitters (E) in a sample (2), wherein the sample (2) is illuminated with an intensity distribution of an illumination light (B) with a local minimum, wherein the illumination light (B) excites emitters (E) in the sample (2) to emit photons or modulates the emission of photons by emitters (E) in the sample (2), characterized in that detection events (22a,22b,22c) are registered with a detector (10), wherein the detection events (22a,22b,22c) each indicate a photon emitted by an emitter (E) in the sample (2) or a plurality of photons emitted by an emitter (E) in the sample (2), wherein weights (w) are assigned to the detection events (22a,22b,22c) on the basis of an analysis of a signal pulse shape (23a,23b,23c) of the respective detection event (22a,22b,22c), and wherein a position of the emitter (E) in the sample (2) is estimated on the basis of the weighted detection events (22a,22b,22c).Method according to Claim 1, characterized in that the position of the emitter (E) in the sample (2) is estimated on the basis of the weighted detection events (22a,22b,22c) and positions (21a,21b,21c) of the minimum of the intensity distribution assigned to the detection events (22a,22b,22c).Method according to Claim 1, characterized in that the position of the emitter (E) in the sample (2) is estimated on the basis of the shapes and / or alignments of the intensity distribution assigned to the weighted detection events (22a,22b,22c) and the detection events (22a,22b,22c).Method according to one of the preceding claims, characterized in that the position of the emitter (E) in the sample (2) is estimated with a maximum likelihood estimator or with an LMS estimator.Method according to one of the preceding claims, characterized in that the illumination light (B) is excitation light which excites the emitters (E) in the sample (E) to emit photons.Method according to one of Claims 1 to 5, characterized in that the detector (10) is a hybrid photodetector.Method according to one of Claims 1 to 5, characterized in that the detector (10) comprises at least one photomultiplier.Method according to one of the preceding claims, characterized in that the weights (w) are determined on the basis of a width of the signal pulse shape (23a,23b,23c) or an area of the signal pulse shape (23a,23b,23c).Method according to one of the preceding claims, characterized in that the analysis of the signal pulse shape (23a,23b,23c) determines a number of photons detected by the detector (10), which indicates the respective detection event (22a,22b,22c).Method according to one of the preceding claims, characterized in that the analysis of the signal pulse shape (23a,23b,23c) and the assignment of the weights (w) to the detection events (22a,22b,22c) are carried out as a function of whether an expected photon rate exceeds a predefined limit value.Method according to one of the preceding claims, characterized in that the position of the emitter (E) is estimated repeatedly one after the other in respective iteration steps, in particular with increasing position accuracy, wherein the analysis of the signal pulse shape (23a,23b,23c) and the assignment of the weights (w) to the detection events (22a,22b,22c) are carried out in at least one first iteration step, wherein the position estimation is carried out in at least one second iteration step, which is carried out after the at least one first iteration step, on the basis of the unweighted detection events (22a,22b,22c).Method according to one of the preceding claims, characterized in that the method has a prelocalization step, wherein the sample (2) is illuminated with illumination light (B) in the prelocalization step and detection events (22a,22b,22c) induced or modulated by the illumination light (B) are detected with the detector (10), wherein weights (w) are assigned to the detection events (10) detected in the prelocalization step on the basis of the analysis of the signal pulse shape (23a,23b,23c) of the detection events (22a,22b,22c), wherein a rough position determination of the emitter (E) to be localized or tracked takes place on the basis of the weighted detection events (22a,22b,22c), and then placing the intensity distribution of the illumination light (B) in the sample (2) based on the rough position determination.Device (100) for locating or tracking emitters (E) in a sample (2), having a computing unit (11) which is designed to analyze signal pulse shapes (23a,23b,23c) of detection events (22a,22b,22c) registered with a detector (10), wherein the detection events (22a,22b,22c) each indicate photons emitted by an emitter (E) in a sample (2) illuminated with an intensity distribution of an illumination light (B) with a local minimum or a plurality of photons emitted by an emitter (E) in the sample (2), wherein the illumination light (B) excites the emitter (E) to emit the photons or modulates the emission of photons by the emitter (E), wherein the arithmetic unit (11) is designed to assign weights (w) to the detection events (22a,22b,22c) on the basis of the analysis of the signal pulse shape (23a,23b,23c) of the respective detection event (22a,22b,22c) and to estimate a position of the emitter (E) in the sample (2) on the basis of the weighted detection events (22a,22b,22c).Light microscope (1) for locating or tracking emitters (E) in a sample (2), comprising - an illumination optical unit (4) which is designed to illuminate a sample (2) with an intensity distribution of illumination light (B) with a local minimum, wherein the illumination light (B) excites emitters (E) in the sample (2) to emit photons or modulates the emission of photons by emitters (E) in the sample (2), - a detector (10), in particular a hybrid photodetector, which is designed to register detection events (22a,22b,22c), wherein the detection events (22a,22b,22c) each indicate a photon emitted by an emitter (E) in the sample (2) or a plurality of photons emitted by an emitter (E) in the sample (2), characterized in that the light microscope (1) comprises a device (100) for locating or tracking emitters (E) in a sample (2) according to claim 13.Computer program comprising program code that causes the emitter locating or tracking device (E) according to claim 13 or the light microscope (1) according to claim 14 to carry out the method according to any one of claims 1 to 12.

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