Methods for locating or tracking emitters

Abstract

Methods for locating or tracking a single emitter (8) using a MINFLUX method or a STED-MINFLUX method in a sample (14), - wherein the sample (14) in a near region (4) of the isolated emitter (8) is exposed to an intensity distribution of an illumination light (12) exhibiting a local intensity minimum, wherein the illumination light (12) is excitation light (13) or emission suppression light, in particular STED light, and wherein the illumination light (12) or other light excites the emitter (8) to emit light, - wherein a metrological recording of one or more measured quantities is carried out, from which the respective current position (1) of the intensity distribution of the illumination light (12) in the sample (14) can be determined, - wherein emissions of the emitter (8) are detected, characterized in that the position of the intensity distribution in the sample (14) fluctuates around a nominal position (2) and that the measurement is carried out in such a way that the current position (1) of the intensity distribution in the sample (14) can be assigned to the detected emissions.

Description

Technical field of the invention

[0001] The invention is directed to a method and a microscope for locating and tracking individual emitters in a sample using the MINFLUX or STED-MINFLUX principle. Compared to the prior art MINFLUX and STED-MINFLUX microscopy methods, the invention is characterized by significantly relaxed requirements for the beam positioning device used to position the illumination light in the sample. State of the art

[0002] A relatively new microscopy technique in the state of the art is MINFLUX nanoscopy. MINFLUX nanoscopy is a localization microscopy technique. The localization of individual particles, especially individual fluorophores, is achieved using structured illumination light distributions. The illumination light is scattered by the particles or excites them to luminescence, especially fluorescence, and in this general sense is considered excitation light.The fundamental feature of MINFLUX nanoscopy is that the illumination of the particles, in particular the excitation of the fluorophores, is carried out in such a way that a particle to be localized or a particle whose movement in a sample is to be tracked is placed at various positions close to or at a minimum of the excitation light distribution, which ideally is a zero point, with the excitation light distribution exhibiting a region of increasing intensity adjacent to the minimum. This ensures that the photons scattered by the particle or the fluorescence photons contain a particularly large amount of information about the current position of the respective particle. This is particularly true for applications in which the movement of fluorophores is to be tracked over time. To obtain this information, the light emitted from the region of the minimum is detected.The tracking of a single particle using an excitation minimum is known, for example, from patent DE 10 2011 055 367 B4, and the localization of a single fluorescent molecule using an intensity distribution of excitation light with a local intensity minimum is known from German patent DE 10 2013 114 860 B3.

[0003] On this basis, a number of refinements for information acquisition have been developed that enable the localization of fluorophores with an uncertainty in the range below 2 nm as well as the tracking of the movement of individual fluorophores both within a range of a few nanometers and within a range of a few micrometers. A detailed, but not exhaustive, description of MINFLUX nanoscopy can be found in F. Balzarotti et al., "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes", Science 355, 606 - 612 (2017), DOI: 10.1126 / science.aak9913. Non-iterative MINFLUX localization and the tracking of individual fluorophores using MINFLUX are experimentally demonstrated. In addition, the concept of iterative MINFLUX is presented. Basically, in order to localize a fluorophore using MINFLUX nanoscopy, the intensity minimum orthe intensity zero is placed at a plurality of positions relative to the position of the fluorophore.

[0004] For this purpose, in a preparatory step, the current position of the fluorophore is estimated with an initial, lower accuracy or determined using an alternative method. This can be done, for example, using conventional localization microscopy (PALM, STORM) or other known state-of-the-art methods. Subsequently, an intensity distribution of excitation light with a central minimum, e.g., in the shape of a donut, as known from STED microscopy, is placed at a known position, which is chosen such that the estimated position of the fluorophore lies close to the minimum of the intensity distribution. The fluorescence response of the fluorophore is measured. The same is usually performed for several additional positions of the intensity distribution located around the estimated position and, if necessary, also for the position corresponding to the estimated position.The set of all such scanning positions forms a set of scanning positions. Using a ratiometric evaluation of the intensities measured at the scanning positions, the position of the fluorophore is determined with greater accuracy. This more precisely determined position can then be used as the starting position for repeating the sequence of the aforementioned steps. In the case of a particle localization, the positions of the minimum of the intensity distribution of excitation light can be placed closer to the estimated position of the fluorophore. This process can be continued iteratively.This method can also be carried out for different spatial directions in a temporal sequence, in particular alternating, so that, for example, the position of the particle in one spatial direction is determined with a first intensity distribution with a minimum extending in a first direction and the position of the particle in a second spatial direction is determined with a second intensity distribution with a minimum extending in a second direction oriented perpendicular to the first direction.

[0005] If the movement of a particle is to be tracked, it may be useful not to decrease the distance between the positions at which the intensity minimum is placed and the position estimated in the previous step from step to step. The method is carried out as described in the above-mentioned publication using a microscope that contains both a piezo scanner and electro-optical deflectors (EOD). Using this combined scanning device, an image field of approximately 20 µm × 20 µm can be addressed. Using the EOD alone, an image field of approximately 2 µm × 2 µm can be addressed, whereby high-speed displacement is only possible in a field of approximately 300 nm × 300 nm. Both when performing localization and when tracking a particle, the scanning positions of a set of scanning positions are controlled exclusively using high-speed EOD scanning.A combination of piezo scanner and EOD can then be used to relocate the center. The movement of individual molecules, which are only mobile within a narrowly defined area—namely, within a region covered by a set of scanning positions—can be achieved using a fixed set of scanning points.

[0006] International Patent Application WO 2018 / 069283 A1 describes various embodiments of MINFLUX methods. Among other things, a method is described in which the sampling positions of a set of sampling points around an initial spatial range are sampled quasi-simultaneously. The positions of the zero point, i.e., the sampling points, are then successively shifted into the initial spatial range depending on the photons of luminescent light registered at each of the positions. In extreme cases, a shift into the initial spatial range can occur even if a single photon has been registered at a sampling position.

[0007] European patent EP 3 372 990 B1 discloses a method in which a set or sets of scanning positions are scanned with a local zero of an intensity distribution of fluorescence-preventing light, which is superimposed, for example, with a Gaussian intensity distribution of excitation light. The position of the fluorophore can then be determined in essentially the same way as with MINFLUX methods, whereby the dependence of the fluorescence intensity on the distance of the fluorophore with respect to the zero of the intensity distribution of the fluorescence-preventing light differs from MINFLUX in that the fluorescence intensity, or the rate at which photons can be detected, decreases with increasing distance. Such methods are referred to in this application as STED-MINFLUX methods.

[0008] In Klaus C. Gwosch et al., "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells," Nat. Methods 17, 217–224 (2020), DOI: 10.1038 / s41592-019-0688-0, and the accompanying "Supplementary Information," a concrete implementation of three-dimensional localization using MINFLUX is described. For this purpose, a 3D donut—i.e., an intensity distribution with a local minimum surrounded by regions of intensity increase in all three spatial directions—is used. This donut is placed at each of a three-dimensional set of scanning positions. A scanning device is used that, in addition to a piezo scanner and EODs, features a lens with adjustable focal length. A static calibration is performed for the scanning device so that a precise mapping between the control voltages of the components and the actual scanning positions of the sample is known.The calibration data is used to precisely address the scanning positions in the sample.

[0009] The MINFLUX implementations described in the prior art, insofar as they are based on shifting an intensity distribution, have in common that the (fluorescence) emission of the particles to be localized is registered at predetermined, defined scanning positions. This means that the position of the illuminating light in the sample represents a measurement parameter, while the (fluorescence) emission registered at this position is the measured variable. Since the dwell time at each scanning position is short (on the order of a few microseconds), this embodiment places high demands on the dynamic properties of the scanning device. In particular, positioning times in the microsecond range are hardly achievable with mechanical beam positioning units or beam scanners alone. Even fast galvanometer scanners have step response times of typically 100 µs to 200 µs.

[0010] For this reason, the MINFLUX embodiments described in the above-mentioned prior art have so far regularly used scanning devices that combine two scanners with complementary properties. For example, galvanometer scanners based on tilting mirrors driven by a galvo motor, which allow the beam to be positioned over larger image fields, are combined with electro-optical deflectors that quickly position the illumination light at the scanning positions. The publication "MINFLUX dissects the unimpeded walking of kinesin-1" (Jan O. Wolff et al., Science 379, 1004-1010 (2023), DOI: 10.1126 / science.ade2650) describes an alternative concept for shifting an intensity distribution with a local minimum.An intensity distribution with a corresponding minimum is generated by irradiating two collimated, coherent light beams into different regions of a lens pupil. The position of the corresponding minimum, which extends in one spatial direction, and the intensity distribution adjacent to the minimum are then deterministically shifted for the MINFLUX scan by changing the phase relationship between the two incident beams. The corresponding device is referred to in the publication as a phase scanner. Larger shifts are achieved using a galvo scanner system and a positioning stage.

[0011] The subsequently published patent DE 10 2022 119 589 B3 discloses a MINFLUX method, a corresponding microscope, and a corresponding computer program. According to the method, a localization is performed iteratively, wherein a systematic deviation of the position determined in a first localization step compared to a position determined in a second step is compensated. At least the second localization step is performed with an intensity distribution of an illumination light exhibiting a local minimum, which is arranged at illumination positions around the position of an emitter determined in the first localization step, wherein the position of the emitter is estimated from the respectively detected emissions.According to one embodiment, the illumination positions are not arranged at discrete points around the emitter, but rather the illumination occurs continuously along an illumination trajectory that includes the emitter. The assignment of light emission to illumination positions can also be achieved by recording the current illumination position as soon as a photon of the light emission is detected. According to another embodiment, a sequence of illumination positions can be run through once or multiple times, or a variable or random sequence of illumination positions can be used. Object of the invention

[0012] It is now the object of the present invention to provide a MINFLUX method or a STED-MINFLUX method that enables localization and / or tracking of isolated emitters in a sample with a high spatial and / or temporal resolution, in which the requirements for the beam positioning device are reduced. Solution

[0013] This object is achieved by the subject matter of independent claims 1 (method) and 16 (MINFLUX microscope or STED-MINFLUX microscope). Advantageous developments of the method are specified in subclaims 2 to 15, and advantageous developments of the MINFLUX microscope are specified in subclaims 17 to 20. Definitions

[0014] 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 emitter movements are to be tracked, 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 to the speed at which the particles or units move in the sample. The time delays can be up to around 10 µs, but are usually in the range of up to a few tens of nanoseconds, frequently in the range of 1 to 10 ns and, if the emission is scattered light, zero. In this application, the term fluorescence emitter is used to describe emitters that act as fluorescent point light sources. Emitters can be, for example, metallic nanoparticles. The more specific 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.

[0015] In the context of the application, an emitter is said to be 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.

[0016] In this application, emission-suppressing light is understood to mean light that prevents, reduces, or completely suppresses the emission of an emitter. In particular, the emission-suppressing light can be stimulation light or STED light, which induces stimulated emission from an electronically excited emitter, thereby converting the emitter (back) to the electronic ground state and thus preventing spontaneous (fluorescence) emission. It is important that the illumination light modulates the emitter's fluorescence emission depending on its intensity, whereby both an amplification of the emission (if the illumination light is excitation light) or an attenuation of the emission (if the scanning light is emission-suppressing light) is possible. If the illumination light is not itself the excitation light, the emitter is additionally illuminated with a separate excitation light that excites the emitter to emit.

[0017] In this application, MINFLUX methods are understood to mean localization and tracking methods for isolated emitters in which light distributions of illuminating light that excites light emissions from the emitter are generated at the focus in the sample, wherein the light distributions have a local minimum, and in which the position of an isolated emitter is determined by detecting light emissions from the emitter for different positions of the minima of the respective light distributions or for different such light distributions in a close range of the emitter, wherein the smaller the distance between the emitter and the minimum of the light distribution, the less light is emitted by the emitter.

[0018] A near field of an emitter is understood to be an area or a volume (for example a circle or a sphere) around a presumed position of an isolated emitter. The presumed position of the emitter can form a center of the area or the volume. The near field has, in particular, dimensions in the size range of the optical diffraction limit, i.e., in particular, the near field extends in one spatial direction (in particular, in two spatial directions or in three spatial directions) no further than a small multiple of the diffraction limit (for a given wavelength of detection of the light emitted by the emitter), in particular ten times, in particular five times, in particular twice, in particular 1.5 times the diffraction limit. In particular, the near field extends in at least one spatial direction no further than the diffraction limit.In particular, the diffraction limit can correspond to the diameter of an Airy disk. For example, in the case of a two-dimensional localization of the emitter, the near field can be a circle with a diameter in the range from 100 nm to 2 µm, in particular 150 nm to 2 µm, more particularly 200 nm to 2 µm, more particularly 250 nm to 2 µm, more particularly 300 nm to 2 µm. According to a further example, in the case of a two-dimensional localization, the near field can be a circle with a diameter in the range from 100 nm to 2 µm, in particular 100 nm to 1.5 µm, more particularly 100 nm to 1 µm, more particularly 100 nm to 500 nm. Further examples are in particular circles with a diameter of 150 nm to 1.5 µm, 150 nm to 1.5 µm, 200 nm to 1.5 µm, 250 nm to 1.5 µm, 300 nm to 1.5 µm, 150 nm to 1 µm, 200 nm to 1 µm, 250 nm to 1 µm, 300 nm to 1 µm, 150 nm to 500 nm, 200 nm to 500 nm, 250 nm to 500 nm or 300 nm to 500 nm.The center of the circle is located at the presumed position of the emitter. In the case of three-dimensional localization, the near field can be a sphere with the diameters mentioned above.

[0019] In this application, STED-MINFLUX methods are understood to mean methods corresponding to the MINFLUX methods as defined above, with the difference that instead of the intensity distribution of excitation light, an intensity distribution of emission suppression light overlapping with excitation light is used.The fact that the intensity distribution of emission suppression light overlaps with excitation light means that the sample in the region of the intensity minimum and the adjacent sections of the intensity increase regions of the emission suppression light, within which an emitter is located, is exposed to excitation light in such a way that an emission of the emitter is excited or would be excited in the absence of emission suppression light, so that the emission suppression light, according to its intensity distribution, affects the excited emission or the excitation of the emission in such a way that a measured emission of an emitter located within the intensity distribution of the emission suppression light decreases with increasing distance from the intensity minimum.If the emission quenching light is STED light and the emitter is a fluorescence emitter, this means that the spontaneous emission of the fluorescence emitter decreases with increasing distance from the intensity minimum of the STED light. The stimulated emission of the emitter, which has the same wavelength as the STED light, does not contribute to the measured emission, as is common in STED microscopy; it is blocked, for example, by a filter. The fact that the intensity distribution of the emission quenching light overlaps with the excitation light does not mean that the excitation light and the emission quenching light are introduced into the sample strictly simultaneously. For example, it is possible, as is common in STED microscopy, to apply a short pulse of excitation light, followed immediately by a pulse of STED light as emission quenching light.The excitation light overlapping the intensity distribution of the emission suppression light can, for example, exhibit an intensity distribution as is common in confocal microscopy, i.e., at least approximately corresponding to an Airy function or a Gaussian function, whose central maximum may coincide with the minimum of the emission suppression light. It can also correspond to a top hat function. Constant excitation in the wide field is also possible.

[0020] The term STED-MINFLUX is used in a generalized sense; it should not be understood that the emission suppression light must be STED light.

[0021] In MINFLUX microscopy and STED-MINFLUX microscopy, the sample is illuminated with focused illumination light. The illumination light forms an intensity distribution with a central intensity minimum, specifically an intensity zero, around a geometric focus in the sample. The position of an individual emitter relative to the central minimum then determines the intensity or photon emission rate of the emitter's light emissions, which are used to localize the emitter. Intensity distributions with a central minimum can be generated, in particular, by phase modulating the illumination light using a phase filter or a spatial light modulator (SLM) in the illumination beam path, focusing the illumination light using an objective lens, and, if necessary, additionally circularly polarizing the illumination light. The intensity distribution then arises at the geometric focus through interference.

[0022] In this application, a donut-shaped intensity distribution is understood to mean a special form of an intensity distribution with a central minimum, which has a minimum that is surrounded by intensity increase regions in all spatial directions within a given plane.

[0023] If this minimum extends along an axis perpendicular to this plane, this minimum is referred to in this application as a 2D minimum. A 2D minimum can be obtained, for example, using a vortex phase plate in the beam path, whereby the minimum can then extend, for example, along the optical axis of a lens.

[0024] In this application, a 3D minimum is understood to be a minimum surrounded by regions of increasing intensity in all spatial directions. A 3D minimum can be obtained, for example, using an annular phase plate or by superimposing two light beams, with the wavefront of one of the two being phase-modulated in each pupil by an annular phase plate and the wavefront of the other being phase-modulated by a vortex phase plate. A 3D minimum can also exhibit a donut-shaped intensity distribution in a given plane.

[0025] In this application, the term "localization" describes a method in which the position of an individual emitter in a sample is determined. In contrast to conventional light microscopy methods, this method does not require optical imaging of the sample. Rather, the MINFLUX localization method according to the invention detects light emissions from the emitter for various positions of the intensity distribution of the illumination light, and from these, the position of the emitter is determined, for example, using a position estimator. This process can be performed sequentially for several individual emitters, and the determined positions of the emitters can be displayed in a localization map, which can produce a high-resolution light microscopy image.

[0026] Tracking an emitter means determining the trajectory of an emitter moving in the sample, particularly by repeatedly locating it in succession.

[0027] In the context of the present application, a beam positioning device is a device designed to shift the intensity distribution of an illuminating light in a sample and the sample relative to each other. As a result, a beam positioning device shifts the intensity distribution of the illuminating light in the sample, viewed from the reference system of the sample. The term "beam positioning device" encompasses, in particular, beam scanners, phase scanners, as known from the above-mentioned publication "MINFLUX dissects the unimpeded walking of kinesin-1," and positioning stages.

[0028] In the context of the present application, a light shifting unit is a device designed to shift an intensity distribution of an illuminating light in a sample by acting on the illuminating light, for example by deflecting it, while the sample remains in a rest position.

[0029] In the context of the present application, a beam scanner is a device designed to shift a beam of illumination light, i.e., an illumination light beam, by means of one or more optical components, such that the focus of the illumination light on or in the sample is shifted in at least one spatial direction. The focus can be shifted in particular in two spatial directions, furthermore in particular in a focal plane that is perpendicular to an optical axis of an objective. The spatial directions can be defined in a wide variety of coordinate systems, e.g., in Cartesian coordinates (e.g., x- and y-directions), in polar coordinates, or in spherical coordinates (e.g., radial and azimuthal directions). The beam scanner can, for example,mechanical scanners, in particular scanners based on tilting mirrors each connected to a galvo motor, electro-optical deflectors or acousto-optical deflectors.

[0030] For the purposes of this application, a galvo scanner is defined as a module with a galvo motor and a tilting mirror connected to it and movable by the galvo motor. A galvo scanner may also comprise elements other than the galvo motor and tilting mirror.

[0031] In the context of this application, a galvoscanner system is understood to mean a scanning unit having one or more galvoscanners, which as a whole is configured to shift and / or deflect a light beam in one or more spatial directions.

[0032] In the context of the present application, a mechanical scanner is a scanner that variably deflects an illumination light, wherein the variation of the deflection occurs by moving at least one optical component, e.g., a mirror. The movement can, in particular, be a rotation, in particular a tilt, about one or more axes of rotation. Accordingly, both a galvo scanner or a galvo scanner system and a microelectromechanically actuated scanner, i.e., a MEMS scanner, are mechanical scanners within the meaning of this application. Likewise, a scanner based on rotatable Risley prisms is a mechanical scanner within the meaning of this application.

[0033] The beam scanner can receive control signals, in particular electrical signals, from a control device that influence the state of the at least one optical component in order to shift the illumination light beam to a desired position. If the beam scanner is a mechanical scanner with at least one movable optical component, the control signals are assigned target positions or target movements of the at least one optical component, which are determined by the control signals. Description of the invention

[0034] A first aspect of the invention relates to a method for locating or tracking an isolated emitter using a MINFLUX method or a STED-MINFLUX method in a sample, with which the position of the individual emitter in a sample is determined and / or the position of the emitter is tracked over time.

[0035] For this purpose, the sample is exposed to an intensity distribution of an illuminating light having a local intensity minimum in a close range of the isolated emitter, with the intensity minimum of the illuminating light being bordered on both sides by intensity increase regions in at least one spatial direction.

[0036] The illumination light can be excitation light, which excites the emitter to emit an emission, in particular a fluorescence emission or a scattered light emission. However, the illumination light can also be emission-suppression light, which prevents, reduces, or completely suppresses an emission from the emitter, i.e., an emission emanating from the emitter. In particular, the emission-suppression light can be stimulation light, which induces a stimulated emission from an electronically excited emitter and thus prevents subsequent (fluorescence) emission from the emitter. If the illumination light is not itself an excitation light, the emitter is additionally illuminated with a separate excitation light.

[0037] Emissions from the emitter are detected. Furthermore, the respective current position of the intensity distribution in the sample is measured, or one or more measured variables are measured, from which the respective current position of the intensity distribution of the illumination light in the sample can be determined or, in preferred embodiments, is determined. The measurement is carried out in such a way that the respective current position of the intensity distribution in the sample can be or is assigned to the detected emissions. In this case, the position of the intensity distribution of the illumination light is generally understood to mean the position of a focal point of the illumination light, at which in particular the local intensity minimum of the intensity distribution of the illumination light is formed.In the following, the term “position of the illuminating light in the sample” is used synonymously with the term “position of the intensity distribution of the illuminating light in the sample”.

[0038] The method according to the invention differs from the MINFLUX methods known from the prior art in that the intensity distribution of the illumination light is not positioned at successive, well-defined scanning positions in the sample or continuously moved along well-defined paths within the sample, but rather the intensity distribution fluctuates non-deterministically in the near field around a nominal position within the sample. This does not preclude the possibility of additionally shifting the intensity distribution relative to the sample in a controlled manner, in particular using beam positioning means or by moving the sample; however, controlled positioning or movement of the intensity distribution within the sample is not required during the implementation of the method according to the invention.The method according to the invention thus offers the advantage that a highly precise and highly stable positioning of the intensity distribution in the sample is not required and the method can also be applied if the beam path of the illumination light comprises means for influencing the illumination light, for example beam deflection means, which do not meet the highest requirements for dynamics, positioning accuracy and positioning stability (as required by conventional MINFLUX methods).

[0039] From the emissions detected during the measurement and assigned to the respective current position of the intensity distribution, the position of the emitter in the sample during the measurement period can be determined either directly or at a later time in a subsequent evaluation step. This can involve both determining the respective current positions of the intensity distribution of the illuminating light in the sample from the recorded measurement variable(s) and assigning the respective current positions of the intensity distribution of the illuminating light in the sample to the respective detected emissions in the subsequent evaluation step. To enable this, the recorded measurement variables and the detected emissions can, for example, be assigned to one another or each registered on a common time axis.If the position of the emitter in the sample is to be determined during the measurement period directly after the measurement, the current positions of the intensity distribution of the illuminating light in the sample are assigned to the detected emissions during the measurement period or directly after the measurement. If the current position of the intensity distribution in the sample is not directly measured, the current positions of the intensity distribution of the illuminating light in the sample are also determined from the recorded measurement value(s) during the measurement period or directly after the measurement. From a continuous measurement at a nominal position, several positions of the emitter belonging to different sub-time periods can also be determined.This also applies if the position of the emitter in the sample, or more precisely, the positions of the emitter, are determined temporally separate from the measurement in a subsequent evaluation step. The multiple determined positions can describe a trajectory of the emitter. If the position of the emitter in the sample can be assumed to be fixed, the multiple determined positions can be used to infer a measurement uncertainty regarding the determination of the position.

[0040] Consecutive measurement cycles can also be performed, with the emitter position being determined for each measurement cycle. After a measurement cycle has been completed, further measurement cycles can be performed on the same emitter to determine its position over time (tracking). Particularly if the emitter can diffuse within the sample or if the emitter moves in a directed manner within the sample, so that the position of the emitter's near area in the sample can or does change over time, it is advantageous to determine the emitter position between successive measurement cycles and to update the nominal position of the intensity distribution of the illumination light. The updated nominal position can correspond to the position determined for the previous measurement cycle.However, the updated nominal position can be derived from the positions determined in several previous measurement cycles, for example, if the emitter moves in a directed manner within the sample. For the subsequent measurement cycle or for each measurement cycle following a measurement cycle, the light intensity of the illumination light can optionally be changed, in particular increased, and / or a property of the fluctuation of the illumination light in the sample can be adjusted; these properties are discussed in more detail below. In this way, for example, an iterative MINFLUX method can be implemented.

[0041] In a preferred embodiment of the method, the nominal position of a first measurement cycle performed according to the invention is a presumed location of the emitter in the sample, which is determined in a pre-localization step. For this purpose, localization using an independent method, particularly with lower accuracy (e.g., raster scanning or PALM / STORM localization), can be used. However, a previous MINFLUX localization, for example, as known from the prior art, can also be used for the pre-localization.Thus, in a preferred embodiment, for example, an iterative MINFLUX measurement can be carried out in which the intensity distribution of the illumination light is positioned at successive, well-defined scanning positions in the sample or is continuously moved along well-defined paths in the sample in each step of the iteration, followed by a measurement or several measurement cycles in which the position of the intensity distribution in the sample fluctuates around a nominal position and in which the emissions are assigned the metrologically recorded, current position of the intensity distribution of the illumination light.

[0042] In another embodiment, particularly applicable in the context of (drug) screening applications, the nominal position may also be a known or predetermined binding site in the sample to which a structure labeled with the emitter is bound or can bind.

[0043] In a preferred embodiment of the method, the fluctuation of the position of the illumination light around a nominal position in the sample is effected by one or more movable components of a beam positioning device, for example, a beam scanner in a beam path of the illumination light. This movable component(s) can be, for example, one or more tilting mirrors of a galvo scanner system, which can also be used for other (image) acquisition modalities or is intended for them. The movable component can be a component of a light displacement unit or a movable sample stage, for example, a piezoelectrically actuated sample stage.

[0044] If the moving component is a tilting mirror or the moving components are tilting mirrors, the current position of the intensity distribution in the sample can be determined, in particular, from the deflection of the tilting mirror or from the amount of deflection of the tilting mirrors. The deflection of the tilting mirror or each tilting mirror can be measured, for example, if the tilting mirrors are part of a galvo scanner, with an integrated rotary encoder or using an auxiliary light beam, possibly one for each mirror, which is also deflected by the corresponding tilting mirror and directed onto a position-sensitive light detector.In particular, if an auxiliary light beam is used for each tilting mirror, an auxiliary light beam can also be directed onto the back of the mirror to measure the deflection. However, the auxiliary light beam is preferably directed onto the front of the mirror, i.e., the side of the mirror that reflects the illuminating light beam. The current position of the intensity distribution of the illuminating light in the sample can then be determined from the measured deflection of the tilting mirror or the measured deflections of the tilting mirrors. When determining the current position of the intensity distribution of the illuminating light in the sample, the imaging properties of the optical device with which the illuminating light beam is directed into the sample and focused must be taken into account; how this can be done is familiar to the person skilled in the art.If the beam scanner has, in addition to one or more tilting mirrors, other movable components that contribute to the fluctuation of the position of the intensity distribution in the sample, their current settings can also be measured and taken into account when determining the current position of the intensity distribution of the illumination light in the sample.

[0045] In a further preferred embodiment, the respective current position of the intensity distribution in the sample can be determined by guiding an auxiliary light beam over all components of the beam scanner that interact to cause the fluctuation of the position of the intensity distribution in the sample, such that the auxiliary light beam experiences a deflection identical to the deflection of the illuminating light beam, wherein the deflection of the auxiliary light beam is measured. The deflections can result in a displacement of the respective light beam at a location of an intermediate image relative to an optical axis of the beam path and an angular change at a location of an intermediate image relative to an optical axis of the beam path.Here, the displacement at a location of an intermediate image corresponds to a tilt at a location of a pupil, and a tilt at a location of an intermediate image corresponds to a displacement at a location of a pupil.

[0046] In a preferred embodiment, the deflection of an auxiliary light beam that is identical to the deflection of the illumination light beam can be achieved by coupling an auxiliary light beam into the beam path of the illumination light beam using a beam splitter of the relevant components of the beam scanner, on the one hand, and outcoupling it from these components using a further beam splitter. After outcoupling, the auxiliary light beam is directed to one or more position-sensitive detectors in such a way that the aforementioned displacement or tilt, or both the aforementioned displacement and tilt, can be determined from the measurement signals. The corresponding position of the intensity distribution of the illumination light in the sample is then determined from the displacement and / or tilt.Beam splitting is based on the fact that the auxiliary light beam and the illumination light beam have certain different optical properties; in particular, they can have different wavelengths or different polarizations. Which quantity or quantities—tilt, offset, or both tilt and offset—are preferably measured depends on the design of the beam scanner, in particular, whether it is constructed as a pupil scanner or, for example, as a quad scanner with four tilting mirrors positioned around an intermediate image position. Quad scanners are described, for example, in the international patent application WO 2010 / 069987 A1.In preferred embodiments in which the components of the beam scanner causing the fluctuation in the position of the illumination light form a quad scanner, the offset at a location of an intermediate image is measured, more preferably both the offset and the tilt are measured. Measuring both variables makes it possible to determine the position of the illumination light in the sample with high precision, taking into account the influence of a displacement of the illumination light beam in the pupil of the objective focusing the illumination light beam. This also applies if the relevant components of the beam scanner form or contain a pupil scanner. In these preferred embodiments, the respective current position of the intensity distribution of the illumination light in the sample is determined from the measured displacement and / or the measured tilt.In this case, the imaging properties of the optical device used to direct and focus the illuminating light beam into the sample must be taken into account; how this can be done is familiar to the person skilled in the art.

[0047] The fact that an auxiliary light beam is guided over all components of the beam scanner which, in cooperation, cause the fluctuation of the position of the intensity distribution in the sample, in such a way that the auxiliary light beam experiences a deflection identical to the deflection of the illuminating light beam, can also be achieved by coupling an auxiliary light beam from the relevant components of the beam scanner at a flat angle into the beam path of the illuminating light beam in such a way that it is deflected by all relevant components of the beam scanner in the same way as the illuminating light beam, and by coupling these components out at a flat angle.

[0048] In a further preferred embodiment, a portion of the light beam emanating from an illuminating light source can be extracted from the light beam emanating from the illuminating light source by means of a beam splitter after it has been guided over all components of the beam scanner that interact to cause the fluctuation in the position of the intensity distribution in the sample. The extracted portion of this light beam is then directed to one or more position-sensitive detectors in such a way that the displacement or tilt, or both the displacement and tilt, that the extracted portion of the light beam has experienced due to the respective components of the beam scanner can be determined from the measurement signals.

[0049] The fluctuation of the position of the illumination light around a nominal position in the sample can be caused by rapid, irregular, or seemingly irregular movements of the mechanical components of the beam positioning device that shift the beam, for example, tilting mirrors in a galvo scanner system. Galvo scanners and galvo scanner systems operated in a closed control loop, like piezo-actuated sample stages, also perform permanent corrective movements due to the continuous tracking by the control loop in order to achieve the desired position of the control loop; these corrective movements are also irregular or appear irregular. The rapid, irregular, or seemingly irregular movements, for example of the tilting mirrors, are referred to as jitter in this application.In the context of this application, the term "jitter" encompasses not only movements of the tilting mirrors about their tilting axis, but also all rapid movements of the tilting mirrors that lead to irregular, or seemingly irregular, deflections of the illuminating light and thus to a fluctuation in the position of the illuminating light in the sample. In the context of this application, the term "jitter" also encompasses corresponding movements of components of other mechanical scanners or corresponding movements of other components of a beam positioning device, such as a positionable sample stage or other light displacement units. The presence of jitter requires that there is an irregular, or seemingly irregular, movement of a component of the microscope, whereby the movement of the component affects the position of the intensity distribution of the illuminating light.Jitter can therefore also occur, for example, when a component that is essentially stationary or cannot be automatically and deterministically moved, such as a deflecting mirror or a lens, actually performs irregular or irregular-appearing movements. Irregular-appearing movements are understood to be movements that exhibit a regularity, but whose regularity is unknown or not recorded. Jitter can also occur, in particular, when a target position of the beam positioning device remains constant over a period of time. In this case, the jitter causes a fluctuation in the position of the intensity distribution of the illuminating light around a target position, i.e., it causes the position of the illuminating light to fluctuate around a nominal position in the sample.

[0050] Rapid, irregular, or seemingly irregular, movements of one or more moving components of a beam positioning device can also be caused by vibrations or sound, such as ambient noise. Jitter can also be caused, for example, by magnetic field fluctuations in the galvo drive of a galvo scanner or, in MEMS scanners, by electric field fluctuations.

[0051] The jitter inherent in the beam positioning device, caused by environmental influences or introduced by a control loop, may, depending on the properties of the imaging system with which the illumination light is imaged or focused into the sample, in particular on its magnification, be sufficient to ensure that an area around the emitter in the sample that is sufficiently large to carry out the method according to the invention is swept over by the illumination light, so that no additional fluctuation in the position of the illumination light in the sample needs to be introduced.

[0052] If the jitter inherent in the beam positioning device, caused by environmental influences and / or introduced by a control loop, which occurs with a basic setting of the control parameters, does not cause a sufficient fluctuation of the position of the illumination light in the sample, the fluctuation can be deliberately amplified.

[0053] For example, the strength of the corrective movements introduced by the control loop depends on the selected control parameters. In PID controllers, for example, large proportional and derivative components in particular lead to rapid control movements with high amplitudes. These increase particularly sharply when the control loop is operated near resonance frequencies. Therefore, in a preferred embodiment of the method, the control parameters are selected such that the position of the intensity distribution of the illumination light fluctuates such that, during the execution of a measurement according to the invention, current positions are reached that have a predetermined or predeterminable minimum distance from the nominal position. The control parameters can also be selected such that the fluctuation has predetermined or predeterminable moments, for example, a predetermined or predeterminable variance.The fluctuation around the nominal position can be approximately normally distributed. In general, the control-based jitter can be conditioned by a targeted adjustment of the control parameters.

[0054] Another way to specifically amplify fluctuations is to actively excite jitter, i.e., to induce excited jitter. For example, sound or ultrasound can be used to specifically influence the beam positioning device or individual components, or a single component of the beam positioning device. The influence can be mediated by the component's mounting.

[0055] In another preferred embodiment of the method, a control input of the beam positioning device is supplied with a random signal, causing a fluctuation in the position of the intensity distribution of the illumination light around the nominal position. The random signal is preferably conditioned to cause a desired fluctuation—that is, a fluctuation with predefined statistical properties—in the position of the intensity distribution of the illumination light around the nominal position. Conditioning the random signal can take into account jitter introduced otherwise, and the relevant control parameters can be both the control parameters of a basic setting of the beam positioning device and specifically predefined control parameters.

[0056] The random signal can be generated, in particular, using an analog or digital signal generator, which allows random signals with well-defined statistical properties, in particular with predetermined or predeterminable spatial distributions, to be generated. The spatial distribution preferably has no preferred direction, at least in the spatial directions considered for the localization of the emitters, thus avoiding the introduction of systematic errors during localization. In particular, the spatial distribution is preferably rotationally symmetrical with respect to an axis containing the nominal position. This axis can, in particular, be the optical axis of a microscope objective used to form the intensity distribution of the illumination light in the sample.

[0057] The spatial distribution can be normally distributed or at least approximately normally distributed around the nominal position. In this case, the intensity distribution covers, in particular, the immediate vicinity of the nominal position in the sample, while positions further away from the nominal position are reached less frequently. This can be advantageous with regard to the information content of detected photons with respect to the position of the emitter to be localized, because the information content of the detected photons is higher the closer the emitter is to the minimum of the intensity distribution of the illuminating light during its excitation, assuming no background photons influence the measurement.

[0058] The spatial distribution can also be a homogeneous distribution or a spatial distribution that has a probability amplitude that is inverse to the normal distribution. For example, the spatial distribution can be a top-hat distribution or a ring-shaped spatial distribution with a central minimum and a maximum value at a defined distance from the nominal position. In these embodiments, the nominal position is then no longer necessarily the location with the highest probability of being located. Such distributions can be particularly advantageous with regard to tracking an emitter.

[0059] A particularly preferred embodiment of the method is characterized in that a position of the emitter is estimated from the positions of the intensity distribution assigned to the emissions. This can be done, for example, in a manner known from the prior art using a position estimator, e.g., a least-mean-squares estimator or a maximum-likelihood estimator. For example, an estimator can correspond to a vector sum or be derived from a vector sum. Generally suitable estimators are known from the prior art, for example, from European patent application EP 3 951 470 A1.

[0060] In particular, in preferred embodiments in which an iterative MINFLUX method is implemented, in particular in which the nominal position is updated after the estimation of the position of the emitter, the fluctuation properties specified above can be adapted from measurement cycle to measurement cycle.For example, a first measurement cycle can be carried out using a homogeneous or annular spatial distribution of the fluctuation of the position of the illumination light around a nominal position in the sample, wherein the fluctuation is caused by the application of random signals to a control input or to control inputs of the beam positioning device, while in a subsequent or several subsequent measurement cycles the fluctuation is caused in each case by a control-based jitter or an excited jitter, for example wherein control parameters of the beam positioning device are adjusted between measurement cycles, and while in, for example, a last measurement cycle an inherent jitter of a mechanical beam scanner which forms the beam positioning device or which is contained in the beam positioning device causes the fluctuation of the position of the illumination light.

[0061] A second aspect of the invention relates to a MINFLUX microscope or a STED-MINFLUX microscope for locating or tracking an isolated emitter in a sample. The microscope has a microscope control device. The microscope control device can, in particular, be configured such that the locating or tracking can be carried out or is carried out according to one of the previously described methods. The MINFLUX microscope or the STED-MINFLUX microscope comprises a light source configured to provide an illumination light and illumination optics configured to expose the sample in a vicinity of the isolated emitter to an intensity distribution of the illumination light having a local intensity minimum.

[0062] The illumination light can be excitation light that excites an emission from the emitter. However, the illumination light can also be emission-suppressing light (in particular STED light), which reduces the emitter's emission depending on its intensity. In this case, the MINFLUX microscope comprises a further light source that provides excitation light that excites the emitter to emit and that is superimposed in the sample with the intensity distribution of the illumination light, thus comprising at least two light sources in total. The MINFLUX microscope can also have a primary light source, in particular a primary laser light source, and a device by means of which both excitation light and emission-suppressing light, in particular STED light, are generated from the primary light. This and similar cases are considered to be cases in which the MINFLUX microscope has at least two light sources.

[0063] The illumination optics comprise at least one microscope objective, with which the illumination light is focused into the sample, and a wavefront modulator, with which the wavefront of the illumination light is modulated so that, upon focusing, a central intensity minimum results with adjacent intensity increase regions on both sides in at least one direction. Wavefront modulators that can be used include, for example, phase plates with a fixed phase delay that varies across a beam cross-section or adjustable wavefront modulators, in particular programmable wavefront modulators (spatial light modulators, SLMs), such as liquid crystal-based, programmable amplitude modulators and, in particular, liquid crystal-based, programmable phase modulators. Corresponding wavefront modulators are known to the person skilled in the art from the prior art of STED microscopy or MINFLUX microscopy.

[0064] The microscope according to the invention further comprises a beam positioning device in the beam path of the illumination light, which is designed to shift the intensity distribution of the illumination light in the sample. The beam positioning device does not have to be dedicated to carrying out the method according to the invention; it can also be a device present in the microscope but intended for other purposes. In particular, the beam positioning device can be part of a beam scanner for image acquisition using raster scanning.

[0065] In a preferred embodiment, the beam positioning device comprises a beam scanner, more preferably in particular a galvo scanner. In a further preferred embodiment, the beam positioning device comprises a galvo scanner system with multiple galvo scanners. In further preferred embodiments, the galvo scanners of the galvo scanner system each have an integrated angular position measurement system, in particular an integrated encoder, for example a rotary encoder.

[0066] The microscope according to the invention comprises a measuring device for acquiring measurement data from which a current position of the intensity distribution of the illumination light in the sample can be determined. A current position of the intensity distribution can belong to a single measurement date or to a set of multiple measurement data. Preferably, the measuring device is configured to acquire measurement data from which the position of the intensity distribution of the illumination light in the sample can be determined with an uncertainty of at most 10 nm, preferably of at most 5 nm, more preferably of at most 2 nm, and particularly preferably of at most 1 nm.

[0067] In a preferred embodiment, the microscope according to the invention has a position determination unit which is configured to determine current positions of the intensity distribution of the illumination light from the measurement data or the sets of measurement data, more preferably to determine the current position of the intensity distribution of the illumination light in the sample with an uncertainty of at most 10 nm, preferably of at most 5 nm, more preferably of at most 2 nm and particularly preferably of at most 1 nm.

[0068] In further preferred embodiments, the integrated angular position measuring systems of the galvo scanners of the beam positioning device are elements of the measuring device for acquiring measurement data from which a current position of the intensity distribution of the illumination light in the sample can be determined. This means that the angular position measuring systems are not only used to control the respective galvo scanners, but the current angular position information is each output or transferred as a measured value in such a way that it serves as the basis for determining the respective current position of the illumination light in the sample. Preferably, the current angular position information is assigned to a time at which the angular position information was acquired. The assignment to a time can be achieved, for example, by writing the angular position information to registers, with adjacent entries having a fixed, short time interval.

[0069] In a particularly preferred embodiment, the measuring device for acquiring measurement data from which a current position of the intensity distribution of the illuminating light in the sample can be determined has a device for generating an auxiliary light beam which is coupled into a beam path of the illuminating light in such a way that the auxiliary light beam is guided over all components of the beam scanner which, in interaction, cause the fluctuation of the position of the intensity distribution in the sample, in such a way that the auxiliary light beam experiences a deflection identical to the deflection of the illuminating light beam, and a measuring device with which the deflection of the auxiliary light beam is measured.

[0070] The auxiliary light beam can be coupled into the beam path of the illumination light beam by, on the one hand, introducing the auxiliary light beam from the relevant components of the beam scanner at a shallow angle into the beam path of the illumination light beam in such a way that the auxiliary light beam is deflected by all relevant components of the beam scanner in the same way as the illumination light beam, and, on the other hand, the auxiliary light beam can be output from these components at a shallow angle. In this embodiment, the auxiliary light can simultaneously match the illumination light or the additional light in both wavelength and polarization. It can therefore be light from the illumination light source or, if the illumination light is emission-suppressing light, from the additional light source.

[0071] Further preferably, the measuring device for acquiring measurement data from which a current position of the intensity distribution of the illumination light in the sample can be determined has a separate auxiliary light source that generates an auxiliary light beam that is easily separable from the illumination light beam and, if the illumination light is emission suppression light, from excitation light, and more preferably also easily separable from the emission of the individual emitters, in particular an auxiliary light beam in a separate wavelength range. If such a separable auxiliary light beam is used, the microscope can, in preferred embodiments, have a beam splitter on the one hand of the relevant components of the beam scanner for coupling the auxiliary light beam into the beam path of the illumination light beam, and on the other hand, a further beam splitter on these components for coupling the auxiliary light beam out of the beam path of the illumination light beam.

[0072] In an alternative embodiment, the measuring device for acquiring measurement data from which a current position of the intensity distribution of the illumination light in the sample can be determined has a beam splitter in the beam path of the illumination light after all components of the beam scanner which, in interaction, cause the fluctuation of the position of the intensity distribution in the sample, with which beam splitter a portion of the light beam emanating from an illumination light source is coupled out of the beam path of the illumination light as an auxiliary light beam.

[0073] In these alternative embodiments, the measuring device for acquiring measurement data from which a current position of the intensity distribution of the illumination light in the sample can be determined further comprises a device for detecting the deflection of the auxiliary light beam. This device comprises, in particular, one or more position-sensitive light detectors, for example, one or more position-sensitive photodiodes (position-sensitive devices, PSDs) or one or more pixelated line or area detectors, e.g., a CCD camera. Preferably, the device comprises further optical elements with which the auxiliary light beam or a portion of the auxiliary light beam is focused onto the position-sensitive detector(s) or is collimated before impinging on the position-sensitive detector(s).Preferably, the measuring device for acquiring measurement data from which a current position of the intensity distribution of the illumination light in the sample can be determined has a beam splitter in the beam path of the auxiliary light beam after coupling it out of the illumination light beam. The beam splitter enables the detection of the deflection of the auxiliary light beam to include detecting a displacement of the auxiliary light beam in an intermediate image plane of the microscope and detecting a tilt of the auxiliary light beam in an intermediate image plane of the microscope with respect to an optical axis.

[0074] A measuring device for acquiring measurement data from which a current position of the intensity distribution of the illuminating light in the sample can be determined can comprise both a device for generating an auxiliary light beam which is coupled into a beam path of the illuminating light in such a way that the auxiliary light beam is guided over all components of the beam scanner which, in interaction, cause the fluctuation of the position of the intensity distribution in the sample, such that the auxiliary light beam experiences a deflection identical to the deflection of the illuminating light beam, and a measuring device with which the deflection of the auxiliary light beam is metrologically recorded, as well as further devices, for example those with which settings of individual elements of the beam scanner are measured, such as integrated angular position measuring systems, in particular those as described above.In general, the presence of one of the aforementioned measuring devices for acquiring measurement data does not preclude the additional presence of other measuring devices for acquiring measurement data in the microscope. If different types of measuring devices are present, they can form a common measuring device. This also applies if multiple measuring devices provide corresponding information, resulting in redundant information. This redundancy can then be used, for example, to reduce the uncertainty in determining the position of the intensity distribution of the illumination light in the sample or to detect malfunctions of the microscope when the measurement data are inconsistent.

[0075] The microscope according to the invention further comprises a detection device which is designed to detect emissions from the emitter.

[0076] The microscope control device is configured to record the respective current position of the intensity distribution of the illumination light in the sample and / or the respective measurement data for each detected emission. Recording measurement data for detected emissions means that the detected emissions can be assigned or are assigned to the respective current positions of the intensity distribution of the illumination light in the sample. Assigning the emissions to the current positions can be achieved, in particular, by, on the one hand, the detection device recording each detected emission in a time-resolved manner, and, on the other hand, the measuring device recording the measurement data from which a current position of the intensity distribution of the illumination light in the sample can be determined.The recording of measurement data for detected emissions can, in particular, mean that the relevant values ​​are stored and associated with each other directly or indirectly, for example, via a common time coordinate. Components of both the detection device and the measurement device can be integral parts of the microscope control system.

[0077] In a preferred embodiment, the microscope control device is designed such that the emissions detected by the detection device are assigned to the respective current positions of the intensity distribution of the illumination light in the sample, further preferably that the emissions detected by the detection device are continuously assigned to the respective current positions of the intensity distribution of the illumination light in the sample while measurements are being carried out.

[0078] Preferably, the detection device is designed to detect individual emission events, preferably to detect individual photons, and the microscope control device is designed to assign the detected individual emission events to a time of detection.

[0079] In a particularly preferred embodiment, the MINFLUX microscope comprises a signal generator for generating a random signal, as well as a control input for the beam positioning device, which is supplied with the random signal from the signal generator. Particularly preferred is the implementation of the signal generator as a digital signal generator that generates random signals with well-defined statistical properties, in particular with predetermined or predeterminable spatial distributions.

[0080] 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. 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.

[0081] 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.

[0082] 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 shows a positional fluctuation of the intensity distribution. Fig. Figure 2 shows a preferential position fluctuation of the intensity distribution. Fig. Figure 3 shows a MINFLUX microscope according to the invention. Fig. Figure 4 shows an alternative MINFLUX microscope according to the invention. Description of the characters

[0083] Fig. Figure 1 shows a fluctuation in the position of the intensity distribution of the illumination light 12 in the sample 14, as can be applied to the method according to the invention. The scatterplot shows the instantaneous positions, i.e., the respective current positions 1, of the intensity distribution, with each point representing an instantaneous position, i.e., a respective current position 1, of the intensity distribution of an equidistantly sampled time series. For the intensity distribution of the illumination light 12 having a central minimum, the current position 1 is, in particular, the current position 1 of the central minimum of the intensity distribution.The majority of the current positions within the inner region 5 lie around the nominal position 2 (which corresponds to the presumed position of the emitter 8 to be localized), whereby the dimension of the inner region 5 lies in accordance with the dimensions for scanning commonly used in MINFLUX microscopy, for example in the range between 30 nm and 300 nm.

[0084] The scatterplot shown corresponds to the radial spatial distribution function 6 shown below the scatterplot, according to which the current positions 1 are normally distributed and rotationally symmetrical around the nominal position 2 located at the coordinate origin 3, i.e., the radial spatial distribution function 6 corresponds to a normal distribution 7. Such a distribution can result to a good approximation, for example, from an inherent, in particular control-based, jitter of a galvo scanner system 32.

[0085] The size of the inner region 5 can be determined, for example, by a corresponding selection of control parameters of a galvo scanner system 32. The size of the inner region 5 and the control parameters can be determined such that, on average over a measurement period, a fixed proportion of all current positions 1, for example a proportion of 95%, is located within the inner region 5. In the example shown, all current positions 1 are located within the near field 4. However, this does not have to be the case in all embodiments. In particular, if the inner region 5 is selected to be large, current positions 1 can also be located outside the near field 4. The emissions registered at the outside current positions 1 can then be disregarded when locating the emitter 8 or evaluated separately, for example with regard to determining a background, and accordingly taken into account when locating the emitter 8.

[0086] Fig. Figure 2 shows an alternative preferred fluctuation of the position of the intensity distribution of the illumination light 12 in the sample 14. As the scatter plot of the current positions 1 of the intensity distribution shows, the density of the points in the center is low and increases with increasing radius up to a maximum value and decreases again with further increasing radius. Fig. 2, the emitter 8 is shown, the position of which in the example shown has a comparatively large distance 9 to the nominal position 2, which corresponds to the position of the emitter 8 assumed before carrying out the method according to the invention; the distance 9 is in Fig. 2 is marked by a connecting line from the nominal position 2, which corresponds to the coordinate origin 3, to the emitter 8. The fluctuation of the position of the intensity distribution of the illumination light 12 around a nominal position 2 in the sample 14 according to such a distribution offers the advantage that the set of current positions 1 used to scan the emitter 8 most certainly includes a sufficient number of positions in each spatial direction of the scan on both sides of the emitter 8 for localization. Such a distribution of the current positions 1 is particularly advantageous when the presumed position of the emitter 8 has a high degree of uncertainty.

[0087] Fig. Figure 3 shows a MINFLUX microscope 40 according to the invention, which is based on a conventional confocal laser scanning microscope. The MINFLUX microscope 40 comprises a light source 11, typically embodied as a laser 10, which emits illumination light 12, which in this case is excitation light 13 for individual emitters 8 contained in the sample 14. The illumination light beam 15 is directed by a beam splitter 16 into a light displacement unit 30, which here is embodied as a beam scanner 31, specifically as a galvoscanner system 32, which enables deflection of the illumination light beam 15 in two spatial directions. For this purpose, the galvo scanner system 32 comprises two tilting mirrors 19, each mounted on a galvo motor 18, whose axes of rotation are perpendicular to each other, i.e. two galvo scanners that deflect the illumination light beam 15 in spatial directions oriented perpendicular to each other.The galvomotors 18 for scanning the sample 14 with the illumination light 12 are controlled via a scan controller integrated into the microscope control unit 29. The galvomotors 18 of the galvoscanners assigned to the tilting mirrors 19 comprise integrated angular position measuring systems 20, specifically encoders 120 here, which can be implemented as rotary encoders 220, with which the angular position of the galvomotors 18 and thus of the tilting mirror 19 is determined. In the example shown, the tilting mirrors 19 are each positioned near a pupil, i.e., near a plane conjugated to the rear pupil of the objective 24. In an alternative arrangement not shown in the figure, a relay optics system can be located between the two scanners, in which case both the tilting mirror 19a and the tilting mirror 19b are preferably each positioned in a pupil.

[0088] The illumination light 12 emerging from the galvoscanner system 32 is imaged into the rear pupil of the objective 24 via an imaging system comprising a scanning lens 22 and a tube lens 23, which focuses the illumination light 12 onto the sample 14. A change in the angle of the tilting mirrors 19 translates into a change in the angle of the illumination light beam 15 in the pupil of the objective 24 and thus into a displacement of the focused illumination light 12 within the sample 14.

[0089] The light 33 emitted by the emitters 8 in the sample 14 is collected by the objective lens 24 and travels in the direction opposite to the illumination light beam 15 to the beam splitter 16, which transmits the emitted light 33 of the emitter 8 and thus separates it from the illumination light 12. The beam splitter 16 can, in particular, be designed as a (dichroic) color splitter. The detection system arranged behind the beam splitter 16 comprises a filter 25 that blocks unwanted stray light components and a lens 26 with which the light 33 emitted by the emitters 8 is focused onto the detector 28 through a (confocal) pinhole 27.

[0090] The MINFLUX microscope 40 differs from a conventional confocal microscope in that a wavefront modulator 21, in the concrete example shown a phase plate 121, is additionally arranged in the illuminating light beam 15, which modulates the wavefront of the illuminating light beam 15 spatially in such a way that the illuminating light 12, when focused with the objective 24, forms an intensity distribution in the sample 14 with a central minimum surrounded by rising regions.

[0091] The illustrated MINFLUX microscope 40 further differs from a conventional confocal microscope in that the microscope comprises a microscope control device 29, which assigns the current angular position of the tilting mirrors 19 to each emission registered by the detector 28. For this purpose, the microscope control device 29 receives, on the one hand, the detector signal 42 of the detector 28 and, on the other hand, the position signals 43 of the galvo motors 18 output by the encoders 20. Thus, in this embodiment, the encoders 20, in conjunction with the microscope control device 29, form a measuring device for acquiring measurement data, from which a current position 1 of the intensity distribution of the illumination light 12 in the sample 14 can be determined.

[0092] To carry out the method according to the invention with the MINFLUX microscope 40 shown, it is exploited that the galvo scanners of the galvo scanner system 32, and thus the galvo scanner system 32 as a whole, have an inherent, for example control-based, jitter, ie they carry out small, random rotational movements which translate into a fluctuation in the position of the intensity distribution of the illumination light 12 in the sample 14, so that the intensity distribution of the illumination light 12 in a close range 4 around the nominal position 2, in particular an inner region 5 around the nominal position 2 of the intensity distribution in the sample 14.

[0093] In Fig. 4 shows a further embodiment of a microscope according to the invention. The microscope is designed as a STED-MINFLUX microscope 41. The STED-MINFLUX microscope 41 comprises a first light source 11a, embodied as a laser 10, for illumination light 12, which here is emission suppression light 44, specifically STED light 45. The microscope further comprises a second light source 11b, also embodied as a laser 10, for excitation light 13. A wavefront modulator 21, embodied as a phase plate 121, is arranged in the beam path of the emission suppression light 44. This wavefront modulator phase-modulates the wavefront of the emission suppression light 44, so that when the emission suppression light 44 is focused by the objective 24, an intensity distribution with a central minimum surrounded by rising regions results.

[0094] The excitation light 13 and the emission suppression light 44 are combined by a beam combiner 47 and directed by a beam splitter 16 onto a galvo scanner system 31. The galvo scanner system 31 in turn comprises two tilting mirrors 19 driven by galvo motors 18. The galvo motors 18 are controlled by a scan controller 48, which generates control signals 49 for the galvo motors 18. The scan controller 48 has a control input 50, which receives the output signal of a signal generator 51. The signal generator 51 is configured to generate a (small) random signal to introduce a fluctuation of the tilting mirrors 19 such that the position of the intensity distribution fluctuates around the nominal position 2; This can be achieved by the random signal being such that it causes a fluctuation of the tilting mirrors 19 around a nominal position associated with the nominal position 2.The random signal preferably has well-defined statistical properties, in particular a predetermined or predeterminable spatial distribution.

[0095] The light beam incident on the first tilting mirror 19a is deflected by the latter onto the second tilting mirror 19b. The second tilting mirror 19b, whose axis is perpendicular to the axis of the first tilting mirror 19a and perpendicular to the direction of the light beam incident on it when the first tilting mirror 19a is in the zero position, is shown perpendicular to the plane of the drawing and offset to the right relative to the first tilting mirror 19a for simplification purposes only, deflects the incident light beam towards a scanning lens 22. The light beam emerging from the galvoscanner system 31 is directed via the scanning lens 22 and a tube lens 23 into the rear pupil of the objective 24 and focused by the objective 24 into the sample 14. As in the Fig.In the embodiment of the MINFLUX microscope 40 shown in Figure 3, the light 33 emitted by the emitters 8 in the sample 14 is collected by the objective lens 24 and travels in a direction opposite to the direction of the excitation and emission suppression light beams to the beam splitter 16, where it is separated from the excitation light 13 and the emission suppression light 44. The detection system arranged behind the beam splitter 16 comprises a filter 25 that blocks unwanted stray light components and a lens 26 with which the light 33 emitted by the emitters 8 is focused onto the detector 28 through a (confocal) pinhole 27.

[0096] In the embodiment shown, the galvo motors 18 do not include integrated angular position measuring systems 20 or do not provide a position signal 43 externally. In order to detect the current angular position of the tilting mirrors 19, a separate angular position measuring system is provided in the embodiment shown, of which, for reasons of clarity, only the components for measuring the position of the tilting mirror 19b are shown. The angle measurement is based on the deflection of an auxiliary light beam 52 by the respective tilting mirror 19 to be monitored and a position measurement of the auxiliary light beam with an auxiliary light detector 53, in particular with a position-sensitive detector 54. For this purpose, the auxiliary light beam 52 emitted by an auxiliary light source 55 is directed onto the back of the respective tilting mirror 19, the deflection of which is to be monitored, and is reflected by the latter onto the corresponding auxiliary light detector 53.Each auxiliary light detector 53 is connected via a first signal line 57a to a first signal input 56a of a microscope control device 29. The microscope control device 29 receives the respective position signals 43 of the auxiliary light detectors. The microscope control device 29 has a second signal input 56b. A second signal line 57b, which connects the detector 28 to the microscope control device 29, is connected to this second signal input 56b. The microscope control device 29 receives the respective detector signals 42 via the second signal line 57b. It is designed to assign the respective current position signals 43 of the auxiliary light detector 53 or the auxiliary light detectors 53 to each of the emission events registered by the detector 28.It is also possible to assign a plurality of position signals 43 to all or some emission events per auxiliary light detector 53, i.e., per tilting mirror 19. This allows, in addition to the current position 1 of the intensity distribution in the sample 14, the respective detected emissions to also be assigned an uncertainty with which the current position 1 in the sample was determined. List of reference symbols 1 current position 2 Nominal position 3 Origin of coordinates 4 Close range 5 Interior 6 radial spatial distribution function 7 Normal distribution 8 emitters 9 Distance 10 lasers 11 Light source 11a first light source 11b second light source 12 Illumination light 13 Excitation light 14 Sample 15 Illumination light beam 16 beam splitters 17 Beam positioning device 18 Galvo motor 19 tilting mirrors 19a first tilting mirror 19b second tilting mirror 20 integrated angular position measuring system 21 Wavefront modulator 22 Scan lens 23 Tube lens 24 lens 25 filters 26 lens 27 pinhole 28 detector 29 Microscope control device 30 light shifting unit 31 beam scanners 32 Galvoscanner system 33 emitted light 40 MINFLUX microscope 41 STED-MINFLUX microscope 42 detector signal 43 Position signal 44 Emission suppression light 45 STED light 46 deflecting mirrors 47 beam combiners 48 scan controllers 49 Control signal 50 Control input 51 Signal generator 52 Auxiliary light beam 53 Auxiliary light detector 54 position-sensitive detector 55 Auxiliary light source 56a first signal input 56b second signal input 57a first signal line 57b second signal line 120 encoders 121 Phase plate 220 rotary encoders

Claims

[1] Method for locating or tracking an isolated emitter (8) by means of a MINFLUX method or a STED-MINFLUX method in a sample (14), - wherein the sample (14) is exposed to an intensity distribution of an illuminating light (12) having a local intensity minimum in a close range (4) of the isolated emitter (8), wherein the illuminating light (12) is excitation light (13) or emission suppression light, in particular STED light, and wherein the illuminating light (12) or another light excites the emitter (8) to emit light, - wherein a metrological recording of one or more measured variables takes place, from which the respective current position (1) of the intensity distribution of the illuminating light (12) in the sample (14) can be determined, - wherein emissions from the emitter (8) are detected, characterized bythat the position of the intensity distribution in the sample (14) fluctuates around a nominal position (2) and that the metrological detection is carried out in such a way that the respective current position (1) of the intensity distribution in the sample (14) can be assigned to the detected emissions. [2] Method according to claim 1, characterized by that the nominal position (2) is a presumed position of the emitter (8) in the sample (14) determined in a previous localization step. [3] Method according to claim 1, characterized by that the nominal position (2) is a known or predetermined binding site in the sample (14) to which a structure labelled with the emitter (8) is bound or can bind. [4] Method according to one of claims 1 to 3, characterized bythat the fluctuation of the position of the intensity distribution in the sample (14) is caused by at least one movable component of a beam positioning device (17) in a beam path of the illumination light (12). [5] Method according to claim 4, characterized by that the fluctuation of the position of the intensity distribution is caused by an inherent jitter of the beam positioning device (17). [6] Method according to claim 4 or 5, characterized by that the fluctuation of the position of the intensity distribution is caused by a control-based jitter of the beam positioning device (17). [7] Method according to claim 6, characterized bythat the control-based jitter is conditioned by a targeted setting of the control parameters, in particular such that the fluctuation has a predetermined variance and / or other predetermined moments and / or that the fluctuation occurs such that current positions (1) are reached which have a predetermined or predeterminable minimum distance from the nominal position (2). [8] Method according to one of the preceding claims, characterized by that the fluctuation of the position around the nominal position (2) is normally distributed. [9] Method according to one of the preceding claims, characterized by that a control input of the beam positioning device (17) is supplied with a random signal. [10] Method according to claim 9, characterized by that the random signal has a predetermined or predeterminable spatial distribution. [11] Method according to claim 10, characterized bythat the spatial distribution is rotationally symmetrical with respect to an axis containing the nominal position (2), wherein the axis is preferably parallel to an optical axis of an objective (24) of a MINLFUX microscope (40) or a STED-MINFLUX microscope (41). [12] Method according to claim 10 or 11, characterized by that the spatial distribution has a local minimum at the nominal position (2). [13] Method according to one of the preceding claims, characterized by that the respective current positions of the intensity distribution of the illumination light (12) in the sample (14) are determined from the measured quantities and assigned to the detected emissions. [14] Method according to claim 13, characterized by that a position of the emitter (8) is estimated from the detected emissions and the associated current positions of the intensity distribution. [15] Method according to claim 14, characterized bythat the nominal position (2) is updated after the estimation of the position of the emitter (8), in particular that the nominal position (2) is shifted to the estimated position of the emitter (8). [16] MINFLUX microscope (40) or STED-MINFLUX microscope (41) for locating or tracking an isolated emitter (8) in a sample (14), in particular according to a method according to one of the preceding claims, comprising - a light source designed to provide an illumination light (12), - an illumination optics designed to expose the sample (14) in a close range (4) of the isolated emitter (8) to an intensity distribution of the illumination light (12) having a local intensity minimum, wherein the illumination light (12) is excitation light (13) or emission suppression light, in particular STED light, and wherein the illumination light (12) or another light excites the emitter (8) to emit light, - a microscope control device designed to control the MINFLUX microscope (40) or STED-MINFLUX microscope (41), - a beam positioning device (17) designed to shift the intensity distribution of the illuminating light (12) in the sample (14), - a detection device designed to detect emissions from the emitter (8), characterized byin that the MINFLUX microscope (40) or STED-MINFLUX microscope (41) has a measuring device for recording measurement data, from which a current position (1) of the intensity distribution of the illumination light (12) in the sample (14) can be determined, wherein the microscope control device is designed to register the respective current position (1) of the intensity distribution of the illumination light (12) in the sample (14) and / or the respective measurement data recorded by the measuring device for a respective detected emission. [17] MINFLUX microscope (40) or STED-MINFLUX microscope (41) according to claim 16, characterized bythat the measuring device is suitable such that the current position (1) of the intensity distribution of the illumination light (12) in the sample (14) can be determined from the recorded measurement data with an uncertainty of at most 10 nm, preferably of at most 5 nm, more preferably of at most 2 nm and particularly preferably of at most 1 nm. [18] MINFLUX microscope (40) or STED-MINFLUX microscope (41) according to claim 16 or 17, characterized by that the measuring device is arranged to detect an angle or a deflection of a movable optical component of the beam positioning device (17). [19] MINFLUX microscope (40) or STED-MINFLUX microscope (41) according to claim 18, characterized by that the measuring device comprises an integrated angular position measuring system, in particular an integrated encoder, further in particular an integrated rotary encoder (20), an auxiliary light beam and / or a position-sensitive auxiliary light detector. [20] MINFLUX microscope (40) or STED-MINFLUX microscope (41) according to one of claims 16 to 19, characterized by that the MINFLUX microscope (40) or the STED-MINFLUX microscope (41) comprises a signal generator for generating a random signal and that a control input of the beam positioning device (17) can be supplied with the random signal.

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