WO2025016759A1 - Minflux or sted minflux microscope having increased temporal-spatial resolution, and corresponding method - Google Patents

Abstract

In order to improve the spatial-temporal resolution of the localization of individual emitters, in particular individual fluorophores, which is achieved when carrying out MINFLUX methods or STED MINFLUX methods by means of a corresponding microscope (1), an improved method and an improved microscope (1) are proposed. A measuring device (13) of the microscope (1) determines each current location or a current direction of the reference light beam (R) deflected or displaced by a beam scanner (10, 18), and a signal (S) indicative of an associated current position of the focus point formed by illumination light (B) in the sample is output as a function of the current location or the current direction. The signal (S) is used to correct the control of the deflection device (9) of the microscope (1) and/or to improve the localization accuracy by taking account of the measured focus positions in an evaluation of MINFLUX or STED MINFLUX measurement data.

Description

MINFLUX OR STED-MINFLUX MICROSCOPE WITH INCREASED TEMPORAL-SPACIAL RESOLUTION AND CORRESPONDING METHOD

Technical field of the invention

The invention is directed to a microscope and to MINFLUX methods or STED-MINFLUX methods that can be carried out with the microscope for determining the location of individual emitters with high temporal and spatial resolution or for tracking emitters with high temporal and spatial resolution. The emitters in question can in particular be individual fluorophores.

State of the art

A microscopy method that is still relatively new in the state of the art is MINFLUX nanoscopy. MINFLUX nanoscopy is a method of localization microscopy. The localization of individual emitters, in particular individual fluorophores, is carried out using structured illumination light distributions, whereby the illumination light is scattered at the emitters or whereby the illumination light excites them to luminescence, in particular to fluorescence, and in such a general sense is an excitation light. The fundamental special feature of MINFLUX nanoscopy is that the illumination of the emitters, in particular the excitation of the fluorophores, is carried out in such a way that an emitter to be localized or an emitter whose movement in a sample is to be tracked is placed at different positions close to one or at a minimum of the excitation light distribution, which is ideally a zero point, whereby the excitation light distribution has an intensity increase region adjacent to the minimum. This ensures that the amount of photons scattered by the emitter or the fluorescence photons or each individual such photon contains a lot of information about the current position of the respective emitter. This is particularly true for applications in which the movement of fluorophores is to be tracked over time. In order to obtain the information, the light emitted from the area of the minimum is detected. Tracking an individual emitter using an excitation minimum is known, for example, from patent DE 10 2011 055 367 B4, and localizing an individual fluorescent molecule using an intensity distribution of excitation light with a local intensity minimum is known from German patent DE 10 2013 114 860 B3. On this basis, a number of refinements for information acquisition have been developed that enable localization of the fluorophores with an uncertainty in the range below 2 nm and tracking of the movement of individual fluorophores both within a range of a few nanometers and within a range of a few micrometers. A detailed but not exhaustive description of MINFLUX nanoscopy can be found in the publication “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes” (F. Balzarotti et al., Science 355, 606-612 (2017), DOI:10.1126/science. aak9913 ). Non-iterative MINFLUX localization and tracking of individual fluorophores using MINFLUX are experimentally demonstrated. The concept of iterative MINFLUX is also presented. Basically, in order to be able to localize a fluorophore using MINFLUX nanoscopy as described in the publication mentioned, the intensity minimum or zero point must be placed at a number of positions relative to the position of the fluorophore. To do this, in a preparatory step, a current position of the fluorophore must be estimated with an initial, lower level of accuracy or must be known. This can be done, for example, using conventional localization microscopy (PALM, STORM) or other known methods. An intensity distribution of excitation light with a central minimum, for example in the shape of a donut, as known from STED microscopy, is then placed at a known position that is chosen such that the estimated position of the fluorophore is close to the minimum of the intensity distribution. The fluorescence response of the fluorophore is measured. The same is usually done for several other positions of the intensity distribution that are placed around the estimated position and, if necessary, also for the position that corresponds to the estimated position. The set of all such scanning positions forms a set of scanning positions. The position of the fluorophore is determined with greater accuracy by means of a ratiometric evaluation of the intensity ratios. This more precisely determined position can now be used as the starting position for repeating the sequence of the aforementioned steps, whereby the positions of the minimum of the intensity distribution of excitation light can be placed closer to the estimated position of the fluorophore in the case of a fluorophore localization. This process can be continued iteratively. If the movement of an emitter is to be tracked, it may be useful not to reduce the distance of the positions at which the intensity minimum is placed from the position estimated in the previous step from step to step. The process is carried out as described in the publication mentioned using a microscope that contains both a piezo scanner and electro-optical deflectors (EOD). Using this combined scanning device, an image field of approximately 20 pm x 20 pm can be addressed. Using the EOD alone, an image field of about 2 pm x 2 pm can be addressed, whereby a high-speed displacement is only possible in a field of about 300 nm x 300 nm is possible. Both when localizing and when tracking a particle, the scanning positions of a set of scanning positions are controlled exclusively using the high-speed EOD scanning. A combination of piezo scanner and EOD can then be used to shift the center. The movement of individual molecules that are only mobile in a narrowly defined area, namely in an area covered by a set of scanning positions, can be carried out using a fixed set of scanning points.

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

The international publication WO 2021/122407 A1 discloses a method for correcting, in particular, short-term, transient interference in a laser scanning microscope and a laser scanning microscope with a device for detecting an interference, which is designed to carry out an interference correction method. The microscope can contain a scanner system that has EOD and a scanner that serves for a comparatively slow but possible coarse positioning of the focused excitation light over a large image field. The scanner can be designed in a quad configuration. In particular, a method for correcting interference during image recordings according to the MINFLUX principle is disclosed. The invention disclosed in the publication solves the problem of achieving a high level of localization accuracy even if interference occurring during data recording according to the MINFLUX method cannot be avoided by determining the size of an interference during data recording for a localization, from which a weighting factor is derived that is taken into account when determining the location of a dye molecule; Alternatively, the data acquisition is interrupted and continued depending on the size of the disturbance. The size of the disturbance can be detected using different types of sensors. In one of the embodiments, the size of the disturbance is detected by passing a measuring light beam provided by an auxiliary light source along a section of the beam path together with the beam of an excitation light and/or a fluorescence prevention light and is finally directed to a position-sensitive detector. In this arrangement, disturbances that are transmitted to the beam-guiding elements, for example of the excitation light, and lead to a change in the position of the focused excitation light in the sample, are also transmitted to the measuring light beam. In a further development, the signal from the measuring device for detecting a disturbance can be used to carry out a beam position correction, in particular by applying a corrected control signal to electro-optical deflectors of a beam deflection device, so that a scanning position of an intensity minimum of the excitation light in the sample is held at its target position. Specifically, a device is described and shown in a figure in which a measuring light beam provided by an auxiliary light source is coupled into the beam path of the excitation light by means of a first dichroic beam splitter and is coupled out again at another point with the help of a further dichroic beam splitter, wherein in the section between the beam splitters the measuring light beam runs in the beam path of the excitation light. Before reaching the first dichroic beam splitter, the excitation light beam runs in the beam direction through an electro-optical deflection device, which has two electro-optical deflectors connected in series to deflect the excitation light beam in the horizontal and vertical directions, and after passing the further dichroic beam splitter through another scanner, for example in a quad configuration, i.e. a special arrangement with four tilting mirrors. The beam path section common to the measuring light beam and the excitation light beam, on the other hand, does not include a deflection device or scanner.

From the publication “Digital light deflection and electro-optical laser scanning for STED nanoscopy” (Jonas Marquard, dissertation, Ruperto-Carola University of Heidelberg, 2017, DOI: 10.11588/heidok.00023956) another microscope is known which has an electro-optical deflection device and a quad scanner in the beam path of an excitation light. In this microscope, the excitation beam path is simultaneously the beam path of a depletion or STED light, which emerges from an optical fiber together with the excitation light. In addition, the electro-optical deflection device consists only of an electro-optical deflector (EOD), which is operated in resonance and is designed to deflect the excitation beam only in one direction, on the fast axis. The publication states that both scanning with an EOD and scanning with a mechanical scanner results in jitter in the sense of a time-varying deviation of the actual position from a target position. The size of the jitter depends on the scanning speed. With a mechanical scanner, the inertia of the mass causes disturbances during fast scanning, while various drifts can occur during slow scanning. The experimental determination of the respective jitter shows that the jitter of the EOD and the mechanical scanner in the existing configuration are in the same order of magnitude, namely in the range of single-digit values in nanometers, and can also be neglected in connection with high-resolution STED measurements.

The publication US 2002/179828 A1 is directed at a device for scanning a sample, with which image data that is largely free of image errors can be generated even when scanning a sample quickly. The device has, among other things, a device for determining an actual signal for each scan point from the setting of a scanning device. It also has a processing unit for calculating a display signal and an image point position from the actual signal and/or the target signal provided by a control unit of the scanning device and the detection signal of a detection device, as well as a processing unit for assigning the display signal to the image point position.

The German patent DE 10 2007 008 009 B3 describes a scanning microscope that has a beam deflection device and a detection device, wherein the detection device detects at least one piece of information about a frequency and/or a phase and/or an amplitude of the beam deflection device. The patent is directed at a method in which the measurement signal of the detection device is processed and, after processing, is used either to control the deflection device or to assign image data to positions in the sample. A direct digital synthesis method is used for the processing; according to the standard technical definition, this is a method in which periodic, band-limited signals are obtained. The method according to the patent solves a problem in particular with fast resonant scanning, namely that a phase shift occurs between the control signal of a resonant scanner and the course of the scanning by the scanner, depending on the control frequency and the quality of the resonator. The detection device can be a device integrated in a scanner. In other embodiments, an auxiliary light beam is directed to the back of a galvanometer mirror. The auxiliary light beam reflected at the back of the mirror and deflected according to the position of the galvanometer mirror is directed to a position-sensitive detector, the signal of which forms the measurement signal.

From the publication “Jitter suppression for resonant galvo based high-throughput laser scanning systems” (Jianian Lin, Opt. Express 28, 26414-26420 (2020), doi: 10.1364/OE.402883) a method and a device for reducing the jitter occurring during resonant scanning are known. A device is described which is designed for the purpose of analyzing the jitter of a beam deflection device. A collimated laser beam is directed onto a mirror of a resonant galvo scanner and from the mirror through a focusing Lens in the direction of a photodiode, in front of which a slit is placed in the focus of the lens, so that the photodiode regularly detects light and emits a signal when the scanner passes through zero, when the light is deflected by the scanner at a right angle. Light from a reference laser is directed via a lens and the scanner's mirror to another detection device consisting of a photodiode and a slit. The reference laser is switched in such a way that a reference signal is only received during a single zero crossing of an oscillation cycle. This reference signal is fed back to the control of the resonant scanner.

The publication US 2021/239452 A1 is directed to a device for measuring an angle of a collimated coherent light beam, a beam stabilization device and a laser scanning microscope with a beam stabilization device. The measuring device aims to measure an angle so precisely that angular changes that lead to deviations in the position of an illumination focus in a sample in the order of 1 nm after focusing through an objective can be detected. A microscope is described which has a laser whose light is collimated and directed to a beam splitter. Part of the light passes through the beam splitter and is focused into a sample through an objective, another part is directed through the beam splitter in the direction of the measuring device for measuring an angle of the collimated coherent light beam. The beam stabilization device and its function are not explained in more detail.

From the publication “Pixel hopping enables fast STED nanoscopy at low light dose” (Britta Vingon, Opt. Express 28, 4516-4528 (2020), DOI: htps://doi.Org/10.1364/OE.385174) a STED microscope with a scanning device that has both a galvo scanner and two electro-optical deflectors (EOD) is known. The EOD and the galvo scanners are arranged in a beam path for excitation and STED light as well as for fluorescence light in such a way that a total shift of the observed spot in the sample results from the sum of the shifts caused by the EOD and galvo scanner. The galvo scanners enable the scanning of a large scan field, the EODs enable fast fine adjustment. In order to be able to scan quickly and accurately at high speed within a large scanning field, the combined scanning system is controlled as follows: A control device sends a control signal to the scanning device, whereby the signal is divided in such a way that the galvo scanners receive a target signal; in addition, an actual signal is tapped from the galvo scanners at a monitoring output, which is provided by the position sensors implemented at the factory; the target signal is not only output to the galvo scanners, but also to comparators, into which, in addition to the target signal, the actual signal tapped at the monitoring output is fed; the difference between the target signal and the The actual signal is sent to the EOD as a correction signal so that, in conjunction with the deflection by the EOD, the target scanning point is reached quickly and precisely, regardless of the inertia-related delay in setting the galvo scanner. The device is used to locally vary the scanning speed, ie the duration during which a pixel is addressed or scanned, when performing a raster scan during a scan.

The publication “MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope” (Schmidt, R. et al., Nat Commun 12, 1478 (2021). htps://doi.org/10.1038/s41467-Q21-21652-z ) now describes the implementation of MINFLUX procedures with a commercially available microscope. In addition to EOD, the microscope has a quad scanner made up of four galvo scanners (two galvo scanners per deflection direction). With the quad scanner, in conjunction with the use of an objective with 100x magnification, an image field of approximately 80 pm x 80 pm can be addressed, so that the microscope is also suitable for taking confocal images or STED microscopic images. When performing a MINFLUX procedure, the sampling points of a single set of sampling points are each addressed using the EOD. The adjustment of the position of the center of a set is then carried out using the galvo scanners. The microscope is used to track biomolecules over a period of time in the range of seconds with a time resolution in the range of 100 microseconds. The publication mentions that the accuracy of the position determination during the tracking of a particle is reduced by the particle moving during the determination of the individual positions.

Investigations by the applicant have now further shown that the position determination when using beam scanners is additionally reduced by the fact that during the measurement of fluorescence at a set of scanning points, rapid irregular or irregular-seeming changes in the state of the beam scanner induce a rapid irregular or at least irregular-seeming movement of the illumination light distribution in the sample. Both these rapid irregular changes in the state of the beam scanner and the rapid irregular movements of the illumination light distribution in the sample are referred to in this application as jitter. The jitter can superimpose a controlled or regulated displacement of the illumination light and it can also occur when the beam scanner is nominally aimed at a fixed position. It can occur in particular when using a beam deflection device that has galvo scanners for addressing a large image field and EOD for addressing the set of scanning points for MINFLUX localization. In this case, in particular rapid irregular or irregular-seeming movements of the mirrors of the galvo scanners that move during the MINFLUX scanning is nominally in a rest position, which induces irregular or irregular-seeming movement of the illumination light. In principle, such jitter can also occur in other beam scanner systems, especially if they are designed to scan a large image field in the microscope in question. Such scanners for large image fields usually have moving components, for example moving mirrors. The effect of the jitter is greater the shorter the effective measurement time at the individual points of the set of scanning points. Consequently, the jitter in the microscopes known from the state of the art limits the spatial-temporal resolution.

task of the invention

The invention is therefore based on the object of providing solutions which, when using a microscope with a beam scanner when carrying out MINFLUX methods or STED-MINFLUX methods, enable an improved spatial-temporal resolution of the localization of individual emitters, in particular individual fluorophores.

Solution

The object of the invention is achieved by a microscope having the features of independent patent claim 1 and by a method having the features of claim 20. Dependent claims 2 to 19 relate to preferred embodiments of the microscope, dependent claims 21 and 22 relate to preferred embodiments of the method.

definitions

In this application, emitters are understood to mean objects which, when illuminated with excitation light, can be considered as point light sources with regard to the measurements according to the invention. The light emitted by the object acting as a point light source can, for example, be scattered light resulting from elastic scattering such as Rayleigh scattering or inelastic scattering such as Raman scattering, or it can be luminescent light, in particular fluorescent light. It is essential for an emitter that it emits light immediately or with a small time delay in response to illumination. When movements of the emitter are to be tracked, the maximum time delay is related to the temporal resolution with which the movements of the light-emitting particles or light-emitting units are to be tracked and to the speed with which the particles or units move in the sample. The time delays can be up to about 10 ps, but are usually in the Range up to several tens of nanoseconds, frequently in the range of 1 to 10 ns and, if the emission is scattered light, at zero. In this application, the term fluorescence emitter is used to describe emitters that act as fluorescence point light sources. Emitters can be, for example, metallic nanoparticles. The more specific term fluorescence emitter includes, for example, individual fluorescent dye molecules or their fluorescent chemical groups. Instead of dyes, other fluorescent units such as quantum dots or up-converting nanoparticles can also be used for marking. In the context of the application, excitation light is therefore understood to mean not only fluorescence excitation light, but generally light that causes light to emanate from an emitter.

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

In this application, emission suppression light is understood to mean light that prevents, reduces or completely suppresses the emission of an emitter. In particular, the emission suppression light can be stimulation light or STED light.

In this application, STED light is understood, as is generally the case, to be light that suppresses fluorescence emission by exciting stimulated emission. In the context of this application, STED light is a special form of emission suppression light.

In this application, MINFLUX methods are used to summarize localization and tracking methods for individual emitters in which light distributions of illumination light that excites light emissions from the emitter are generated at the focus in the sample, the light distributions having a local minimum, and in which the position of an individual emitter is determined by detecting light emissions from the emitter for different positions of the minima of the respective light distributions or for different such light distributions in a close range of the emitter, the smaller the distance between the emitter and the minimum of the light distribution, the less light is emitted by the emitter.

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

The term STED-MINFLUX is used in a generalized sense and should not be understood to mean that the emission suppression light must be STED light.

In MINFLUX microscopy and STED-MINFLUX microscopy, the sample is illuminated with focused illumination light, whereby the illumination light forms an intensity distribution with a central intensity minimum, in particular an intensity zero, around a geometric focus in the sample. The position of an individual emitter relative to the central minimum then determines the intensity or the photon emission rate of the emitter's light emissions, which are used to determine the position. Intensity distributions with a central minimum can be achieved in particular by phase modulation of the illumination light by a phase filter or a spatial light modulator (SLM) in the illumination beam path, focusing the illumination light using an objective lens and If necessary, additional circular polarization of the illumination light can be generated. The intensity distribution is then created at the geometric focus by interference.

When the present application refers to the intensity distribution of the illumination light being shifted at scanning positions, this means that the intensity distribution of the illumination light is shifted relative to the sample such that the focal point at which the central minimum is formed is arranged at a respective scanning position.

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

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

In this application, a 3D minimum is understood to mean a minimum that is surrounded by areas of increasing intensity in all spatial directions. A 3D minimum can be obtained, for example, by superimposing two light beams, whereby in each pupil the wave front of one of the two is phase modulated by means of a so-called annular phase plate and that of the other is phase modulated by means of a vortex phase plate. A 3D minimum can also have a donut-shaped intensity distribution in a given plane.

In this application, the term localization describes a method in which a position of an individual emitter in a sample is determined. In contrast to conventional light microscopic methods, in particular, no optical imaging of the sample is required. Rather, in the MINFLUX localization method according to the invention, light emissions from the emitter are detected for different positions of the intensity distribution of the illumination light and the position of the emitter is determined from these, for example calculated using a position estimator. This process can be carried out successively for several individual emitters, and the determined positions can be displayed in a localization map, which can produce a high-resolution light microscopic image. Both localizations that are carried out using a MINFLUX method and those that are carried out using a STED-MINFLUX method are referred to as MINFLUX localizations for the purposes of this application.

Tracking an emitter means determining the trajectory of an emitter moving in the sample, in particular by repeatedly localizing it in time.

In the context of the present specification, a beam scanner is a device that variably deflects or shifts a light beam. A shift in a light beam can be achieved, for example, by means of a beam scanner composed of several individual beam scanners, each of which variably deflects the light beam. A beam scanner can in particular be designed to shift a beam of the illumination light, i.e. an illumination light beam, by means of an optical component or by means of several optical components, so that the focus of the illumination light on or in the sample is shifted in at least one spatial direction. The focus can be shifted in particular in two spatial directions, further in particular in a focal plane that is perpendicular to an optical axis of an objective. The spatial directions can be defined in a wide variety of coordinate systems, e.g. in Cartesian coordinates (e.g. x and y directions), in polar coordinates or in spherical coordinates (e.g. radial and azimuthal directions). The beam scanner can comprise, for example, mechanical scanners, in particular scanners based on tilting mirrors each connected to a galvo motor, electro-optical deflectors or acousto-optical deflectors.

In the context of this application, a galvo scanner is understood to mean a module with a galvo drive and a tilting mirror connected to it and movable by the galvo drive. The drive can be designed in such a way that the angular positions of the tilting mirror can be adjusted, or in such a way that the tilting mirror performs a periodic rotary movement. In the context of this application, the term galvo scanner therefore includes resonant galvo scanners and galvo scanners in which the angular positions of the tilting mirror can be adjusted; these are referred to as adjustable galvo scanners in the context of this application. The tilting mirror can be flat or curved, for example concavely curved. A galvo scanner can also have elements other than a galvo motor and tilting mirror.

In the context of this application, a galvoscanner system is defined as a scanning unit that has one or more galvoscanners, which as a whole is designed to shift and/or deflect a light beam in one or more spatial directions. Accordingly, in In the context of this application, a MEMS scanner system is a scanning unit having one or more MEMS scanners, and a piezo scanner system is a scanning unit having one or more piezo scanners, each of which is configured as a whole to shift and/or deflect a light beam in one or more spatial directions.

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

In the context of the present application, the deflection device refers to the totality of all beam scanners in the beam path of the illumination light.

Description of the Invention

The microscope according to the invention is designed to carry out a MINFLUX method and/or a STED-MINFLUX method. In the MINFLUX method according to the invention and/or the STED-MINFLUX method according to the invention, the position of an individual emitter in a sample is determined and/or an emitter is tracked.

The microscope according to the invention for carrying out a MINFLUX method and/or a STED-MINFLUX method has a light source for generating an illumination light. It also has optical elements that form an illumination light beam path through which the illumination light is guided as an illumination light beam from the light source into a sample area. In the illumination light beam path, it has optical means for focusing the illumination light into a focus area in a sample in the sample area such that in the focus area of the illumination light, an intensity minimum of the illumination light in a focus point is bordered on both sides by intensity increase areas in at least one direction. It also has a deflection device in the beam path of the illumination light, which comprises a first beam scanner. The deflection device is designed to position the focus point in the sample at several positions in the sample. The microscope has a detection device for detecting emissions from the focus area of the illumination light. From state-of-the-art microscopes for performing MINFLUX methods or STED-MINFLUX The microscope according to the invention differs from the method by a measuring device for detecting a position and/or a direction of a reference light beam which is deflected and/or displaced by the first beam scanner, wherein the measuring device is designed to output a signal at a signal output as a function of the position and/or the direction of the reference light beam, which signal is indicative of a position of the focal point in the sample.

The optical means for focusing the illumination light into a focus area in a sample may in particular comprise an objective lens.

The fact that in the focus area of the illumination light, intensity increase areas border on both sides of an intensity minimum of the illumination light in a focus point in at least one direction includes in particular the cases in which a 2D minimum is formed in the focus area in the sample, that a 3D minimum is formed and that in a plane that contains the optical axis and thus the focus point, a leaf-shaped intensity minimum, ideally a zero point, with adjacent intensity increase areas is formed. The optical means for focusing the illumination light in a focus area in a sample comprise one or more elements that cause or cause intensity increase areas to border on both sides of an intensity minimum of the illumination light in a focus point in at least one direction in the focus area of the illumination light. For example, such an element can comprise a vortex phase plate to form a 2D minimum, or an annular phase plate to form a 3D minimum, or a phase plate with a phase jump of 180° along a diameter to form a 1D minimum. Corresponding elements influencing the phase can also be implemented as adjustable spatial light modulators. Adjustable amplitude modulators, for example, can also be used to generate corresponding intensity distributions in the focus area.

As explained above, the microscope according to the invention has a deflection device in the beam path of the illumination light, which has a first beam scanner. This first beam scanner is designed to position the focus point in the sample. The first beam scanner can have several individual beam scanners, i.e. several individual deflection units or displacement units, for example a galvo scanner system according to the definition given above. The first beam scanner can also be the only beam scanner that the deflection device of the microscope has. The term "the first beam scanner" serves to make it easier to distinguish in cases in which the deflection device of the microscope has several beam scanners. The fact that the first beam scanner is designed Positioning the focus point in the sample does not mean that the positioning of the focus point in the sample is carried out exclusively by means of the first beam scanner, but it means that the current position of the focus point in the sample is at least partly determined by the current setting of the first beam scanner. The fact that the focus point is positioned in the sample by the first beam scanner also means that the current setting of the first beam scanner changes randomly, so that the current position of the focus point in the sample changes according to the random change in the setting of the first beam scanner.

The first beam scanner can have a mechanical scanner or be designed entirely as a mechanical scanner. The mechanical scanner of the first beam scanner can in particular have a deflection unit with at least one connected mirror that deflects all of the illumination light. Alternatively or additionally, this mechanical scanner can have one or more movable, in particular rotatable elements through which the illumination light shines, such as a prism or several prisms. Such prism-based mechanical scanners are known, for example, from the publication “Laser scanners with rotational Risley prisms: Exact scan patterns” (Duma, Virgil-Florin and Schitea, Alexandru; Proceedings of the Romanian Academy - Series A; Volume 19, Number 1/2018, pp. 53-60). The mechanical scanner of the first beam scanner can, for example, contain MEMS scanners (MEMS: Micro-Electro-Mechanical Systems), piezo scanners or galvo scanners or can be designed as a MEMS scanner system, piezo scanner system or galvo scanner system. Piezo scanners and MEMS scanners can be designed such that a mirror can be tilted about two mutually orthogonal axes. The first beam scanner can in particular have or be designed from resonant mechanical scanners with at least one connected mirror that deflects all of the illumination light, for example resonant or adjustable galvo scanners or resonant or adjustable MEMS scanners. In the context of this application, adjustable scanners are understood to mean scanners that enable targeted addressing of focus positions, i.e. in which the displacement and/or deflection they cause can be directly adjusted.

The first beam scanner can comprise electro-optical deflectors (EOD) or acousto-optical deflectors (AOD) or can be formed entirely from EOD or AOD or from EOD and AOD. In preferred embodiments, the first beam scanner is formed from mechanical scanners, in particular from adjustable galvo scanners, piezo scanners and/or MEMS scanners, and non-mechanical scanners, in particular AOD or EOD. In order to be able to carry out a MINFLUX or STED-MINFLUX method using an objective through which the illumination light is guided into the sample, it is important that the illumination light beam is only tilted by the deflection device, i.e. by the first beam scanner or by the first beam scanner and further beam scanners, in the illumination light beam in the pupil of the objective and is not displaced or at least displaced as little as possible, since a displacement of the illumination light beam would change the shape of the intensity distribution of the illumination light in the focus area and thus indirectly also the position of the focus point, i.e. the local minimum of the intensity distribution. Beam scanners that deflect the illumination light beam in such a way that the above-mentioned condition is met can, for example, have a mirror that can be tilted about two axes and is located in a pupil, i.e. in a plane in which the pupil of the objective is imaged, or they can have two mirrors that can each be tilted about one axis and are each placed in a pupil. In a preferred arrangement in the microscope according to the invention, the first beam scanner has two separate tiltable mirrors for shifting the focus point in the sample in one direction; more preferably, it has two tiltable mirrors for each of two mutually orthogonal shift directions. The latter arrangement is known from the prior art (see, for example, WO 2021/122407 A1) as a scanner in quad configuration or also as a quad scanner. Mechanical scanners based on galvo scanners are preferred when large image fields are to be addressed, for example in order to be able to record a confocal overview image or to be able to shift the focal point of the illumination light to different areas within the larger image field, among other things because they typically enable a larger usable scanning area compared to other scanner types due to their properties such as their usable beam diameter and their maximum deflection angle. Another preferred embodiment with two separate tiltable mirrors for shifting the focal point in the sample in one direction has two MEMS scanners, whereby the mirrors of the MEMS scanners can each be tilted about two mutually orthogonal axes. Compared to a quad scanner, this embodiment requires fewer reflective surfaces.

In preferred embodiments, the deflection device of the microscope can have further beam scanners in addition to the first beam scanner. All beam scanners, the first beam scanner and the further beam scanners, can be designed as mechanical scanners. In preferred embodiments, the microscope can have a further beam scanner in the beam path of the illumination light, which is preferably designed and configured to shift the focal point of the illumination light in the sample in conjunction with the deflection or shifting by the first beam scanner. It is possible that different scanning positions are addressed or different scanning paths are scanned by combining controlled deflections by the first beam scanner and controlled deflections by the further beam scanner, and also that different scanning positions are addressed or different scanning paths are scanned by only controlled deflections by the further beam scanner, while the first beam scanner is controlled in such a way that it is, as far as achievable, in a static position, that is, in the case of a mechanical scanner, that it is, as far as achievable, in a rest position, or that it is controlled in such a way that it specifically carries out very small setting changes, in the case of a mechanical scanner, very small movements, which, in the case of an unchanged setting of the further beam scanner, would cause a shift of the focal point of the illumination light in the sample around a fixed position. Such control of very small movements can have the advantage of reducing irregularities of a shift of the focus point in the sample compared to irregularities that occur when a static adjustment of the first beam scanner is sought. This is especially true if the first beam scanner comprises adjustable galvo scanners and/or adjustable MEMS scanners.

The deflection device is preferably designed such that the focal point can be quickly displaced within a close range with dimensions that correspond approximately to the extent of a diffraction-limited illumination spot. The fact that the focal point can be quickly displaced also includes cases in which the rapid displacement of the focal point in the sample is induced by rapid irregular or irregular-seeming changes in the state of the deflection device, for example the first beam scanner. Furthermore, the deflection device is preferably designed to displace the focal point of the illumination light in the sample over larger distances, for example over 10 pm or 20 pm or 100 pm or 500 pm. In preferred embodiments, the deflection unit therefore has a plurality of beam scanners, wherein one of the beam scanners, preferably the further beam scanner, is configured to quickly shift the focal point of the illumination light so that, for example, a set of scanning positions for a MINFLUX localization can be addressed or covered in less than 1 ms, and another of the beam scanners, preferably the first beam scanner, is configured to shift the focal point of the illumination light in the sample over larger distances, for example over 10 pm or 20 pm or 100 pm or 500 pm.

In preferred embodiments, the further beam scanner, i.e. a different beam scanner than the first beam scanner, is an adjustable beam scanner. Further preferably, the further beam scanner has a high bandwidth and is designed to quickly adjust the focal point of the illumination light on a set of scanning positions for a MINFLUX localization. to shift. In further preferred embodiments, the bandwidth of the further beam scanner is greater than the bandwidth of the first beam scanner, for example twice as large, five times as large, ten times as large or a hundred or more than a hundred times as large. Accordingly, the response times of the deflection unit, i.e. the time periods that a scanner requires to be moved from one angular position to another angular position, are preferably smaller than those of the first beam scanner, for example they are only 50%, 20%, 10%, 1% or less than 1% of that of the first beam scanner.

If the deflection device has a first beam scanner and a further beam scanner, in preferred embodiments the further beam scanner has one or more AODs or EODs or is formed from one or more AODs or EODs. AODs and EODs have the advantage that the adjustment of the deflection angle is not limited by the inertia. Both types of deflectors thus enable extremely fast displacement to any point within the range that can be addressed by the further beam scanner. The bandwidth of an EOD in conjunction with suitable control electronics can be, for example, 1 MHz or 5 MHz or higher and thus enables the focus point to be shifted within less than 1 ps.

The additional beam scanner of the microscope can also have a mechanical scanner or be designed entirely as a mechanical scanner. The mechanical scanner of the additional beam scanner can in particular have a deflection unit with at least one connected mirror that deflects all of the illumination light. The mechanical scanner of the additional beam scanner can, for example, contain MEMS scanners, piezo scanners or galvo scanners or can be designed as a MEMS scanner system, piezo scanner system or galvo scanner system. Piezo scanners and MEMS scanners in particular can be designed so that a mirror can be tilted about two mutually orthogonal axes.

If the further beam scanner of the microscope has mechanical scanners with a deflection unit with at least one continuous mirror that deflects all of the illumination light, mechanical scanners with a small aperture can preferably be selected for this purpose. The microscope then preferably has an objective. The continuous mirror that deflects all of the illumination light can preferably be arranged in a pupil. The microscope can then have an expansion unit in the illumination light beam after the further beam scanner, with which the illumination light beam is adapted to the aperture of an objective. A mirror of a mechanical scanner with a small aperture can be small and therefore have a low mass and consequently a low moment of inertia, so that the bandwidth of the mechanical scanner system of the additional beam scanner can be much larger than the bandwidth of the first beam scanner and the response time of the additional beam scanner can be much smaller than that of the first beam scanner. If the mechanical scanners of the additional beam scanner are resonant scanners or if the mechanical scanner of the additional beam scanner is a resonant scanner, it preferably has a high resonance frequency or these preferably have a high resonance frequency. Due to the nature of the system, such resonant scanners enable significantly higher displacement speeds than adjustable scanners with the same usable aperture, but only along certain paths. For example, using two resonant scanners of identical frequency with axes that are perpendicular to each other after imaging into the sample, the focus point can be shifted along circular paths; if the frequencies differ, other paths result, for example Lissajous paths result with integer frequency ratios. Instead of two resonant scanners, the microscope can also have one resonant scanner with two mutually perpendicular tilt axes, in particular such a MEMS scanner.

In a further preferred embodiment, the further beam scanner comprises a resonant mechanical scanner, which can in particular be placed in a pupil and which displaces the illumination light beam in the sample in a first direction, and an adjustable mechanical scanner which displaces the illumination light beam in the sample in a second direction orthogonal to the first direction. In a particularly preferred embodiment, the microscope additionally has an expansion unit as described above. With such an embodiment, scanning for MINFLUX localization can be carried out particularly quickly along a path that corresponds to a scanning path of a raster image recording.

In the embodiments of the microscope which have a first beam scanner, a further beam scanner, an objective and an expansion unit, the aperture of the further beam scanner and the expansion unit and the aperture of the objective are preferably coordinated with one another, more preferably coordinated with one another in such a way.

In other preferred embodiments, the further beam scanner may be or comprise a resonantly operated EOD.

With a resonantly operated beam scanner, the focal point of the illumination light can be shifted particularly quickly. The microscope according to the invention further comprises a detection device for detecting light emitted from the focus area of the illumination light, i.e. for detecting emissions from the focus area of the illumination light. The detection device is designed to detect emissions in such a way that an emission can be assigned to a position of the focus point in the sample. For this purpose, the detection device preferably comprises a detector that can detect individual photons, for example a photomultiplier tube (PMT), a single-photon avalanche diode (SPAD) or a SPAD array; with a SPAD array that spatially resolves a diffraction pattern of an emitter in the detection plane, an emission can be assigned not only to a location of the focus point in the sample, but also to a location in the detection plane.

As already explained above, the microscope according to the invention differs from microscopes known from the prior art in that the microscope according to the invention has a measuring device for detecting a position and/or a direction of a reference light beam that is deflected and/or displaced by the first beam scanner, wherein the measuring device is designed to output a signal at a signal output depending on the position and/or the direction, which is indicative of a position of the focal point in the sample. The reference light beam can be a light beam that was split off from the light beam emanating from the illumination light source, or it can emanate from a reference light source. The fact that the reference light beam is deflected and/or shifted by the first beam scanner means that the deflection and/or shifting of the reference light beam is deflected and/or shifted by the same deflecting element or elements as the illumination light beam when the focal point of the illumination light is positioned in the sample by the first beam scanner. If the first beam scanner is a mechanical scanner with a continuous mirror that deflects all of the illumination light, it means that the reference light beam and the illumination light beam are deflected and/or shifted by the same mirror surface or surfaces. The fact that the signal is indicative of a position of the focal point in the sample means that a position of the focal point in the sample can be determined from the signal, at least in conjunction with other known values. The signal can, for example, be a measure of the deviation from a target position of a mechanical scanner that corresponds to a target position of the focal point in the sample. Then the position of the focal point in the sample can be determined from the known target position and the signal, which then represents a deviation from the target position, whereby the imaging properties of the microscope are taken into account in a manner known to the person skilled in the art. However, the signal can also be a direct measure of a position of the mechanical Scanner, which corresponds to the position of a focal point in the sample. The measuring device preferably has a signal output. In a preferred embodiment, the signal output is connected via an analog line of the fast, adjustable scanner, in particular an EOD scanner, which can be part of the first beam scanner or which forms the additional beam scanner. Then, after appropriate amplification, the signal of the measuring device is fed back to the fast beam scanner as an analog control signal.

The measuring device preferably has a detector that is position-sensitive or spatially resolving at one end of the reference light beam path. A detector of the measuring device is also preferably a position-sensitive diode, in particular a diode that is position-sensitive in two spatial directions. Position-sensitive diodes have the particular advantage that they have a high temporal resolution and thus allow fast data acquisition and fast control, for example of an EOD scanner of the deflection device, for example with a frequency of several hundred kilohertz.

In a preferred embodiment, the measuring device is designed to detect a direction of the reference light beam deflected by the first beam scanner by focusing the reference light beam or a portion of the reference light beam on the detector. If, for example, the reference light beam is deflected by the first beam scanner, i.e. if the first beam scanner imposes an angle on the reference light beam to an optical axis of the beam scanner, which the latter has in a zero position, then the imprinted angle in particular determines the position of the focus on the spatially resolving detector of the measuring device. This applies both when the reference light beam emerges from the first beam scanner convergently, as is the case, for example, with a pupil scanner in which the scanning lens is an integral part of the scanner, and in the case where the reference light beam emerges from the first beam scanner collimated and is then focused outside the illumination beam path.

In a preferred embodiment, the measuring device is designed to detect a position of the reference light beam deflected and/or displaced by the first beam scanner by directing the reference light beam or a portion of the reference light beam in a collimated manner to the or another position-sensitive or spatially resolving detector.

Further properties of the measuring device can be found in the descriptions of the method below as well as in the descriptions of the figures. Preferably, the reference light beam propagates along a reference light beam path, wherein the reference light beam path comprises the first beam scanner.

In a preferred embodiment, the microscope comprises a reference light source for generating the reference light beam. In preferred embodiments, the reference light source is a laser. Further properties of the reference light source emerge from the description of the method according to the invention given further below in connection.

The microscope according to the invention preferably has a control unit which is designed to control the first beam scanner or the further beam scanner(s) or the first beam scanner and the further beam scanner(s) in such a way that the focal point is positioned in the sample, for example at scanning positions of a set of scanning points of a MINFLUX localization or is displaced along a scanning path for a MINFLUX localization. The first beam scanner can receive control signals from the control unit, in particular electrical signals, which influence a state of the at least one deflection unit in order to displace or position the illumination light beam. If the first beam scanner is a mechanical scanner with at least one movable optical component, the control signals are assigned target positions or target movements of the at least one optical component, which are determined by the control signals. The control unit is preferably further designed, in particular, to control the first beam scanner in such a way that the focal point in the sample is positioned and/or shifted within a larger image field, for example in such a way that, for example, a confocal raster image is recorded. The control unit can in particular be an integral component of a computing and control unit. The computing and control unit is preferably also designed in particular to receive and/or evaluate and/or store signals from the measuring device and the detection device. The signal output of the measuring device is preferably connected to the control unit of the deflection device, wherein the connection can be direct or indirect, mediated by further elements of the computing and control unit, of which it can be an integral component. In a further preferred embodiment, the computing and control unit is designed to determine control and regulating signals for the deflection device on the basis of the signals from the measuring device and to output them to the deflection device.

In the MINFLUX method according to the invention and in the STED-MINFLUX method according to the invention, the position of a single emitter in a sample is determined or a single emitter in a sample is tracked so that a trajectory of the emitter can be determined For this purpose, an illumination light is focused into the sample in such a way that in the focus area of the illumination light, an intensity minimum of the illumination light in a focus point is bordered on both sides by areas of intensity increase in at least one direction, the focus point is positioned at several positions in the sample by means of a deflection device which has a first beam scanner, an emitter in the focus area of the illumination light is excited by the illumination light or by another light to emit luminescence or scattered light as emitted light, the light emitted from the focus area of the illumination light is detected by a detection device, measurement values of the detection device are assigned to the scanning positions, and a position or trajectory of an emitter is determined from the measurement values and the assigned scanning positions.

The MINFLUX method according to the invention and the STED-MINFLUX method according to the invention differ from MINFLUX methods and STED-MINFLUX methods known from the prior art in that in the MINFLUX method according to the invention and in the STED-MINFLUX method according to the invention a reference light beam is deflected and/or displaced by the first beam scanner, and that a measuring device detects a position and/or a direction of the deflected and/or displaced reference light beam and, depending on the position and/or the direction, outputs a signal at a signal output that is indicative of a position of the focal point in the sample.

The positioning of the focal point in the sample can be achieved by sequentially addressing different positions in the sample using the deflection device. The set of addressed scanning positions can form or contain a continuous path of scanning positions, for example a circular path around the presumed position of the emitter; the path can also run in a grid-like manner or form a Lissajous path, a rosette or a loop. It is also possible for the deflection device to address only one scanning position and for the sequential positioning to various actual scanning positions to result from random setting changes of the first beam scanner. In both cases, the positioning of the focus point is then carried out sequentially at different actual scanning positions, whereby each actual scanning position, i.e. each position of the focus point, does not correspond exactly to the respective addressed scanning position, but deviates from it by an amount that depends on a difference between the respective actual setting of the first beam scanner and the respective target setting, i.e. the respective setting of the first beam scanner associated with the addressed scanning position. According to the invention, a position and/or a direction of the reference light beam deflected and/or displaced by the first beam scanner is detected by means of a measuring device; the associated measuring signal, which is indicative of a respective position of the focus point in the Sample, ie from which the respective position of the focus point in the sample can be determined, is output at a signal output.

In a preferred embodiment of the invention, the focal point in the sample is positioned sequentially at different scanning positions by means of a deflection device comprising a first beam scanner. The set of addressed scanning positions can form or contain a continuous path of scanning positions, for example a circular path around the presumed position of the emitter; the path can also run in a grid-like manner or form a Lissajous path, a rosette or a loop.

As stated above, in the method according to the invention, an emitter is excited by the illumination light or by another light in the focus area of the illumination light to emit luminescence or scattered light. If the excitation is carried out by the illumination light, the illumination light can be the only light that is irradiated into the sample when the method is carried out. With such a method, the position of an emitter at which illumination light is elastically scattered, such as a gold bead, as well as a luminescent particle, for example a fluorescent bead or in particular a fluorophore, can be determined and a corresponding emitter can be tracked.

In addition to the illumination light, further light can also be irradiated into the sample. In one embodiment, the further light can be a fluorescence prevention light, in particular a STED light, with which the emission of free, i.e. non-stimulated, fluorescence from an area adjacent to the focus area of the illumination light and/or from outer areas of the focus area, of which it is known from previous position determinations or position estimates that the fluorophore whose position is to be determined is not located in them, is suppressed. In this case, the fluorescence prevention light can therefore be used in particular to suppress background light that would reduce the quality of the position determination. For this purpose, the fluorescence prevention light can form a wide STED donut, which is generated, for example, by means of a vortex phase plate with a multiple increase in the modulation of the phase of the wave front from 0 to 2TT over one revolution along the circumferential direction. It can also form a 3D donut, which can be created using an annular phase plate, for example. The additional light can then rest in the sample, so that the illumination light is displaced relative to the additional light, or the additional light can be displaced together with the illumination light, but not necessarily in the same way. In another embodiment of the method according to the invention, the illumination light can be a luminescence prevention light, in particular a STED light, i.e. the method according to the invention can be carried out as a STED-MINFLUX method according to the definition given in the relevant section. In STED-MINFLUX methods, the emitter, the position of which is to be determined or which is to be tracked, is excited with additional light as excitation light. The additional light can be light focused together with the illumination light through an objective, wherein the shape of the intensity distribution can correspond at least approximately to an Airy function or a Gaussian function or a top-hat function. The intensity distribution preferably has a wide range in its center - wide in comparison with the distances of the scanning positions of the illumination light to which the illumination light is shifted - in which the intensity does not change or changes only slightly. This condition can be met, for example, by focusing the additional light using part of the aperture of the objective, i.e. not the full aperture. In this embodiment, too, the additional light can rest in the sample so that the illumination light is displaced relative to the additional light, or the additional light can be displaced together with the illumination light, but not necessarily in the same way.

The light emissions of the emitter are detected by a detection device. This can, for example, have a point detector, such as an avalanche photodiode (APD), a photomultiplier or a hybrid detector, or a spatially resolving detector such as a camera or a so-called APD array. In particular, the detection device also comprises evaluation electronics. The detection device is designed in particular to detect individual photons emanating from the emitter and to register them in such a way that the scanning positions associated with the light emissions can be assigned.

There is now a specific relationship between the position and/or direction of the reference light beam deflected and/or shifted by the first beam scanner and the position and/or direction of the illumination light beam after the scanner, whereby it is necessary to take into account how the directions and/or positions of the reference light beam and the illumination light beam relate to one another. There is also a specific relationship between the position and/or direction of the illumination light beam after the scanner and the position of the focal point in the sample. The detection of the position and/or direction of the reference light beam therefore enables the determination of the respective actual scanning position, i.e. the respective actual position of the focal point in the sample, at the respective measurement time, i.e. at the respective time of excitation of the emission detected by the detection device. This, and thus the signal of the Measuring device can be used in two ways, which can also be combined with each other, to increase the spatial and temporal resolution.

One way is that the detection takes place in connection with the use of the signal from the measuring device in such a way that a correction value is determined from the signal in real time, by which the control signal of the deflection device or at least one beam scanner of the deflection device is corrected. For example, the signal from the measuring device can, after appropriate amplification, be fed back as an analog control signal to the deflection device as a whole or to a beam scanner that forms part of the deflection device. In this case, the correction value is determined by an amplification unit, for example an operational amplifier tailored to the application. The reference light beam and the measuring device are therefore used to control the deflection device. The control can be implemented using control strategies known in the art, for example in the form of a PID controller (PID: proportional-integral-differential). The use of the measurement signal for control can be particularly advantageous when the deflection device has a further adjustable beam scanner that allows random access to scanning points, which has shorter, in particular much shorter response times, for example only 20%, 10%, 1% or less than 1% of that of the first beam scanner, than the first beam scanner, i.e. a fast beam scanner. The same applies if the first beam scanner itself has different units with correspondingly different response times, fast and slow units, in which case the control of the deflection device takes place by feeding the measurement signal or a signal derived from the measurement signal back to the fast unit or units. Accordingly, the control is preferably carried out in such a way that fast corrections of the position of the focal point in the sample are effected by the further, fast beam scanner or by the fast unit or units of the first beam scanner. This does not exclude the possibility of slower corrections being made using or co-using the first beam scanner or the slow unit or units for correction. However, it is particularly simple to control the first beam scanner or the slow units of the first beam scanner and to regulate only the other beam scanner or the fast unit or units of the first beam scanner. The position and/or direction of the reference light beam can be detected with a bandwidth or time resolution of the detection that is adapted to the bandwidth or response time of the other beam scanner. The bandwidth with which the position and/or direction of the reference light beam is detected can in particular also be higher than the bandwidth of the other beam scanner or the fast units of the first beam scanner. for example twice as high or more than twice as high. In this case, the bandwidth of the control signal is preferably reduced compared to the bandwidth of the detection of the position and/or the direction. In particular, if the bandwidth of the additional beam scanner or the fast units of the first beam scanner is at least as large as that of the measuring device, the control can advantageously be implemented particularly simply as a proportional control of the deflection unit.

The other type is that the detection is carried out in such a way that actual scanning positions are determined from the signal of the measuring device. For this purpose, the signal of the measuring device for detecting the position and/or the direction or a signal that is derived from a control signal of the deflection device and the signal of the measuring device is preferably stored in a storage device in such a way that it can be assigned to a measurement signal of the detection device for detecting the light emitted from the focus area. This can be achieved, for example, by storing both the signal of the measuring device or the signal derived from it and the signal of the detection device, each provided with a time stamp.

Both types can be advantageously combined, especially if the bandwidth of the detection of the position and/or direction of the reference light beam is higher than the bandwidth of the other beam scanner.

Determining the position of the emitter or tracking the emitter by determining a trajectory can now be done according to the methods that are usually used when carrying out MINFLUX methods or STED-MINFLUX methods. This also applies to the selection of the sampling positions at which the focus point is to be positioned.

If the deflection device is controlled based on the signal from the measuring device, an advantage arises from the fact that the actual scanning positions are actually scanned with greater accuracy. This is especially true if the first beam scanner is controlled during a sequential shift of the focal point in the sample in such a way that the focal point would not change its position if the first beam scanner were perfect. Investigations by the applicant using a typical microscope configuration with a quad scanner have shown that the jitter of all individual galvo scanners of the nominally stationary quad scanner can result in shifts in the focal point in the sample with a stroke of several nanometers, with the noise power spectrum having large values in the frequency range of several kilohertz. This can result in an (additional) uncertainty of several nanometers when determining the current position of the emitter, particularly when tracking emitters, regardless of the uncertainty caused by the movement of the emitter. Since measurements with uncertainties in the range of 1 nm are basically possible using the MINFLUX method, this uncertainty due to the jitter is therefore significant. Using another, particularly fast, adjustable beam scanner, for example an AOD or in particular an EOD, the position of the focal point can now be corrected according to typical bandwidths, for example within about 1 ps or less, provided that the bandwidth of the measurement of the position and/or the position is sufficiently large. This measurement with a correspondingly high bandwidth or time resolution can be realized using means known in the prior art, as will be explained below. With a smaller measurement bandwidth, this limits the time constant of the correction. By means of the measuring device and, for example, the EOD of the deflection device, it can be achieved, for example, that a scanning position can be addressed with an uncertainty of less than 1 nm, which significantly reduces the uncertainty of determining the position during fast scanning or, conversely, makes it possible to achieve a high level of accuracy even during very fast measurements, as is particularly necessary when tracking a fast-moving emitter.

It is also possible for the first beam scanner to comprise several, in particular different scanners, for example a galvo scanner system and an EOD system. In these cases, the signal from the measuring device for detecting the position and/or direction of the reference light beam can be used to control the deflection device in such a way that only one or individual scanners of the first beam scanner are controlled, while the control signal of other scanners is not dependent on the signal from the measuring device. In the specific example in which the first beam scanner has a galvo scanner system and an EOD system, the measuring device measures a deflection and/or displacement that depends on both the setting of the galvo scanner system and the setting of the EOD system, but the measurement signal is nevertheless fed back to the EOD system in this example.

The measurement signal of the measuring device for determining the position and/or the position or the signal derived from it can alternatively or additionally be used in another way to increase the spatial-temporal resolution, namely by taking into account the actual focus points at the time of excitation of the emission when determining the position or trajectory of the emitter from the emissions detected by the detection device, i.e. the focus points determined on the basis of the measurement of the position and/or the direction of the reference light beam. If this other type of accuracy improvement is used exclusively, it is particularly suitable for evaluating data that has already been recorded. During data recording, the values that correspond to the target positions can then be used as the coordinates of the scanning positions, as in the prior art. For example, using methods known from the prior art, a position of the emitter can then be determined in real time, based on which subsequent scanning positions can be determined, to which the focus point is moved in the further course of the measurement in order to determine the position of the emitter more precisely or to track the emitter. In this case, measuring the position and/or direction of the reference light beam after the scanner does not have a direct effect on the rest of the measurement process. However, a scanning position can now be determined with greater accuracy from the measured position and/or position of the reference light beam than from the data from controlling the deflection device. If the actual scanning positions determined from the measurement, i.e. the positions of the focal point in the sample, are used as a basis for determining the position of the emitter instead of the target positions, the position of the emitter is also determined with greater accuracy in this evaluation than if the target positions are used as a basis.

In principle, however, the actual positions of the focal point in the sample at the time of excitation of the emission, i.e. the positions determined on the basis of the measurement of the position and/or direction of the reference light beam, can be taken into account during data acquisition in order to determine the position of the emitter in real time or to track the emitter. For this purpose, the routine for evaluating the data in real time can be modified accordingly and adapted hardware such as a powerful Field Programmable Gate Array (FPGA) can be used. The actual scanning positions can be determined from the measurement signal of the measuring device, for example in a separate unit and sent from there to an FPGA to control the deflection unit, or the signal from the measuring device can be sent to an FPGA to control the deflection unit and fully evaluated there.

A combination of both types of use of the signal from the measuring device, i.e. the use for controlling the deflection device and for determining the scanning positions, i.e. the positions of the focal point in the sample, is also advantageously possible. This applies in particular when the bandwidth of the measuring device is higher than the bandwidth of the deflection unit. In this case in particular, even if the deflection unit is controlled using the signal from the measuring device, there remains a deviation between the actual scanning position and the target position, which is recorded using the measuring device. This deviation is reduced compared to the deviation in the absence of control. The The data can now be evaluated in real time in the same way as when the signal from the measuring device is used exclusively to determine the scanning positions, by using the target positions as a basis for determining the position of the emitter, or by taking into account the actual focus points, i.e. the focus points determined on the basis of the measurement of the position and/or direction of the reference light beam at the time of excitation of the emission, during data acquisition.

In preferred embodiments of the method, a reference light beam is generated by means of a reference light source and is guided over the first beam scanner.

The reference light source can be, for example, a laser, a light-emitting diode or a laser diode. In particular, if the reference light source is a broadband light source, then generating the reference light beam can include filtering the wavelengths.

In some embodiments, it is preferred that the light of the reference light beam does not comprise a wavelength range that contains wavelengths that are used in the microscope associated with the method according to the invention for the microscopic images of samples to be examined. For example, in a preferred embodiment, the wavelength range of the reference light beam can exclusively comprise wavelengths above the largest wavelength used when carrying out microscopy methods with the microscope, for example a largest STED laser wavelength or a largest fluorescence wavelength to be detected; alternatively, the wavelength range of the reference light source can exclusively comprise wavelengths below the smallest wavelength used for carrying out microscopy methods, which can, for example, be the smallest wavelength used for fluorescence excitation or for activating or deactivating switchable fluorophores. In particular, in a preferred embodiment, the wavelength range of the reference light beam does not overlap with that of the illumination light beam.

The choice of wavelength and other properties, such as the polarization of the reference light beam, is fundamentally interrelated with the specific design of the first beam scanner and the choice of the entire beam path. If the first beam scanner comprises AOD and/or EOD, boundary conditions arise for the properties of the reference light beam. For example, the deflection angle that the first beam scanner imposes on a light beam can be dependent on polarization, as is regularly the case with a single EOD, or it can be dependent on the wavelength, as is regularly the case with a single AOD. Accordingly, if the deflection unit has EOD and the reference light beam is guided through the deflection unit, preferably that the reference light beam guided through or over the first beam scanner has the same polarization as the illumination light beam, but preferably a different wavelength, thereby enabling the reference light beam to be separated from the illumination light beam by means of a color splitter. This preferred embodiment is one in which the wavelength range of the reference light beam does not overlap with that of the illumination light beam. However, even when using EOD in the first beam scanner, it is not absolutely necessary that the wavelength ranges of the illumination light beam and the reference light beam do not overlap.

In order to guide the reference light beam over the first beam scanner, it can be introduced into the beam path of the illumination light beam at a shallow angle on one side of the first beam scanner and correspondingly be guided out of the beam path of the illumination light at a shallow angle on the other side of the first beam scanner. In this embodiment, the reference light beam is separated completely independently of differences in internal properties such as wavelength or polarization. The reference light beam basically experiences the same deflections as the illumination light beam, insofar as these are caused by the first beam scanner. The angles at which the reference light beam can be introduced so that it is guided over the first beam scanner result from the parameters of the first beam scanner, for example from the dimensions of mirrors and the structure of the beam path in the area of the first beam scanner, which can have relay optics, for example.

In another embodiment, the reference light beam can be coupled, for example by means of a mirror, into an edge region of the beam path of the illumination light parallel to the optical axis of the beam path of the illumination light, which is present when the deflection device is in a neutral position. This edge region of the beam path cannot then be used by the illumination light.

In a preferred embodiment, the reference light beam is coupled into the beam path of the illumination light by means of a beam splitter element, for example by means of a dichroic beam splitter. This is possible in particular if the wavelength ranges of the reference light beam and the illumination light beam do not overlap, and is particularly advantageous if the reference light beam does not have a wavelength used for the actual microscopic images. With this type of coupling, the reference light beam is preferably parallel to the optical axis of the beam path of the Illumination light, which is present when the deflection device is in a neutral position, is coupled in. More preferably, the coupling takes place on this optical axis.

The reference light beam can be coupled in or introduced at a shallow angle such that the illumination light beam and the reference light beam pass through the first beam scanner in the same direction, that is, the illumination light source and the reference light source are on the same side of the scanner, or that the illumination light beam and the reference light beam pass through the first beam scanner in opposite directions, that is, the illumination light source and the reference light source are on opposite sides of the first beam scanner.

The output or the decoupling can take place in the same way as the coupling. The decoupling does not necessarily have to take place using the same method as the coupling; combinations of different methods are also possible. For example, the coupling can take place using a mirror in an edge area and the decoupling can take place using a beam splitter. The output preferably takes place when the reference light beam was introduced at an angle, also without using another optical element that would be touched by the illumination light. If the reference light is coupled onto the optical axis using a beam splitter, the decoupling also takes place using a beam splitter. The types of beam splitters can differ on both sides of the first beam scanner; in preferred embodiments they are the same. In a preferred embodiment, the reference light beam is coupled onto the optical axis using a dichroic beam splitter and also decoupled again using a dichroic beam splitter.

If a reference light beam is used which, due to its properties, for example due to its polarization, cannot be deflected by another beam scanner or several other beam scanners of the deflection device, the reference light beam can also be coupled or introduced into the beam path of the illumination light beam in such a way that the reference light beam path includes the other beam scanner(s) in question in addition to the first beam scanner.

The reference light beam can be coupled or introduced into the beam path of the illumination light beam as a collimated, convergent or divergent light beam, or the reference light can have two different components, for example a collimated and a convergent component, when coupled or introduced. If the reference light has such different components, it is preferred that it additionally either differs from the illuminating light in another property, such as wavelength or polarization, or that the components are coupled in separately from one another. "Separated from one another" can mean, for example, spatially separated and parallel or at different angles or temporally separated, e.g. by coupling in short pulses intermittently. When using several different components, it is essential that the components can be detected separately after the scanner.

The reference light beam can further be coupled or introduced in such a way that, in the beam path section that it has in common with the illumination light beam, it has a state corresponding to that of the illumination light beam with regard to its convergence or divergence or in particular with regard to a position of a focus within this beam path section.

In a preferred embodiment, the reference light or a portion of the reference light is collimated and coupled onto the optical axis by means of a beam splitter and, after the first beam scanner, is coupled out again at a location where the reference light beam is also collimated. The position of such locations can easily be determined by a person skilled in the art in a given beam path.

There are no imaging optical elements within a quad scanner, such as is used in the applicant's microscopes, i.e. between the first and last tilting mirror of a quad scanner. Accordingly, when using a quad scanner, a collimated coupling or introduction can take place adjacent to one of the outer mirrors along the beam path and the coupling or exit can take place adjacent to the outer mirror opposite along the beam path. According to the functional principle of a quad scanner (see e.g. DE 103 39 134 A1 or WO 2010/069 987 A1), a quad scanner in a microscope having an objective deflects an illuminating light beam in such a way that for each deflection angle the axis of the illuminating light beam intersects the optical axis of the objective in the pupil, i.e. the rear focal plane of the objective. In a typical microscope setup with a quad scanner, a first optical element, for example a lens, can be arranged in the beam path of the illumination light in front of the scanner, which focuses collimated illumination light hitting the lens into the scanner, and a second optical element, for example a lens, can be arranged after the quad scanner, which collimates the illumination light again after the focus, so that it enters the lens in a collimated state, whereby an angle of the illumination light beam to the optical axis of the lens, and thus the position of the Focus of the illumination light in the sample depends on the position of the quad scanner. If the lens and the second optical element form a telecentric system, the condition that the axis of the illumination light beam intersects the optical axis of the lens for all scanner settings in the rear focal plane or the pupil is met if, for all different scanner settings, the illumination light beams after the last mirror of the scanner and before the second optical element run parallel to each other and to the optical axis, i.e. if the scanner only offsets the illumination light beam but does not tilt it. The change in the position of the focal point of the illumination light in the sample by the scanner is then directly dependent on the change in the offset of the illumination light caused by the scanner. The same change in offset is now impressed on a reference light beam. If the reference light beam is coupled into the beam path of the illumination light or introduced at a shallow angle in such a way that it passes through the entire scanner as a collimated beam, the offset of the reference light beam and thus the change in the offset of the illumination light beam can be determined in a simple manner by directing or decoupleing the reference light beam in the direction of the reference light beam after the scanner and before the first or second optical element, depending on whether the reference light beam propagates in the direction of the illumination light beam or against it, and collimating it onto a position-sensitive or spatially resolving detector, which is an essential element of the measuring device for detecting - here - the position of the reference light beam after the scanner. Alternatively or additionally, the reference light beam can be directed or decoupled in the direction of the reference light beam after the scanner and before the first or second optical element and focused onto a position-sensitive or spatially resolving detector, which is then an essential element of the measuring device for detecting the - here - direction of the reference light beam. The position of the focus would not change if the scanner were functioning ideally. It is a measure of the change in angle that the scanner imposes on the reference light beam and thus also on the illumination light beam. This change in angle, which is recorded with the measuring device, leads to a change in the position of the illumination light beam in the rear focal plane, i.e. the pupil of the objective. Recording this change in position makes it possible to draw conclusions about the change in the quality of the excitation light distribution and a shift in the focus point in the sample resulting from the change in position in the pupil.

Advantageous developments of the invention result from the patent claims, the description and the drawings and the associated explanations of the drawings. The described advantages of features and/or combinations of features of the invention are merely exemplary and can be effective alternatively or cumulatively. The following applies with regard to the disclosure content (but not the scope of protection) of the original application documents and the patent: Further features can be found in the drawings - in particular the relative arrangements and active 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 are 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, but this does not apply to the independent patent claims of the granted patent.

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 only the purpose of making the patent claims easier to understand.

Short description of the characters

Fig. 1 shows an embodiment of a microscope according to the invention with a reference light source.

Fig. 2 shows an embodiment of a microscope according to the invention, in which a

Reference light beam is split from light from an illumination light source.

Fig. 3 shows an embodiment of a microscope according to the invention, in which a

Reference light beam from a reference light source runs in the opposite direction to the illumination light beam.

Fig. 4 shows an embodiment of a microscope according to the invention, which has a measuring device whose signal is fed back analogously to a beam scanner.

Fig. 5 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a tilt.

Fig. 6 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a tilt with an enlarged measuring range. Fig. 7 shows a section of a further embodiment of a microscope according to the invention with a measuring device for determining a tilt with an enlarged measuring range and improved sensitivity.

Fig. 8 shows a part of the measuring device of the microscope shown in Fig. 7 in detail.

Fig. 9 shows exemplary measurement data that can be recorded with the measuring device of the microscope shown in Fig. 7.

Fig. 10 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a displacement.

Description of the characters

In Fig. 1, Fig. 2 and in Fig. 3 each an embodiment of the microscope 1 according to the invention is shown. In the embodiments shown, the microscope 1 has a light source 2 for generating an illumination light. The illumination light emanating from this light source 2 forms an illumination light beam B, which is guided into a sample region 7 by optical elements that form an illumination light beam path, so that a focus is formed in a sample 8 introduced into the sample region 7 such that in the focus region of the illumination light, an intensity minimum of the illumination light in a focus point is bordered on both sides in at least one direction by intensity increase regions, which can specifically mean that a 2D donut or a 3D donut is formed in the sample. The sample region 7 is formed on an adjustable sample table 20. The embodiments of the microscope 1 shown in these figures each have a fast, adjustable scanner, which is specifically an EOD scanner 11 here, and they all have a mechanical beam scanner 10, and in all embodiments shown the mechanical scanner 10 and the EOD scanner 11 form a deflection unit 9. All embodiments of the microscope 1 shown in these figures have a light modulator, which is specifically designed as a phase filter 3 here. A static phase plate, for example a vortex phase plate or an annular phase plate, or adjustable phase modulators can be used as the phase filter 3. Adjustable phase modulators in particular can be reflective elements. For reasons of simplicity, a transmissive phase filter 3 is shown in all figures. The embodiments of the microscope 1 shown in the figures mentioned each have a main beam splitter 4, which allows the illumination light to pass through, but redirects emissions 19 originating from the sample 8 to a detection device 12. The embodiments of the microscope 1 shown in the figures mentioned each have a mechanical Scanner 10 which deflects the illumination light beam B, an X/4 plate 5 for adjusting a polarization of the illumination light with a view to forming an intensity minimum of the lowest possible intensity and an objective 6 which focuses the illumination light beam B into the sample 8 in the sample region 7.

The embodiments of the microscope 1 shown in the above-mentioned Figures 1 to 3 each further comprise a measuring device 13 which is set up to detect a position and/or a direction of a reference light beam R which is deflected and/or displaced by a first beam scanner 18. In the embodiments of the microscope 1 shown, the measuring device 13 each comprises a signal output 13a via which a signal S, which is indicative of a position of the focal point in the sample, is output to a computing and control unit 17. The output preferably takes place via a fixed control line, not explicitly shown in each case. The computing and control unit 17 can, for example, comprise FPGAs (Field Programmable Gate Arrays) and a memory device, neither of which is shown separately. In particular, the computing and control unit 17 is designed such that position values corresponding to the signal S or a position of the focal point determined from the signal or the deviation of the position of the focal point from a respective associated target position can be stored in the storage device, wherein the position values can be assigned in time or stored in a time-associated manner to measured values of the detection device 12 for detecting emissions from the focus area of the illumination light. In the embodiments of the microscope 1 shown, the computing and control unit 17 receives measured values from the detection device 12, which is symbolized in Figures 1 to 3 by a dotted line. Another dotted line indicates that a control signal generated in the computing and control unit 17 is passed on to the EOD scanner 11. The control signal is selected such that a scan is carried out for carrying out a MINFLUX method or a STED-MINFLUX method. In this case, a correction is determined on the basis of the signal S, i.e. there is feedback or regulation of the deflection device 9, specifically in particular of the EOD scanner 11. This feedback and regulation is not necessary in all embodiments of the invention, but it is advantageous in order to improve the quality of the data acquisition when carrying out a MINFLUX method or a STED-MINFLUX method. However, the signal S can not only be used to improve the data acquisition, which is usually associated with a first data evaluation carried out in parallel with the data acquisition, but it can also be used to improve a data evaluation to be carried out after the completion of a data acquisition by taking into account the actual scanning positions when determining the position of an emitter, that is, the respective actual positions of the focal point in the sample are used as a basis. Another dotted line indicates that a control signal, which is generated in the computing and control unit 17, is passed on to the mechanical beam scanner 10. This control signal can be static during the implementation of a localization using a MINFLUX method or a STED-MINFLUX method, or it can vary over time. When determining the control signal for the mechanical scanner 10, the signal S of the measuring device 13 can also be taken into account.

By means of the adjustable sample table 20 shown in the figures, it can be ensured that the deflection device 9 can be operated during the implementation of a MINFLUX method or a STED-MINFLUX method with settings close to a zero position, in particular close to a zero position of the mechanical scanner 10, by regularly pushing the sample 8 into the center of the area that can be scanned by the deflection device 9 using the sample table 20. This reduces the requirements for the measuring device 13, since the measuring range to be recorded of the position and/or a direction of the reference light beam R is reduced. Accordingly, the method according to the invention can advantageously be carried out accordingly.

In the embodiment of the microscope 1 shown in Fig. 1, the mechanical beam scanner 10 forms the first beam scanner 18 and the first beam scanner 18 forms a deflection device 9 together with the EOD scanner 11. In this embodiment, the mechanical beam scanner 10 alone is the first beam scanner 18, by which the reference light beam R is deflected and/or shifted. In this embodiment, the microscope 1 has a reference light source 16. The reference light beam R is coupled by means of a beam splitter, which is referred to below as the second beam splitter 15, onto an optical axis of the microscope 1, along which the illumination light beam B propagates when the deflection device 9 is set to a zero position. The reference light beam R is shown offset slightly parallel for reasons of better representation. The reference light beam R propagates after the beam splitter 15 along the illumination beam path and is deflected and/or shifted in the same way as the illumination light beam B according to the setting of the first beam scanner 18, which is formed by the mechanical scanner 10. After the first beam scanner 18 there is another beam splitter, which is referred to here as the first beam splitter 14. The first beam splitter 14 separates the reference light beam R from the illumination light beam B and directs it in the direction of the measuring device 13. Both the first beam splitter 14 and the second beam splitter 15 are set to a difference between the reference light beam R and the illumination light beam B. tuned, for example to different polarizations or different wavelengths.

In the embodiment of the microscope 1 shown in Fig. 2, the EOD scanner 11 and the mechanical beam scanner 10 form the first beam scanner 18, wherein the first beam scanner 18, formed from the EOD scanner 11 and the mechanical beam scanner 10, forms the entire deflection device 9 here. In this embodiment, the fast, adjustable scanner, specifically the EOD scanner 11, is a component of the first beam scanner 18, by which the reference light beam R is deflected and/or shifted. The microscope 1 shown in Fig. 2 has a first beam splitter 14 in the illumination beam path after the mechanical scanner 10, which separates the reference light beam R from the illumination light beam B and directs it in the direction of the measuring device 13. In the specific embodiment shown, the reference light beam R is generated by the light source 2 for the illumination light and, together with the illumination light beam B, passes through all optical elements as a common light beam up to the first beam splitter 14. The reference light beam R thus undergoes identical deflections and/or displacements as the illumination light beam B up to the first beam splitter 14. The beam splitter 14 now splits off a portion, for example half, of the light beam emanating from the light source. Since the reference light beam R deflected and/or displaced by the first beam scanner 18 has undergone phase modulation in this embodiment of the microscope 1, an embodiment of the measuring device 13 that is coordinated with this is preferably used in this embodiment of the microscope 1.

In the embodiment of the microscope 1 shown in Fig. 3, as in the embodiment of the microscope 1 shown in Fig. 1, the mechanical beam scanner 10 forms the first beam scanner 18 and the first beam scanner 18 forms a deflection device 9 together with the EOD scanner 11. The embodiment of the microscope 1 shown has a reference light source 16. In contrast to the embodiment of the microscope 1 shown in Fig. 1, the reference light beam R, which emanates from the reference light source 16, is not coupled into the beam path of the illumination light by means of a beam splitter, but the reference light beam R is introduced into the illumination light beam path at a flat angle, which is not shown at an accurate angle in the figure for reasons of simplicity, such that the reference light beam R and the illumination light beam B are deflected and/or shifted in a corresponding manner by the first beam scanner 18, which is identical here to the mechanical beam scanner 10. Accordingly, the reference light beam R is guided out of the beam path of the illumination light at a flat angle on the other side of the first beam scanner 18. Even in a zero position of the mechanical scanner 10, the angle at which the Reference light beam R is introduced may not be identical to the angle at which the reference light beam R is guided out on the other side. Rather, the relationship between these two angles depends on which imaging elements the first beam scanner 18 comprises. A further difference from the embodiment of the microscope 1 shown in Fig. 1 is that the reference light beam R is guided through the first beam scanner 18 in the opposite direction to the illumination light beam B.

The properties of the embodiments of Fig. 1 and Fig. 3 could also be combined. The reference light beam R could therefore be introduced at a flat angle, but in the direction of the illumination light beam B, or conversely it could be coupled in by means of a beam splitter in the opposite direction to the illumination light.

The embodiment shown in Fig. 4 corresponds in most respects to that shown in Fig. 3. The reference light source 16 and the measuring device 13 are shown in slightly different positions. In contrast to the embodiment shown in Fig. 3, the embodiment shown in Fig. 4 has an analog line 21, by means of which an analog signal that the measuring device 13 generates in conjunction with an analog amplifier is fed back to the EOD scanner 11 as a control signal. The analog amplifier can also be regarded as an integral component of the measuring device 13 and is therefore not shown separately. The measuring device 13 can in particular be based on a position-sensitive diode.

Figures 1 to 4 are schematic representations in which not all elements that are typically present, for example lenses for focusing or collimating the illumination light beam B or the reference light beam R, are also shown.

Fig. 5 shows a section of an embodiment of the microscope 1. The section contains the reference light source 16, the second beam splitter 15, the mechanical beam scanner 10, which here forms the first beam scanner 18, and the first beam splitter 14. In addition to these elements of a reference light beam path, the section shown contains the X/4 plate 5, the objective 6, the sample area 7 with the sample 8, which is placed on the adjustable sample table 20. The aforementioned elements are arranged in the same way as in Fig. 1, to which reference is hereby made. In contrast to the representation in Fig. 1, an entrance lens 22 and an exit lens 23 are shown here, which delimit the mechanical scanner 10 and are to be understood here as elements of the mechanical scanner 10; This does not mean that the lenses 22,23 form a common component, but it simply means that in the terminology of this application they are functional components of the mechanical beam scanner 10 and thus of the first beam scanner 18. No beam-forming element, in particular no lens, is shown between the exit lens 23 and the objective 6. While Figures 1 to 4 are schematic representations in which not all elements that are typically present, for example lenses for focusing or collimating the illumination light beam B or the reference light beam R, are also shown, Fig. 5 is to be understood in such a way that in the embodiment of the microscope 1 shown there is actually no beam-forming element, in particular no lens, between the exit lens 23 and the objective 6. The microscope 1 in this embodiment has a measuring device 13 with a signal output 13a. The measuring device 13 of this embodiment has a lens 25. The reference light beam R is coupled into the illumination beam path by means of the second beam splitter 15. The reference light beam R is collimated at the second beam splitter 15. The reference light beam R and the illumination light beam B enter the first beam scanner 18 collimated at the entrance lens 22 and exit the first beam scanner 18 at the exit lens 23, also collimated. When exiting the beam scanner 18, the illumination light beam B and the reference light beam R have been tilted in the same way by the first beam scanner 18. This does not mean that they exit the first beam scanner 18 at the same tilt angle, since, for example, the illumination light beam B in particular can enter tilted against the optical axis of the additional beam scanner, which the latter has in a zero position, according to a setting of a further beam scanner not shown here, for example an EOD scanner. The fact that a beam is tilted does not exclude the possibility that it is also displaced. In the example shown, a displacement would cause an axis of the illumination light beam B to pass through the rear aperture of the objective 6 at different points depending on the displacement. The illumination light beam B passes through the λ/4 plate 5 after the first beam splitter and is focused by the objective 6 into the sample 8 in the sample area 7. The position of the focus point in the sample 8 is determined by the overall tilt imposed on the illumination light beam B, which results additively from the tilt with which the illumination light beam B entered the first beam scanner 18 and the tilt imposed by the first beam scanner 18, and by the properties of the objective 6, in particular by its focal length. The reference light beam R is coupled out of the illumination beam path by the first beam splitter 14 and directed in the direction of an embodiment of the measuring device 13. The measuring device 13 has a lens 25 which focuses the reference light beam R collimated in front of the lens 25 onto a detector of the measuring device 13, specifically onto a position-sensitive diode 24. The position of the focal point of the reference light beam R depends on the tilting imposed on the reference light beam R by the first beam scanner 18 and on the properties of the lens 25, in particular on its focal length. The position of the lens 25 in the reference light beam path and the distance of the detector, here the position-sensitive diode 24, from the lens 25 are adjusted to the focal length of the lens 25. Depending on the adjustment, the measuring device 13 achieves a high sensitivity in determining the tilting and a smaller measuring range in which tilting can be measured, or a lower sensitivity and a larger measuring range.

Fig. 6 shows a section of another embodiment of the microscope 1, which corresponds to that shown in Fig. 5 with regard to the guidance of the illumination light beam B and the reference light beam R up to the first beam splitter 14. In order to be able to cover a larger measuring range of the tilts while maintaining high sensitivity for determining the tilt, the measuring device 13 has a diffractive optical element (DOE) 26 on the one hand and several position-sensitive diodes 24 arranged next to one another on the other. The DOE 26 splits the incident reference light beam R into several partial beams. Each of the partial beams is impressed with an identical tilt by the first scanner 18, so that in principle each of the partial beams is suitable for measuring the impressed tilt. In the situation shown, the reference light beam R is not tilted by the first beam scanner 18, so that the 0th order partial beam falls centrally on the central position-sensitive diode 24. In the case of a stronger tilt, the 0th order beam now runs out of the area of the central position-sensitive diode 24, while either the 1st order partial beam or the -1st order partial beam runs into the area of the central position-sensitive diode 24, depending on the direction of the tilt. In general, the splitting and detection with several position-sensitive diodes 24 ensures that small changes in the tilt can be measured even with large tilts of the reference light beam R. The individual signals of the several diodes 13 can also be combined for this purpose. For the sake of simplicity, only a splitting of the reference light beam R in one spatial direction is shown. In fact, the DOE 26 can preferably be a 2D DOE, in which case the position-sensitive diodes 24 preferably form a two-dimensional arrangement in the detection plane, so that tilts in two spatial directions, i.e. tilts that lead to displacements of the illumination light in the sample 8 in two spatial directions, can be determined by means of the measuring devices 13. At least three position-sensitive diodes 24 are required for a two-dimensional arrangement. However, more than three position-sensitive diodes 24 can preferably also be arranged, for example on a Cartesian or hexagonal grid. A corresponding splitting as shown in Fig. 6 can also be used in a corresponding manner to determine displacements of the reference light beam R.

Fig. 7 shows a section of another embodiment of the microscope 1, in which the measuring device 13 is also designed to detect a larger measuring range of the tilt or displacement of the reference light beam R while simultaneously improving sensitivity. The measuring device 13 here has a third beam splitter 27, e.g. a 50/50 beam splitter, for splitting the reference light beam R into two partial beams. One of the partial beams reaches a position-sensitive diode 24 as in the embodiment according to Fig. 5. The other partial beam is reflected via a mirror 28 to an arrangement 29 which comprises a first transmission grating 30a, a second transmission grating 30b, a first measuring sensor 31a and a second measuring sensor 31b. The measuring sensors 31a, 31b can be designed as photodiodes, for example. Fig. 7 shows the arrangement in cross section in the plane of the optical axis of the incident reference light beam R, wherein the optical axis is defined by the zero position of the reference light beam R.

In Fig. 8, the arrangement 29 is shown in detail in a plane perpendicular to the optical axis of the reference light beam R. The first transmission grating 30a and the second transmission grating 30b are arranged on opposite sides with respect to this optical axis. The first measuring sensor 31a is arranged in the light path of the reference light beam R behind the first transmission grating 30a and the second measuring sensor 31b is arranged in the light path of the reference light beam R behind the second transmission grating 30b. The reference light beam R falls on both the first transmission grating 30a and the second transmission grating 30b. The transmission gratings 30a each have a periodic stripe pattern with alternating areas of high transmissivity (white in Fig. 8) and low transmissivity (black in Fig. 8). The stripe patterns of the first transmission grating 30a and the second transmission grating are offset from one another. In Fig. 8, a deflection direction A is also shown in which the reference light beam R migrates on the arrangement 29 when the tilt or displacement of the reference light beam R changes.

Fig. 9 shows first measurement data 32 of the position-sensitive diode 24 of the measuring device 13 shown in Fig. 7 and second measurement data 33 of the measuring sensors 31a, 31b of the arrangement 29. The deflection of the reference light beam R in the deflection direction A is shown on the abscissa, and the corresponding sensor signal is shown on the ordinate. The signal of the position-sensitive diode 24 shown in the first measurement data 32 increases monotonically over the measurement range shown, but has a comparatively low sensitivity. The second measurement data 33 consists of two phase-shifted sinusoidal signals of the first measuring sensor 31a and the second measuring sensor 31b, which arise when the reference light beam R travels over the respective transmission gratings 30a, 30b. From the two signals, the position of the reference light beam R can be determined with significantly greater accuracy or sensitivity to small deflections/tilts than with the position-sensitive diode 24, for example by forming a difference or a ratio of the two sensor signals. This type of position determination is known from the prior art for so-called position encoders. Due to the periodic stripe pattern, the position signal of the arrangement is not unambiguous over the entire measuring range, ie the position relative to a stripe can be determined very precisely, but it is not known over which stripe the reference light beam is arranged. This unambiguousness can, however, advantageously be restored on the basis of the first measurement data 32 obtained from the position-sensitive diode 24.

Due to the combination of the position-sensitive diode 24 with the arrangement 29 of transmission gratings 30a, 30b and measuring sensors 31a, 31b, the position of the reference light beam R can be determined with high sensitivity over a large measuring range. This is particularly advantageous if, in a MINFLUX or STED-MINFLUX method, the image field of the mechanical beam scanner 10 is not centered on the currently estimated emitter position after each measuring step, so that larger deflections of the mechanical beam scanner 10 and thus larger tilting and/or displacements of the reference light beam R are required. These relationships also apply to the embodiment shown in Fig. 6.

The measuring device 13 shown in Fig. 7 can be extended to a position determination in two spatial directions by, for example, using a two-dimensional position-sensitive diode 24 and arranging a further fourth beam splitter behind the third beam splitter 27, which splits the reference light, wherein one of the resulting partial beams is guided to a further arrangement 29, the transmission gratings 30a, 30b of which are perpendicular to the transmission gratings 30a, 30b of the other arrangement 29, so that they detect a deflection perpendicular to the deflection direction A shown in Fig. 8 (not shown).

Fig. 10 shows an arrangement in which a reference light beam R is collimated and directed to a detector. Specifically, the arrangement shown can be used to determine a displacement imposed on the reference light beam R by the first scanner 18. The arrangement has essentially the same components as the arrangement shown in Fig. 5. In contrast to the arrangement according to Fig. 5, in Fig. 10 the lens 23 is not part of the first scanner 18, but is located exclusively in the illumination beam path. with the function of a tube lens. The reference light beam R is collimated and directed to the second beam splitter 15 and coupled into the illumination light beam path. The lens 22, which is functionally an element of the first beam scanner 18 in the sense of this application, focuses the reference light beam R. The convergent reference light beam R is coupled out of the illumination light beam path by means of the first beam splitter 14. A focus F of the reference light beam R is formed between the first beam splitter 14 and a lens 25. The fact that the first beam scanner 18 shifts the reference light beam R means that the position of the focus F is shifted along a perpendicular to an optical axis of the arrangement in a zero position of the first scanner 18. This is indicated in the figure by the double arrow. The reference light beam R is now collimated by the lens 25 of the measuring device 13. The collimated reference light beam R then runs at an angle to the aforementioned optical axis, the angle being dependent on the displacement of the focus F of the reference light beam R. The detector of the measuring device 13, the position-sensitive diode 24, is located at a distance which is very large in relation to the focal length of the lens 25, so that the point of incidence of the collimated reference light beam R depends sensitively on the position of the focus F relative to the optical axis, and thus on the displacement to be measured.

list of reference symbols

1 microscope

2 light sources

3 phase filters

4 main beam splitters

5 lambda/4 plate

6 lens

7 sample area

8 samples

9 deflection device

10 mechanical beam scanners

11 EOD scanners

12 detection device

13 measuring device

13a signal output

14 first beam splitter

15 second beam splitter

16 reference light source

17 computing and control unit

18 first beam scanner

19 emissions

20 adjustable sample table

21 analog line

22 entrance lens

23 exit lens

24 position-sensitive diodes

25 lens

26 diffractive optical element (DOE)

27 third beam splitter

28 mirrors

29 arrangement

30a, 30b transmission grating

31a, 31b measuring sensor

32 First measurement data 33 Second measurement data

A deflection direction

B Illumination light beam

R reference light beam

S Signal

F Focus of the reference light beam

Claims

patent claims 1. Microscope (1) for carrying out a MINFLUX method and/or a STED-MINFLUX method comprising - a light source (2) for generating an illumination light, - optical elements which form an illumination light beam path through which the illumination light is guided as an illumination light beam (B) from the light source (2) into a sample area (7), - in the illumination light beam path, optical means for focusing the illumination light into a focus region in a sample (8) in the sample region (7) such that in the focus region of the illumination light, an intensity minimum of the illumination light in a focus point is bordered on both sides by intensity increase regions in at least one direction, - a deflection device (9) comprising a first beam scanner (18) in the beam path of the illumination light, which is designed to position the focus point in the sample (8) at several positions in the sample (8), and - a detection device (12) for detecting emissions (19) from the focus area of the illumination light, characterized in that the microscope (1) - a measuring device (13) for detecting a position and/or a direction of a reference light beam (R) which is deflected and/or displaced by the first beam scanner (18), wherein the measuring device (13) is designed to output a signal at a signal output (13a) as a function of the position and/or the direction, which signal is indicative of a position of the focal point in the sample (8). 2. Microscope (1) according to claim 1, characterized in that the reference light beam (R) propagates along a reference light beam path, wherein the reference light beam path comprises the first beam scanner (18). 3. Microscope (1) according to claim 1 or 2, characterized in that the first beam scanner (18) has a mechanical beam scanner (10), in particular a mechanical beam scanner (10) with a continuous mirror deflecting the entire illumination light, further in particular a resonant or an adjustable galvo scanner or a resonant or an adjustable MEMS scanner or a piezo scanner. 4. Microscope (1) according to one of the preceding claims, characterized in that the first beam scanner (18) has electro-optical deflectors (EOD) and/or acousto-optical deflectors, preferably electro-optical deflectors (EOD) or acousto-optical deflectors. 5. Microscope (1) according to one of the preceding claims, characterized in that the reference light beam path comprises a first beam splitter (14) after the first beam scanner (18), which separates the reference light beam path from the illumination light beam path. 6. Microscope (1) according to claim 5, characterized in that the reference light beam (R) is collimated at the location of the first beam splitter (14). 7. Microscope (1) according to one of the preceding claims, characterized in that the reference light beam path from the illumination light source (2) to the first beam splitter (14) is identical to the illumination light beam path. 8. Microscope (1) according to one of claims 1 to 6, characterized in that the microscope (1) has a reference light source (16) for generating the reference light beam (R). 9. Microscope (1) according to claim 8, characterized in that the microscope (1) has a further beam scanner in addition to the first beam scanner (18), wherein the further beam scanner preferably has electro-optical deflectors (EOD) and/or acousto-optical deflectors. 10. Microscope (1) according to claim 9, characterized in that the signal output (13a) of the measuring device (13) is connected to the further beam scanner via an analog line (21), wherein the signal of the measuring device (13) is fed back to the further beam scanner for controlling the deflection device (9). 11. Microscope (1) according to one of claims 8 to 10, characterized in that the reference light beam path comprises a second beam splitter (15) in front of the first beam scanner (18), which combines the reference light beam path with that of the illumination light. 12. Microscope (1) according to claim 11, characterized in that the reference light beam (R) is collimated at the location of the second beam splitter (15). 13. Microscope (1) according to one of claims 8 to 10, characterized in that the reference light beam (R) is guided to the first beam scanner (18) at an angle to the illumination light beam, in particular is introduced into the illumination light beam path in front of the first beam scanner (18) at a shallow angle, such that the reference light beam (R) and the illumination light beam are deflected and/or displaced by the first beam scanner (18) in a manner corresponding to one another. 14. Microscope (1) according to one of the preceding claims, characterized in that the signal output (13a) of the measuring device (13) is connected to a storage device and that position values corresponding to the signal or the position of the focal point or the deviation of the position of the focal point from the respectively associated target position can be stored in the storage device. 15. Microscope (1) according to claim 14, characterized in that the position values in the storage device can be assigned in time or stored in a time-associated manner to measured values of the detection device (12) for detecting emissions (19) from the focus area of the illumination light. 16. Microscope (1) according to one of the preceding claims, characterized in that the signal output (13a) of the measuring device (13) is connected to a control unit of the deflection device (9). 17. Microscope (1) according to one of the preceding claims, characterized in that the measuring device (13) has a detector which is position-sensitive or spatially resolving, preferably a position-sensitive diode (24), in particular a diode which is position-sensitive in two spatial directions, at one end of the reference light beam path. 18. Microscope (1) according to claim 3, characterized in that the measuring device (13) is designed to detect a direction of the reference light beam (R) deflected and/or displaced by the first beam scanner (18) by focusing the reference light beam (R) or a portion of the reference light beam (R) on the detector. 19. Microscope (1) according to claim 3 or 4, characterized in that the measuring device (13) is set up to detect a position of the reference light beam (R) deflected and/or displaced by the first beam scanner (18) by directing the reference light beam (R) or a portion of the reference light beam (R) in a collimated manner to the or a further position-sensitive or spatially resolving detector. 20. MINFLUX method or STED-MINFLUX method for determining the position of a single emitter in a sample (8) or for tracking a single emitter in a sample (8), in which - an illumination light is focused into the sample (8) in such a way that in the focus area of the illumination light, an intensity minimum of the illumination light in a focus point is bordered on both sides by intensity increase areas in at least one direction, - the focus point is positioned at several positions in the sample (8) by means of a deflection device (9) having a first beam scanner (18), - an emitter is stimulated by the illumination light or by another light in the focus area of the illumination light to emit emissions (19), - the emissions (19) from the focus area of the illumination light are detected, whereby the emissions are assigned to the respective positions of the focus point in the sample (8), - a position or a trajectory of an emitter is determined from the emissions (19) and the associated positions of the focal point in the sample (8), characterized in that - a reference light beam (R) is deflected and/or displaced by the first beam scanner (18), and that a measuring device (13) detects a position and/or a direction of the deflected and/or displaced reference light beam (R) and, depending on the position and/or the direction, outputs a signal at a signal output (13a) which is indicative of a respective position of the focal point in the sample (8). 21. Method according to claim 20, characterized in that a correction value is determined from the signal in real time, by which the control signal of the deflection device (9) is corrected 22. Method according to claim 20 or 21, characterized in that the signal or a signal which is derived from the signal and a control signal of the deflection device (9) is stored in a storage device in such a way that it can be assigned to a measurement signal of the detection device (12) for detecting the light emitted from the focus area.