Methods for locating and tracking emitters in a sample

Abstract

The invention relates to a method for locating or tracking individual emitters (2) in a sample (38) according to the MINFLUX principle, wherein the sample (38) is illuminated at several illumination positions in a close-range area around the emitter (2) from different illumination directions (4, 5) with modulating light (3) that has a linearly or planarly intensity minimum. From the emissions of the emitter (2) detected at the illumination positions, a position (1) of the emitter (1) in the sample (38) is calculated. The invention further relates to a light microscope for carrying out the method.

Description

Technical field of the invention

[0001] The invention relates to a method and a light microscope for locating and tracking individual emitters in a sample using the MINFLUX or STED-MINFLUX principle. Compared to the MINFLUX methods known from the prior art, the method and the light microscope according to the invention allow the three-dimensional localization of emitters in a sample without having to illuminate the sample at different illumination planes. State of the art

[0002] The term MINFLUX methods refers to localization and tracking methods for isolated emitters in which light distributions of illuminating light are generated at the focus in the sample, which excites light emissions from the emitter, whereby the light distributions exhibit a local minimum, and in which the position of an isolated emitter is determined by detecting light emissions from the emitter for various positions of the minima of the respective light distributions or for various such light distributions in a close range of the emitter. In particular, this method exploits 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. At the minimum itself, the emitter should be excited to emit as little as possible, so that the increase in emission with increasing distance from the minimum of the light distribution is as large as possible in relation to the emission.Due to the latter fact, MINFLUX methods are particularly photon-efficient, especially compared to localization methods based on spatially resolved emission detection, such as PALM or STORM microscopy. This means that with the same number of detected photons, MINFLUX methods achieve particularly high localization accuracy. This allows the emitters to be localized or tracked with the desired accuracy to be exposed to relatively little light compared to other localization methods, thus resulting in less bleaching.

[0003] The term "MINFLUX" was introduced by Balzarotti et al. in "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes," Science 355 (6325), 606-612 (2017), and previously in arXiv:1611.03401 [physics.optics] (2016). The method described therein implements the MINFLUX principle by identifying small structures containing stochastically blinking fluorophores in a wide-field image and positioning them as precisely as possible in the center of the wide-field image. Subsequently, the central region is scanned using an electro-optical scanner with a donut-shaped excitation light distribution in the center and at other points that form a symmetric pattern of illumination positions around the center. From the photon numbers registered for the individual illumination positions, the positions of the individually blinking fluorophores of the structures are then determined to within a few nanometers using a maximum likelihood estimator.For the same number of detected photons, the accuracy increases the smaller the area defined by scanning positions in which the fluorophore to be localized is located. Therefore, it is advantageous to run MINFLUX iteratively on individual fluorophores, reducing the defined area step by step.

[0004] European patent EP 3 372 990 B1 describes a related method, now known as "STED-MINFLUX." As with a MINFLUX method, an isolated emitter is illuminated with a light distribution having a central intensity minimum (preferably a zero) and regions of increasing intensity surrounding this minimum. Also as with MINFLUX, this minimum is placed at a plurality of sampling points around the presumed location of the isolated molecule, and a fluorescence emission is detected for each sampling point. The position of the emitter is estimated with high precision from the fluorescence intensity values ​​or photon counts thus obtained. In contrast to MINFLUX, however, the light distribution is not a distribution of excitation light, but rather a distribution of fluorescence-inhibiting light, in particular stimulation or STED light.This is applied together with (additional) excitation light, whereby the intensity distribution of the excitation light does not exhibit a central local minimum. While with MINFLUX, the fluorescence emission is higher when the isolated emitter is farther away from the central minimum of the intensity distribution, the opposite is true with this method.

[0005] US 2012 / 0104279 A1 discloses a microscope with a segmented chromatic beam-shaping plate arranged in the rear objective aperture, which can be used to generate a donut-shaped intensity distribution in the sample, e.g., for STED microscopy. The beam-shaping plate consists of segments of a λ / 2 plate, with the fast axes of the adjacent segments being at an angle of 45° to each other. Additionally, a method for investigating the orientation of a dipole moment of a fluorophore using an embodiment of the beam-shaping plate is described.

[0006] WO 2020 / 128106 A1 describes a method for point-like illumination of a sample in a MINFLUX microscope. The sample is illuminated sequentially at the illumination points of a predefined illumination point pattern, with each illumination point being assigned an individual light source, e.g., a fiber end of an optical fiber. In this way, different positions in the sample can be illuminated with the illumination light without a beam scanner, in particular with the central local minimum of an excitation intensity distribution, in order to implement a MINFLUX method. The fiber ends can, in particular, be offset parallel to the optical axis to create three-dimensional illumination patterns.

[0007] Some of the embodiments described in WO 2020 / 128106 A1 are also reproduced in DE 10 2022 119 304 A1 and DE 10 2022 119 327 B4. Both documents relate to MINFLUX methods with a pre-localization and a main localization. According to DE 10 2022 119 304 A1, a maximum extent of the illumination pattern used in the main localization is set or specified depending on a position uncertainty of the position estimated in the pre-localization. According to DE 10 2022 119 327 B4, several iterations of an illumination sequence are performed during the main localization, with the maximum extent of the illumination pattern being kept constant.

[0008] The publication "Pulsed Interleaved MINFLUX" by LA Masullo et al., Nano Lett. 2021, 21, 840-846, describes a MINFLUX method in which excitation light pulses are coupled into optical fibers of different lengths so that the light pulses are delayed relative to each other. The light of the light pulses is directed by respective optics in parallel beam paths in such a way that, after beam shaping by a vortex phase plate and focusing by an objective lens, the zero point of a donut-shaped intensity distribution of the excitation light is shifted sequentially to illumination points of an illumination pattern without deflection of the light by a scanner.DE 10 2019 008 304 B3 describes a fluorescence microscope (in particular a STED microscope or a MINFLUX microscope) with an active wavefront modulator in a beam path section common for illumination and detection, wherein a beam path is formed using a telescope such that the effective beam diameter is small at the point of separation of the beam paths for detection and illumination.

[0009] CN 118010692 A describes a MINFLUX method in which an intensity distribution of excitation light with a zero surface parallel to the optical axis is rotated around the optical axis between two mutually perpendicular arrangements by means of an electro-optical modulator and additionally displaced laterally by means of an electro-optical deflector.

[0010] DE 10 2015 105 018 A1 also describes intensity distributions with a central zero area rotated at different angular positions around the optical axis and their use in STED microscopy.

[0011] All MINFLUX methods known from the prior art have in common that three-dimensional localization of the emitters requires illuminating the sample at illumination positions not only within one illumination plane (especially the focal plane), but also at illumination positions distributed in all spatial directions. For example, the publication DE 10 2021 100 564 A1 discloses a MINFLUX method for high-resolution localization of an emitter in three spatial directions. In an axial localization step, the intensity minimum of the excitation light is placed at two axial positions, and in a lateral localization step, the intensity minimum is placed exclusively in one scanning pattern plane.

[0012] Patent application WO 2021 / 111187 A2 discloses a method for the three-dimensional localization of light-emitting objects, in which the object is illuminated along a first projection direction with a plurality of first light distributions having a common intensity zero, and the light distributions are shifted such that the light emission of the object is minimized, so that the resulting position of the light distributions characterizes the position of the object in two spatial directions. Furthermore, in the method, the light emitted by the object is shaped with an optical module so that the shape of the light emission depends on the axial position of the object, and the axial position of the object is determined from the spatial distribution and the total intensity of the light emissions.In a further embodiment of the method, the object is illuminated along a second projection direction with a plurality of second light distributions having a common intensity zero with the first light distributions, and the light distributions are shifted until the light emission of the object becomes minimal, so that the resulting position of the light distributions characterizes the position of the object in all three spatial directions.

[0013] Patent application US 2023 / 0101017 A1 discloses a method and a light microscope for parallelized MINFLUX microscopy. A programmable micromirror array is used to generate multiple independently positionable light distributions in the sample, each with an intensity minimum, in order to simultaneously localize multiple emitters in the sample using a MINFLUX method. The document also proposes generating light distributions inclined relative to the optical axis through interference of beams of different diffraction orders, which are generated using locally limited optical gratings presented on the micromirror array. The light distributions are then spatially superimposed to recreate a light distribution with a minimum along the optical axis—similar to that of a bottle beam—enabling three-dimensional localization of the emitter.

[0014] The state of the art describes various technical implementations for rapid focus shifting in the axial direction, but these are technically challenging and expensive. In "Fast response varifocal lenses using KTa 1-x Nb x O3 crystals and a simulation method with electrostrictive calculations“, Appl. Opt. 51 (10), 1532-1539 (2012), T. Imai et al. describe a now commercially available Kerr lens based on a potassium tantalate niobate (KTN) single crystal, the focal length of which can be shifted by almost 90 mm on a time scale of a few microseconds, which, however, requires switching voltages of several 100 V.

[0015] EP 4 167 010 A1 discloses a device comprising a polarization-switching optical element capable of switching a linear polarization of an incident light beam between two output polarizations according to a switching signal, and a focusing optical element that axially shifts the focus position of the focused light beam depending on the output polarization of the polarization-switching optical element. According to a preferred embodiment, the polarization-switching optical element is configured as a Pockels cell, and the focusing optical element is configured as a birefringent lens.

[0016] Focal length-tunable lenses can also be designed as liquid lenses, in which a liquid optical medium is enclosed by transparent membranes. By varying the distance between the membranes while maintaining a constant liquid volume, the curvature of the membrane surfaces can be varied from concave to convex, and thus the focal length of the lens can be varied over a wide range [see, for example, M. Blum et al., "Compact optical design solutions using focus tunable lenses", SPIE Optical Design and Engineering IV, Proceedings Vol. 8167, 81670W (2011)]. With a moving coil drive, such arrangements can achieve switching times in the range of a few milliseconds. A major disadvantage of tunable liquid lenses, however, is that they typically exhibit gravity-induced coma aberrations when operated in an upright position.

[0017] Another class of electrically tunable lenses are liquid crystal lenses, whose focal length results from a spatially varying refractive index of differently aligned liquid crystals, with the alignment being controlled by applied electric fields [see, for example, H.-C. Lin and Y.-H. Lin in “An electrically tunable-focusing liquid crystal lens with a low voltage and simple electrodes”, Opt. Expr. 20 (3), 2045-2052 (2012)]. Object of the invention

[0018] It is now the object of the present invention to provide a method and a light microscope which allows a localization of emitters in a sample according to a MINFLUX method or STED-MINFLUX method also in three dimensions, wherein the illumination positions can all be arranged in one illumination plane, in particular in a focal plane. Solution

[0019] This object is achieved by the subject matter of independent claims 1 and 17. Advantageous developments of the method are specified in subclaims 2 to 16, and advantageous developments of the light microscope are specified in subclaims 18 to 22. Definitions

[0020] In this application, emitters are understood to be objects that, when illuminated with excitation light, can be considered point light sources with regard to the measurements according to the invention. The light emitted by the object acting as a point light source can, for example, be scattered light resulting from elastic scattering such as Rayleigh scattering or inelastic scattering such as Raman scattering, or it can be luminescent light, in particular fluorescent light. It is essential for an emitter that it emits light immediately or with a short time delay in response to illumination.When tracking emitter movements, the maximum time delay is related to the temporal resolution with which the movements of the light-emitting particles or light-emitting units are to be tracked and the speed at which the particles or units move within the sample. Time delays can be as low as about 10 µs, but are typically in the range of up to a few tens of nanoseconds, often in the range of 1 to 10 ns, and, if the emission is scattered light, zero.

[0021] Emitters can be, for example, metallic nanoparticles. The term "fluorescence emitter" includes, for example, individual fluorescent dye molecules or their fluorescent chemical groups. Instead of dyes, other fluorescent units such as quantum dots or upconverting nanoparticles can also be used for labeling. Accordingly, in the context of this application, excitation light is understood not only to mean fluorescence excitation light, but generally to mean light that causes light to emanate from an emitter.

[0022] In the context of the application, an emitter is referred to as isolated if a distance to a nearest neighbouring emitter, from which it is optically indistinguishable, is at least as large as a distance corresponding to the resolution of the optical arrangement with which the emissions are detected when carrying out the method.

[0023] In this application, modulation light is understood to mean light that enhances or attenuates the light emission of an emitter in any desired manner. The modulation light can therefore excite, enhance, reduce / inhibit, or completely suppress the light emission. This definition includes the possibility that the modulation light can also be excitation light, which stimulates the emitter to emit light and can also replace the excitation light as a separate light. Alternatively, the modulation light can be emission-suppressing light that attenuates the emitter's emission, in particular stimulation or STED light, which induces stimulated emission from an electronically excited state of the emitter, thereby converting the emitter (back) to the electronic ground state and thus preventing spontaneous (fluorescence) emission.

[0024] In this application, an illumination plane is understood to be a plane in which the excitation and / or modulation light is positioned when the sample is illuminated. The term "position" refers to the nominal (geometric) position of the light focus in the sample. For a Gaussian intensity distribution, the position thus corresponds to the point of maximum intensity. The intensity distributions of the modulation light discussed in this application regularly exhibit linearly or areally extended intensity minima at the location of the geometric focus; for these intensity distributions, too, the geometric focus point is considered the position of the intensity distribution.

[0025] In this application, an illumination line is understood to mean the corresponding case of the illumination plane reduced to one dimension, i.e. a straight line in an illumination plane. Description of the invention

[0026] A first aspect of the invention relates to a method for locating or tracking an isolated emitter in a sample. In this method, the sample is illuminated through an objective lens, with the illumination being performed with excitation light, which excites the isolated emitter to emit light, and with modulation light, which attenuates, amplifies, or excites the emitter's light emission. The modulation light can, in particular, be excitation light or emission or fluorescence suppression light, as is known, for example, from STED microscopy.

[0027] According to the invention, the modulated light is used to form an intensity distribution in the sample which has an intensity minimum extending along a minimum line or a minimum surface, in particular a zero line or a zero surface. Perpendicular to the minimum line or the minimum surface, the intensity distribution has regions of intensity increase. Methods for forming such intensity distributions in the sample are well known to those skilled in the art from the prior art, including STED microscopy and MINFLUX microscopy. For example, a light beam whose wavefront is subjected to an azimuthal phase gradient of 0 to 2π (or a multiple of 2π) (for example by arranging a vortex phase plate in a light beam of the modulated light) forms an annular intensity distribution with a zero line along the optical axis of the objective when focused with the objective in the sample.

[0028] If, on the other hand, one half of the wavefront of the light beam is delayed by π compared to the other half of the wavefront (for example by arranging a split phase plate in a light beam of the modulation light, which has a phase jump of π at the splitting line and perpendicular to it), an intensity distribution with two intensity maxima laterally offset from the geometric focus is formed in the sample when focusing with the objective, which are separated by a zero plane containing the optical axis.

[0029] Ideally, the gradient in the intensity increase regions in the direction of the minimum line or in a direction extending along the minimum surface is homogeneous, i.e. the intensity of the modulated light is constant at a given distance from the minimum line or the minimum surface in the direction of the minimum line or in the extension direction. This facilitates the calculation of the position of the emitter in the sample, which is described in more detail below. In practice, a homogeneous gradient can only be realized approximately and only over a limited range of the intensity distribution; when using objectives with a high numerical aperture, this range usually extends from one to a few micrometers. Optionally, this range can be extended to over 100 micrometers by executing the modulated light as a Bessel light beam. See, for example, Yu et al., “Super-resolution deep imaging with hollow Bessel beam STED microscopy”, Laser Photon. Rev.10 (1), 147-152 (2016).

[0030] In one step of the method according to the invention, the intensity distribution of the modulation light is positioned at several illumination positions in a close range around the emitter, and light emissions from the emitter are detected at the illumination positions.

[0031] The term "illumination position" refers to a fixed reference point with respect to the intensity distribution of the modulated light. The illumination positions can be discrete positions at which the intensity distribution is positioned sequentially, but they can also be points along a continuous path in the sample. In either case, the emitter's light emissions are detected at the illumination positions, i.e., emission events or light intensities and positions are assigned to one another. For this purpose, the intensity distribution can be positioned at an illumination position, and an amount of light emission at this illumination position can be measured for the duration of an integration time; then, the illumination position is a parameter of the measurement, and the light emission is the measured value.Conversely, the current illumination position can also be measured as soon as an emission event is registered; then the illumination position is the measured value.

[0032] The near-field region has, in particular, dimensions in the range of the optical diffraction limit, with the near-field region being centered, in particular, at the (presumed) position of the emitter. In particular, the near-field region extends in one spatial direction over no more than a small multiple of the diffraction limit (at a given wavelength of the light emanating from the emitter), more particularly ten times, more particularly five times, more particularly twice, more particularly 1.5 times the diffraction limit. In particular, the diffraction limit can correspond to the diameter of an Airy disk.

[0033] In a further step of the method according to the invention, a position of the emitter in the sample is calculated from the emissions detected at the illumination positions. The method is similar to the MINFLUX and STED-MINFLUX methods known from the prior art. However, it differs from these in that the sample is illuminated with the modulated light from different illumination directions while the light emissions are being detected. The illumination can take place at different inclination angles, i.e., at different angles formed by the minimum line or the minimum area of ​​the intensity distribution of the modulated light with the sample-side (xy) focal plane or the optical (z) axis of the objective. The illumination with the modulated light can additionally or alternatively also take place at different azimuth angles, i.e.,by rotating the minimum line or the minimum surface of the intensity distribution of the modulated light around the optical (z-)axis. A complete specification of the illumination direction in which the sample is illuminated with the modulated light comprises the angle pair (φ, θ) (azimuth angle, inclination angle) in three-dimensional space.

[0034] According to the invention, the light emissions of the emitter are detected under illumination with the modulated light from at least two different illumination directions, wherein the light emissions are assigned or can be assigned to the different illumination directions of the modulated light. For example, light emissions can first be collected for a first illumination direction and then for a second illumination direction. It is also possible to detect the light emissions alternating with the illumination directions.

[0035] The assignment of light emissions to the illumination direction can be achieved, for example, by recording the respective azimuth and / or inclination angle for each light emission. Since illumination usually occurs from a small number of illumination directions, an index indicating the illumination direction is particularly suitable for assignment.

[0036] The number of illumination directions under which the emitter's light emissions are detected determines the number of spatial directions in which the emitter's position in the sample can be calculated. When the sample is illuminated from two illumination directions, the emitter can be located in two dimensions; when illuminated from three illumination directions, it can be located in all three dimensions. Furthermore, it may be useful to detect light emissions under additional illumination directions to reduce the uncertainty with which the emitter's position in the sample can be determined.

[0037] The illumination directions preferably have inclination angles between 20° and 60°, particularly preferably between 30° and 45°, relative to the optical axis. Inclination angles in this range are technically easy to implement and are optimal with regard to the localization of the emitter in the direction of the optical axis. In the azimuthal direction, the illumination directions are preferably arranged uniformly around the optical axis, for example, at φ = 0°, 180°, and for two illumination directions, or at φ = 0°, 120°, and 240° for three illumination directions.

[0038] In a particularly preferred embodiment of the method, the modulation light acts as excitation light, and in the sense of the present invention, it represents both the modulation light and the excitation light. It is therefore not necessary for the modulation light and the excitation light to be different from one another; rather, both functions "excitation of an emission of the emitter" and "modulation of the emission" can also be fulfilled by the same light from a common light source.

[0039] According to a preferred embodiment of the method, the illumination positions are located on an illumination line oriented, in particular, perpendicular to the optical axis or in an illumination plane oriented, in particular, perpendicular to the optical axis. The illumination plane is preferably a lateral (xy) plane, in particular the (sample-side) focal plane of the objective.

[0040] According to a preferred embodiment of the method, calculating the position of the emitter in the sample involves calculating the distance to the illumination line or the illumination plane, including information on which side of the illumination line or the illumination plane the emitter is located. Therefore, the distance is normally considered to be signed. In particular, the distance to the illumination plane corresponds to the z-coordinate of the emitter if the illumination plane is the sample-side (xy) focal plane of the objective.In this embodiment, a particular advantage of the method according to the invention compared to known MINFLUX methods from the prior art is evident: Even for localization of the emitter in all spatial directions, it is not necessary to shift the intensity distribution in the direction of the optical axis, so that the method can be carried out with a beam scanner of a conventional laser scanning microscope, for example a galvo scanner.

[0041] In one embodiment of the method, the tilt and azimuth angles characterizing the various illumination directions are measured in a calibration step prior to the method, and the position of the emitter in the sample is calculated using the measured angles. The illumination directions, each defined by a tilt and an azimuth angle, can be determined from images of the intensity distribution generated by scanning the intensity distribution with a light-scattering nanoparticle in the sample using raster scanning. This type of experimental determination of the intensity distribution in the sample is familiar to the skilled person, for example, from STED microscopy.

[0042] If the tilt angles can be set with sufficient reproducibility, are stable over time, and are not distorted by sample-induced aberrations, the calibration step can be performed once, and the tilt and azimuth angles characterizing the illumination directions can be saved for later use. Alternatively, the illumination directions can be determined immediately before performing the method according to the invention. For this purpose, light-scattering nanoparticles can be introduced directly into the sample as auxiliary objects, so that the illumination directions are determined under the same conditions under which the method for localizing the emitters is performed.

[0043] According to a preferred embodiment of the method, apparent positions of the emitter are calculated from subsets of light emissions, each detected under illumination from the same illumination directions of the modulated light, and the position of the emitter in the sample is calculated from the apparent positions. In particular, in this embodiment, the position of the emitter in the sample can be calculated in at least one more spatial direction than the apparent positions.

[0044] For illustration, the two-dimensional case is shown here in which an emitter and the minimum line of the light distribution of the modulation light are all located in the xz-plane, i.e. y = 0. The emitter is illuminated successively from two illumination directions in the xz-plane (φ1 = 0° and φ2 = 180°), whereby the inclination angles θ1, θ2 can be the same or different in magnitude, but are inclined in the opposite direction to the optical axis.

[0045] For each of the two illumination directions, the sample is illuminated at several illumination positions in the focal plane (i.e., at z = 0) around the emitter, and the position of the emitter is localized from the light emission detected at the illumination positions. For this purpose, the position estimators known from the state of the art in MINFLUX microscopy can be used. However, these position estimators do not provide the actual position of the emitter in the sample, but rather projections of its actual position (x, 0, z) into the (xy) focal plane in the direction of the minimum line of the intensity distribution. The x-coordinate of these apparent positions (x i , 0) deviate from the x-coordinate of the actual position (x, 0, z) the more the greater the distance of the emitter from the focal plane.

[0046] Fig. 4, the following relationships between the inclination angles θ1, θ2, the apparent positions x1 and x2 and the position of the emitter (x, z) can be directly derived: z tan θ1=x1−x z tan θ2=x−x2

[0047] This calculates the position (x, z) of the emitter in the sample as (x,z)=(x1 tan θ2+x2 tan θ1tan θ1+tan θ2,x1−x2tan θ1+tan θ2).

[0048] To locate the emitter in three spatial directions, it is necessary to illuminate the emitter from at least one additional illumination direction and to determine another apparent position. In one embodiment of the method, the emitter is illuminated from three azimuth angles evenly spaced around the optical axis: φ1 = 0°, φ2 = 120°, φ3 = 240° at a constant inclination angle θ. (The inclination angle in this example is, by convention, the angle between the minimum line of the intensity distribution and the xy plane.) For the three illumination directions, the following applies according to Fig. 5: z=(x−x1)tan θ for φ1=0° z sin φ2=(y−y2)tan θ for φ2=120° z sin φ3=(y−y3)tan θ for φ3=240°

[0049] Solving this system of equations for the coordinates of the emitter in the sample yields: (x,y,z)=(x1(sin φ2−sin φ3)+y2−y1sin φ2−sin φ3,y2sin φ2−y1 sin φ3 sin φ2−sin φ3,(y2−y1)tan θsin φ2−sin φ3)

[0050] The accuracy of position determination can be further improved by illuminating the sample at different angles. The expert will then solve the overdetermined system of equations approximately, for example, by minimizing the sum of squares of the errors.

[0051] In a further preferred embodiment, the method is applied iteratively, with the positional uncertainty of the calculated emitter position decreasing from one iteration to the next. This takes advantage of the fact that the apparent positions approach each other and the actual position of the emitter in the sample as the distance of the emitter from the illumination line or the illumination plane decreases. Specifically, this embodiment of the method comprises the following steps: i. Detecting light emissions at multiple illumination positions of the modulation light in a close range around the emitter and calculating the position of the emitter in the sample from the light emissions detected at the illumination positions in a first iteration of the method, ii. Shifting the illumination line or the illumination plane so that the distance of the emitter from the illumination line or the illumination plane is reduced, iii. Detecting light emissions at multiple illumination positions of the modulation light in a close range around the emitter and calculating the position of the emitter in the sample from the light emissions detected at the illumination positions in a second iteration of the method, wherein the position of the emitter is calculated with a reduced position error compared to the first iteration.

[0052] The advantage of this iterative embodiment of the method is that precise localization of the emitter is possible even if the inclination and azimuth angles characterizing the illumination directions are unknown or not exactly known. Then, in the first iteration (step i.), a position of the emitter can be calculated using inaccurate or even estimated inclination and azimuth angles. This position may be subject to a large error, but allows a displacement of the illumination line or the illumination plane, thus reducing the distance of the emitter from the illumination line or the illumination plane (step ii.). In the subsequent second iteration (step iii.) the apparent positions are closer together according to the above, so that despite the only approximately known inclination and azimuth angles, the position of the emitter can be calculated with a reduced position error compared to the first iteration.

[0053] Optionally, further iterations can be performed, with the apparent positions converging toward the actual position of the emitter (and thus to each other) in the sample. Further iterations can be performed, in particular, until the distance between the apparent positions or the position uncertainty with which the position of the emitter is calculated falls below a threshold value or no longer decreases compared to the previous iteration. This threshold value for the position uncertainty is preferably 10 nm, more preferably 5 nm, and most preferably 2 nm.

[0054] The direction of illumination under which the sample is illuminated with the intensity distribution of the modulated light can be changed, for example, by shifting a phase or intensity distribution of the modulated light in a rear pupil of the objective. For example, the center of a helical phase distribution, which leads to the formation of a donut mode in the sample when focused by the objective, can be shifted relative to the center of the rear pupil of the objective. The magnitude of the shift determines the angle of inclination of the minimum line or the minimum area of ​​the intensity distribution of the modulated light relative to the optical axis, and the direction of the shift determines the direction in which the intensity distribution in the sample is tilted.Alternatively, the modulation light beam can be shifted in the rear pupil of the lens so that the pupil of the lens is no longer illuminated homogeneously, but only partially and / or with an intensity gradient.

[0055] According to a preferred embodiment of the method, the phase distribution or intensity distribution of the modulated light in a pupil of the objective is shifted using a beam scanner. For this purpose, the beam scanner can, in particular, have two deflection elements that deflect the modulated light beam in the same deflection direction in order to independently adjust the position and angle of incidence of the modulated light beam in the pupil of the objective.

[0056] According to a further preferred embodiment of the method, the phase distribution or intensity distribution of the modulated light in a pupil of the objective is varied using a programmable light modulator (spatial light modulator, SLM) in order to illuminate the sample with the modulated light from different illumination directions. Pixelated liquid crystal modulators operated in phase or amplitude modulation mode can be used as light modulators in a beam path of the modulated light. The center of the phase pattern can be shifted on the active surface of the light modulator by appropriately controlling the light modulator, so that the phase pattern is shifted in a pupil of the objective.Alternatively, the phase pattern can be superimposed by the light modulator with various (linear) phase gradients, whereby the modulated light beam is deflected in different directions depending on the direction of the phase gradient and shifted within a pupil of the objective. Shifting the modulated light beam within a pupil of the objective can also be achieved using a programmable deformable mirror in the beam path of the modulated light.

[0057] According to another preferred embodiment of the method, the sample is illuminated with the modulated light from different illumination directions by alternately illuminating the sample with different beams of the modulated light. The beams can differ in the phase pattern imprinted on their wavefronts or in the position of the beams within a pupil of the objective. It is crucial that the different beams of the modulated light illuminate the sample from different illumination directions when focused by the objective.For example, the modulated light beam can be directed alternatively through one of two different light paths using a polarization rotator, in particular a Pockels cell, and a downstream polarization beam splitter. The light paths contain different (vortex) phase plates or light modulators, or those arranged differently relative to the modulated light beam. The modulated light beams from both light paths can be recombined using a second polarization beam splitter and directed along a common light path to the objective. By (rapidly) switching the modulated light between orthogonal polarization states with the polarization rotator, the sample can be alternately illuminated in different illumination directions.

[0058] The method according to the invention can be carried out once or multiple times on an individual emitter. In a preferred embodiment for single particle tracking, the method is repeatedly carried out on a single emitter or on multiple emitters, each of which binds individually one after the other to an object in the sample, and a three-dimensional trajectory of the emitter or object is created from the calculated positions of the emitter or emitters. The trajectory of the single emitter itself can be of interest, but the emitter(s) can also merely serve as markers for the object whose trajectory is to be determined. The object can in particular be marked one after the other with a plurality of emitters (of the same type) in order to extend the length of the trajectory beyond the photobleaching of an emitter.The person skilled in the art can find corresponding methods for serially staining an object in a sample in the state of the art for PAINT microscopy.

[0059] In a further preferred embodiment of the method, a plurality of individual emitters are located in the sample, and a visual representation is generated from the calculated positions of the emitters. Suitable methods for generating a visual representation from the positions of individual emitters are familiar to those skilled in the art from the prior art for PALM, STORM, and MINFLUX microscopy. The visual representation can, in particular, be a projection of the positions onto a two-dimensional image plane or a two-dimensional representation in two directions, in which the third spatial direction is color-coded. The visual representation can also be a stereoscopic spatial representation, in particular generated by projection onto a (partially) transparent projection surface or by displaying it in data glasses.

[0060] This embodiment can also be modified so that a plurality of emitters bind transiently to a plurality of binding sites on a structure in the sample, whereby the binding sites can be occupied by only one emitter at any one time. Swapping the emitters at the binding sites allows for the detection of more light emissions at the binding sites than is possible with a single, permanently bound emitter, whose light emission is limited due to photobleaching. Due to the increased amount of light emissions, the localization of the individual binding sites with emitters can be achieved with improved accuracy.

[0061] A second aspect of the invention relates to a light microscope for locating or tracking an isolated emitter in a sample, in particular for carrying out a method according to one of the preceding claims. For this purpose, the light microscope comprises - a light source for excitation light, which is designed to excite the isolated emitter in the sample to emit light, - a light source for modulated light designed to attenuate, amplify or excite the light emission of the emitter in the sample, - an objective lens designed to illuminate the sample with the excitation light and the modulation light, - optical means designed to form an intensity distribution of the modulated light in the sample such that the intensity distribution has an intensity minimum extending along a minimum line or a minimum area, in particular a zero line or a zero area, - a scanner designed to position the intensity distribution of the modulation light in the sample at different illumination positions in a close range around the emitter and - a detection device configured to detect light emissions from the emitter to the illumination positions.

[0062] The scanner serves to shift the intensity distribution of the modulated light relative to the sample. In this respect, the scanner can also be a sample scanner that shifts the sample relative to a fixed intensity distribution of the modulated light. The scanner can also be arranged in a common beam path for the excitation light and the modulated light, shifting both beams together.

[0063] The light microscope according to the invention is characterized in that it comprises means designed to illuminate the sample with the modulation light from different illumination directions, and in that it comprises a computing unit designed to calculate a position of the emitter in the sample from the light emissions detected when the sample is illuminated with the modulation light under different illumination directions.

[0064] In a preferred embodiment of the light microscope, the modulation light is simultaneously the excitation light, and the light microscope has a common light source for the modulation light and the excitation light.

[0065] A further embodiment of the light microscope is characterized in that the computing unit is configured to calculate an apparent position of the emitter from each of the light emissions detected when the sample is illuminated at the same angle of the modulated light, and to calculate the position of the emitter in the sample from the apparent positions. Preferably, the computing unit is configured to calculate the position of the emitter in an additional spatial direction from the apparent positions calculated in one or two spatial directions.

[0066] In a further preferred embodiment of the light microscope, the light microscope comprises a light modulator with a control device configured to shift a phase or intensity distribution of the modulated light in a rear pupil of the objective. In this embodiment of the microscope, the light modulator serves to illuminate the sample with the modulated light from different illumination directions.

[0067] In a further preferred embodiment of the light microscope, the scanner of the light microscope comprises two deflection elements configured to deflect the modulated light beam in the same deflection direction. The scanner has a control device configured to shift the modulated light beam into the pupil of the objective, regardless of the angle of incidence. In this embodiment of the microscope, the scanner serves to illuminate the sample with the modulated light from different illumination directions.

[0068] Advantageous developments of the invention emerge from the patent claims, the description, the drawings, and the associated 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. Regarding 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.

[0069] The combination of features of different embodiments of the invention or of features of different patent claims is also possible, deviating from the chosen references of the patent claims, and is hereby encouraged. This also applies to features 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 claims of the granted patent.

[0070] 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. Short description of the characters Fig. 1 illustrates the inventive idea of ​​the inventive method. Fig. 2 shows a flowchart of an embodiment of the method. Fig. 3 shows a flowchart of another embodiment of the method. Fig. 4 shows the calculation of the emitter position from apparent positions in two dimensions. Fig. 5 shows an extension to three dimensions. Fig. 6 shows an embodiment for generating inclined intensity distributions. Fig. Figure 7 shows a simulation for generating tilted intensity distributions. Fig. 8 shows a microscope according to the invention. Fig. 9 shows a microscope according to the invention. Fig. 10 shows a microscope according to the invention. Description of the characters

[0071] In Fig. Figure 1 shows the principle by which the position 1 of an emitter 2 is determined by illuminating it with modulated light 3 from different illumination directions 4, 5. The modulated light 3 is embodied here as excitation light 6, which excites the emitter 2 to emit light.

[0072] The modulated light 3 creates an intensity distribution 7 in the sample, which has a minimum line 9, here represented as a zero line 8. The figure shows a section through the xz plane, where the z axis corresponds to the optical axis 10 (of the lens not shown here) and the xy plane perpendicular to the plane of the drawing corresponds to the illumination plane 11. When the illumination direction 4, 5 is changed, the intensity distribution 7 of the modulated light 3 is tilted relative to the optical axis 10 and the illumination plane 11, but not shifted perpendicular to the illumination plane 11. A device for shifting the intensity distribution 7 in the direction of the optical axis 10 in the sample is therefore not required.

[0073] In the embodiment shown, light emissions are detected for each of the illumination directions 4, 5 at different lateral positions of the modulation light 3, and a position of the emitter is calculated from these light emissions. This procedure corresponds to the MINFLUX method known from the prior art. However, due to the inclined intensity distributions 7, the two determined positions of the emitter 2 are not its actual position 1, but rather apparent positions 12, 13, which are projections of position 1 in the illumination plane 11 along the illumination directions 4, 5. The actual position 1 of the emitter 2 in the x- and z-directions can be calculated from the apparent positions 12, 13 and the angles 14, 15.

[0074] Fig. Figure 2 shows a general embodiment of the method in a flow chart. In the first step 16, the intensity distribution of the modulated light is adjusted in a first illumination direction. In the second step 17, the sample is illuminated at illumination positions in the illumination plane (here the xy plane), with light emissions from the emitter at the illumination positions being detected and recorded as data points (N i , (x i , y i )) 18. Where N i for the position (x i , y i ) detected light quantity or the emission events recorded at these positions. Substeps 19 of illuminating and detecting the light emissions are performed repeatedly.

[0075] In the third step 20, the intensity distribution of the modulated light is adjusted in a second illumination direction. In the fourth step 21, the sample is illuminated at illumination positions in the illumination plane, with light emissions from the emitter at the illumination positions being detected and recorded as data points (N j , (x j , y j )) 18 are stored. The substeps 19 of illuminating and detecting the light emissions are again carried out repeatedly.

[0076] In the fifth step 22, the position (x, y, z) 1 of the emitter in the sample is calculated from the data points 18 detected in the second step 17 and the fourth step 21. The calculation of position 1 can be performed using all data points 18 simultaneously with a suitable estimator.

[0077] Alternatively, the data points 18 for each of the illumination directions can first be evaluated separately and then calculated in the final calculation step 22 to determine the position 1 of the emitter. The corresponding flow chart is shown in Fig. 3. The steps of the procedure largely correspond to those in Fig. 1, however, after the data acquisition steps 17 and 21, calculation steps 23 are inserted, in which an apparent position 12 or 13 is calculated from the previously acquired data points. The final calculation of the emitter's position 1 in step 22 is then performed using the apparent positions 12 and 13 and no longer the original data points 18.

[0078] Fig. 4 shows the calculation of the position 1 of the emitter 2 in two dimensions x and z from the apparent positions 12, 13 in the illumination plane 11. The apparent positions 12, 13 are obtained when the emitter 2 is positioned as in Fig. 1 is localized using a MINFLUX method, whereby the sample is illuminated not in the direction of the optical axis 10, but with intensity distributions of the modulated light inclined relative to the optical axis 10, i.e., under different illumination directions 4, 5 (indicated by the inclination angles 14, 15). The following relationships between the inclination angles (θ1, θ2) 14, 15, the apparent positions (x1, x2) 12, 13, and the position (x, z) 1 of the emitter 2 can be directly derived from the figure: z tan θ1=x1−x z tan θ2=x−x2 Solving the system of equations results in reconstruction formula 24, which can be used to calculate position 1 of emitter 2 in the xz plane. The reconstruction is shown for the general case where the inclination angles 14, 15 differ not only in direction but also in magnitude. For the case where the magnitudes of the inclination angles 14, 15 are equal (i.e., |θ1| = |θ2|), reconstruction formula 24 reduces to the symmetric case, in which the x-coordinate is the mean of the apparent positions x = (x1 + x2) / 2.

[0079] Based on the illustration in Fig. 5, a relationship can be derived between the apparent position 12 of the emitter 2 in the focal plane 11 and the actual position 1 of the emitter 2 in three-dimensional space when the emitter 2 is localized with modulation light from the illumination direction 4. For a given azimuth angle 25 φ l and inclination angle 14 θ ithe lighting direction 4 applies according to drawing: z sin φi=(y−yi)tan θi or z cos φi=(x−xi)tan θi

[0080] For example, if the emitter is located at three azimuth angles 25 (φ1 = 0°, φ2 = 120°, φ3 = 240°) evenly arranged around the optical axis 10 and at a constant inclination angle θ, and the apparent positions 12 (x1, y1), (x2, y2) and (x3, y3) are determined, the position 1 of the emitter 2 can be calculated from the apparent positions according to the reconstruction formula 24.

[0081] Fig. Figure 6 schematically shows how different illumination directions 4 can be generated by shifting the modulated light beam 26 in a pupil 27 of the objective. Shown is a modulated light beam 26 with a helical phase pattern 28, which exhibits a phase increase from 0 to 2π and a phase jump 29 in the azimuthal direction.

[0082] Line A shows a displacement of the phase pattern 28 relative to the center 30 of the pupil 27 in the -y direction, which tilts the intensity distribution 7 of the modulated light 3 in the xz section of the sample, but remains vertical perpendicular to it in the yz section. The tilt of the intensity distribution 7 can be accompanied by a (slight) translational displacement in the y direction.

[0083] In line B, a displacement of the phase pattern 28 relative to the center 30 of the pupil 27 in the -x direction is shown, whereby the intensity distribution 7 of the modulation light 3 is tilted in the yz section in the sample, but remains upright perpendicular to it in the xz section.

[0084] In line C, a displacement of the phase pattern 28 relative to the center 30 of the pupil 27 in the +y direction is shown, whereby the intensity distribution 7 of the modulation light 3 in the xz section is inclined in a direction opposite to the case shown in line A.

[0085] In line D, a displacement of the phase pattern 28 relative to the center 30 of the pupil 27 in the +x direction is shown, whereby the intensity distribution 7 of the modulation light 3 in the yz section is inclined in a direction opposite to the case shown in line B.

[0086] Fig. Figure 7 shows how the intensity distribution of the modulation light 3 in the sample inclines with increasing displacement 31 of the phase pattern 28 in the pupil 27 relative to the optical axis 10 (simulation). The modulation light 3 entering the pupil 27 has, as shown in Fig. 6 a helical phase pattern 28 with an azimuthal phase increase 32 from 0 to 2π and a phase jump 29. When focused by the lens, an annular intensity distribution 7 of the modulated light 3 results, wherein the zero line 8 coincides with the optical axis 10 when the phase pattern 28 is centered in the pupil 27 (line A). With a slight displacement 31 of the phase pattern 28 in the y-direction relative to the center 30 of the pupil 27, the zero line 8 of the intensity distribution 7 begins to incline in the x-direction relative to the optical axis 10, with a simultaneous translation of the intensity distribution 7 in the direction of the displacement 31 of the phase pattern 28 (i.e. in the y-direction) occurring (line B); the zero line is thereby shifted into the plane defined by y = -100 nm.With a larger displacement 31 of the phase pattern 28 relative to the center 30 of the pupil 27, the inclination of the zero line 8 of the intensity distribution 7 in the x-direction and the translation in the y-direction increase (line C). The intensity distributions 7 shown are calculated for homogeneous illumination of the pupil 27, i.e., a constant intensity of the modulation light 3 across the pupil 27.

[0087] Fig. Figure 8 shows a light microscope according to the invention, specifically an embodiment as a STED-MINFLUX microscope. The light microscope comprises a light source 33 for excitation light 6 and a light source 33 for modulation light 3, which here is emission suppression light or stimulation light 34 and suppresses the light emission of the emitters in the sample 38. The modulation light 3 is guided by mirrors over a light modulator 36 controlled by a programmable wavefront controller 35, which imposes a variable phase pattern on the wavefront of the modulation light 3, so that the modulation light 3 forms an intensity distribution with a linear or areal intensity minimum when focused with the objective 37 in the sample 38.The wavefront controller 35 is configured to display the phase pattern presented on the light modulator 36 in different positions relative to the incident modulation light beam 26, so that the phase pattern is centered at different positions in the pupil of the objective 37 and the intensity distribution of the modulation light 3 is inclined relative to the optical axis of the objective (according to the illustrations in FIGS. Fig. 6 and Fig. 7).

[0088] The excitation light 6 and the modulation light 3 are coupled into the scanner 41 by a beam combiner 39 and the main beam splitter 40. The scanner 41 is designed as a galvo scanner and enables deflection of the combined excitation and modulation light beams in two spatial directions. For this purpose, the scanner 41 comprises two tilting mirrors 43, each mounted on a galvo motor 42, whose axes of rotation are perpendicular to each other—i.e., two galvo scanners that deflect the light beam in mutually perpendicular spatial directions. The galvo motors 42 are controlled by a scan controller 44 to position the excitation light 6 and the modulation light 3 in the sample 38. In the example shown, the tilting mirrors 43 are each positioned near a pupil, i.e., near a plane conjugate to the rear pupil of the objective 37.In an alternative arrangement, not shown in the figure, a relay optics can be located between the two galvo scanners, in which case both tilting mirrors 43 are preferably placed in one pupil each.

[0089] The light emerging from the scanner 41 is imaged via a scanning lens 45 and a tube lens 46 into the rear pupil of the objective 37, which focuses the excitation light 6 onto the sample 38. A change in the angle of the tilting mirrors 43 translates into a change in the angle of the light beam in the pupil of the objective 37 and thus into a displacement of the focused excitation light 6 and modulation light 3 in the sample 38.

[0090] Emission light 47 emitted by the emitters in the sample 38 is collected by the objective lens 37 and travels in the excitation and modulation light beams in opposite directions to the main beam splitter 40, which transmits the emission light 47 and thus separates it from the excitation light 6 and the modulation light 3. The main beam splitter 40 can, in particular, be designed as a (dichroic) color splitter. The detection system arranged behind the main beam splitter 40 comprises a filter 48 that blocks unwanted stray light components and a lens 49 with which the emission light 47 emitted by the emitters is focused onto the detector 51 through a (confocal) pinhole 50.

[0091] The illustrated STED-MINFLUX microscope differs from a conventional confocal microscope and from (STED-)MINFLUX microscopes known from the prior art in that the microscope comprises a computing unit 52 which calculates a position of an emitter in the sample 38 from detection signals 53 generated by the detector 51, position signals 54 provided by the scan controller 44 and identification signals 55 representing the phase patterns presented by the light modulator 36.

[0092] Fig. Figure 9 shows another light microscope according to the invention, here an embodiment as a MINFLUX microscope. Compared to the Fig. In the STED-MINFLUX microscope shown in Figure 8, the excitation light 6 provided by the light source 33 is simultaneously the modulation light 3. Instead of a light modulator, the microscope comprises a (vortex) phase plate 56 in the light beam of the modulation light 3, which imposes a helical phase pattern on the wavefront of the modulation light 3, so that the modulation light 3 forms a donut-shaped intensity distribution with a linearly extended intensity minimum in the sample 38 when focused with the objective 37. The scanner 41 is designed as a quad scanner 57, i.e., it comprises pairs of tilting mirrors 43 and 59 for each of the two deflection directions, which are driven by pairs of galvomotors 42 and 58, respectively. The two tilting mirrors 43 and 59 per deflection direction make it possible to adjust both the position and the inclination at which the light beam falls into the rear pupil 27 of the objective 37 independently of each other.By shifting the light beam, an image of the phase plate 56 can be translated in the pupil 27, allowing the illumination direction of the modulated light 3 in the sample 38 to be varied. The illumination direction is adjusted here via the scan controller 44, which transmits both a position signal 54 and an identification signal 55 encoding the illumination direction to the computing unit 52. If an inclination of the intensity distribution of the modulated light 3 in the sample 38 in one direction is sufficient, an X2Y scanner can be used instead of a quad scanner, which has two tilting mirrors for only one of the two deflection directions.

[0093] The remaining components of the microscope correspond to those of the Fig. 8 microscope shown.

[0094] Fig. Figure 10 shows another light microscope according to the invention. As in the Fig.In the embodiment shown in Figure 8, the excitation light 6 is also simultaneously the modulation light 3. The MINFLUX microscope shown is characterized in that the excitation light 6 is provided by two separately switchable light sources 33, which are combined with a beam combiner 39 designed as a polarization beam splitter 60. A phase plate 55 is arranged in each of the modulation light beams 26, which imprint helical phase patterns on the wavefronts of the modulation light beams 26, so that the modulation light 3 of the modulation light beams 26 forms donut-shaped intensity distributions with linear intensity minima in the sample 38 when focused with the objective 37. In the embodiment shown, the phase plates 56 can be positioned independently of one another and adjusted such that the phase plates 56 are imaged at different positions in the rear pupil 27 of the objective 37.Subsequently, the sample 38 can be illuminated with the modulation light 3 under different illumination directions, depending on which of the two light sources 33 the sample 38 is switched on with.

[0095] In this embodiment, the scan controller 44 of the light microscope generates, in addition to control signals 61 for controlling the galvomotors 42 of the scanner 41, control signals 62 for switching the light sources 33, so that the sample 38 can be illuminated alternately with the two light sources 33 and under different illumination directions. In addition to the position signal 54, the scan controller 44 also transmits an identification signal 55 encoding the currently activated light source 33 or the illumination direction to the processing unit 52. List of reference symbols 1 position 2 emitters 3 Modulation light 4 Lighting direction 5 Lighting direction 6 Excitation light 7 Intensity distribution 8 Zero line 9 Minimal line 10 optical axis 11 Lighting level 12 apparent position 13 apparent position 14 angles 15 angles 16 Step 1 17 2nd step 18 data points 19 sub-steps 20 Step 3 21 Step 4 22 Step 5 23 Calculation step 24 Reconstruction formula 25 azimuth angles 26 Modulation light beam 27 pupil 28 phase patterns 29 Phase jump 30 Center 31 Relocation 32 Phase rise 33 Light source 34 Stimulation light 35 wavefront controllers 36 Light modulator 37 lens 38 Sample 39 beam combiners 40 main beam splitters 41 scanners 42 Galvomotor 43 tilting mirrors 44 scan controllers 45 scan lens 46 Tube lens 47 Emission light 48 filters 49 lens 50 pinhole 51 Detector 52 computing unit 53 Detection signal 54 Position signal 55 Identification signal 56 Phase plate 57 Quad Scanner 58 Galvomotor 59 Tilting mirrors 60 polarization beam splitters 61 Control signal 62 control signal

Claims

[1] Method for locating or tracking an isolated emitter (2) in a sample (38), wherein the sample (38) is viewed through an objective (37) - with excitation light (6) which excites the isolated emitter (2) to emit light, and - is illuminated with modulation light (3) which attenuates, amplifies or excites the light emission of the emitter (2), wherein the modulation light (3) forms an intensity distribution (7) in the sample (38) with an intensity minimum extended along a minimum line (9) or a minimum area, in particular with a zero line (8) or a zero area, comprising the steps: - detecting light emissions from the emitter (2) to several illumination positions of the modulation light (3) in a close range around the emitter (2), - Calculating a position (1) of the emitter in the sample (38) from the emissions detected at the illumination positions, characterized bythat the sample (38) is illuminated with the modulation light (3) during the detection of the light emissions under different illumination directions (4, 5), wherein the light emissions are assigned or can be assigned to the different illumination directions (4, 5), wherein the intensity distribution (7) is tilted relative to an optical axis (10) when the illumination direction (4, 5) changes. [2] Method according to claim 1, characterized by that the modulation light (3) is simultaneously the excitation light (6). [3] Method according to one of claims 1 or 2, characterized by that the illumination positions (4, 5) lie on an illumination line oriented in particular perpendicular to the optical axis (10) or in an illumination plane (11) oriented in particular perpendicular to the optical axis (10). [4] Method according to claim 3, characterized bythat calculating the position (1) of the emitter (2) comprises calculating the distance to the illumination line or the illumination plane (11). [5] Method according to one of claims 1 to 4, characterized by that the illumination directions (4, 5) under which the sample (38) is illuminated with the modulation light (3) are measured in a calibration step preceding the method and that the position (1) of the emitter (2) in the sample (38) is calculated using the measured illumination directions (4, 5). [6] Method according to one of claims 1 to 5, characterized by that apparent positions (12, 13) of the emitter (2) are calculated from subsets of light emissions which are detected when the sample (38) is illuminated with the modulation light (3) from the same illumination direction (4, 5), and that the position (1) of the emitter (2) in the sample (38) is calculated from the apparent positions (12, 13). [7] Method according to claim 6, characterized by that the position (1) of the emitter (2) in the sample (38) is calculated in at least one spatial direction more than the apparent positions (12, 13). [8] Method according to claim 7, characterized by , - that the detection of light emissions at several illumination positions of the modulation light (3) in a close range around the emitter (2) and the calculation of the position (1) of the emitter (2) in the sample (38) from the light emissions detected at the illumination positions are carried out in a first iteration of the method, - that the illumination line or the illumination plane (11) is displaced so that the distance of the emitter (2) from the illumination line or the illumination plane (11) is reduced, and - that the detection of light emissions at a plurality of illumination positions of the modulation light (3) in a close range around the emitter (2) and the calculation of the position (1) of the emitter (2) in the sample (38) from the light emissions detected at the illumination positions are carried out in a second iteration of the method, wherein the position (1) of the emitter (2) is calculated with a position error that is reduced compared to the first iteration. [9] Method according to claim 8, characterized by that further iterations of the method are carried out until the distance between the apparent positions (12, 13) or the position uncertainty with which the position (1) of the emitter (2) is calculated falls below a limit value or does not decrease further compared to the previous iteration. [10] Method according to one of claims 1 to 9, characterized bythat the illumination direction under which the sample (38) is illuminated with the modulation light (3) is changed by shifting (31) a phase or intensity distribution of the modulation light (3) in a rear pupil (27) of the objective (37). [11] Method according to claim 10, characterized by that the displacement (31) is adjusted with a beam scanner (41). [12] Method according to one of claims 1 to 11, characterized by that the illumination direction under which the sample (38) is illuminated with the modulation light (3) is changed using a programmable light modulator (36). [13] Method according to one of claims 1 to 11, characterized by that the illumination direction under which the sample is illuminated with the modulation light (3) is changed by alternately illuminating the sample with different beams of the modulation light (3). [14] Method according to one of claims 1 to 13, characterized by that the method is carried out repeatedly on a single emitter (2) or on several emitters (2), each of which binds individually one after the other to an object in the sample (38), and that a three-dimensional trajectory of the emitter (1) or of the object is created from the calculated positions (1) of the emitter (2) or the emitters (2). [15] Method according to one of claims 1 to 13, characterized by that the method is carried out with a plurality of isolated emitters (2) in the sample (38) and that a pictorial representation is generated from the calculated positions (1) of the emitters (2). [16] Method according to claim 15, characterized by that a plurality of emitters (2) bind transiently to a plurality of binding sites on a structure in the sample (38), wherein the binding sites can be occupied by only one emitter (2) at any one time. [17] Light microscope for locating or tracking an isolated emitter (1) in a sample, in particular for carrying out a method according to one of the preceding claims, comprising - a light source (33) for excitation light (6) which is designed to excite the isolated emitter (1) in the sample (38) to emit light, - a light source for modulation light (3) which is designed to attenuate, amplify or excite the light emission of the emitter (1) in the sample (38), - an objective lens (37) designed to illuminate the sample (38) with the excitation light (6) and the modulation light (3), - optical means designed to form an intensity distribution (7) of the modulation light (3) in the sample (38) such that the intensity distribution (7) has an intensity minimum extending along a minimum line (9) or a minimum area, in particular a zero line (8) or a zero area, - a scanner (41) designed to position the intensity distribution (7) of the modulation light (3) in the sample (38) at different illumination positions in a close range around the emitter (2), - a detection device (51) designed to detect light emissions from the emitter (2) to the illumination positions, characterized byin that the light microscope has means which are designed to illuminate the sample (38) with the modulation light (3) from different illumination directions (4, 5) and to tilt the intensity distribution (7) with respect to an optical axis (10) when the illumination direction (4, 5) changes, and in that the microscope has a computing unit (52) which is designed to calculate a position (1) of the emitter (2) in the sample (38) from the light emissions detected when the sample (38) is illuminated with the modulation light (3) under different illumination directions (4, 5). [18] Light microscope according to claim 17, characterized by that the modulation light (3) is simultaneously the excitation light (6) and that the light microscope has a common light source (33) as a light source for the modulation light (3) and as a light source for the excitation light (6). [19] Light microscope according to claim 17 or 18, characterized byin that the computing unit (52) is designed to calculate an apparent position (12, 13) of the emitter (2) from light emissions which are detected when the sample (38) is illuminated under the same illumination directions (4, 5) of the modulation light (3), and to calculate the position (1) of the emitter (2) in the sample (38) from the apparent positions (12, 13). [20] Light microscope according to claim 19, characterized by that the computing unit (52) is configured to calculate the position (1) of the emitter (2) in an additional spatial direction from the apparent positions (12, 13) calculated in one or two spatial directions. [21] Light microscope according to one of claims 17 to 20, characterized by that the optical means comprise a light modulator (36) with a control device which is designed to shift a phase or intensity distribution of the modulation light (3) in a rear pupil (27) of the objective (37). [22] Light microscope according to one of claims 17 to 21, characterized by in that the scanner (41) has two deflection elements which are designed to deflect the modulation light beam (3) in the same deflection direction, wherein the scanner (41) has a control device (44) which is designed to displace the modulation light beam (3) in the rear pupil (27) of the objective (37) independently of the angle of incidence into the pupil (27).

Images