METHOD, DEVICE AND COMPUTER PROGRAM FOR LOCALIZING SINGLE EMISSORS IN A SAMPLE

Abstract

Method for localizing isolated emitters (E) in a sample (2), comprising a prelocalization with the steps - Illuminating the sample (2) with illumination light (B), wherein the illumination light (B) induces or modulates light emissions (D) from a single emitter (E) in the sample (2); - Detecting the light emissions (D) of the emitter (E); - Estimating the position (S) of the emitter (E) in the sample (2) from the detected light emissions (D); and subsequent principal localization using the steps - Illuminating the sample (2) with an intensity distribution (I) of the or another illumination light (B) exhibiting a local minimum (M) at illumination positions (10) arranged around the position (S) of the emitter (E) estimated in the prelocalization; - Capturing the light emissions of the emitter (E) for the respective illumination positions (10); - Determining the position (S) of the emitter (E) in the sample (2) from the light emissions (D) detected for the illumination positions (10), characterized in that the illumination positions (10) for the main localization are determined before illuminating the sample (2) with the intensity distribution (I), wherein the illumination positions (10) form an illumination pattern (11) having a maximum extent (L), the maximum extent (L) depending on a position uncertainty (σ). r ) the position (S) estimated in the pre-localization is set or determined.

Description

Technical field of the invention

[0001] The invention relates to a method, a device and a computer program for localizing isolated emitters in a sample according to the MINFLUX principle and to the use of a fiber bundle in the method according to the invention. State of the art

[0002] The term "MINFLUX microscopy" or "MINFLUX method" refers to specific localization and tracking techniques for isolated emitters. These techniques generate a light distribution of illuminating light at the focus in the sample, inducing or modulating the emitter's light emission. The light distribution exhibits a local minimum. The position of an isolated emitter is determined by detecting light emissions from the emitter, taking advantage of the fact that the smaller the distance between the emitter and the minimum of the light distribution, the less light is emitted by the emitter. Due to this latter fact, MINFLUX methods are particularly photon-efficient, especially compared to so-called PALM / STORM localization techniques.In addition, certain versions of the method also have the advantage that the emitters to be located or tracked are exposed to relatively little light compared to other localization methods and are therefore less bleached.

[0003] The individual light-emitting emitters are typically fluorophores, and the illumination light is typically excitation light, which excites the fluorophores, causing them to emit fluorescent light. Alternatively, the light-emitting emitters can also be light-scattering particles, such as gold nanoparticles.

[0004] The light distribution with the local minimum can be particularly 2D donut-shaped or 3D donut-shaped.

[0005] A method of the type described above was described in patent application DE 10 2011 055 367 A1 for single-molecule tracking. According to the method disclosed therein, the position of a single fluorophore is tracked over time by applying an excitation light distribution with a local minimum to the fluorophore in such a way that the fluorescence emission rate is minimized.

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

[0007] The term "MINFLUX" was used for the first time in the publication "Balzarotti F, Eilers Y, Gwosch KC, Gynnå AH, Westphal V, Stefani FD, Elf J, Hell SW. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science. 2017 Feb 10;355(6325):606-612". The method described therein implements the MINFLUX principle in concrete terms. A single fluorophore is first pre-localized by scanning with a first Gaussian excitation light distribution. A second donut-shaped excitation light distribution is then placed at points that form a symmetric pattern of illumination positions around the fluorophore position estimated in the pre-localization. From the photon numbers registered for the individual illumination positions, the position of the fluorophore is then determined to within a few nanometers using a maximum likelihood estimator.

[0008] Further variants and embodiments of 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.

[0009] The publication "Gwosch KC, Pape JK, Balzarotti F, Hoess P, Ellenberg J, Ries J, Hell SW. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat Methods. 2020 Feb;17(2):217-224" describes iterative 2D and 3D MINFLUX localization methods. In several iterative steps, the sample is illuminated at illumination positions with the minimum of a donut-shaped excitation light distribution. The illumination positions form a symmetric illumination pattern centered around the fluorophore position estimated in the previous step. In each iterative step, the illumination positions are placed more closely around the currently estimated fluorophore position. This allows for very high positioning accuracy to be achieved in just a few steps.

[0010] Another iterative MINFLUX localization and tracking method using a modified position estimator and based on a commercial microscope setup is described in “Schmidt R, Weihs T, Wurm CA, Jansen I, Rehman J, Sahl SJ, Hell SW. MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nat Commun. 2021 Mar 5;12(1):1478”.

[0011] The light that induces or modulates the emitter's light emission can, for example, also be STED (stimulated emission depletion) light. For example, patent applications DE 10 2017 104 736 A1 and EP 3 372 989 A1 describe MINFLUX-like methods based on the superposition of an excitation light distribution with a local maximum and a STED light distribution with a local minimum. The sample is scanned by shifting the STED distribution with the STED minimum, and the position of the fluorophore is determined from the measured fluorescence intensity values ​​at various positions of the STED intensity distribution. In contrast to the previously described MINFLUX methods, the fluorophore emits more light with decreasing distance from the local minimum.

[0012] Patent application DE 10 2021 100 564 A1 describes an iterative 3D MINFLUX method in which lateral and axial localizations are performed in a specific sequence. Furthermore, a dependency between the size of the illumination pattern and position uncertainty in the individual iteration steps is explained.

[0013] DE 10 2020 127 320 B3 discloses a MINFLUX method in which a scanning rule is defined on the basis of pixel signals of a previously recorded raster image of the sample.

[0014] The iterative MINFLUX methods described above reliably deliver very high position accuracy, but have the disadvantage of requiring powerful processors and control units that combine computationally intensive live position estimation with feedback control of beam deflection devices such as electro-optical deflectors.

[0015] The patent application WO 2020 / 128106 A1 and the publication "Masullo LA, Steiner F, Zähringer J, Lopez LF, Bohlen J, Richter L, Cole F, Tinnefeld P, Stefani FD. Pulsed Interleaved MINFLUX. Nano Letters 2021, 21 (1), 840-846" describe, among other things, embodiments of MINFLUX localization methods in which the positions at which the sample is illuminated with the minimum of the excitation light distribution are fixed by arrangements of optical fibers, wherein the excitation light is generated by a pulsed laser, and wherein individual excitation light pulses are emitted at different times through the different fiber ends of the optical fibers.

[0016] The publication “Masullo LA, Lopez LF, Stefani FD. A common framework for singlemolecule localization using sequential structured illumination. Biophysical Reports 2022 2(1), 100036” describes a variant of the MINFLUX technique called RASTMIN, in which a small area within a microscopic field of view containing a single emitter is scanned in a Cartesian grid with the minimum of a donut-shaped excitation light distribution, whereby the position of the emitter is determined from the detected light intensities.

[0017] The publication "Slenders E, Vicidomini G. ISM-FLUX: single-step MINFLUX with an array detector. bioRxiv; 2022. DOI: 10.1101 / 2022.04.19.488747" describes a MINFLUX method in which the light emitted by a single fluorophore is detected position-dependently using an array detector to determine the position of the fluorophore in a single localization step non-iteratively, without repositioning the illumination pattern and without pre-localization.

[0018] However, position determination without pre-localization has the disadvantage that photoactivation is necessary to obtain individual light-emitting fluorophores in the image field. This limits the applicability of the method, since not all fluorophores are photoactivatable.

[0019] DE 10 2016 117 096 A1 describes a small-field microscopy technique in which the sample is illuminated with an intensity distribution of illuminating light, e.g., STED light, with a local minimum and scanned across a small image field. The size of the image field is set to a maximum of 75% of the distance between the maxima of the intensity distribution to protect fluorophores adjacent to the image field from bleaching due to high light intensities. Object of the invention

[0020] Based on the disadvantages of the prior art described above, the object of the present invention is to provide a photon-efficient, computationally and control-technically simple and universally applicable localization method for isolated emitters. Solution

[0021] This object is achieved by the subject matter of independent claims 1, 30, 33 and 34. Advantageous embodiments of the invention are specified in subclaims 2 to 29 and 31 to 32 and are described below. Description of the invention

[0022] A first aspect of the invention relates to a method for localizing isolated emitters in a sample, comprising a pre-localization with the steps - illuminating the sample with illuminating light, wherein the illuminating light induces or modulates light emissions from an isolated emitter in the sample; - Detecting the light emissions of the emitter; - Estimating the position of the emitter in the sample from the detected light emissions; and a subsequent main localization with the steps - illuminating the sample with an intensity distribution of the or another illuminating light having a local minimum in at least one spatial direction at illumination positions arranged around the position of the emitter estimated in the pre-localization; - Recording the light emissions of the emitter for the respective lighting positions; - Determine the position of the emitter in the sample from the light emissions recorded for the illumination positions.

[0023] According to the invention, the illumination positions for the main localization are determined before illuminating the sample with the intensity distribution, wherein the illumination positions form an illumination pattern having a maximum extent, wherein the maximum extent is set or determined depending on a position uncertainty of the position estimated in the pre-localization.

[0024] According to the present invention, the illumination positions are fixed for the main localization (in particular at the beginning of the main localization). In other words, the illumination positions are in particular changeable, but the illumination pattern of the illumination positions is no longer changed during the main localization. During the main localization, the illumination positions remain fixed relative to the sample. In particular, only a single main localization is performed to localize the emitter. The fixing of the illumination positions has the advantage over iterative MINFLUX methods known from the prior art, in which the illumination pattern is adjusted in each iteration step, that the control of the light microscope does not require the otherwise required feedback loop and can thus be designed more simply.

[0025] The term emitter refers to molecules, molecular complexes, or particles that emit light when illuminated with illuminating light. The emitted light can be, in particular, fluorescent light, Rayleigh scattered light, or RAMAN scattered light. An emitter can be viewed as a point light source, particularly in diffraction-limited imaging with a light microscope, and therefore has an extension in the range of the diffraction limit of light microscopy or below. The emitters can be, for example, individual fluorophores (fluorescent dyes), molecules or molecular complexes labeled with one or more fluorophores, or so-called quantum dots. The fluorescent dyes can be bound to the molecules by covalent or non-covalent interactions. Biological macromolecules such as proteins, for example, are often detected by binding to antibodies, which in turn are covalently linked to fluorescent dyes.Furthermore, an emitter within the meaning of the invention can also be, for example, a light-scattering nanoparticle, such as a gold nanoparticle.

[0026] Isolated emitters refer to emitters that are optically separable or optically resolvable. This can mean that the emitters are spaced apart at a distance above the diffraction limit of light microscopy. Alternatively, it is also possible for the light emissions of one emitter to be recorded during a time interval in which a neighboring emitter does not emit light, e.g., because it is in a dark state (in the case of fluorophores). In this way, emitters that are spaced apart below the diffraction limit but blink asynchronously can be resolved using light microscopy. This is known, for example, from stochastic localization methods such as PALM / STORM.Finally, it is also possible to resolve emitters that are separated below the diffraction limit but emit light at different wavelengths using a light microscope by spectrally separating the emitted light, or to excite two emitters with different excitation spectra at different wavelengths to optically separate the emitters. Likewise, emitters with different emission lifetimes can be differentiated from one another by measuring the lifetime (e.g., by time-resolved single-photon counting) and thus detected separately. All of these embodiments fall under the term "isolated emitters."A sample with isolated emitters can be obtained in particular by adjusting the conditions for labeling the sample with fluorescent dyes in such a way that a desired labeling density of individual molecules in the sample is achieved, by targeted photoactivation of fluorescent dyes and / or by adjusting the physicochemical properties of the sample environment (e.g. by reducing agents, oxidizing agents and certain enzymes in the sample) so that a certain blinking rate of the fluorescent dyes is achieved.

[0027] The emitters to be localized are arranged stationary in the sample, particularly during the localization process, with respect to the timescale of the experiment. In this case, the method according to the invention is therefore not a tracking method for tracking the position of an emitter moving within the sample. However, if a sufficiently fast pre-localization method is used, the method can also be designed as a tracking method for tracking the position of an emitter moving within the sample.

[0028] The sample is illuminated with the illumination light, in particular using a light microscope, and in particular through a lens of the light microscope. The illumination light is focused onto the sample, in particular during the main localization and optionally also during the pre-localization.

[0029] The illumination light is, in particular, excitation light, which excites the emitters to fluoresce. Excitation to fluorescence is an example of the induction of light emission by the illumination light. Alternatively, the illumination light can also modulate, e.g., inhibit, the light emission. Examples of this include STED light, which depopulates the excited state of fluorophores through stimulated emission, and switching light, which can, for example, convert fluorophores from the excited state to a dark state, such as a triplet state. Illumination light that modulates light emission is used, in particular, in main localization, and, furthermore, in particular, in combination with excitation light. In pre-localization, only excitation light is used as illumination light.

[0030] During main localization, the sample is illuminated with an intensity distribution of the illumination light that has a local minimum. The local minimum is located in particular at the geometric focus of the illumination light in the sample and is ideally formed as an intensity zero. The local minimum is surrounded by regions of increasing intensity in at least one spatial direction and can be point-shaped, i.e. surrounded by regions of increasing intensity in two or three spatial directions. In particular, the intensity distribution can be a 2D donut or a 3D donut (also referred to as a bottle beam). Alternatively, the local minimum can extend along a line or a plane, i.e. extend along a first spatial direction and be surrounded by regions of increasing intensity in a further second spatial direction (which is in particular perpendicular to the first spatial direction).An intensity distribution with a zero point extending along a plane can be generated, for example, by modulating a linearly polarized light beam with a segmented phase pattern with a phase jump of π. In all the cases described above, a phase plate or a so-called spatial light modulator (SLM) can be used to phase modulate the illumination light. Corresponding methods are known from the state of the art for STED and MINFLUX microscopy. Using the intensity distribution with the local minimum, position determination during main localization can be carried out according to the MINFLUX principle, i.e. it exploits the fact that the light emissions of an isolated emitter are lower the closer the emitter is to the local minimum.

[0031] The illumination positions are the positions at which the local minimum of the illumination light is located. The local minimum is positioned at these positions during the main localization, particularly sequentially, and the corresponding light emissions are recorded. If the local minimum is positioned at the illumination positions in a temporal sequence, a fixed sequence of illumination positions can be run through once or multiple times. Alternatively, a variable or even random sequence of illumination positions is also possible. The displacement of the local minimum relative to the sample can occur stepwise or continuously. With continuous movement, the assignment of the recorded light emissions to an illumination position can be carried out in a similar way to continuous scanning in scanning microscopy, for example.by defining appropriate time intervals (so-called dwell times) and assigning the light emissions to a mean illumination position during the corresponding time interval. The displacement of the local intensity minimum relative to the sample can be achieved, in particular, by beam displacement (e.g., electro-optically or using a galvanometric scanner), sample displacement (e.g., using a sample holder movable by a piezoelectric actuator), or by controlling specific point light sources such as the ends of an optical fiber.

[0032] For one-dimensional localization of the emitter, at least two spaced-apart illumination positions are required. For two-dimensional localization of the emitter, at least three illumination positions are needed in a plane perpendicular to the optical axis of the illumination light (focal plane). Three-dimensional localization requires at least four illumination positions, of which at least two illumination positions are spaced along the optical axis (i.e., not both are in the focal plane). However, significantly more illumination positions can also be used, as is known, for example, from the so-called RASTMIN technique.

[0033] The emitter's light emissions are detected by a detector, e.g., a point detector (such as an avalanche photodiode, APD, a photomultiplier, or a so-called hybrid detector) or an area detector with multiple detector elements or pixels (e.g., a CCD or CMOS camera or an APD array). In the case of an area detector, the individual detector elements can be read out separately. When detecting the light emissions, individual emitted photons can be registered, particularly with time resolution. Evaluation electronics (e.g., a TCSPC module) can be provided for this purpose. APDs with the capability of single-photon counting are also called SPADs (single-photon avalanche photodiodes). If individual photons are detected, a photon rate, for example, can be calculated, which serves as a measure of the emitter's light emission.

[0034] The same detector can be used to detect the light emissions in the pre-localization and the main localization, or different detectors can be used, e.g. a camera for the pre-localization and a point detector or an array of photodiodes for the main localization.

[0035] Estimating the emitter position during pre-localization can be performed in various ways. For example, if a detector with multiple detector elements is used during pre-localization for position-dependent detection of the light emissions in the detection plane, the position can be estimated by determining a two-dimensional intensity histogram and determining the centroid of this histogram, or by determining a moment (in particular, the first moment of the distribution of light intensities). If the sample is scanned during pre-localization with a (e.g., Gaussian) focus of the illumination light, the position can be determined based on the scan position for which maximum light emission occurs.

[0036] For position determination according to the MINFLUX principle in the main localization, a position estimator is used. Estimators known from the state of the art can be used (see, for example, "Balzarotti F, Eilers Y, Gwosch KC, Gynnå AH, Westphal V, Stefani FD, Elf J, Hell SW. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science. 2017 Feb 10;355(6325):606-612").

[0037] The maximum extent (which is defined, in particular, by the diameter of a circle for a polygonal illumination pattern and by the length and / or width of the grid area for a Cartesian grid) is chosen to be as small as possible, since the localization accuracy of the position determined in the main localization depends on this maximum extent. At the same time, however, the maximum extent must be chosen large enough to ensure that the emitter to be localized is, with a high probability, located within a capture range of the localization method. The capture range is the area in which the emitter must be located in order to be unambiguously localized using the method. This means, in particular, that a maximum-likelihood position estimator usable for the corresponding localization method exhibits a clear maximum when the emitter is located within the capture range.The size of the capture area can also depend, in particular, on the type of emission light detection used. For example, the capture area can be increased by a detector with multiple detector elements arranged in a detection plane.

[0038] By taking positional uncertainty into account when selecting the maximum extent of the illumination pattern, the emitter can be located with sufficient accuracy in the main localization with a high degree of probability in a single step (i.e., non-iteratively, unlike the prior art) using illumination positions that are fixed for this step or overall. This allows for much simpler control electronics without feedback loops compared to iterative MINFLUX methods of the prior art. In particular, a higher positional uncertainty of the estimated position during pre-localization leads to a larger maximum extent of the illumination pattern.

[0039] According to one embodiment, the method according to the invention is carried out several times for different emitters, wherein the position of the emitters is determined one after the other, and wherein an image of structures of the sample is created from the determined positions.

[0040] According to one embodiment, the illumination light is excitation light, which induces the light emissions of the emitter. Thus, as in the originally described MINFLUX method, the sample is illuminated with an intensity distribution of excitation light with a local minimum, in particular a zero.

[0041] According to a further embodiment, the illumination positions are arranged on a scanning circle or a scanning sphere around the estimated position of the emitter, with the maximum extent corresponding to the diameter of the scanning circle or the scanning sphere. 2D MINFLUX localization can be realized by arranging the positions on a scanning circle with a suitable diameter, with the positional accuracy of the localization depending on the diameter. For 3D localization, illumination points on a scanning sphere can be used. Alternatively, in the 3D case, the illumination positions can also be arranged on a scanning ellipsoid, with the maximum extent then being defined by the length of the longer major axis of the ellipsoid.

[0042] According to a further embodiment, the illumination pattern is a grid, in particular a Cartesian grid or a hexagonal grid. The term "grid" describes a two- or three-dimensional regular arrangement of illumination points that covers an area of ​​the sample. In particular, neighboring illumination points are equidistant from one another. A Cartesian grid of illumination points is used, for example, in the so-called RASTMIN technique.

[0043] According to one embodiment, the positional uncertainty is determined from the detected light intensities, wherein the maximum extent of the illumination pattern is adjusted according to the determined positional uncertainty. The positional uncertainty is determined, in particular automatically, by means of a computing unit coupled to or integrated into the light microscope. In particular, the positional uncertainty is determined from a number of photons emitted by the emitter recorded during pre-localization. In camera-based localization methods (e.g., PALM / STORM and related methods), the positional uncertainty decreases the more photons are detected from an emitter. If this positional uncertainty is expressed, for example, as the standard deviation of a spatial coordinate, it is inversely proportional to the square root of the total number of photons.Furthermore, the position uncertainty in camera-based methods also depends on the pixel size, the background, and the width of the point spread function. The so-called Cramer-Rao lower bound can be used as the lower limit of the position uncertainty, i.e., the best possible localization precision with any unbiased position estimator.

[0044] By determining the position uncertainty, the maximum extent can be set to particularly well-optimized values ​​in order to achieve the most accurate position estimate possible in a single localization step combined with the information from the pre-localization.

[0045] According to a further embodiment, the maximum extent of the illumination pattern is set to a predetermined value, with the pre-localization being performed in such a way that a positional uncertainty value is obtained that is associated with the predetermined value of the maximum extent. The predetermined value of the maximum extent can be fixed by means of a device, e.g., by using an optical fiber with multiple fiber ends arranged in a pattern. Alternatively, the value of the maximum extent can be fundamentally variable, but can be set for each primary location or a group of primary locations.

[0046] According to a further embodiment, the pre-localization steps are repeated, or a time interval during which the pre-localization is performed is adjusted, until the position uncertainty of the estimated position falls below a predetermined or predeterminable limit. In particular, the pre-localization is performed until the emitter has emitted a predetermined number of photons.

[0047] According to a further embodiment, the maximum extent of the illumination pattern is determined according to the condition L = a · σ r set or fixed, where L denotes the maximum extension, where σ rdenotes the position uncertainty of the position estimated in the pre-localization, and where a denotes a proportionality factor. In particular, the position uncertainty is a standard deviation or a variance of a localization distribution of the emitter. In particular, the proportionality factor a is greater than zero.

[0048] According to a further embodiment, the proportionality factor a has a value in the range from 1 to 3. This means that the maximum extent of the illumination pattern is in particular one to three times the standard deviation of the localization distribution.

[0049] According to a further embodiment, the position uncertainty of the position estimated in the pre-localization is 50 nm or less, in particular 20 nm or less, further in particular 10 nm or less.

[0050] According to a further embodiment, the maximum extent of the illumination pattern is 100 nm or less, in particular 50 nm or less.

[0051] According to a further embodiment, the position estimated in the pre-localization has a greater position uncertainty than the position of the emitter determined in the main localization.

[0052] According to a further embodiment, the estimation of the emitter's position during pre-localization is performed using a non-iterative method. The position is thus estimated only once within a pre-localization after exposure to the illumination light and detection of the light emissions. This means, in particular, that the illumination of the sample with an adapted illumination pattern is not repeated after an initial position estimation in order to refine the position estimate, as is the case, for example, with iterative MINFLUX methods described in the prior art. This reduces the computational effort and increases the speed of the method.

[0053] According to a further embodiment, the sample is scanned during pre-localization using a focus or an intensity minimum of the illumination light, in particular creating a scanned image of the sample. A scanned image is an image with pixels, with each pixel being assigned a light intensity detected for a specific position of the focus or minimum.

[0054] According to a further embodiment, a wide-field image of the sample is generated during the pre-localization step. A wide-field image is an image obtained by substantially homogeneously illuminating the sample with the illumination light and, in particular, by detecting the light emissions from emitters in the sample with a detector having multiple detector elements, such as a camera.

[0055] According to a further embodiment, the positions of a plurality of emitters are estimated in the pre-localization, wherein a first emitter is selected from the plurality of emitters, and wherein the position of the first emitter is determined in the main localization. For this purpose, a detector with a plurality of detector elements, e.g. a camera, can be used. In particular, the positions of the plurality of emitters are estimated using a stochastic localization method. The term “stochastic localization method” refers to a method in which a localization map of the particles with a resolution below the diffraction limit (so-calledAbbe limit, which is determined by the wavelength of the light and the numerical aperture of the objective), whereby the particles or emitters coupled to the particles stochastically switch between an emitting state and a non-emitting state. The conditions are set in particular so that those particles or emitters that are each located in a localization in the emitting state have a distance above the diffraction limit. The stochastic transition between the non-emitting state and the emitting state can, for example, be induced by illuminating the sample with activation light, as is known from the so-called PALM technique (photoactivated localization microscopy). Alternatively, the chemical conditions in the sample can be set so that the particles or emitters blink at a desired frequency, i.e.spontaneously switch between the emitting and the non-emitting state. This is exploited, for example, in the so-called dSTORM technique (direct stochastic optical reconstruction microscopy) and in the SOFI technique (superresolution optical fluctuation imaging). In the latter technique, an autocorrelation function is used, particularly during data evaluation, to separate individual emitters from one another. In particular, the first emitter is automatically selected from among several emitters. Alternatively, manual selection by the user of the light microscope is also possible. The selection can be random or based on a predefined criterion. One such criterion can be the number of photons captured and registered for the respective emitter, which, among other things, determines the positional uncertainty.

[0056] According to a further embodiment, a total photon number of photons to be registered is determined for the emitter, wherein the total photon number is divided into a first photon number and a second photon number, and wherein the position of the emitter in the pre-localization is estimated when the light emissions detected in the pre-localization reach the first photon number.

[0057] In this way, the position of an emitter can be determined in a particularly photon-efficient manner with high positioning accuracy, taking advantage of certain advantages of prelocalization. For example, prelocalization can be particularly fast, allowing a desired combination of speed and accuracy to be achieved by dividing the photon budget.

[0058] Depending on the application, particularly depending on the requirements for measurement speed and positioning accuracy, the total number of photons can be approximately in the range of 40 to 1000 photons. The ratio of the first photon number to the second photon number is in particular 1:10 to 10:1, more particularly 1:5 to 5:1, more particularly 1:2 to 2:1.

[0059] According to a further embodiment, the position of the emitter in the main localization is determined when the light emissions detected in the main localization reach the second photon number.

[0060] According to a further embodiment, the light emissions of the emitter are detected during pre-localization using a detector having a plurality of detector elements, in particular a camera or an array of photodiodes. The detector elements detect the light emissions, in particular in a position-dependent manner, in a detection plane. The detection plane is in particular confocal to a focal plane in the sample, which contains a geometric focus of the illumination light, i.e., the detection plane is an image plane relative to the focal plane in the sample. In particular, the detector elements of the detector can be read individually.

[0061] The detection of the light emissions with the detector having a large number of detector elements during the pre-localization enables a relatively fast position estimation with sufficient accuracy (and adjustable by the number of detected photons) in order to subsequently perform a main localization in a single step with a fixed maximum extent of the illumination pattern.

[0062] According to a further embodiment, the position of the emitter estimated during the pre-localization is determined using a position estimator, e.g. a least-mean-square estimator or a maximum-likelihood estimator, on the basis of the light emissions detected by the detector elements.

[0063] According to a further embodiment, the position of the emitter estimated during the pre-localization is determined by a moment determination, in particular by determining the first moment (in particular a weighted mean) of a light intensity distribution, on the basis of the light emissions detected by the detector elements.

[0064] According to another embodiment, a function is fitted to the data obtained from the light emissions detected by the detector elements, wherein the position estimated during pre-localization is determined from the fitted function. For example, a function fit of the data to a two-dimensional Gaussian function can be performed, wherein the position of the maximum and the width of the Gaussian function are adjusted until the deviation of the data from the function reaches a minimum.

[0065] According to a further embodiment, a centroid of a distribution of light emissions detected by the detector elements (i.e. in particular a spatial distribution in the detection plane) is determined, wherein the position of the emitter is estimated on the basis of the centroid.

[0066] According to a further embodiment, the light emissions of the emitter are detected during the primary localization with a detector having a plurality of detector elements, in particular an array of photodiodes. The detector elements detect the light emissions in a position-dependent manner in a detection plane. In particular, the detection plane is confocal to a focal plane in the sample, which contains a geometric focus of the illumination light, i.e., the detector elements are arranged in an image plane associated with the focal plane. In particular, the detector elements of the detector can be read individually.

[0067] This has the advantage that the capture range of the localization (i.e., the area within which an emitter to be localized must be located in order to be unambiguously localized using the MINFLUX method described above) is expanded by the additional information provided by position-resolved detection. This allows the emitter to be localized using a non-iterative method even if the actual position of the emitter lies outside the illumination pattern. This enables localization in a single main localization step with fixed illumination positions. The method can therefore be implemented particularly on a simpler light microscope with control electronics without feedback loops.

[0068] According to a further embodiment, individual photons emitted by the emitter and detected by the detector elements of the detector used in the pre-localization and / or the main localization are registered. For this purpose, an electronic evaluation unit such as a so-called TCSPC (time-correlated single photon counting) module can be used.

[0069] According to one embodiment, the light emissions during the pre-localization are detected with a detector having a plurality of detector elements, wherein the light emissions during the main localization are detected with a point detector (e.g., an avalanche photodiode (APD), a photomultiplier, or a hybrid detector).

[0070] According to a further embodiment, the light emissions during pre-localization are detected with a point detector, while the light emissions during main localization are detected with a detector having a plurality of detector elements. In particular, the detector elements are individually readable. Further, the detector elements are configured to register individual photons emitted by the emitter and detected by the detector elements. Further, the detector with the plurality of detector elements is an array of photodiodes.

[0071] According to a further embodiment, the light emissions during the pre-localization are detected with a first detector having a plurality of detector elements, wherein the light emissions during the main localization are detected with a second detector having a plurality of detector elements. In particular, the first detector is a camera and / or the second detector is a detector with individually readable detector elements, further in particular wherein the detector elements are designed to register individual photons emitted by the emitter and detected by the detector elements, further in particular wherein the second detector is an array of photodiodes (e.g., a SPAD array).

[0072] According to a further embodiment, the arrangement of the illumination positions is fixedly predetermined by at least one light source and / or by an illumination optics during the localization of the emitter, in particular during the main localization. "Fixedly predetermined" means in particular that the relative positioning of the illumination positions to one another is fixed, but a shift of the illumination pattern formed by the illumination positions relative to the sample is possible.

[0073] This has the particular advantage that the sample can be illuminated at the illumination positions with the local minimum of the intensity distribution of the illumination light without the need for rapid beam deflection devices, such as electro-optical deflectors. This allows for a less complex and more cost-effective MINFLUX microscope. The method according to the invention is particularly well suited for the use of a fixed illumination pattern, since the position determination according to the MINFLUX principle is non-iterative.

[0074] According to a further embodiment, the sample is illuminated with the illumination light at the illumination positions via respective point light sources. The point light sources can be implemented using separate light sources (e.g., lasers), or the light from one light source can be split to form the point light sources, e.g., via an array of beam splitters, via micromirrors, in particular individually controllable and movable ones, or by coupling the light from one light source into multiple optical fibers.

[0075] According to a further embodiment, the point light sources are formed by respective fiber ends of optical fibers from which the illumination light emerges.

[0076] According to a further embodiment, at least two groups of optical fibers are provided, wherein one of the groups is selected in the main localization depending on the position uncertainty of the position estimated in the pre-localization, and wherein the sample is illuminated in the main localization using the selected group of optical fibers at the illumination positions, so that the maximum extent of the illumination pattern is set by the selection of the group. For example, the groups of optical fibers can be arranged concentrically around a common center point of a fiber bundle.

[0077] This embodiment has the advantage that the maximum extension can be easily changed without beam deflection units, such as electro-optical deflectors, without adjusting optical components (e.g. an adjustable telescope optics).

[0078] According to a further embodiment, the point light sources are formed by reflecting an input light beam off one or more micromirrors of a micromirror array. Such micromirror arrays, e.g., so-called digital mirror devices (DMDs), enable very flexible splitting and targeted deflection of an input light beam. According to a further embodiment, the illumination positions, in particular the illumination pattern, are adjustable between different main locations. The adjustment can be achieved in particular by setting a magnification factor, further in particular by selecting or changing a lens or by adjusting a zoom lens. Furthermore, it is particularly possible to realize the adjustment by adjusting a micromirror array or by selectively using different optical fibers, in particular a fiber bundle.

[0079] According to a further embodiment, the sample is illuminated by means of light pulses of the illumination light, in particular wherein successive light pulses are assigned to different illumination positions. The latter embodiment can also be referred to as pulse-interleaved illumination. In this variant of the method, the sample is illuminated, in particular at the illumination positions of an illumination pattern, by a respective fiber end of an optical fiber. The laser beam of a pulsed laser can be split between various optical fibers of different lengths in order to achieve the desired time delay of the light pulses. The laser beam can be split before being coupled into the optical fibers, e.g., by means of beam splitters. During the main localization, the sample is then illuminated at the illumination positions, in particular repeatedly, i.e.The pulse sequence is repeated multiple times to obtain a sufficient number of emitted photons. The term "light pulses" refers to amounts of light that propagate from a light source at specific time intervals, while no light is emitted from the light source between the time intervals. The light pulses can be periodic or aperiodic, i.e., occur at regular or irregular intervals. Furthermore, the term "light pulse" used in the context of this disclosure is not restricted to a specific time scale.

[0080] According to a further embodiment, the intensity distribution of the illumination light has at least three local maxima arranged around the local minimum, wherein an angular position of the maxima changes along an optical axis defined by a propagation direction of the illumination light. The angular position refers to a position in a plane perpendicular to the optical axis. Such an intensity distribution, also referred to as a "trenut", can be generated in particular by phase modulation of the illumination light with a combination of a vortex phase and a trefoil phase in a plane conjugated to the pupil of the objective, in particular with a so-called spatial light modulator (SLM) which has individually controllable pixels. The phase shift of the trefoil phase can, for example,10% to 200%, in particular 20% to 150%, further in particular 30% to 120%, further in particular 80% to 110%, further in particular 100%, of the phase shift of the vortex phase. The ratio of the phase shifts particularly influences the lateral position of the maxima and the spacing of the maxima along the optical axis – the greater the phase shift of the trefoil phase, the further the maxima shift outward and the greater the length of the maxima in an axial direction (parallel to the optical axis).

[0081] Using the trenut intensity distribution, depth information about the emitter position can advantageously be obtained by illuminating the intensity distribution in a single focal plane. Within a certain range, the accuracy of axial position determination increases with the maximum extent of an illumination pattern in which the sample is illuminated at the illumination positions with the trenut intensity distribution. In this case, no additional axial information is obtained by iteratively reducing the illumination pattern. Therefore, this intensity distribution can be used particularly advantageously in the non-iterative, single-step method according to the invention.

[0082] In particular, the three or more maxima of the intensity distribution are evenly distributed around the local minimum. In particular, the angular positions of the three or more maxima along the optical axis change over a designated axial detection range (along the optical axis) by an amount corresponding to 180° divided by the number of maxima. In particular, the number of illumination points is matched to the number and position of the maxima. This allows the emitter to be illuminated with an intensity that is a function of the axial position of the emitter with a maximum range of values. In particular, the number of illumination points is twice the number of maxima.

[0083] In particular, when determining the position of the emitter, the light emissions for at least two subsets of the illumination points of the illumination pattern are evaluated separately. An estimated value is determined for each of the two subsets, e.g. by forming a vector sum weighted with the number of photons recorded for the respective illumination points, whereby the summed vectors indicate the illumination positions of the respective subset of the illumination pattern. Subsequently, the difference between the estimated values ​​can be calculated, for example. This difference can be used in particular as an uncalibrated estimator (i.e. as an estimator with a systematic error dependent on the actual position of the emitter) for the lateral position of the emitter in the focal plane. Calibration of the estimator (i.e. a correction of the systematic error) can be carried out, for example, using simulations.The difference calculation described above allows for particularly precise position determination, especially when using an intensity distribution with three or more maxima whose angular position changes along the optical axis (trenut distribution). Furthermore, the capture range can also be increased in this way using other intensity distributions (e.g., a 2D donut). When using a trenut distribution, the difference between the light emissions recorded for the two subsets (here, photon numbers) can be used as an uncalibrated estimator for the axial position.

[0084] A second aspect of the invention relates to a light microscope for locating isolated emitters in a sample, in particular according to the method according to the first aspect, comprising - at least one light source and / or an illumination optics designed to illuminate the sample with illumination light, wherein the illumination light induces or modulates light emissions of an emitter in the sample, - a light modulator designed to generate an intensity distribution of the illuminating light or another illuminating light in the sample having a local minimum, - at least one detector designed to detect light emissions from the emitter, - a computing unit designed to estimate and / or determine a position of the emitter in the sample from detected light emissions, and - a control unit configured to control the light source and / or the illumination optics of the light microscope during a pre-localization such that the light source and / or the illumination optics illuminate the sample with the illumination light, to control the detector such that the detector detects the light emissions of the emitter, and to control the computing unit such that the computing unit estimates the position of the emitter in the sample from the light emissions detected by the detector, and to control the light source and / or the illumination optics and / or the light modulator during a subsequent main localization such that the light source and / or the illumination optics illuminate the sample with an intensity distribution of the or another illumination light having a local minimum at illumination positions defined for the main localization before illuminating the sample with the intensity distribution,wherein the illumination positions are arranged around the position of the emitter estimated in the pre-localization, wherein the illumination positions form an illumination pattern having a maximum extent that is set or determined depending on a positional uncertainty of the position estimated in the pre-localization, to control the detector or a further detector such that the detector or the further detector detects the light emissions of the emitter for the illumination positions, respectively, and wherein the computing unit is configured to determine the position of the emitter in the sample from the light emissions detected for the illumination positions.

[0085] According to one embodiment of the light microscope, the control unit is designed to adjust or set the maximum extent of the illumination pattern depending on the position uncertainty of the position estimated in the pre-localization.

[0086] The light source comprises in particular one or more lasers.

[0087] The light modulator is, in particular, a phase plate or a so-called spatial light modulator with individually controllable pixels. In particular, the light modulator is designed to modulate a phase distribution of the illumination light, in particular in a plane conjugate to the pupil of the objective (i.e., a Fourier plane relative to the image plane), so that the intensity distribution with the local minimum is obtained at the focus in the sample.

[0088] According to one embodiment of the light microscope, the detector has a plurality of detector elements. In particular, the detector elements can be read individually. In particular, the detector or an evaluation electronics unit coupled to the detector is configured to register individual photons emitted by the emitter and detected by the detector elements.

[0089] According to a further embodiment, the light microscope has a first detector configured to detect the light emissions of the emitter during pre-localization, wherein the light microscope has a further second detector configured to detect the light emissions of the emitter during main localization. In particular, both the first detector and the second detector have a plurality of detector elements, wherein furthermore, in particular, the detector elements of the second detector are individually readable, and / or furthermore, in particular, the second detector is configured to register individual photons emitted by the emitter and detected by the detector elements of the second detector.

[0090] According to a further embodiment, the light source comprises a plurality of optical fibers, wherein the optical fibers have respective fiber ends, and wherein the optical fibers are designed to illuminate the sample via the fiber ends at the illumination positions with illumination light coupled into the optical fibers.

[0091] According to a further embodiment, the optical fibers form a fiber bundle.

[0092] According to a further embodiment, the optical fibers are arranged symmetrically around a center of the fiber bundle in a plane perpendicular to a longitudinal extension direction of the fiber bundle.

[0093] According to a further embodiment, the light source or an illumination optics of the light microscope predetermines the arrangement of the illumination positions during the localization of the emitter, in particular during the main localization. This means, in particular, that the relative positioning of the illumination positions to one another is fixed, but a displacement of the illumination pattern formed by the illumination positions relative to the sample is possible. For this purpose in particular, the light microscope can, for example, have a galvanometric scanner configured to displace an illumination light beam relative to the sample.

[0094] According to a further embodiment, the light microscope has a plurality of point light sources, wherein the point light sources are designed to illuminate the sample with the illumination light at respective illumination positions. In particular, the light microscope has a plurality of light sources, e.g., lasers, wherein the light sources each form point light sources. In particular, the light microscope has at least one beam splitter or an arrangement of, in particular individually controllable and movable, micromirrors, which is designed to split the light from the light source to form the point light sources. In particular, alternatively, the fiber ends of the optical fibers form the point light sources.

[0095] According to a further embodiment, the light microscope has at least a first group of optical fibers and a second group of optical fibers, wherein the control unit is designed to select the first group or the second group in the main localization depending on the position uncertainty of the position estimated in the pre-localization and to control the light source such that the sample in the main localization is illuminated according to the selection either by means of the first group of optical fibers at first illumination positions that form a first illumination pattern that has a first maximum extent or by means of the second group of optical fibers at second illumination positions that form a second illumination pattern that has a second maximum extent that differs from the first maximum extent.For example, the first and second groups of optical fibers may be arranged concentrically around a common center point of a fiber bundle.

[0096] According to a further embodiment, the light microscope comprises a first group of optical fibers and a second group of optical fibers, wherein the optical fibers of the first group have a first distance from a center of the fiber bundle in a plane perpendicular to a longitudinal extension direction of the fiber bundle, and wherein the optical fibers of the second group have a second distance from the center which differs from the first distance.

[0097] According to a further embodiment, the light microscope comprises a micromirror arrangement with a plurality of mirrors, wherein the point light sources are formed by reflection of an input light beam at one or more of the mirrors.

[0098] According to a further embodiment, the control unit of the light microscope is designed to control a lens revolver, a zoom optics, the micromirror arrangement or the optical fibers such that the illumination positions, in particular the illumination pattern, can be adjusted between different main localizations, in particular by setting a magnification factor.

[0099] According to a further embodiment, the light source comprises a pulsed laser designed to illuminate the sample using light pulses of the illumination light, in particular wherein successive light pulses are assigned to different illumination positions. The light microscope is particularly configured such that the light pulses are coupled into optical fibers of different lengths, wherein the optical fibers are designed to illuminate the sample at respective illumination positions. The term "pulsed laser" particularly also encompasses devices in which a continuous laser light source is combined with a pulse generation device (e.g., based on an AOM).

[0100] According to a further embodiment, at least some of the optical fibers have different lengths. In particular, all optical fibers can have different lengths. If multiple groups of optical fibers are provided, the optical fibers of a respective group can, in particular, have different lengths. If laser pulses are coupled into the optical fibers of such a fiber bundle, pulse-interleaved illumination can be easily implemented at the illumination positions.

[0101] A third aspect of the invention relates to a computer program comprising instructions that cause the light microscope according to the second aspect to carry out the method according to the first aspect.

[0102] A fourth aspect of the invention relates to the use of a fiber bundle having a plurality of optical fibers in a method according to the first aspect, wherein the sample is illuminated with the illumination light via respective fiber ends of the optical fibers at the illumination positions.

[0103] According to a further embodiment, the optical fibers are arranged symmetrically around a center of the fiber bundle in a plane perpendicular to a longitudinal extension direction of the fiber bundle.

[0104] According to an embodiment of the fourth aspect, the fiber bundle comprises a first group of optical fibers and a second group of optical fibers, wherein the optical fibers of the first group have a first distance from a center of the fiber bundle in a plane perpendicular to a longitudinal extension direction of the fiber bundle, and wherein the optical fibers of the second group have a second distance from the center that differs from the first distance. The groups of optical fibers are arranged, in particular, concentrically around the center. Such arrangements make it possible to selectively couple the illumination light into the groups of optical fibers and thus adjust the maximum extent of the illumination pattern.

[0105] According to a further embodiment of the fourth aspect, at least some of the optical fibers have different lengths. In particular, all optical fibers can have different lengths. If multiple groups of optical fibers are provided, the optical fibers of a respective group can, in particular, have different lengths. If laser pulses are coupled into the optical fibers of such a fiber bundle, pulse-interleaved illumination can be easily implemented at the illumination positions.

[0106] Further features of the light microscope according to the second aspect, the computer program according to the third aspect and the use according to the fourth aspect result from the above-described features of the method according to the first aspect.

[0107] Advantageous developments of the invention will become apparent from the patent claims, the description, the drawings, and the accompanying explanations of the drawings. The described advantages of features and / or combinations of features of the invention are merely exemplary and may be effective alternatively or cumulatively.

[0108] With regard to the disclosure content (but not the scope of protection) of the original application documents and the patent, the following applies: Further features can be found in the drawings – in particular the relative arrangements and operative connections shown. The combination of features of different embodiments of the invention or of features of different patent claims is also possible, deviating from the selected references of the patent claims, and is hereby suggested. This also applies to features that are shown in separate drawings or mentioned in their description. These features can also be combined with features of different patent claims. Likewise, features listed in the patent claims can be omitted for further embodiments of the invention; however, this does not apply to the independent patent claims of the granted patent.

[0109] The reference signs contained in the patent claims do not represent a limitation of the scope of the subject-matter protected by the patent claims. They serve solely to make the patent claims easier to understand.

[0110] In the following, exemplary embodiments of the invention are described with reference to the figures. These do not limit the subject matter of this disclosure or the scope of protection. Short description of the characters Fig. 1 shows schematically an embodiment of the method according to the invention; Fig. 2 shows a light microscope according to the invention with a detector having several detector elements; Fig. 3 shows a light microscope according to the invention with two detectors, each with several detector elements; Fig. 4 shows part of a light microscope according to the invention with a fiber bundle; Fig. 5 shows an illumination beam path of another light microscope according to the invention with a fiber bundle; Fig. 6 shows another light microscope according to the invention with a fiber bundle; Fig. Figure 7 shows another example of a fiber bundle with several concentric groups of optical fibers; Fig. Figure 8 illustrates the beam displacement of an illumination pattern using a galvo scanner; Fig. 9 illustrates the adjustment of the maximum extent of the illumination pattern using a zoom lens; Fig. 10 shows part of another light microscope according to the invention with beam splitters for generating point light sources; Fig. 11 shows a light source and part of the beam path of a light microscope according to the invention with a micromirror arrangement. Description of the characters

[0111] Fig. Figure 1 schematically shows the sequence of an exemplary method according to the invention for localizing an isolated emitter E, e.g., a single molecule labeled with a fluorescent dye, in a sample 2. The emitter E emits fluorescent light (light emissions D) when the sample 2 is irradiated with illumination light B (here, excitation light). To excite the fluorescent dye, the sample 2 can be illuminated with the illumination light B, e.g., using wide-field illumination.

[0112] The position of the emitter E is first determined in a pre-localization ( Fig. 1 above). For this purpose, the light emissions D are detected by the detector elements 7 of a detector 5 in a detection plane depending on their position. The detector 5 can be, for example, a camera (such as a CCD camera or a CMOS camera) that detects the light intensities emitted by the emitter E in a position-dependent manner. These light intensities are Fig. 1 in the form of grayscale levels assigned to the individual detector elements 7. From the light intensities, the position of the emitter E in the sample 2 can be estimated using a computing unit 8, e.g. (as known from PALM / STORM microscopy) by determining a centroid of the light intensity distribution on the detector 5, by fitting a two-dimensional Gaussian distribution to the light intensity distribution, by using a maximum likelihood position estimator, or by moment determination.

[0113] In a subsequent main localization ( Fig. 1 below), the position of the emitter E in the sample 2 is then determined with high accuracy according to the MINFLUX principle. For this purpose, the sample 2 is illuminated with an intensity distribution I of the illumination light B (here the excitation light) with a local minimum M (e.g., a 2D donut or a 3D donut). The local minimum M is placed, in particular successively, at illumination positions 10 in the sample 2, which form an illumination pattern 11. In the embodiment according to Fig. 1, a hexagonal illumination pattern 11 is used, which is arranged symmetrically around the position S estimated during pre-localization. However, alternatively, for example, a Cartesian or hexagonal grid of illumination positions 10 can also be used, as known from the so-called RASTMIN method. For each illumination position 10, the light emissions D of the emitter E are recorded, with particular attention being paid to registering individual photons. Then, by means of a computing unit 8 of the light microscope 1, the position of the emitter E is determined from the light emissions D recorded and registered for the individual illumination positions 10, e.g., using a maximum likelihood position estimator or a moment determination.

[0114] The illumination pattern 11 has a maximum extension L, which is Fig. 1 corresponds to the diameter of a scanning circle A around the estimated position S. In particular, the size of the intensity distribution I is not shown to scale compared to the size of the scanning circle A.

[0115] According to the invention, the maximum extension L is determined as a function of a position uncertainty σ R of the position S estimated in the pre-localization. In particular, the maximum extent L is larger the more uncertain the position estimate is. By adjusting the maximum extent L to the position uncertainty σ R The pre-localization makes it possible to determine the position of the emitter E according to the MINFLUX principle in a single step with fixed illumination positions 10 (i.e., in contrast to methods known from the state of the art, non-iteratively).

[0116] The dependence between position uncertainty σR and the maximum extent arises from the fact that the area in sample 2 in which the emitter E is actually located with a certain probability is larger, the more uncertain the position estimate during pre-localization is. The capture area of ​​the main localization (MINFLUX localization) must then be at least as large as the area in which the emitter E is located with the given probability. A larger capture area can be achieved by a larger maximum extent L of the illumination pattern 11.

[0117] Instead of achieving a high accuracy through several iteration steps of the main localization, as in some MINFLUX methods known from the prior art, regardless of the accuracy of the pre-localization, the method according to the invention carries out a more accurate pre-localization compared to the prior art in order to obtain sufficient accuracy overall in the subsequent one-step main localization.

[0118] The position uncertainty σ R can be adjusted, especially during pre-localization, but also during main localization, by the number of captured and registered photons (the more photons are captured and registered, the lower the position uncertainty σ R). Therefore, in the method according to the invention, in particular, a photon budget is divided between the pre-localization and the main localization in order to obtain a desired accuracy with the non-iterative, one-step MINFLUX method.

[0119] A non-iterative MINFLUX method has the advantage that a much simpler microscope control without feedback loops can be used.

[0120] The position uncertainty σ R can be determined from data obtained in the pre-localization and the maximum extent L can be calculated based on the determined position uncertainty σ R Alternatively, the maximum extent L can be set first and the pre-localization can then be carried out in such a way that a desired position uncertainty σ matching the maximum extent L is obtained. Re.g. by capturing and registering a certain number of photons.

[0121] Fig. 2 shows an embodiment of a light microscope 1 for locating an isolated emitter E in a sample 2 according to the method according to the invention. The light microscope 1 is a MINFLUX microscope having a light source 3 with a laser 31 that generates an illuminating light beam of illuminating light B. The illuminating light beam passes through a first beam deflection unit 12 and a second beam deflection unit 13, e.g., two electro-optical deflectors (EODs), which form part of an illuminating optics 15 and deflect the illuminating light beam in a first direction and a second direction orthogonal to the first direction in a plane extending perpendicular to a propagation direction of the illuminating light beam (also referred to as the x-direction and y-direction), when the first beam deflection unit 12 and the second beam deflection unit 13 receive a corresponding control signal from the control unit 9.

[0122] Furthermore, the illumination light beam is modulated, in particular phase-modulated, by a light modulator 4 of the illumination optics 15 in order to generate an intensity distribution I with a local minimum M (in particular a 2D donut or a 3D donut) at the focus in the sample 2. The light modulator 4 can, as in Fig. 2, are transmitted by the illumination light beam. Alternatively, the illumination light beam can also be refracted by a diffraction grating of the light modulator 4 or reflected by a surface of the light modulator 4 and thereby phase-modulated.

[0123] The illumination optics 15 further comprises a dichroic beam splitter 26 which reflects the phase-modulated illumination light beam and an objective 14 which focuses the illumination light beam into the sample 2.

[0124] The isolated emitter E in the sample 2, excited by the illumination light B, emits fluorescent light (light emissions D), which, due to its wavelength, passes through the dichroic beam splitter 26 and reaches a detector 5 of the light microscope 1. The detector 5 has a plurality of detector elements 7 arranged in a detection plane extending perpendicular to the propagation direction of the light emissions D.

[0125] The individual detector elements 7 detect individual photons emitted by the emitter E, which are registered by a processing unit 8 of the light microscope 1 or the detector 5. The detector 5 can be, for example, a camera or a SPAD array.

[0126] When carrying out the method according to the invention, the sample 2 is first illuminated with the illumination light B in order to carry out the pre-localization. For this purpose, wide-field illumination of the sample 2 can be provided, for example, by means of the light source 3 or a further light source (not shown). Alternatively, the sample 2 can be scanned with focused illumination light B during the pre-localization, e.g. by means of the first beam deflection unit 12 and the second beam deflection unit 13 or with a galvanometric scanner (not shown). As in the subsequent main localization, the intensity distribution I with the local minimum M can be used, or the sample 2 can be scanned with a different light distribution, for example a regular, approximately Gaussian focus.In the latter case, it is advantageous if the light modulator has 4 individually controllable pixels in order to be able to switch between an intensity distribution I with a local minimum M and a regular focus by changing the displayed phase pattern.

[0127] Optionally, activation light can be applied (focused or wide-field) simultaneously with or before the excitation light during pre-localization to activate photoactivatable fluorophores.

[0128] From the photons (light emissions D) detected by the detector elements 7 of the detector 5 during pre-localization and registered by the computing unit 8, the computing unit 8 estimates the position S of the emitter E, e.g., by determining a centroid of a distribution of light intensities (or photon numbers) detected by the detector elements 7. Alternatively, the position estimation by the computing unit 8 can also be performed, e.g., by fitting a Gaussian function to the light intensity distribution of the detected light, by means of a maximum likelihood estimator, or by moment determination.

[0129] In the following main localization, the sample 2 is illuminated with the intensity distribution I with the local minimum M at fixed illumination positions 10, which form an illumination pattern 11, which depends on the position uncertainty σ R the pre-localization has a corresponding maximum extension L (see Fig. 1). The first beam deflection unit 12 and the second beam deflection unit 13 position the focus of the illumination light beam in the focal plane in the sample 2 at the illumination positions 10. For each illumination position 10, light emissions D are detected by the detector elements 7 of the detector 5. The computing unit 8 then determines the position of the emitter E from the light emissions D, e.g., using a maximum likelihood estimator.

[0130] Optionally, the computing unit 8 can also calculate the position uncertainty σ R and based on the calculated position uncertainty σ R set the maximum extent L of the illumination pattern 11. Alternatively, a predetermined value of the maximum extent L can be stored in the computing unit 8 or in an external memory connected to the computing unit 8. The computing unit 8 can then, for example, calculate a required position uncertainty σ from the predetermined maximum extent L.R Finally, the computing unit 8 can calculate the required position uncertainty σ R calculate a minimum number of photons that must be captured and registered during pre-localization in order to achieve the required position uncertainty σ R During pre-localization, the computing unit 8 can then determine whether the minimum number of photons has been reached. If this is the case, the control unit 9 can control the light microscope 1 such that the pre-localization is terminated and the main localization is initiated.

[0131] Fig. Figure 3 shows a further embodiment of the light microscope 1 according to the invention (MINFLUX microscope). This is analogous to the one shown in Fig. 2, but has a first detector 5 and a second detector 6 in the detection beam path, each with a plurality of detector elements 7 arranged in a detection plane. The detection light, i.e. the light emissions D, are separated by a beam splitter 30. This can be a neutral beam splitter 30 which splits the detection light in a predetermined ratio (e.g. 1:1) regardless of its properties. Alternatively, the beam splitter 30 can also be a polarization beam splitter, for example. In this case, a polarization switching element (not shown), e.g. a Pockels cell, can be arranged in front of the beam splitter 30 in order to guide the detection light selectively to the first detector 5 or the second detector 6 by means of a control signal depending on its polarization direction.

[0132] For example, the first detector 5 can be optimized for pre-localization and the second detector 6 can be optimized for main localization. The first detector 5 can be a CCD or CMOS camera, for example. In the context of pre-localization, this has the advantage that individual emitters E can be localized relatively quickly in a relatively large image field. The second detector 6 can be a SPAD array. For main localization according to the MINFLUX principle, this has the particular advantage that single-photon counting is possible for MINFLUX localization with an extended capture range, meaning that emitters E can be unambiguously localized in a larger area.

[0133] Fig. 4 shows part of a light microscope 1 for carrying out the method according to the invention according to a further exemplary embodiment with a light source 3 having a plurality of point light sources 16, which are designed as fiber ends 18 of optical fibers 17 of a fiber bundle 19. The light source 3 has a plurality of lasers 31 for generating respective input light beams 22 of illumination light B, as well as a plurality of lenses 27, which function as fiber couplers 28 to couple the input light beams 22 into respective optical fibers 17 of the fiber bundle 19. The light beams emerging at the fiber ends 18 of the optical fibers 17 then pass through an illumination optics 15. In doing so, they are collimated and tilted relative to one another by means of a further lens 27, which is designed as a collimator 33.At the intersection point of the collimated light beams, a light modulator 4 is arranged perpendicular to an optical axis O, which phase-modulates the light beams using a phase pattern in order to generate a respective intensity distribution I with a local minimum M for each of the light beams at the focus in the sample 2. The light beams are then jointly focused into the sample 2 by an objective lens 14 (in . Fig. 4 not shown).

[0134] Intensity modulators 32, e.g., acousto-optical modulators (AOM), can optionally be arranged between the lasers 31 and the fiber couplers 28 in order to selectively modulate the intensity of input light beams 22 by the control unit 9, in particular such that only one input light beam 22 is coupled into a respective optical fiber 17, while the other input light beams 22 are completely shaded by the intensity modulators 32. In this way, the sample 2 can be illuminated successively at the illumination positions 10 with the illumination light B from the individual optical fibers 17.

[0135] Another possibility to illuminate the sample 2 with illumination light B from optical fibers 17 one after the other at the illumination positions 10 is shown in Fig. 5. Here, a single laser 31 designed as a pulse laser generates the illumination light B in the form of light pulses LP. The illumination light B is then split into different light beams by means of beam splitters 30 and mirrors 29, which are then coupled into respective optical fibers 17 of a fiber bundle 19 via lenses 27 designed as fiber couplers 28. The individual optical fibers 17 are of different lengths, which in Fig. 5 is schematically represented by a different number of loops 17a. In this way, time-shifted light pulses LP1, LP2, LP3, LP4 are generated, which propagate through the respective optical fibers 17. The phase modulation of the light beams to generate temporally and spatially shifted intensity distributions I with local minima M can be carried out analogously to the Fig. 4 by means of a light modulator 4 of an illumination optics 15, which is located at an intersection point of collimated light beams tilted relative to one another, which are generated by a common collimator 3 from the light emerging from the fiber ends 18.

[0136] In Fig. 6 shows a further embodiment of a light microscope 1 according to the invention, which uses fiber ends 18 of optical fibers 17 as point light sources 16. Similar to the embodiment according to Fig. 5, the light from a laser 31 configured as a pulsed laser is split between several optical fibers 17 of different lengths (indicated by the loops 17a), here by means of a common fiber coupler 28. In contrast to this embodiment, however, the light emerging from the fiber ends 18 at lenses 27 configured as fiber couplers 28 is combined with beam splitters 30 and mirrors 29 of an illumination optics 15. The beam splitters 30 and mirrors 29 are located in particular in a pupil plane that is conjugated to the entrance pupil (rear aperture) of the objective 14. This plane is therefore a Fourier plane relative to the image plane (which is conjugated to the focal plane in the sample 2). The beam splitters 30 and mirrors 29 are adjusted so that the individual light beams are deflected at different angles on the reflecting surfaces of the beam splitters 30 and mirrors 29.This creates the desired lateral offset between the light beams in the focal plane to illuminate the sample 2 at the illumination positions 10. In this embodiment, the combined light beams are jointly modulated by a light modulator 4, which is also positioned in a plane conjugate to the pupil plane or at a short distance from this plane. The optical fibers 17 of different lengths create, as described above for the system shown in . Fig. 5, a pulse sequence of light pulses LP of the illumination light B is described, so that the illumination positions 10 are illuminated in a temporal sequence with the illumination light B. The remaining components of the light microscope 1 are in particular analogous to the one shown in Fig. 1 and described above. Regarding the structure and function of these components, reference is made to the relevant sections of this figure description.

[0137] Fig. 7 shows an example of a fiber bundle 19 with a first group G1, a second group G2, and a third group G3 of optical fibers 17, which are arranged concentrically around a center Z of the fiber bundle 19. The first group G1 is at the greatest distance from the center, followed by the second group G2 and the third group G3. By selectively coupling illumination light B into the groups G1, G2, G3 of optical fibers 17 or by shading certain groups G1, G2, G3 (e.g., as described above using intensity modulators such as AOMs), the maximum extent L of the illumination pattern 11 can be adjusted.

[0138] Fig. Fig. 8 shows a further embodiment of a light microscope 1 according to the invention with optical fibers 17 and a galvanometric scanner 34 for the lateral displacement of the illumination pattern 11 predetermined by the optical fibers 17. The light source 3 and the illumination optics 15 of the light microscope 1 can be designed analogously to the Fig. 4 or Fig. 5, and the phase modulation of the individual light beams takes place in particular as described for these exemplary embodiments, i.e. the light beams emerging from the fiber ends 18 are collimated and tilted relative to one another by a collimator 33, with the light modulator 4 for phase modulating the light beams being located at the intersection point of the collimated light beams. The fiber ends 18 are arranged here in an image plane BE, and the light modulator 4 is located in a conjugate plane KE to the entrance pupil of the objective 14, which is a Fourier plane with respect to the image plane BE. The light beams phase-modulated by the light modulator 4 are focused by a lens 27 (scan lens) into an intermediate image plane ZBE conjugate to the image plane BE, which intermediate image plane ZBE is located between two adjustable mirrors of the galvanometric scanner 34.The light beams are collectively deflected by the mirrors of the galvanometric scanner 34. They then pass through a tube lens 35 and are focused by the objective 14 onto the sample 2.

[0139] In Fig. 9 shows a zoom lens 25 for adjusting a magnification factor as part of an illumination lens 15. Such a lens allows the size of an illumination pattern 11 of illumination positions 10 to be adjusted, i.e., in particular, the maximum extent L of the illumination pattern 11 to be adjusted. The zoom lens 25 is located in the illumination beam path behind a collimator 33, which collimates and tilts the light rays emerging from fiber ends 18 of optical fibers 17 of a fiber bundle 19, which function as point light sources 16. It consists of several lenses 27 and an adjustable lens 37. The light modulator 4 for phase modulating the illumination light B is arranged between the adjustable lens 37 and another lens 27 of the zoom lens 25.The collimated light beams emanating from the adjustable lens 37 intersect in a plane in which the light modulator 4 is arranged and are jointly phase-modulated. By moving the adjustable lens 37 along the optical axis O, the distance between the illumination points 10 in the intermediate image plane ZBE can be adjusted.

[0140] Fig. 10 shows part of a beam path of a light microscope 1 according to the invention, in which point light sources 16 are generated without optical fibers 17 by splitting an input light beam 22 of the illumination light B generated by a light source 3 (not shown) into partial beams by an illumination optics 15 with beam splitters 30 and mirrors 29, and the partial beams are phase modulated by a light modulator 4 in order to generate intensity distributions with a local minimum M at the focus in the sample 2. Intensity modulators 32, e.g. acousto-optical modulators, are arranged behind the light modulator 4, which control the intensity of the individual partial light beams, in particular such that the illumination positions 10 are illuminated in chronological sequence, with only one partial beam reaching the sample at a time, while the other partial beams are completely shaded by the respective intensity modulators 32.Alternatively, a sequence of light pulses LP can of course also be generated in the last-described embodiment if a pulse laser is used to generate the illumination light B and the different light paths of the partial beams have correspondingly different lengths.

[0141] Fig. 11 shows a light source 3 and part of a beam path with illumination optics 15 of a light microscope 1 according to the invention according to a further embodiment. An input light beam 22, emanating from a laser (not shown), for example, is directed onto a micromirror arrangement 23 with a plurality of movable micromirrors 24. The micromirrors 24 can be individually electrically controlled in order to rotate the respective micromirror 24 about a respective axis of rotation. By reflecting the input light beam 22 at the micromirrors 24, partial light beams are created. By appropriately rotating the micromirrors 24, the partial light beams are optionally coupled into the beam path of the light microscope 1 or directed to an absorber 39. By controlling the micromirrors 24 at different times, the sample 2 (in Fig. 11 not shown) successively at different illumination positions 10, forming an illumination pattern 11. By selecting appropriate micromirrors 24, via which the illumination light B is coupled into the beam path of the light microscope 1, the maximum extent L of the illumination pattern 11 can also be adjusted.

[0142] In the beam path behind the micromirror 24 there is a microlens arrangement 40 comprising a plurality of microlenses 41. The microlens arrangement 40 is positioned such that each microlens 41 is assigned to a micromirror 24 and focuses the illumination light B emanating from it into an intermediate image plane ZBE, creating point light sources 16. Behind the intermediate image plane ZBE, a collimator 33 is arranged in the diverging beam path, which collimates and tilts the light of the individual partial light beams so that the collimated partial light beams intersect in a plane KE conjugate to the pupil of the objective 14 (not shown) (i.e., a Fourier plane with respect to the intermediate image plane ZBE).At this position there is a light modulator 4 which modulates the phase of the illumination light B of the partial light beams together in order to form an intensity distribution I with a local minimum M for each partial light beam at the focus in the sample 2.

[0143] This beam path corresponds in particular to the one in Fig. 4 and described above. Alternatively, it is of course possible to use the same structure as in Fig. 6, it is also possible here to combine the partial beams by means of beam splitters 30 and mirrors 29 and to modulate the phase of the combined light beam with the light modulator 4. List of reference symbols 1 light microscope 2 Sample 3 Light source 4 Light modulator 5 Detector, first detector 6 Second detector 7 Detector element 8 computing unit 9 Control unit 10 Lighting position 11 lighting patterns 12 First beam deflection unit 13 Second beam deflection unit 14 Lens 15 Lighting optics 16 point light source 17 optical fibers 17a Loop 18 fiber ends 19 fiber bundles 22 Input light beam 23 micromirror array 24 micromirrors 25 zoom optics 26 Dichroic beam splitter 27 lens 28 fiber couplers 29 mirrors 30 beam splitters 31 lasers 32 intensity modulator 33 Collimator 34 scanners 35 tube lens 37 Adjustable lens 39 absorbers 40 microlens array 41 Microlens A scanning circuit B Illumination light D Light emissions E Emitter G1 First Group G2 Second Group G3 Third Group I Intensity distribution BE image plane KE Conjugate plane ZBE intermediate image plane L Maximum extension LP light pulse M Minimum O Optical axis S Position Z Center

Claims

[1] Method for localizing isolated emitters (E) in a sample (2), comprising a pre-localization with the steps - illuminating the sample (2) with illuminating light (B), wherein the illuminating light (B) induces or modulates light emissions (D) of an isolated emitter (E) in the sample (2); - detecting the light emissions (D) of the emitter (E); - Estimating the position (S) of the emitter (E) in the sample (2) from the detected light emissions (D); and a subsequent main localization with the steps - illuminating the sample (2) with an intensity distribution (I) of the or another illuminating light (B) having a local minimum (M) at illumination positions (10) arranged around the position (S) of the emitter (E) estimated in the pre-localization; - detecting the light emissions of the emitter (E) for the respective lighting positions (10); - determining the position (S) of the emitter (E) in the sample (2) from the light emissions (D) detected for the illumination positions (10), characterized by that the illumination positions (10) for the main localization are determined before illuminating the sample (2) with the intensity distribution (I), wherein the illumination positions (10) form an illumination pattern (11) having a maximum extent (L), wherein the maximum extent (L) depends on a position uncertainty (σ r ) of the position (S) estimated in the pre-localization is set or determined. [2] Method according to claim 1, characterized by that the illumination light (B) is excitation light which induces the light emissions (D) of the emitter (E). [3] Method according to claim 1 or 2, characterized bythat the illumination positions (10) are arranged on a scanning circle (A) or a scanning sphere around the estimated position (S) of the emitter (E), wherein the maximum extent (L) corresponds to the diameter of the scanning circle (A) or the scanning sphere. [4] Method according to claim 1 or 2, characterized by that the illumination pattern (11) is a grid (R), in particular a Cartesian grid or a hexagonal grid. [5] Method according to one of claims 1 to 4, characterized by that the position uncertainty (σ r ) is determined from the detected light intensities (D), wherein the maximum extent (L) of the illumination pattern (11) is determined according to the determined position uncertainty (σ r ), in particular where the position uncertainty (σ r ) is determined from a number of photons emitted by the emitter (E) registered in the pre-localization. [6] Method according to one of claims 1 to 4, characterized by that the maximum extent (L) of the illumination pattern (11) is set to a predetermined value, wherein the pre-localization is carried out in such a way that a value of the position uncertainty (σ r ) to which the specified value of the maximum extension (L) is assigned. [7] Method according to claim 6, characterized by that the pre-localization steps are repeated or a time interval during which the pre-localization is carried out is adjusted until the position uncertainty (σ r ) of the estimated position (S) falls below a predetermined or predeterminable limit value. [8] Method according to one of the preceding claims, characterized by that the maximum extension (L) according to the condition L = a · σ r is set or fixed, where L denotes the maximum extension, where σ rdenotes the position uncertainty of the position estimated in the pre-localization, and where a denotes a proportionality factor, in particular where the position uncertainty (σ r ) is a standard deviation or a variance of a localization distribution of the emitter (E). [9] Method according to claim 8, characterized by that the proportionality factor (a) has a value in the range from 1 to 3. [10] Method according to one of the preceding claims, characterized by that the position uncertainty (σ r ) of the position (S) estimated in the pre-localization is 50 nm or less, in particular 20 nm or less, further in particular 10 nm or less. [11] Method according to one of the preceding claims, characterized by that the position estimated in the pre-localization (S) has a larger position uncertainty (σ r ) than the position of the emitter (E) determined in the main localization. [12] Method according to one of the preceding claims, characterized by that the estimation of the position (S) of the emitter (E) in the pre-localization is carried out using a non-iterative method. [13] Method according to one of the preceding claims, characterized by that the sample (2) is scanned in the pre-localization with a focus or an intensity minimum (M) of the illumination light (B), wherein in particular a scanned image of the sample (2) is created, or that a wide-field image of the sample (2) is generated in the pre-localization. [14] Method according to one of the preceding claims, characterized by that in the pre-localization the positions (S) of several emitters (E) are estimated, in particular with a stochastic localization method, wherein a first emitter is selected from the several emitters (E), in particular automatically, and wherein the position of the first emitter is determined in the main localization. [15] Method according to one of the preceding claims, characterized by that a total photon number of photons to be registered is determined for the emitter (E), the total photon number being divided into a first photon number and a second photon number, the position (S) of the emitter (E) in the pre-localization being estimated when the light emissions (D) detected in the pre-localization reach the first photon number. [16] Method according to claim 15, characterized by that the position (S) of the emitter (E) in the main localization is determined when the light emissions (D) detected in the main localization reach the second photon number. [17] Method according to one of the preceding claims, characterized bythat the light emissions (D) of the emitter (E) are detected during pre-localization with a detector (5) having a plurality of detector elements (7), wherein in particular the detector elements (7) detect the light emissions (D) in a detection plane depending on the position. [18] Method according to claim 17, characterized by that the position (S) of the emitter (E) estimated during the pre-localization is determined with a position estimator or by moment determination on the basis of the light emissions (D) detected by the detector elements (7). [19] Method according to claim 17, characterized by that a function is fitted to data obtained from the light emissions (D) detected by the detector elements (7), wherein the position (S) estimated during the pre-localization is determined from the fitted function. [20] Method according to one of the preceding claims, characterized bythat the light emissions (D) of the emitter (E) are detected during the main localization with a detector (5) having a plurality of detector elements (7), wherein in particular the detector elements (7) detect the light emissions in a detection plane depending on the position. [21] Method according to one of claims 17 to 20, characterized by that individual photons emitted by the emitter (P) and detected by the detector elements (7) of the detector (5) are registered. [22] Method according to one of the preceding claims, characterized by that the arrangement of the illumination positions (10) is fixedly predetermined by at least one light source (3) and / or by an illumination optics (15) during the localization of the emitter (E). [23] Method according to claim 22, characterized by that the sample (2) is illuminated with the illumination light (B) at the illumination positions (10) via respective point light sources (16). [24] Method according to claim 23, characterized by that the point light sources (16) are formed by respective fiber ends (18) of optical fibers (17) from which the illumination light (B) emerges. [25] Method according to claim 24, characterized by that at least two groups (G1,G2,G3) of optical fibers (17) are provided, wherein in the main localization depending on the position uncertainty (σ r ) of the position (S) estimated in the pre-localization, one of the groups (G1, G2, G3) is selected, and wherein the sample (2) is illuminated in the main localization by means of the selected group of optical fibers (17) at the illumination positions (10), so that the maximum extent (L) of the illumination pattern (11) is set by the selection of the group. [26] Method according to claim 24, characterized bythat the point light sources (16) are formed by reflection of an input light beam (22) at one or more micromirrors (24) of a micromirror arrangement (23). [27] Method according to one of claims 22 to 26, characterized by that the illumination positions (10) are adjustable between different main locations, in particular by setting a magnification factor, further in particular by selecting or changing a lens (14) or by setting a zoom lens (25), by setting a micromirror arrangement (23) or by selectively using different optical fibers (17). [28] Method according to one of the preceding claims, characterized by that the sample (2) is illuminated by means of light pulses (LP) of the illumination light (B), in particular wherein successive light pulses (LP) are assigned to different illumination positions (10). [29] Method according to one of the preceding claims, characterized by that the intensity distribution of the illumination light (B) has at least three maxima arranged around the local minimum (M), wherein an angular position of the maxima changes along an optical axis (O) defined by a propagation direction of the illumination light (B). [30] Light microscope (1) for locating isolated emitters (E) in a sample (2), in particular according to the method according to one of claims 1 to 29, comprising - a light source (3) designed to illuminate the sample (2) with illumination light (B), wherein the illumination light (B) induces or modulates light emissions (D) of an emitter (E) in the sample (2), - a light modulator (4) which is designed to generate in the sample (2) an intensity distribution (I) of the illuminating light (B) or of another illuminating light (B) having a local minimum (M) in at least one spatial direction, - at least one detector (5) designed to detect light emissions (D) of the emitter (P), - a computing unit (8) designed to estimate and / or determine a position (S) of the emitter (P) in the sample (2) from detected light emissions (D), - a control unit (9) which is designed to i. during a pre-localization, to control the light source (3) such that the light source (3) illuminates the sample (2) with the illumination light (B), to control the detector (5) such that the detector (5) detects the light emissions (D) of the emitter (P) and to control the computing unit (8) such that the computing unit (8) estimates the position (S) of the emitter (P) in the sample (2) from the light emissions (D) detected by the detector (5), and ii. during a subsequent main localization, to control the light source (3) and the light modulator (4) such that the light source (3) illuminates the sample (2) with an intensity distribution (I) of the or another illumination light (B) having a local minimum (M) at illumination positions (10) determined for the main localization and before illuminating the sample (2) with the intensity distribution (I), wherein the illumination positions (10) are arranged around the position (S) of the emitter (E) estimated in the pre-localization, wherein the illumination positions (10) form an illumination pattern (11) having a maximum extent (L) which depends on a position uncertainty (σ r) of the position (S) estimated in the pre-localization is set or fixed, and to control the detector (5) or a further detector such that the detector (5) or the further detector detects the light emissions (D) of the emitter (E) for each of the illumination positions (10), wherein the computing unit (8) is designed to determine the position (S) of the emitter (E) in the sample (2) from the light emissions (D) detected for the illumination positions (10). [31] Light microscope (1) according to claim 30, characterized by that the detector (5) has a plurality of detector elements (7), in particular individually readable. [32] Light microscope (1) according to claim 30 or 31, characterized byin that the light source (3) has a plurality of optical fibers (17), wherein the optical fibers (17) have respective fiber ends (18), and wherein the optical fibers (17) are designed to illuminate the sample (2) via the fiber ends (18) at the illumination positions (10) with illumination light (B) coupled into the optical fibers (17). [33] Computer program comprising instructions which cause the light microscope (1) according to one of claims 30 to 32 to carry out the method according to one of claims 1 to 29. [34] Use of a fiber bundle (19) with a plurality of optical fibers (17) in a method according to one of claims 1 to 29, wherein the sample (2) is illuminated with the illumination light (B) via respective fiber ends (18) of the optical fibers (17) at the illumination positions (10).

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