Upgrade kit, Minflux microscope, methods and computer programs for locating or tracking emitters in a sample

Abstract

The invention relates to an upgrade kit (10) for upgrading a light microscope (1) having a detector (3) to a MINFLUX microscope (100), wherein components of the upgrade kit (10) are designed to couple an illuminating light beam (B) into an illuminating beam path (4) of the light microscope (1), wherein the upgrade kit (10) has a first beam scanning device (11) and / or a beam shaping device (12) and / or a stabilization system (13). The invention further relates to a MINFLUX microscope (100), a method for upgrading a light microscope (1), a method for controlling a MINFLUX microscope (100), a method for locating or tracking emitters (E) in a sample (2), and computer programs for carrying out the methods.

Description

Technical field of the invention

[0001] The invention relates to an upgrade kit for upgrading a light microscope to a MINFLUX microscope for locating or tracking emitters in a sample using a MINFLUX method, as well as to the MINFLUX microscope upgraded with the upgrade kit. Further subjects of the invention are a method for upgrading a light microscope to a MINFLUX microscope, a method for controlling a MINFLUX microscope, a method for locating or tracking emitters using the MINFLUX principle (MINFLUX method), as well as a computer program for controlling the MINFLUX microscope and a computer program for locating or tracking emitters in a sample (for implementing the MINFLUX method). State of the art

[0002] In contrast to classical imaging techniques of light microscopy, light microscopy localization methods calculate the positions of individual emitters (e.g., fluorophores or molecules labeled with fluorophores) based on detected light emissions. A localization map can be created from the positions of a large number of emitters, usually determined one after the other, which visualizes the distribution of the emitters in the sample. Such localization maps can have a resolution significantly better than the light microscopic diffraction limit, e.g., in the range of 1 nm to 5 nm.

[0003] The term "individually" here means that at a given point in time, light-emitting emitters whose emission light is indistinguishable are at a distance above the diffraction limit, so that their emission light can be separated. This can be achieved, for example, in the case of permanently light-emitting emitters by a labeling density of the sample below a limit value. If the emitters flash asynchronously, for example, a sufficient distance can be achieved at any given point in time even with a higher labeling density. For this purpose, the sample environment (e.g. buffer, embedding medium) can be chemically configured to result in a desired flashing rate. Smaller distances are also tolerable when different types of emitters are present whose emission light can be optically separated, e.g. due to different emission spectra or emission lifetimes. Finally, in exceptional cases, special evaluation methods (e.g.It may also be possible (e.g., using statistical or time-resolved methods) to jointly localize groups of several closely adjacent emitters. A specific position can be determined for each emitter, or an average position of several emitters can be determined.

[0004] Individual emitters moving within the sample can be tracked by several rapid, consecutive localizations within the sample (tracking). The corresponding measurement data can be displayed, for example, in the form of a trajectory.

[0005] In the so-called MINFLUX technique, a sample containing a single emitter (e.g., a single fluorophore, a fluorophore-labeled molecule, or a light-scattering particle) is illuminated with an intensity distribution of excitation light at illumination positions in a range around a roughly estimated position of a single emitter. The intensity distribution has a central intensity minimum (ideally, an intensity zero). For each illumination position, the light emissions (in particular, single photons) of the individual emitter are recorded. A new position estimate of the emitter is then calculated from the light emissions and the corresponding illumination positions. The intensity distribution can, for example, be a so-called 2D donut, a so-called bottle beam, or a superposition of these light distributions, which are known from the field of STED microscopy.

[0006] Various variants of the MINFLUX technique are described, for example, in the publications F. Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612 and KC Gwosch et al. (2020) MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224, as well as in the patent documents US 9,719,928 B1, US 10,900,901 B2, US 10,908,089 B2 and US 10,962,479 B2.

[0007] Due to the excitation light distribution with an intensity minimum, the MINFLUX technique allows for localization precision in the single-digit nanometer range with a significantly lower number of emitted photons compared to the so-called PALM / STORM technique. This can be explained by the fact that the emitter is illuminated with less excitation light the closer the minimum of the excitation light distribution is to the actual emitter position.

[0008] Patent publication US 11,255,791 B2 describes, among other things, a variant of the MINFLUX technique in which the sample is illuminated with a combination of an excitation light distribution with a local maximum and a STED (stimulated emission depletion) light distribution with a local minimum. The position of an individual emitter is also calculated from the light emissions recorded for different illumination positions, but the closer the minimum of the STED light distribution (and the maximum of the excitation light distribution superimposed with the minimum) is to the actual emitter position, the more light is emitted. Therefore, the advantage of particularly high photon efficiency is not achieved here.

[0009] Patent application US 2023 / 0236401 A1 describes a method for localizing an emitter by switching between different orientations of an excitation light distribution with a local minimum. This light distribution can be generated, for example, using the principle of conical diffraction, which is described, for example, in the publication "Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy" by C. Fallet, J. Caron, S. Oddos, JY. Tinevez, L. Moisan, GY Sirat, PO Braitbart, and SL Shorte (2014, Proc. of SPIE Vol. 9169, 916905-1 - 916905-6).

[0010] A special localization technique using an excitation light distribution with a local maximum and a STED light distribution with a local minimum is described in the publication M. Weber et al. (2021) MINSTED fluorescence localization and nanoscopy, Nat. Photonics 15, 361-366 and the patent application WO 2023 / 006176 A1 and is known as MINSTED. The combination of the excitation light distribution and the STED light distribution is moved along a continuous trajectory around an estimated emitter position. For each detected photon, the corresponding position of the minimum of the STED light distribution is determined, and the trajectory is adjusted based on the detected photon. The excitation and STED intensities can also be increased.

[0011] The publication R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478, describes an implementation of the MINFLUX concept by connecting optical components to a commercial fluorescence microscope. The illumination light beam is coupled into the microscope stand via an external quad scanner. Additionally, an infrared laser beam from a stabilization system is connected to the stand via a camera port. This allows the user to combine conventional fluorescence microscopy techniques with MINFLUX localization microscopy and tracking with a spatial resolution of a few nanometers and a temporal resolution of a few microseconds on the same microscope platform. In this way, biological sample structures, in particular, can be observed across multiple spatial and temporal scales.However, the additional components connected to the microscope stand are relatively complex, expensive, and require a large amount of space. In addition to electro-optical deflectors and an external quad scanner, the additional components also include a detection beam path including an avalanche photodiode. Object of the invention

[0012] The disadvantages of the state of the art described above give rise to the task of upgrading a conventional light microscope to a MINFLUX microscope in the simplest, most cost-effective and space-saving way possible. Solution

[0013] This problem is solved by the subject matter of the independent claims. Advantageous further developments are specified in the subclaims and are described below. Description of the invention

[0014] A first aspect of the invention relates to an upgrade kit for upgrading a light microscope having a detector for detecting light emissions from emitters in a sample to a MINFLUX microscope. Components of the upgrade kit are configured to couple an illuminating light beam into an illuminating beam path of the light microscope, wherein the illuminating light beam is configured to excite the emitters in the sample to emit light or to modulate the light emission of the emitters caused by the excitation light.

[0015] The term "upgrade kit" describes a set of components that can be attached to or installed in an existing light microscope. The components can be installed or installed in a connected manner (e.g., mechanically coupled to a support structure and / or enclosed in a housing), but multiple components can also be installed or installed separately and, if necessary, at different positions on the light microscope. The upgrade kit can be delivered as such or together with the light microscope. In the latter case, the upgrade process may already be partially or fully completed upon delivery; the MINFLUX microscope resulting from the upgrade process may therefore be partially or fully pre-assembled.

[0016] In the context of this specification, a MINFLUX microscope is understood to mean a microscope system designed to perform a MINFLUX method.

[0017] A MINFLUX method in the context of this specification is a method for locating or tracking emitters in a sample, in which the sample is illuminated with an intensity distribution of an illuminating light having a local minimum, wherein the illuminating light excites emitters in the sample to emit photons or modulates the emission of photons by emitters in the sample, and wherein a position of the emitter is estimated based on detected light emissions from an emitter. The illuminating light can be excitation light that excites the emitters in the sample to luminescence, in particular fluorescence, or is reflected and / or scattered by the emitters. Alternatively (if the illuminating light modulates the emission of photons by the emitters), the illuminating light can also be, for example, STED light. In this case, the sample is illuminated in particular with additional excitation light, e.g. with a “regular” (i.e.Gaussian-shaped) focus, whose intensity maximum overlaps in particular with the minimum of the STED distribution.

[0018] 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. An emitter can therefore be, for example, a light-reflecting nanoparticle, a quantum dot, a fluorescent dye molecule (fluorophore), or a molecule or nanoparticle labeled with one or more fluorescent dye molecules.Depending on the size of the molecule and the distance between the fluorophores, a molecule labeled with multiple fluorophores or a nanoparticle labeled with multiple fluorophores can of course also have multiple emitters in the sense of the definition used here.

[0019] In the context of this specification, "individual" emitters are understood to be emitters that can be optically separated from one another using light microscopy techniques. This can be achieved by a labeling density of the sample that results in an average distance between the emitters above the diffraction limit. Alternatively, asynchronously flashing emitters can be used if the sample conditions (in particular the composition of the sample buffer and embedding medium) are adjusted so that the average distances between the light-emitting emitters are above the diffraction limit at all times. Finally, different emitters that are otherwise optically distinguishable can, under certain circumstances, also be separated if they are separated below the diffraction limit, e.g., based on their characteristic emission spectrum or their emission lifetime.Optionally, a spatially limited area of the sample can be illuminated with activation light in order to convert emitters in this area from an inactive state in which the emitters do not emit light when irradiated with excitation light, to an active state in which the emitters emit light when irradiated with excitation light.

[0020] In a localization method, a high-resolution image of structures in the sample can be determined by successively determining the positions of a plurality of emitters in the sample, in particular with a spatial resolution, ie a localization precision, of 1 nm to 10 nm, more particularly 1 nm to 5 nm.

[0021] A tracking method is characterized by the fact that light emissions from an emitter moving in the sample are recorded several times in succession in order to estimate the respective positions of the emitter. From the positions of the emitter, a trajectory can then be determined which describes the movement of the emitter in the sample.

[0022] In a MINFLUX method according to the invention, the local minimum of the intensity distribution of the illumination light can be placed at illumination positions in a near range of the approximate position of an individual emitter, determined in particular in a search step, and photons emitted by the emitter can be detected for each illumination position. The near range can, in particular, have an extent on the order of magnitude of the optical diffraction limit. The detected photon numbers (or photon rates) and the associated illumination positions can form input values of a position estimator (e.g., a least-mean-square estimator or a maximum-likelihood estimator), which is then used to determine an updated position estimate for the emitter.This process can be repeated iteratively by placing the intensity distribution of the illumination light at updated illumination positions in a range close to the updated position estimate and again detecting photons for each position. In particular, a radius of an illumination pattern formed by the illumination positions around the previously estimated position can be reduced. Optionally, the light intensity of the illumination light can also be increased. The iteration steps can be continued, for example, until the emitter stops emitting light or until a photon limit or a threshold of the localization precision is reached. In a MINFLUX tracking method, one or more iterations can be repeated at short time intervals to track the trajectory of a moving emitter.In particular, lighting patterns specifically tailored to a tracking method can be used.

[0023] In the MINFLUX method, the intensity distribution of the illumination light can be shifted in an area around a pre-estimated position of a single emitter using a beam scanner (e.g., electro-optical deflectors or galvo scanners). Embodiments known from the prior art include, for example, the step-by-step shifting of the intensity distribution to three to six positions located on a circle around the previously estimated position, using electro-optical deflectors (see, for example, Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612, KC Gwosch et al. (2020) MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 and R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun.12 (1), 1478) or a continuous circular movement of the intensity distribution around the previously estimated position (see, for example, US 2023 / 0008453 A1). Value pairs consisting of position vectors of the illumination positions and corresponding photon numbers measured at the illumination positions can then be inserted into the position estimator. When traversing a continuous path, the position vectors of positions on the path at which the local minimum of the intensity distribution was located during a specific detection event can be used instead of the position vectors of the previously known illumination positions. This position can be derived, for example, from control data of the beam scanner or from a measurement using a position sensor. Alternatively, a temporal modulation of the light emission signal can be analyzed while traversing the continuous path, as described, for example, in US 2023 / 0008453 A1, in order to estimate the position of the emitter.

[0024] According to a further possible embodiment of the MINFLUX method, the position of an emitter can be estimated from light emissions that are assigned to different shapes and / or orientations of the intensity distribution of the illumination light. When illuminating the sample with the illumination light, instead of shifting the intensity distribution (see above) or in addition to this, the shape and / or orientation of the intensity distribution can be adjusted so that an emitter to be located or tracked in the sample is exposed to different intensities of the illumination light depending on its actual position in the sample (which is not known in advance or is only known inaccurately). In concrete terms, this can be done, for example:an intensity distribution with two opposite intensity maxima separated by a planar intensity minimum intersecting the geometric focus (in particular a zero surface of the intensity), which can be rotated around the geometric focus, in particular without shifting the geometric focus relative to the sample, in order to expose the emitter to different illumination intensities depending on its position. Such intensity distributions can be achieved, for example, by phase modulation of the illumination light beam with a phase pattern that exhibits a linear phase jump. Switching the intensity distribution to change the shape and / or orientation can be achieved, for example, with a controllable light modulator (SLM) or electro-optical elements. The light emissions of the emitter are recorded for each shape and / or orientation of the intensity distribution. On this basis, the position of the emitter in the sample is estimated.A corresponding procedure is described, for example, in US 2023 / 0236401 A1.

[0025] According to the invention, the upgrade kit comprises the following: - a first beam scanning device which is designed to displace the illuminating light beam so that a focus of the illuminating light beam in the sample can be displaced by means of the first beam scanning device, and / or - a beam-shaping device designed to shape the illuminating light beam such that an intensity distribution of the illuminating light with a local minimum is formed or adjusted at a focus of the illuminating light beam in the sample, and / or - a stabilization system configured to detect a disturbance acting on the MINFLUX microscope and / or the sample and to compensate for the detected disturbance or to correct an estimated position of an emitter based on the detected disturbance or to interrupt or abort localization or tracking of an emitter based on the detected disturbance.

[0026] In contrast to the prior art, the invention proposes an upgrade kit that does not include a detector for detecting light emissions from individual emitters in the sample for localizing or tracking the emitters (using a MINFLUX method). Instead, the existing detector of the light microscope to be upgraded is advantageously used. This reduces the complexity and cost of the upgrade kit as well as the complexity of the upgrade process, and the additional space required can be minimized. The existing detector can be used particularly advantageously for detecting light emissions in the MINFLUX method if it is a single-photon detector such as an avalanche photodiode (APD) or a hybrid photodetector. Such detectors are used, for example, in some confocal laser scanning microscopes.

[0027] Because the detector of the light microscope to be upgraded is used, only the illumination light beam needs to be coupled into the light microscope using the components of the upgrade kit. The illumination light beam can, in particular, comprise the excitation light for the individual emitters in the sample, i.e., induce light emissions from the emitters. The excitation light can, for example, excite the emitters to luminescence, in particular fluorescence, or the excitation light can be scattered or reflected by the emitters. Alternatively, the illumination light can also be, for example, inhibition light (e.g., STED light or switching light), which modulates the light emissions from the emitters. In this case, an additional excitation light beam can be provided (by the components of the upgrade kit or by the light microscope). Optionally, a light beam for the stabilization system, for example, can also be coupled into the light microscope.

[0028] The upgrade kit may in particular comprise a first beam scanning device for the illumination light beam, a beam shaping device for the illumination light beam, a stabilization system, combinations of two of these components, or all three components.

[0029] The beam scanning device can be used in the MINFLUX method to shift the focus of the illuminating light beam in the sample, whereby light emissions from an emitter can then be detected at the various positions of the focus using the detector of the light microscope. In particular, the first beam scanning device is a fast scanning device, e.g. comprising at least one acousto-optical or electro-optical deflector. The first beam scanning device can be used in addition to a second beam scanning device of the light microscope for the MINFLUX method. In this case, for example, the second beam scanning device (e.g. comprising at least one galvo scanner) can be used for the slower coarse positioning of the focus, while the first beam scanning device is used for fast positioning during a MINFLUX measurement sequence. Under certain circumstances, an upgrade kit without the first beam scanning device can also be useful. This can be the case, for example.This could be the case, for example, if the focus of the illumination light is shifted in the sample during a MINFLUX measurement sequence by the existing second beam scanning device of the light microscope, e.g., using galvo scanners on a continuous path. Furthermore, the first beam scanning device can be dispensed with, in particular, if the focus of the illumination light is not shifted during the MINFLUX measurement sequence, but merely the shape and / or arrangement of the intensity distribution of the illumination light at the focus is adjusted.

[0030] The beam-shaping device can be configured to shape the illuminating light beam (e.g., by phase and / or amplitude modulation) such that an intensity distribution of the illuminating light with a local minimum, in particular an intensity zero, is created at the focus formed by the objective of the light microscope. Regions of increasing intensity can border the local minimum in at least one spatial direction. The intensity distribution can be arranged symmetrically around a central intensity minimum located at the geometric focus, such as in a so-called donut or bottle beam.

[0031] The beam shaping device can be, for example, a phase plate with a fixed phase pattern or a controllable light modulator with an adjustable phase pattern.

[0032] The beam-shaping device can be omitted, especially if the light microscope already has one. This can be the case, for example, with STED microscopes with a phase plate or light modulator for the STED light, if the illumination light can be coupled into the light microscope in such a way that it is modulated by the phase plate or light modulator.

[0033] Alternatively, the beam shaping device of the upgrade kit can also be used to adapt an already existing intensity distribution of the illumination light with a local minimum (which is generated, for example, by components of the light microscope), ie in particular to adapt its shape and / or orientation.

[0034] Of course, the beam-shaping device can also modulate the illumination light beam to form the intensity distribution with the local minimum, as well as adjust its shape and / or orientation. For example, a light distribution with two regions of high light intensity separated by an area of minimum intensity intersecting the geometric focus can be rotated using the beam-shaping device (e.g., by phase modulation), so that the orientation of the regions of high intensity is adjusted. The light emissions from an emitter can then be recorded for the different orientations and used for position estimation.

[0035] The stabilization system serves primarily to compensate for or correct the effects of disturbances on position estimation. This is particularly important given the spatial resolution in the single-digit nanometer range achievable with MINFLUX methods.

[0036] The disturbance can be caused, for example, by displacements between the sample and the objective, by drift or other movements in the sample, or by the effects of external influences (e.g., temperature changes, mechanical shocks, sound) on components of the light microscope or the upgrade kit. Numerous designs of disturbance detection and disturbance compensation / correction are possible for the stabilization system. Regardless of the type of detection, the stabilization system can, for example, control an actuator connected to a sample stage to keep the position of the sample stable, e.g., using a control loop. In particular, if the light microscope already has a stabilization system or a separate stabilization system is available, the upgrade kit does not necessarily have to have a stabilization system.

[0037] According to one embodiment, at least one component of the upgrade kit can be connected directly or indirectly to the detector of the light microscope, so that the at least one component can be controlled based on light emissions detected by the detector. "Connectable" means that a transmission of data and / or control commands between the detector and the at least one component of the upgrade kit is enabled. The connection can be, for example, an electrical or optical data connection. The data or control commands can be transmitted in analog or digital form.

[0038] In this way, the detected light emissions can be advantageously used, for example, to control the first beam scanning device or the beam shaping device, for example, to implement an iterative MINFLUX method. If the stabilization system can be controlled based on the detector signals or detector data, a simpler stabilization system can be implemented, for example, by using the detector for measurements on the sample, based on which the sample or the measurement is stabilized.

[0039] According to a further embodiment, the upgrade kit comprises a control unit that can be connected to the detector of the light microscope directly or, for example, via a computing unit, wherein the control unit is designed to control the at least one component on the basis of light emissions detected by the detector.

[0040] The control unit can, for example, control the first beam scanning device, the beam shaping device, or the stabilization system, or several of these components together. Of course, multiple control units of the upgrade kit can also be connected directly or indirectly to the detector of the light microscope.

[0041] The control unit can be integrated into the corresponding component, e.g. a so-called driver of the first scanning device (such as a galvo driver of a galvo scanning device, a driver of an acousto-optical deflector or a driver of an electro-optical deflector) or a control electronics of a beam shaping device (e.g. a controllable light modulator such as a liquid crystal SLM).

[0042] Alternatively, the control unit connectable to the detector of the light microscope may be an external control unit (with respect to the controlled component of the upgrade kit), e.g., a microcontroller, FPGA or ASIC or a personal computer, which in turn is connected to the control electronics of the first scanning device, the beam shaping device and / or the stabilization system.

[0043] According to one embodiment, the control unit is a microcontroller, FPGA, or ASIC. This has the advantage that control processes can be executed particularly effectively and with minimal time delay.

[0044] According to a further embodiment, the at least one component connectable to the detector comprises the first beam scanning device, wherein the first beam scanning device is controllable, in particular by means of the control unit, such that the focus of the illuminating light beam (at which the local minimum of the intensity distribution of the illuminating light is located) in the sample is displaced to illumination positions that form an illumination pattern around a position of the emitter estimated on the basis of light emissions of an emitter in the sample detected by means of the detector.

[0045] With such a coupling, for example, an iterative MINFLUX method can be advantageously implemented, in which the illumination positions are placed in several iteration steps around the position of the emitter estimated in the previous iteration step.

[0046] The illumination positions form an illumination pattern around an estimated emitter position. The estimated position may, but does not necessarily, form the center of the illumination pattern. Furthermore, the estimated position can optionally be part of the illumination pattern itself. The illumination pattern can also include a continuous trajectory of the focus (i.e., the local minimum).

[0047] According to a further embodiment, the at least one component connectable to the detector comprises the beam-shaping device, wherein the beam-shaping device is controllable, in particular by means of the control unit, such that the intensity distribution of the illumination light in the sample is adapted, in particular such that a shape of the intensity distribution, a position of the intensity distribution in the sample (e.g. a focus position in the axial direction, ie in the direction of the optical axis of the objective) and / or an orientation of the intensity distribution is adapted.

[0048] In this way, for example, an intensity distribution with two maxima separated by an areal minimum can be rotated around the geometric focus depending on the detected light emissions and / or the position of the emitter estimated from them.

[0049] According to a further embodiment, the upgrade kit comprises an illumination light source designed to generate the illumination light beam, or the upgrade kit comprises a connection element designed to couple an illumination light beam generated by an external illumination light source into an illumination beam path of the light microscope.

[0050] The illumination light source can be, for example, a laser source. The connection element can be, for example, a fiber coupler for coupling a laser source via an optical fiber.

[0051] According to a further embodiment, the upgrade kit comprises a support structure designed to align components of the upgrade kit at a defined position relative to the illumination beam path of the light microscope.

[0052] According to a further embodiment, the upgrade kit comprises a support structure which is designed to pre-align the illumination light source and / or the connection element relative to the first beam scanning device and / or to the beam shaping device.

[0053] This increases the user-friendliness of the upgrade kit because the user does not have to perform complex adjustments between the light source or connection element and the beam scanning device or beam shaping device.

[0054] The support structure can comprise, for example, a perforated plate or a so-called optical breadboard. The support structure can also be formed by a housing or connected to a housing, wherein the housing encloses at least one component of the upgrade kit.

[0055] According to a further embodiment, the upgrade kit comprises at least one optical element arranged between the illumination light source or the connection element and the first beam scanning device and / or the beam shaping device, wherein the at least one optical element is configured to focus, expand, and / or guide the illumination light beam. In particular, the at least one optical element is connected to the support structure.

[0056] The at least one optical element can be, for example, a lens, a lens system, a mirror, or a prism. Lenses can, for example, form an optical relay that images the illumination light. For example, in certain beam scanners, such as electro-optical scanners, it may be advantageous or necessary for a pupil plane of the illumination light, which is conjugate to a rear aperture of an objective lens that focuses the illumination light, to extend within the beam scanner. The same can apply, for example, to certain beam-shaping devices, such as SLMs.

[0057] According to a further embodiment, the upgrade kit comprises a computing unit connectable to the detector of the light microscope, wherein the computing unit is designed to estimate a position of the emitter on the basis of light emissions of an emitter in the sample detected by the detector for different positions of a local minimum of an intensity distribution of the illumination light in the sample or for different shapes and / or orientations of an intensity distribution of the illumination light with a local minimum.

[0058] With this embodiment, a dedicated computing unit for position estimation is provided as part of the upgrade kit. Although position estimation could, in principle, also be performed using a standard computer coupled to the light microscope, in practice this can lead to long computing times and problems with microscope control. Therefore, the separate computing unit increases the user-friendliness and improves the performance of the MINFLUX microscope obtained through the upgrade.

[0059] According to another embodiment, the computing unit is a microcontroller, a field programmable gate array (FPGA), or an application-specific integrated circuit (ASIC). Such computing units are particularly well suited for the computational steps required for position estimation and are capable of executing them reliably and at high speed.

[0060] The computing unit can also form a unit with the above-mentioned control unit or be implemented by the same processor.

[0061] According to a further embodiment, the upgrade kit comprises a coupling element which is designed to couple at least one component of the upgrade kit to a light input of the light microscope, so that the illumination light beam is coupled into the illumination beam path of the light microscope.

[0062] In particular, the light input has a predetermined orientation relative to an objective of the light microscope, i.e. the light path between the light input and the objective is pre-adjusted so that the components of the upgrade kit can also be given a desired orientation relative to the objective by adjusting them relative to the coupling element.

[0063] According to a further embodiment, an intermediate image of the light microscope has a defined position relative to the light input. In particular, the light input is fully corrected and / or normalized.

[0064] According to a further embodiment, the coupling element is designed for the stationary mounting of components of the upgrade kit, in particular the housing of the upgrade kit, at the light input of the light microscope. According to a further embodiment, the upgrade kit has a support structure designed to align components of the upgrade kit, in particular the illumination light source, the connection element, the first beam scanning device, and / or the beam shaping device, relative to the coupling element at a defined position, i.e., pre-adjusted.

[0065] According to a further embodiment, the upgrade kit comprises a housing, wherein the housing surrounds components of the upgrade kit, in particular in a light-tight manner.

[0066] In particular, the housing surrounds all components of the upgrade kit.

[0067] Alternatively, individual or all components of the upgrade kit can be attached separately to the light microscope or installed in the light microscope, e.g. in suitable predefined installation positions that are easily accessible, e.g. by opening a flap or unscrewing a housing part of the light microscope.

[0068] According to a further embodiment, the upgrade kit comprises a polarization rotator configured to adjust a polarization direction of the illumination light beam such that a phase and / or an amplitude of the illumination light beam can be modulated by means of the beam-shaping device or another beam-shaping device. A defined polarization direction of the illumination light beam may be necessary, for example, for phase modulation using a liquid crystal SLM.

[0069] According to a further embodiment, the upgrade kit comprises a polarizer configured to linearly polarize the illumination light beam. Linear polarization may be necessary, in particular, for scanning the illumination light beam by means of the first beam scanning device and / or for modulating the illumination light beam by means of the beam-shaping device.

[0070] According to a further embodiment, the first beam scanning device is configured to deflect the illumination light beam such that a focus of the illumination light beam formed by an objective of the light microscope is deflected in the sample perpendicular to an optical axis of the objective. In other words, the first beam scanning device scans the illumination light beam laterally or in the xy direction.

[0071] According to a further embodiment, the first beam scanning device comprises at least one acousto-optical deflector. Like electro-optical deflectors, acousto-optical deflectors have a very high scanning speed, are less complex in design, require fewer additional optical components, and can therefore be combined with an existing light microscope in a particularly space-saving manner. Acousto-optical deflectors comprise, in particular, a birefringent crystal into which acoustic waves are coupled by means of a transducer, resulting in a deflection of a light beam passing through the crystal.

[0072] In particular, the acousto-optical deflector operates in the so-called Bragg regime. A control unit allows the frequency of the acoustic wave and thus the deflection angle to be adjusted.

[0073] In particular, the first beam scanning device comprises a first acousto-optical deflector that deflects the illumination light beam in a first direction (e.g., the x-direction), and a second acousto-optical deflector that deflects the illumination light beam in a second direction perpendicular to the first direction (e.g., the y-direction). The first acousto-optical deflector and the second acousto-optical deflector are arranged, in particular, one behind the other in the beam path of the illumination light beam.

[0074] According to a further embodiment, the first beam scanning device comprises at least one electro-optical deflector.

[0075] Acousto-optical and electro-optical scanning devices offer the advantage of high scanning speed, which is often difficult or impossible to achieve with mechanical beam scanners (e.g., galvo scanners), such as those already present in the light microscope being upgraded. This allows for more MINFLUX localizations per unit of time, which is particularly advantageous for tracking individual emitters.

[0076] According to a further embodiment, the first beam scanning device comprises a deflection element and at least one actuator, wherein the deflection element is designed to deflect the illumination light beam and the at least one actuator is coupled to the deflection element such that the deflection element can be displaced by means of the actuator, so that the illumination light beam is displaced. In other words, the first beam scanning device is a mechanical scanning device with moving parts. The deflection element can be, for example, a reflective element (in particular a mirror or a prism) or a refractive element (in particular a lens), and the actuator can comprise, for example, a galvo drive, at least one piezoelectric element, a motor, or an inductive element (in particular a coil).

[0077] According to one embodiment, the first beam scanning device is a galvo scanning device, wherein the actuator is a galvo drive and the deflection element is a mirror. Typically, a galvo scanning device has at least a first galvo drive and a first deflection element coupled to the first galvo drive for deflecting the illumination light beam in the x-direction, and a second galvo drive and a second deflection element coupled to the second galvo drive for deflecting the illumination light beam in the y-direction (perpendicular to the x-direction). Two or more pairs of galvo drives and deflection elements can also be provided per deflection direction, e.g., in so-called quad scanners or x2y scanners.

[0078] According to a further embodiment, the first beam scanning device is a Risley prism scanning device, wherein the deflection element is a prism, and wherein the actuator is a motor configured to rotate the deflection element about a rotation axis, wherein the rotation axis is parallel to an optical axis of the illuminating light beam. In particular, the first scanning device comprises a first prism and a second prism, as well as a first motor and a second motor, wherein the first motor is configured to rotate the first prism about the rotation axis, and wherein the second motor is configured to rotate the second prism about the rotation axis independently of the rotation of the first prism by the first motor. With such a scanning device, the illuminating light beam can be shifted to points in a ring around the optical axis / rotation axis.

[0079] According to a further embodiment, the first beam scanning device is a piezoelectric scanning device, wherein the actuator comprises a stack of piezoelectric elements, and wherein the deflection element is formed by a reflective surface at a tip of the stack. The deflection element is designed and arranged such that the deflection element can be displaced by applying an electrical voltage to the piezoelectric elements, in particular on a circular path around a rotation axis associated with the deflection element. Furthermore, the first scanning device can comprise an optical system having at least one lens, wherein the optical system acts on the illumination light beam deflected by the deflection element.

[0080] According to a further embodiment, the actuator of the first beam scanning device is a motor, and the deflection element of the first beam scanning device is a lens. The motor is configured to rotate the lens about a rotation axis that is offset parallel to an optical axis of the lens, so that the optical axis rotates about the rotation axis. In this way, the illumination light beam is displaced along a circular path. In particular, the first scanning device has at least one further lens in the light path behind the deflection element, which is configured to focus the deflected illumination light beam.

[0081] According to a further embodiment, the actuator of the first beam scanning device comprises at least one inductive element, in particular a coil, for generating a magnetic field. The deflection element of the first beam scanning device is a lens, and the deflection element is coupled to a magneto-responsive element, so that the magneto-responsive element is displaced with the deflection element by a magnetic field generated by the at least one inductive element, and the illumination light beam is deflected by the displaced deflection element. In particular, the first scanning device comprises at least one further lens in the light path behind the deflection element, which lens is designed to focus the deflected illumination light beam.

[0082] The three last-described embodiments of a beam scanning device have the particular advantage that the illuminating light beam can be moved along a circular path with these devices in a relatively simple manner and sometimes at high speed.

[0083] According to a further embodiment, the at least one actuator is designed to displace the deflection element such that the illumination light beam is displaced on a circular path.

[0084] If the focus of the illuminating light beam (i.e., the local minimum of the intensity distribution of the illuminating light) is shifted along a circular path within the sample as part of a MINFLUX method, the position of an individual emitter can be estimated, for example, based on a temporal modulation of the light emissions. This can be done, for example, using Fourier or phasor analysis or a function fit.

[0085] According to a further embodiment, the deflection element has a modulation pattern which is designed to spatially modulate the illuminating light beam in its phase and / or in its amplitude, in particular such that an intensity distribution with a local minimum is formed at a focus of the illuminating light beam.

[0086] The modulation pattern can be, for example, a vortex-shaped phase pattern (phase clock) for generating an illuminating light donut, a ring-shaped phase pattern with a phase jump of π for generating an illuminating light bottle beam, or a phase pattern with a linear phase jump of π for generating an intensity distribution with two intensity maxima separated by a planar intensity minimum intersecting the geometric focus.

[0087] The modulation pattern can, for example, be imprinted on a reflective surface at the top of a stack of piezoelectric elements.

[0088] A deflection element with a modulation pattern has the advantage that the first beam scanning device is simultaneously used to shape the illumination light beam into an intensity distribution with a local minimum. This is particularly advantageous for the upgrade kit according to the invention because it reduces the installation space and the complexity of the additional components.

[0089] According to a further embodiment, the first beam scanning device has a maximum scan rate of 5 kHz or more, in particular 10 kHz or more, further in particular 100 kHz or more, further in particular 1 MHz or more, even further in particular 10 MHz or more.

[0090] According to a further embodiment, the upgrade kit comprises an axial beam scanning device which is designed to deflect the illuminating light beam such that a focus of the illuminating light beam formed by an objective of the light microscope is deflected in the sample by means of the axial beam scanning device parallel to an optical axis of the objective (ie in the direction of the optical axis).

[0091] According to a further embodiment, the axial beam scanning device comprises a deformable mirror, a controllable phase modulator or a, in particular electro-optical, lens with variable focus.

[0092] With an upgrade kit containing an axial beam scanning device, a conventional light microscope can be advantageously upgraded to a 3D MINFLUX microscope.

[0093] According to a further embodiment, the beam-shaping device comprises a light modulator which is designed to spatially modulate the illuminating light beam in its phase and / or in its amplitude.

[0094] According to a further embodiment, the light modulator is a phase filter (e.g. a phase plate) or a spatial light modulator (SLM) controllable by a control unit.

[0095] According to a further embodiment, the beam-shaping device comprises a biaxial crystal, a first polarization switching unit and a second polarization switching unit, wherein the first polarization switching unit is arranged in an illumination beam path in front of the biaxial crystal, and wherein the second polarization switching unit is arranged in the illumination beam path behind the biaxial crystal.

[0096] With such a beam shaping device, intensity distributions of different shapes and / or orientations can be generated at the focus of the objective of the light microscope, depending on the switching state of the first polarization switching unit and the second polarization switching unit, and it is possible to quickly switch between them by switching the polarization. This principle is described, for example, in patent application US 2023 / 0236401 A1 and in the literature cited therein. For example, intensity distributions with two intensity maxima separated by a planar intensity zero can be generated using a combination of input polarization and output polarization of 45° / -45°, -45° / 45°, 90° / 0°, or 0° / 90°, whereby the combinations 45° / -45°, -45° / -45° and 90° / 0°, 0° / 90° result in mutually orthogonal courses of the planar minimum.

[0097] According to a further embodiment, the first polarization switching unit and the second polarization switching unit each have at least one electro-optical polarization switch, in particular at least two electro-optical polarization switches each.

[0098] According to a further embodiment, at least one component of the upgrade kit is designed to couple a measuring light beam for the stabilization system into the light microscope in addition to the illumination light beam.

[0099] This measuring light beam can be scattered or reflected by reference particles in the sample, for example, and the scattered or reflected light can be detected by a detector, e.g., to determine the current position of the sample relative to the objective of the light microscope. If it is determined that the current position does not correspond to the desired position, an actuator on a sample stage can be controlled, for example, to correct the position.

[0100] Such a stabilization system is described, for example, in patent application US 2022 / 0011559 A1 and in the publication R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478.

[0101] The measurement light beam can be, for example, a linearly polarized infrared laser beam coupled via a polarization beam splitter and focused into a rear aperture of the objective to illuminate the sample in the wide field. A λ / 4 plate can be arranged in front of the objective, which converts the linearly polarized measurement light into circularly polarized measurement light and converts the returning light scattered by gold nanoparticles into linearly polarized light that is orthogonal to the initial polarization. This can be separated from the measurement light illuminating the sample at the polarization beam splitter and imaged onto a camera.

[0102] According to a further embodiment, the at least one component of the upgrade kit is designed to couple the measuring light beam together with the illuminating light beam into the illumination beam path of the light microscope. This means that the measuring light beam and the illuminating light beam, in particular, pass through the coupling element connected to the light microscope together. This embodiment has the advantage that the complexity and the installation space required for upgrading the light microscope can be further reduced. For this implementation, particular care must be taken to ensure that the measuring light beam is compatible with the beam splitters present in the illuminating beam path of the light microscope so that it reaches the objective of the light microscope. This is particularly relevant if the measuring light beam has a wavelength in the infrared range.For example, an edge filter, a bandpass filter or similar could be used as a beam splitter in the light microscope to separate longer-wavelength fluorescent light into a detection beam path compared to the excitation light, while even longer-wavelength reflected or scattered measurement light passes back through the illumination beam path to the components of the upgrade kit.

[0103] According to a further embodiment, the upgrade kit comprises a beam splitter for installation in the light microscope, wherein the beam splitter is designed to separate light emissions emanating from the sample from the illumination light and the measurement light, in particular wherein the beam splitter is an edge filter.

[0104] According to a further embodiment, the upgrade kit comprises a beam combiner configured to combine the measurement light beam and the illumination light beam. The beam combiner can be, for example, a dichroic beam splitter / combiner or a polarization beam splitter / combiner. The beam combiner can be arranged, in particular, in the beam path of the illumination light beam downstream of the first beam scanning device and / or downstream of the beam shaping device, so that only the illumination light beam, but not the measurement light beam, is shifted by the first beam scanning device and / or shaped by the beam shaping device.

[0105] According to a further embodiment, the upgrade kit comprises an optical system designed to focus the measuring light beam so that the measuring light beam illuminates the sample in the wide field. For this purpose, the measuring light beam must be focused into a plane conjugate to the rear aperture of the objective. The optical system can be, for example, a system of lenses, in particular an optical relay.

[0106] According to a further embodiment, the measuring light beam generates measurement detection light emanating from the sample, e.g., scattered light, reflected light, or luminescence light (in particular, fluorescent light). This measurement detection light can be generated, for example, by interaction with the sample itself (e.g., with reference particles or reference markers contained in the sample) or with a structure associated with the sample (such as a slide, a coverslip, an embedding medium, or an immersion medium).

[0107] Depending on the type of measurement detection light and the technical specifications of the light microscope, the measurement detection light can be detected by a detector of the light microscope (especially by the same detector used to detect the light emissions from the emitters for the MINFLUX method), or the measurement detection light can be redirected, particularly in the illumination beam path, back to the components of the upgrade kit to be detected by a separate measurement sensor (different from the detector of the light microscope). Finally, it is also possible to provide a dedicated measurement detection beam path for the measurement detection light. The measurement detection light can also be detected, for example, by a non-descanned detector.

[0108] According to a further embodiment, the stabilization system comprises a measuring sensor configured to detect the disturbance. The measuring sensor can, for example, be an optical detector that detects the measurement detection light generated by the measuring light, e.g., light scattered or reflected by reference particles in the sample. Alternatively, the measuring sensor can also detect external influences such as temperature changes, sound, or mechanical shocks.

[0109] According to a further embodiment, the upgrade kit comprises a control unit, in particular connected to the measuring sensor, wherein the control unit is designed to control a compensation unit of the light microscope, in particular on the basis of signals received from the measuring sensor, wherein the compensation unit is designed to compensate for the disturbance, or to control a computing and / or control unit, wherein the computing and / or control unit is designed to correct position data of an emitter on the basis of the disturbance, or to abort or interrupt a localization of an emitter in the sample.

[0110] According to a further embodiment, the upgrade kit comprises an optics designed to image the collimated illumination light beam onto a first scanning mirror of a second beam scanning device of the light microscope, wherein the first scanning mirror is arranged in a pupil plane conjugated to a rear aperture of the objective of the light microscope.

[0111] If the light microscope has a second beam scanning device, e.g. a galvanometer scanning device, this can ensure in particular that the illuminating light beam coupled into the light microscope by the components of the upgrade kit passes through the rear aperture of the objective at a desired position. In particular, the first beam scanning device of the upgrade kit can also deflect the illuminating light beam in a pupil plane conjugate to the rear aperture, which is imaged by the optics onto the first scanning mirror and imaged by optical elements of the light microscope, in particular a scanning lens and a tube lens, into the rear aperture of the objective. This is particularly advantageous for a MINFLUX method in which the illuminating light beam is phase modulated in order to ideally form an intensity distribution with a central intensity zero at the focus in the sample.In this case, a lack of centering of the illuminating light beam in the rear aperture (pupil) of the objective lens results in imperfect destructive interference at the center of the intensity distribution, so that the local minimum is no longer a zero. This additional intensity at the minimum can lead to inaccuracies in the position estimation.

[0112] Therefore, the above-described embodiment increases the accuracy of position estimation by the MINFLUX microscope obtained by upgrading.

[0113] The second beam scanning device, which can be used, for example, in the light microscope to acquire a raster image, can be used, in particular, in a MINFLUX process for coarse positioning of the illumination light beam and / or for pre-localization. For rapid control of illumination positions during the MINFLUX process, the first beam scanning device of the upgrade kit, if available, is used, in particular.

[0114] According to a further embodiment, the second beam scanning device of the light microscope further comprises a second scanning mirror and a third scanning mirror configured to deflect the illumination light beam in a second direction perpendicular to a first direction in which the first scanning mirror deflects the illumination light beam.

[0115] The first scanning mirror can be arranged in the light path in front of or behind the second scanning mirror and the third scanning mirror.

[0116] According to a further embodiment, the second scanning device has a fourth scanning mirror configured to deflect the illumination beam path in the same direction as the first scanning mirror. The second scanning device, in particular, has an adjustment motor configured to selectively move the first scanning mirror or the fourth scanning mirror into the light path of the illumination light beam. The fourth scanning mirror can, for example, be part of a particularly fast resonant scanner, while the first scanning mirror can, for example, be part of a slower galvo scanner that covers a larger image field.

[0117] A second aspect of the invention relates to a MINFLUX microscope comprising the components of the upgrade kit according to the first aspect, an objective and a detector (in particular comprised in a light microscope), wherein the objective is designed to focus the illuminating light beam (in particular coupled into the light microscope) modulated in its phase and / or amplitude into a sample, so that an intensity distribution of the illuminating light with a local minimum is formed in the sample, and wherein the detector is designed to detect light emissions from emitters in a sample.

[0118] According to one embodiment of the MINFLUX microscope, the MINFLUX microscope (i.e., in particular, the components of the upgrade kit or the light microscope) comprises a computing unit, wherein the computing unit is configured to estimate a position of an emitter in the sample based on light emissions detected by the detector for different positions of the local minimum or for different shapes and / or orientations of the intensity distribution.

[0119] According to another embodiment of the MINFLUX microscope, the light microscope is a confocal laser scanning microscope or a STED microscope.

[0120] According to a further embodiment of the MINFLUX microscope, the detector of the light microscope comprises an avalanche photodiode or a hybrid photodetector.

[0121] According to a further embodiment of the MINFLUX microscope, the light microscope has a second beam scanning device which is designed to shift the illumination light beam coupled into the illumination beam path of the light microscope relative to the sample.

[0122] According to a further embodiment, the second beam scanning device comprises a first scanning mirror which is designed to deflect the illumination light beam in a first direction.

[0123] According to a further embodiment, the first scanning mirror is arranged in a pupil plane conjugated to a rear aperture of the objective of the light microscope.

[0124] According to a further embodiment, the second beam scanning device comprises a second scanning mirror and a third scanning mirror configured to deflect the illumination light beam in a second direction perpendicular to a first direction in which the first scanning mirror deflects the illumination light beam.

[0125] The first scanning mirror can be arranged in the light path in front of or behind the second scanning mirror and the third scanning mirror.

[0126] According to a further embodiment, the second scanning device has a fourth scanning mirror configured to deflect the illumination beam path in the same direction as the first scanning mirror. The second scanning device, in particular, has an adjustment motor configured to selectively move the first scanning mirror or the fourth scanning mirror into the light path of the illumination light beam. The fourth scanning mirror can, for example, be part of a particularly fast resonant scanner, while the first scanning mirror can, for example, be part of a slower galvo scanner that covers a larger image field.

[0127] A third aspect of the invention relates to a method for upgrading a light microscope having a detector for detecting light emissions from emitters in a sample to a MINFLUX microscope by means of an upgrade kit according to the first aspect, wherein an illuminating light beam is coupled into an illuminating beam path of the light microscope by means of at least one component of the upgrade kit.

[0128] According to one embodiment of the method for upgrading a light microscope, the light microscope is supplemented with a software component or a software function of the light microscope is activated, wherein the software component or the software function is designed to control at least one component of the upgrade kit and / or the detector of the light microscope. The software component or software function can, for example, be or comprise a computer program or a sub-module of a computer program that is installed on a memory unit of a local computing unit (which is connected to the light microscope or the MINFLUX microscope) or, for example, on a cloud server to which a computing unit of the light microscope or the MINFLUX microscope can be connected via a data connection. The software component or function can accordingly be executed, for example, on the local computing unit or on the cloud server.

[0129] The software component or software function may, for example, comprise or be comprised by the computer program according to the sixth aspect and / or the computer program according to the seventh aspect.

[0130] A fourth aspect of the invention relates to a method for controlling a MINFLUX microscope according to the second aspect, wherein at least one component of the upgrade kit according to the first aspect is controlled based on light emissions detected by the detector.

[0131] A fifth aspect of the invention relates to a method for locating or tracking emitters in a sample by means of a MINFLUX microscope according to the second aspect, wherein a sample is illuminated by means of the illuminating light beam, wherein the illuminating light forms an intensity distribution with a local minimum in the sample, and wherein a position of the emitter is estimated on the basis of light emissions of an emitter in the sample detected by means of the detector of the light microscope.

[0132] A sixth aspect of the invention relates to a computer program comprising program instructions that cause a control unit to execute the method according to the fourth aspect.

[0133] A seventh aspect of the invention relates to a computer program comprising program instructions that cause the MINFLUX microscope according to the second aspect to carry out the method according to the fifth aspect.

[0134] The computer programs according to the sixth and seventh aspects can be executed either locally on a computing unit connected to or included in the light microscope or the MINFLUX microscope, or, for example, on a cloud server that is or can be connected to a computing unit of the light microscope or the MINFLUX microscope.

[0135] An eighth aspect of the invention relates to a system comprising the upgrade kit according to the first aspect and the computer program according to the sixth aspect and / or the computer program according to the seventh aspect.

[0136] Further embodiments of the methods according to the third, fourth and fifth aspects as well as the computer programs according to the sixth and seventh aspects and the system according to the eighth aspect emerge from the above description of the upgrade kit according to the first aspect and the MINFLUX microscope according to the second aspect.

[0137] Advantageous developments of the invention will become apparent from the patent claims, the description, the drawings, and the accompanying explanations of the drawings. The described advantages of features and / or combinations of features of the invention are merely exemplary and may be effective alternatively or cumulatively.

[0138] With regard to the disclosure content (but not the scope of protection) of the original application documents and the patent, the following applies: Further features can be found in the drawings – in particular the relative arrangements and operative connections shown. The combination of features of different embodiments of the invention or of features of different patent claims is also possible, deviating from the selected references of the patent claims, and is hereby suggested. This also applies to features that are shown in separate drawings or mentioned in their description. These features can also be combined with features of different patent claims. Likewise, features listed in the patent claims can be omitted for further embodiments of the invention; however, this does not apply to the independent patent claims of the granted patent.

[0139] 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.

[0140] In the following, exemplary embodiments of the invention are described with reference to figures. These do not limit the subject matter of this disclosure or the scope of protection. Short description of the characters Fig. 1 shows a MINFLUX microscope obtained by upgrading using the upgrade kit according to the invention; Fig. 2 shows an embodiment of a stabilization system; Fig. 3 shows another alternative embodiment of an upgrade kit with a stabilization system; Fig. 4 shows an embodiment of a beam shaping device. Description of the characters

[0141] Fig. 1 shows a light microscope 1 upgraded to a MINFLUX microscope 100 with an upgrade kit 10 according to the invention according to an embodiment.

[0142] The light microscope 1 is located in the upper part of the Fig. 1 and can be a confocal laser scanning microscope.

[0143] The light microscope 1 has a light input 5 for coupling an illuminating light beam B into an illuminating beam path 4 of the light microscope 1. The illuminating light is coupled into the illuminating beam path 4 via a beam splitter 38, in particular a dichroic beam splitter.

[0144] The illumination light then passes through a second beam scanning device 7 and a scanning lens 73 and is guided by two light deflecting elements 9 (e.g. mirrors) to an objective 6, which focuses the illumination light into a sample 2.

[0145] The focus F of the illumination light can be scanned across the sample 2 using the second beam scanning device 7. The second beam scanning device 7 comprises a first scanning mirror 71, which deflects the illumination light beam B along a first spatial direction, as well as a second scanning mirror 74 and a third scanning mirror 76, which jointly deflect the illumination light beam B along a second spatial direction, wherein the second spatial direction is perpendicular to the first spatial direction. The first scanning mirror 71, the second scanning mirror 74, and the third scanning mirror 76 are each coupled to a first drive 72, a second drive 75, and a third drive 77, respectively. The drives 72, 75, 77 can, in particular, be galvanometer drives. In this case, the second scanning device is a galvanometer scanning device.The illuminating light beam B is first deflected by the second scanning mirror 74, then by the third scanning mirror 76, and finally by the first scanning mirror 71. The second scanning mirror 74 and the third scanning mirror 76 can, in particular, be controlled such that the illuminating light beam B always strikes the same point on the first scanning mirror 71, regardless of its deflection (although at different angles depending on the deflection in the second spatial direction). The first scanning mirror 71 is arranged in a pupil plane P, which is conjugated to a rear aperture R of the objective 6. The pupil plane P is imaged, in particular, by the scanning lens 73 and a tube lens (not shown here) into the rear aperture R of the objective 6. In the focal plane in the sample 2, different angles of the illuminating light beam B lead to different lateral displacements of the focus F of the illuminating light beam B.

[0146] Light emissions emanating from sample 2, e.g., luminescence, particularly fluorescence, or reflected or scattered light, are descanned by the second scanning device 7, transmitted by the beam splitter 38, and passed via two light deflection elements 9, e.g., mirrors, to the detector 3, which detects the light emissions. A computing unit 8 of the light microscope 1 is connected to the detector 3. This computing unit can, for example, calculate a confocal image from light emissions detected during scanning across sample 2, but can also be used, if necessary, in the MINFLUX microscope obtained by upgrading to estimate the position of an emitter.

[0147] According to the invention, the light microscope 1 can be upgraded to a MINFLUX microscope 100 by connecting the coupling element 19 of the upgrade kit 10 to the light connection 5 in order to couple the illumination light beam B into the illumination beam path 4 of the light microscope 1 and by connecting the stabilization system 13 (also part of the upgrade kit 10) to a camera input 39 of the light microscope 1.

[0148] In the illustrated embodiment, the upgrade kit 10 is enclosed by a housing 20, with the exception of the stabilization system 13, and has a connection element 16 for connecting an external illumination light source 15 and coupling the illumination light beam B generated by the illumination light source 15 (e.g., a laser) into the beam path of the upgrade kit 10. Alternatively, the illumination light source 15 could also be arranged within the housing 20. The components of the upgrade kit 10 (with the exception of the stabilization system 13) are further connected to a support structure 17, which provides the components with a defined position and alignment relative to the illumination beam path 4 of the light microscope 1.

[0149] The illumination light beam B is guided by light deflection elements 9, in particular mirrors, via a first beam scanning device 11, a beam shaping device 12 and an optional axial scanning device 21 to the coupling element 19, wherein the first beam scanning device 11 is designed to shift the illumination light beam B in two mutually orthogonal directions and the beam shaping device 12 is designed to spatially modulate the phase and / or amplitude of the illumination light beam B, so that an intensity distribution of the illumination light B with a local minimum, in particular an intensity zero (in particular at the geometric focus), is formed at the focus F of the illumination light beam B in the sample 2.

[0150] In the example shown, the first beam scanning device 11 is designed as a pair of, in particular acousto-optical, deflectors 32a, 32b, which deflect the illumination light B in two mutually orthogonal directions (in particular x and y).

[0151] Acousto-optical deflectors are particularly advantageous for the upgrade kit according to the invention because, in contrast to electro-optical deflectors, they require relatively short beam paths and fewer optical components and therefore require only a small installation space.

[0152] Alternatively, electro-optical deflectors can also be used. In this case, the illumination light B must be linearly polarized, which can be achieved either by the illumination light source 15 itself or by a polarization rotator. The polarization direction of the illumination light B must also be rotated between the deflector 32a and the deflector 32b. Furthermore, the electro-optical deflectors are each positioned in a pupil plane P, which, when connected to the light microscope 1, is conjugated to the rear aperture R of the objective 6. This can be achieved by lens systems such as optical relays.

[0153] The beam forming device 12 is in the embodiment according to Fig. 1 is designed as a controllable spatial light modulator 22 (spatial light modulator, SLM) for polarization-dependent phase modulation of the illumination light beam B. For example, a vortex-shaped phase pattern for generating a donut-shaped intensity distribution or an annular phase jump for generating a bottle-beam-shaped intensity distribution can be displayed on the light modulator 22. The polarization direction of the illumination light beam B required for the phase modulation can be adjusted, for example, using a polarization rotator (not shown). The light modulator 22 is arranged in a pupil plane P that is conjugate to the rear aperture R of the objective 6.

[0154] The optional axial scanning device 21 is in the Fig. 1, the deformable mirror 34 is configured as a deformable mirror. The axial scanning device 21 allows the position of the focus F of the illumination light beam B in the sample 2 to be shifted in the axial direction (along the optical axis of the objective 6), in particular to implement a 3D MINFLUX method. The deformable mirror 34 is also positioned in a pupil plane P conjugated to the rear aperture R of the objective 6. The lenses 31a, 31b project the pupil plane P associated with the light modulator 22 onto the deformable mirror 34.

[0155] Finally, the lens 31c, which is matched to the optics of the light microscope 1, ensures that the pupil planes P of the light modulator 22 and the deformable mirror 34 are imaged onto the pupil plane P of the first scanning mirror 71 and the rear aperture R of the objective 6. The upgrade kit 1 thus comprises an optics 27 that images the collimated illumination light beam B onto the first scanning mirror 71 of the second beam scanning device 7 of the light microscope 1, wherein the first scanning mirror 71 is arranged in a pupil plane P conjugated to a rear aperture R of the objective 6 of the light microscope 1. This has the particular advantage that, regardless of the position of the focus F of the illumination light beam B in the sample 2, the zero point of the intensity distribution is maintained, which improves the position estimation.

[0156] The Fig. The upgrade kit shown in Figure 1 further comprises a control unit 14 and a computing unit 18, each of which is connected to the detector 3 of the light microscope 1.

[0157] The computing unit 18 is designed to estimate the position of an emitter in the sample 2 on the basis of light emissions of an emitter detected by the detector 3, in particular in a MINFLUX method.

[0158] The control unit 14 controls the first beam scanning device 11 (i.e., the two deflectors 32a, 32b), the beam shaping device 12, and the axial beam scanning device 21 and is also connected to the detector 3 and the computing unit 18, so that the light emissions detected by the detector 3 and / or the position of an emitter estimated by the computing unit 18 based on the light emissions can be taken into account in the control. This allows, for example, an iterative 2D or 3D MINFLUX method to be implemented, in which the local minimum of the intensity distribution is positioned in an iteration step depending on the position of the emitter estimated in the previous iteration step.

[0159] The stabilization system 13 couples according to the Fig. 1, a measuring light beam M is introduced into the light microscope 1 via a camera input 39. The stabilization system 13 is further connected to a compensation unit 36, which in turn is designed to displace the sample 2 depending on data from a measuring sensor 35 of the stabilization system 13 or data from a detector of the light microscope 1 in order to stabilize the sample 2 against interference. The measuring light beam M can, for example, be reflected or scattered by reference particles in the sample 2, wherein the reflected or scattered light is detected by the measuring sensor 35 or the detector.

[0160] An embodiment of the stabilization system 13 is shown in Fig. 2 shown.

[0161] The stabilization system 13 accordingly comprises a measuring light source 28 for generating the measuring light beam M. The measuring light source 28 can be, for example, an infrared laser. The measuring light beam M is linearly polarized (by the measuring light source 28 or a polarizer not shown here) and is transmitted by the polarization beam splitter 29. The λ / 4 plate 41 converts the linear polarization of the measuring light beam M into a circular polarization. The circularly polarized measuring light beam M is then coupled into the light microscope 1 via a camera connection 39. By means of the lenses 31d, 31e, the measuring light beam M is focused into a focal plane F, and the focus F of the measuring light beam M is imaged into the rear aperture R of the objective 6 of the light microscope 1, so that the measuring light illuminates the sample 2 in the wide field. According to this exemplary embodiment, reference particles are arranged in the sample 2 to scatter or reflect the measuring light. The scattered orThe reflected light returns through the objective lens 6 into the beam path of the light microscope 1 and via the camera connection 39 into the stabilization system 13. In doing so, it passes through the λ / 4 plate 41 and acquires a linear polarization that is orthogonal to the polarization direction of the measuring light beam M. Therefore, the reflected or scattered light is reflected by the polarization beam splitter 29 and captured by a measuring sensor 35, e.g., a camera. The measuring sensor 35 is connected to a stabilization control unit 33, which analyzes the signal from the measuring sensor 35 and, on the basis of the analysis, controls the compensation unit 36 coupled to the sample 2 (e.g., via a sample holder and a slide) in such a way that disturbances are compensated for and the sample 2 is stabilized.

[0162] Of course, the polarization beam splitter 29 can alternatively be designed and arranged such that it reflects the light of the measuring light source 28 and transmits the light scattered or reflected by the sample.

[0163] In particular, an optional spatial filter can be arranged between the polarization beam splitter 29 and the measuring sensor 35, which can facilitate the analysis of the disturbances by means of the signal of the measuring sensor 35.

[0164] Fig. 3 shows a compared to Fig. 1 and Fig. 2 alternative embodiment of an upgrade kit 10 according to the invention with stabilization system 13.

[0165] In contrast to the Fig. In the upgrade kit 10 shown in Figure 1, in addition to the illumination light source 15 connected to the upgrade kit via a first connection element 16a, a measuring light source 28 is connected to the upgrade kit 10 via a second connection element 16b, so that the illumination light beam B and the measuring light beam M are coupled into a common beam path. The illumination light beam B and the measuring light beam M are combined via a beam combiner 40, which is arranged in the beam path in front of the coupling element 19, so that the illumination light beam B is coupled into the light microscope 1 together with the measuring light beam M.

[0166] The additional in Fig. 2 shown components of the stabilization system 13, e.g. an additional polarization beam splitter 29, a measuring sensor 35 and a λ / 4 plate 41 can, but need not, also be included in the stabilization system 13 according to Fig. 3 should be included.

[0167] At the Fig. 3, it is important to ensure that the beam splitter 38 of the light microscope 1 (see Fig. 1) is reflective for the measuring light beam M and, if applicable, the measuring light reflected or scattered by the sample 2, while being transparent for the light emissions emanating from the emitters in the sample 2 (in particular, fluorescent light), so that the light emissions, but not the measuring light, reach the detector 3 of the light microscope 1. This can be achieved, for example, by designing the beam splitter 38 as a notch filter. If required, a corresponding beam splitter 38 can be provided as part of the upgrade kit 10 and installed in the light microscope 1.

[0168] In the Fig. 3, no axial beam scanning device 21 is shown for clarity. Of course, the embodiment shown in Fig. 1, the upgrade kit 10 including the axial beam scanning device 21 can be combined with a second connection element 16b for coupling the measuring light beam M and with the beam combiner 40.

[0169] In Fig. 4 shows a further embodiment of a beam shaping device 12 which can be used as a component of the upgrade kit 10 according to the invention as an alternative to the light modulator 22 ( Fig. 1) can be used at the corresponding position in the beam path.

[0170] The beam-shaping device 12 comprises a first (linear) polarizer 121, a first polarization switching unit 24, a lens 31f, a biaxial crystal 23, another lens 31g, a second polarization switching unit 25, and a second (linear) polarizer 122. These components are traversed by the illumination light beam B in the order mentioned. The first polarizer 121 and the second polarizer 122 have mutually orthogonal polarization directions. The first polarization switching unit 24 comprises a first polarization switch 24a and a second polarization switch 24b, and the second polarization switching unit 25 comprises a third polarization switch 25a and a fourth polarization switch 25b. The first polarization switch 24a and the second polarization switch 24b can each be designed, for example, as Pockels cells, wherein the fast axes of the Pockels cells are arranged at an angle of 45° to each other.The same applies to the third polarization switch 25a and the fourth polarization switch 25b. The lens 31f focuses the illumination light beam B onto the biaxial crystal 23, and the lens 31g collimates the illumination light beam B emanating from the biaxial crystal 23.

[0171] The input polarization can be adjusted with the first polarization switching unit 24 by controlling the first polarization switch 24a and / or the second polarization switch 24b via the control unit 14. Accordingly, the illumination light beam B is shaped by the biaxial crystal 23 through conical diffraction. The output polarization of the illumination light beam B can then be adjusted with the second polarization switching unit 25 by controlling the third polarization switch 25a and / or the fourth polarization switch 25b via the control unit 14. In this way, an intensity distribution with a local minimum, in particular a zero, can be generated at the focus F of the illumination light beam B in the sample 2, in particular to implement a MINFLUX method.

[0172] With the beam shaping device 12 according to Fig.4, it is also possible to quickly switch between different intensity distributions by controlling the polarization switches 24a, 24b, 25a, 25b, e.g., between light distributions with two intensity maxima separated by a minimum extending along a surface, whereby, for example, switching can be made between different positions of the surface of minimum intensity. In a MINFLUX method, this can replace the displacement of a point-like intensity minimum; thus, instead of shifting the focus with a beam scanner, the shape or orientation of the intensity distribution can be switched. List of reference symbols 1 light microscope 2 Sample 3 Detector 4 Illumination beam path 5 Light input 6 Lens 7 Second beam scanning device 8 computing unit 9 Light deflection element 10 Upgrade kit 11 First beam scanning device 12 Beam shaping device 13 Stabilization system 14 Control unit 15 Illumination light source 16, 16a, 16b connecting element 17 Support structure 18 computing unit 19 Coupling element 20 housings 21 Axial beam scanning device 22 Light modulator 23 Biaxial crystal 24 First polarization switching unit 24a First polarization switch 24b Second polarization switch 25 Second polarization switching unit 25a Third polarization switch 25b Fourth polarization switch 27 Optics 28 Measuring light source 29 polarization beam splitters 31a-31g lens 32a,32b Deflector 33 Stabilization control unit 34 Deformable Mirror 35 measuring sensor 36 compensation unit 38 beam splitters 39 Camera input 40 beam combiners 41 λ / 4 plate 71 First scanning mirror 72 First drive 73 Scan lens 74 Second scanning mirror 75 Second drive 76 Third scanning mirror 77 Third drive 100 MINFLUX microscope 121 First polarizer 122 Second polarizer B Illumination light beam F Focus M measuring light beam P Pupillary plane R Rear aperture QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Zitierte Patentliteratur

[0000] US 9,719,928 B1

[0006] US 10,900,901 B2

[0006] US 10,908,089 B2

[0006] US 10,962,479 B2

[0006] US 11,255,791 B2

[0008] US 2023 / 0236401 A1 [0009, 0024, 0096] WO 2023 / 006176 A1

[0010] US 2023 / 0008453 A1

[0023] US 2022 / 0011559 A1

[0100] Zitierte Nicht-Patentliteratur

[0000] F. Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612

[0006] K. C. Gwosch et al. (2020) MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 [0006, 0023] Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy“ von C. Fallet, J. Caron, S. Oddos, J-Y. Tinevez, L. Moisan, G.Y. Sirat, P.O Braitbart und S. L. Shorte (2014, Proc. Of SPIE Vol. 9169, 916905-1 - 916905-6

[0009] M. Weber et al. (2021) MINSTED fluorescence localization and nanoscopy, Nat. Photonics 15, 361-366

[0010] R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478 [0011, 0023] Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612

[0023] R. Schmidt et al. (2021

[0100]

Claims

[1] Upgrade kit (10) for upgrading a light microscope (1) having a detector (3) for detecting light emissions from emitters (E) in a sample (2) to a MINFLUX microscope (100), wherein components of the upgrade kit (10) are designed to couple an illuminating light beam (B) into an illuminating beam path (4) of the light microscope (1), wherein the illuminating light beam (B) is designed to excite the emitters (E) in the sample (2) to emit light or to modulate a light emission of the emitters (E) caused by excitation light, wherein the upgrade kit (10) comprises the following: - a first beam scanning device (11) which is designed to displace the illuminating light beam (B) so that a focus (F) of the illuminating light beam (B) in the sample (2) can be displaced by means of the first beam scanning device (11), and / or - a beam-shaping device (12) which is designed to shape the illuminating light beam (B) such that an intensity distribution of the illuminating light with a local minimum is formed or adapted at a focus (F) of the illuminating light beam (B) in the sample (2), and / or - a stabilization system (13) designed to detect a disturbance acting on the MINFLUX microscope (100) and / or the sample (2) and to compensate for the detected disturbance or to correct an estimated position of an emitter (E) on the basis of the detected disturbance or to interrupt or abort a localization or tracking of an emitter (E) on the basis of the detected disturbance. [2] Upgrade kit (10) according to claim 1, characterized bythat at least one component of the upgrade kit (10) can be connected directly or indirectly to the detector (3) of the light microscope (1), so that the at least one component can be controlled on the basis of light emissions detected by the detector (3). [3] Upgrade kit (10) according to claim 2, characterized by that the upgrade kit (10) has a control unit (14) which can be connected to the detector (3) of the light microscope (1), wherein the control unit (14) is designed to control the at least one component on the basis of light emissions detected by means of the detector (3). [4] Upgrade kit (10) according to claim 2 or 3, characterized byin that the at least one component connectable to the detector (3) comprises the first beam scanning device (11), wherein the first beam scanning device (11) is controllable, in particular by means of the control unit (14), in such a way that the focus (F) of the illuminating light beam (B) in the sample (2) is displaced to illumination positions which form an illumination pattern around a position of the emitter (E) estimated on the basis of light emissions of an emitter (E) in the sample (2) detected by means of the detector (3). [5] Upgrade kit (10) according to claim 2 or 3, characterized byin that the at least one component connectable to the detector (3) comprises the beam-shaping device (12), wherein the beam-shaping device (12), in particular by means of the control unit (14), is controllable such that the intensity distribution of the illumination light in the sample (2) is adapted, in particular such that a shape, a position of the intensity distribution in the sample (2) and / or an orientation of the intensity distribution is adapted. [6] Upgrade kit (10) according to one of the preceding claims, characterized by that the upgrade kit (10) has an illuminating light source (15) which is designed to generate the illuminating light beam (B), or a connecting element (16) which is designed to couple an illuminating light beam (B) generated by an external illuminating light source (15) into an illuminating beam path (4) of the light microscope (1). [7] Upgrade kit (10) according to one of the preceding claims, characterized by in that the upgrade kit (10) has a support structure (17) which is designed to align components of the upgrade kit (10) relative to the illumination beam path (4) of the light microscope (1) at a defined position and / or which is designed to align the illumination light source (15) and / or the connection element (16) relative to the first beam scanning device (11) and / or to the beam shaping device (12) at a defined position. [8] Upgrade kit (10) according to one of the preceding claims, characterized byin that the upgrade kit (10) has a computing unit (18) that can be connected to the detector (3) of the light microscope (1), wherein the computing unit (18) is designed to estimate a position of the emitter (E) on the basis of light emissions of an emitter (E) in the sample (2) detected by the detector (3) for different positions of a local minimum of an intensity distribution of the illumination light in the sample (2) or for different shapes and / or orientations of an intensity distribution of the illumination light (B) with a local minimum. [9] Upgrade kit (10) according to one of the preceding claims, characterized by that the upgrade kit (10) has a coupling element (19) which is designed to couple at least one component of the upgrade kit (10) to a light input (5) of the light microscope (1), so that the illuminating light beam (B) is coupled into the illuminating beam path (4) of the light microscope (1). [10] Upgrade kit (10) according to one of the preceding claims, characterized by that the upgrade kit (10) has a housing (20), wherein the housing (20) surrounds components of the upgrade kit (10), in particular in a light-tight manner. [11] Upgrade kit (10) according to one of the preceding claims, characterized by that the first beam scanning device (11) has a maximum scanning rate of 5 kHz or more, in particular 10 kHz or more, further in particular 100 kHz or more, further in particular 1 MHz or more, even further in particular 10 MHz or more. [12] Upgrade kit (10) according to one of the preceding claims, characterized by that the first beam scanning device (11) comprises at least one acousto-optical deflector. [13] Upgrade kit (10) according to one of the preceding claims, characterized byin that the upgrade kit has an axial beam scanning device (21) which is designed to deflect the illuminating light beam (B) in such a way that a focus (F) of the illuminating light beam (B) formed by an objective (6) of the light microscope (1) in the sample (2) is deflected by means of the axial beam scanning device (21) parallel to an optical axis (O) of the objective (6). [14] Upgrade kit (10) according to one of claims 1 to 13, characterized by that the beam-shaping device (12) has a light modulator (22) which is designed to spatially modulate the illuminating light beam (B) in its phase and / or in its amplitude. [15] Upgrade kit (10) according to one of claims 1 to 14, characterized byin that the beam-shaping device (12) comprises a biaxial crystal (23), a first polarization switching unit (24) and a second polarization switching unit (25), wherein the first polarization switching unit (24) is arranged in an illumination beam path in front of the biaxial crystal (23), and wherein the second polarization switching unit (25) is arranged in the illumination beam path behind the biaxial crystal (23). [16] Upgrade kit (10) according to one of the preceding claims, characterized by that at least one component of the upgrade kit (10) is designed to couple a measuring light beam (M) for the stabilization system (13) into the light microscope (1) in addition to the illuminating light beam (B). [17] Upgrade kit (10) according to claim 16, characterized bythat the at least one component of the upgrade kit (10) is designed to couple the measuring light beam (M) together with the illuminating light beam (B) into the illuminating beam path (4) of the light microscope (1), in particular wherein the upgrade kit has a beam combiner (40) which is designed to combine the measuring light beam (M) and the illuminating light beam (B). [18] Upgrade kit (10) according to one of the preceding claims, characterized by that the stabilization system (13) has a measuring sensor (26) which is designed to detect the disturbance. [19] Upgrade kit according to one of the preceding claims, characterized byin that the upgrade kit has an optic (27) which is designed to image the collimated illuminating light beam (B) onto a first scanning mirror (71) of a second beam scanning device (7) of the light microscope (1), wherein the first scanning mirror (71) is arranged in a pupil plane (P) conjugated to a rear aperture (R) of the objective (6) of the light microscope (1). [20] MINFLUX microscope (100) comprising the components of the upgrade kit (10) according to one of claims 1 to 19 and a light microscope (1), wherein the light microscope (1) has an objective (6) and a detector (3), wherein the objective (6) is designed to focus the illuminating light beam (B) coupled into the light microscope (1) and modulated in its phase and / or amplitude into a sample (2) so that an intensity distribution of the illuminating light with a local minimum is formed in the sample (2), and wherein the detector (3) is designed to detect light emissions from emitters (E) in a sample (2). [21] MINFLUX microscope (100) according to claim 20, characterized byin that the MINFLUX microscope (100) has a computing unit (8), wherein the computing unit (8) is designed to estimate a position of an emitter (E) in the sample (2) on the basis of light emissions detected by the detector (3) for different positions of the local minimum or for different shapes and / or orientations of the intensity distribution. [22] MINFLUX microscope (100) according to claim 20 or 21, characterized by that the light microscope (1) is a confocal laser scanning microscope or a STED microscope. [23] MINFLUX microscope (100) according to one of claims 20 to 22, characterized by that the detector (3) of the light microscope has an avalanche photodiode or a hybrid photodetector. [24] MINFLUX microscope (100) according to one of claims 20 to 23, characterized bythat the light microscope (1) has a second beam scanning device (7) which is designed to shift the illuminating light beam (B) coupled into the illuminating beam path (4) of the light microscope (1). [25] Method for upgrading a light microscope (1) with a detector (3) for detecting light emissions from emitters (E) in a sample (2) to a MINFLUX microscope (100) by means of an upgrade kit (10) according to one of claims 1 to 19, wherein an illuminating light beam (B) is coupled into an illuminating beam path (4) of the light microscope (1) by means of at least one component of the upgrade kit (10). [26] Method according to claim 25, characterized bythat the light microscope (1) is supplemented by a software component or a software function of the light microscope (1) is enabled, wherein the software component is designed to control at least one component of the upgrade kit (10) and / or the detector (3) of the light microscope (1). [27] Method for controlling a MINFLUX microscope (100) according to one of claims 20 to 24, wherein at least one component of the upgrade kit (10) according to one of claims 1 to 19 is controlled on the basis of light emissions detected by the detector (3) of the light microscope (1). [28] Method for locating or tracking emitters (E) in a sample (2) by means of a MINFLUX microscope (100) according to one of claims 20 to 24, wherein a sample (2) is illuminated by means of the illuminating light beam (B), wherein the illuminating light forms an intensity distribution with a local minimum in the sample (2), and wherein a position of the emitter (E) is estimated on the basis of light emissions of an emitter (E) in the sample (2) detected by means of the detector (3) of the light microscope (1). [29] A computer program comprising program instructions which cause a control unit (14) to carry out the method according to claim 27. [30] A computer program comprising program instructions that cause the MINFLUX microscope (100) according to any one of claims 20 to 24 to carry out the method according to claim 28.

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