Minflux or STED minflux microscope having increased temporal-spatial resolution, and corresponding method

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

[0001] MINFLUX OR STED-MINFLUX MICROSCOPE WITH ENHANCED SPatio-temporal resolution and corresponding method

[0002] Technical field of the invention

[0003] The invention is directed to a microscope and MINFLUX methods or STED-MINFLUX methods to be performed with the microscope for the temporally and spatially high-resolution determination of the location of individual emitters or for the temporally and spatially high-resolution tracking of emitters. The emitters in question can, in particular, be individual fluorophores.

[0004] State of the art

[0005] A relatively new microscopy technique in the state of the art is MINFLUX nanoscopy. MINFLUX nanoscopy is a localization microscopy technique. The localization of individual emitters, particularly individual fluorophores, is achieved using structured illumination light distributions. The illumination light is scattered by the emitters or excites them to luminescence, particularly fluorescence, and in this general sense is considered excitation light.The fundamental 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 various positions close to a minimum of the excitation light distribution, which ideally is a zero point, with the excitation light distribution exhibiting a region of increasing intensity adjacent to the minimum. This ensures that the quantity of photons scattered by the emitter or the fluorescence photons, or each individual such photon, contains a particularly large amount 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. To obtain this information, the light emitted from the region of the minimum is detected.Tracking a single emitter using an excitation minimum is known, for example, from patent DE 10 2011 055 367 B4, and localizing a single fluorescent molecule using an intensity distribution of excitation light with a local intensity minimum is known from German patent DE 10 2013 114 860 B3. Based on this foundation, a number of refinements for information acquisition have emerged that enable localization of fluorophores with an uncertainty in the range below 2 nm and tracking 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. Furthermore, the concept of iterative MINFLUX is presented. In principle, in order to localize a fluorophore using MINFLUX nanoscopy as described in the cited publication, the intensity minimum or zero must be placed at a number of positions relative to the fluorophore. For this purpose, the current position of the fluorophore must be estimated with an initial, lower degree of accuracy in a preparatory step, or it must be known. This can be done, for example, using conventional localization microscopy (PALM, STORM) or other known methods.Subsequently, an intensity distribution of excitation light with a central minimum, for example, in the shape of a donut, as known from STED microscopy, is placed at a known position, which is chosen such that the estimated position of the fluorophore is close to the minimum of the intensity distribution. The fluorescence response of the fluorophore is measured. The same is usually performed for several additional positions of the intensity distribution located around the estimated position and, if necessary, also for the position corresponding to the estimated position. The set of all such sampling positions forms a set of sampling positions. Using a ratiometric evaluation of the intensity ratios, the position of the fluorophore is determined with greater accuracy.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, in the case of a fluorophore localization, can be placed closer to the estimated position of the fluorophore. This process can be continued iteratively. If the movement of an emitter is to be tracked, it may be useful not to decrease the distance between the positions at which the intensity minimum is placed and the position estimated in the previous step from step to step. The process is carried out as described in the cited publication using a microscope that contains both a piezo scanner and electro-optical deflectors (EOD). Using this combined scanning device, an image field of approximately 20 pm x 20 pm can be addressed.Using the EOD alone, an image field of approximately 2 pm x 2 pm can be addressed, with high-speed displacement only possible within a field of approximately 300 nm x 300 nm. Both when performing localization and when tracking a particle, the scanning positions of a set of scanning positions are controlled exclusively using high-speed EOD scanning. A combination of piezo scanner and EOD can then be used to shift the center. The movement of individual molecules, which are only mobile within a narrowly defined area—namely, within the area covered by a set of scanning positions—can be achieved using a fixed set of scanning points.

[0006] European patent EP 3 372 990 B1 discloses a method in which a set or sets of scanning positions are scanned with a local zero of an intensity distribution of fluorescence-preventing light, which is superimposed, for example, with a Gaussian intensity distribution of excitation light. The position of the fluorophore can then be determined in essentially the same way as with MINFLUX methods, whereby the dependence of the fluorescence intensity on the distance of the fluorophore with respect to the zero of the intensity distribution of the fluorescence-preventing light differs from MINFLUX in that the fluorescence intensity, or the rate at which photons can be detected, decreases with increasing distance. Such methods are referred to in this application as STED-MINFLUX methods.

[0007] International Patent Application 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 interference, which is configured to carry out an interference correction method. The microscope can include a scanner system that has an 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 implemented in a quad configuration. In particular, a method for correcting interference during image acquisition according to the MINFLUX principle is disclosed.The invention disclosed in the published patent application achieves the object of achieving high localization accuracy even when interference occurring during data acquisition using the MINFLUX method cannot be avoided. This is achieved by determining the magnitude of an interference during data acquisition for a localization, from which a weighting factor is derived that is taken into account when determining the location of a dye molecule. Alternatively, data acquisition is interrupted and continued depending on the magnitude of the interference. The magnitude of the interference can be detected using various types of sensors. In one embodiment, the magnitude of the interference 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-preventing light and finally directing it to a position-sensitive detector.In this arrangement, disturbances transmitted to the beam-guiding elements, for example, of the excitation light, which 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 perform 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 a minimum intensity of the excitation light in the sample is maintained at its target position.Specifically, a device is described and illustrated 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 decoupled again at a different point by means of a further dichroic beam splitter. 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 passes through an electro-optical deflection device, which has two consecutively connected electro-optical deflectors for deflecting the excitation light beam in the horizontal and vertical directions, respectively. After passing through the further dichroic beam splitter, it passes through another scanner, for example in a quad configuration—a special arrangement with four tilting mirrors.The beam path section common to the measuring light beam and the excitation light beam, however, does not include a deflection device or scanner.

[0008] 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 that 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. Furthermore, the electro-optical deflection device consists only of an electro-optical deflector (EOD) that operates in resonance and is designed to deflect the excitation beam only in one direction, along the fast axis.The publication explains that both scanning with an EOD and scanning with a mechanical scanner results in jitter, a time-varying deviation of the actual position from a target position. The magnitude of the jitter depends on the scanning speed. With a mechanical scanner, mass inertia causes disturbances during fast scanning, while various drifts can occur during slow scanning. Experimental determination of the respective jitter shows that the jitter of the EOD and the mechanical scanner, in the existing configuration, is of the same order of magnitude, namely in the single-digit range in nanometers, and can be neglected even in the context of high-resolution STED measurements.

[0009] Publication US 2002 / 179828 A1 is directed to a device for scanning a sample, which allows for the generation of image data largely free of image defects even during rapid scanning of a sample. The device includes, among other things, a device for determining an actual signal for each scan point from the settings of a scanning device. It further includes 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.

[0010] German patent DE 10 2007 008 009 B3 describes a scanning microscope having 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 to a method in which the measurement signal from 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 processing, which, according to the standard technical definition, is a method in which periodic, band-limited signals are obtained.The patented method solves a particular problem in fast resonant scanning, namely the phase shift that occurs between the control signal of a resonant scanner and the scanning pattern of the scanner, depending on the control frequency and the quality of the resonator. The detection device can be a device integrated into a scanner. In other embodiments, an auxiliary light beam is directed onto 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, whose signal forms the measurement signal.

[0011] 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 that 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 towards a photodiode. A slit is placed in front of the photodiode at the focus of the lens so that the photodiode regularly detects light and outputs a signal at a zero crossing of the scanner, during which 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.

[0012] Publication US 2021 / 239452 A1 relates to a device for measuring the 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 with such precision that angular changes that, after focusing through an objective, lead to deviations in the position of an illumination focus in a sample on the order of 1 nm can be detected. A microscope is described that has a laser whose collimated light is directed to a beam splitter. Part of the light passes through the beam splitter and is focused through an objective into a sample; another part is directed through the beam splitter toward the measuring device for measuring the angle of the collimated coherent light beam. The beam stabilization device and its function are not explained in detail.

[0013] 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 includes both a galvo scanner and two electro-optical deflectors (EODs) 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, such that the total displacement of the observed spot in the sample results from the sum of the displacements caused by the EOD and the galvo scanner. The galvo scanners enable the scanning of a large scan field, while the EODs enable rapid fine adjustment.In order to be able to scan quickly and accurately at high speed within a large scan 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 factory-implemented position sensors; 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 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 accurately, regardless of the inertial delay in the adjustment of the galvo scanners.The device is used to strongly vary the scanning speed, i.e. the duration during which a pixel is addressed or scanned, locally when performing a raster scan during a scan.

[0014] 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). https: / / doi.org / 10.1038 / s41467-Q21-21652-z ) describes the implementation of MINFLUX methods using a commercially available microscope. In addition to EOD, the microscope features a quad scanner consisting of four galvo scanners (two galvo scanners per deflection direction). The quad scanner, in conjunction with a 100x magnification objective, can address an image field of approximately 80 pm x 80 pm, making the microscope suitable for acquiring confocal images or STED microscopy. When performing a MINFLUX procedure, the sample points of a single set of sample points are each addressed using the EOD. The position of the center of each set is then adjusted using the galvo scanners.The microscope tracks biomolecules over a period of seconds with a time resolution in the range of 100 microseconds. The publication addresses the fact that the accuracy of position determination during particle tracking is reduced by the particle's displacement during the determination of individual positions.

[0015] Investigations by the applicant have now further shown that position determination using beam scanners is further reduced by the fact that, during the measurement of fluorescence at a set of scanning points, rapid, irregular, or irregular-appearing changes in the state of the beam scanner induce rapid, irregular, or at least irregular-appearing movements in the illumination light distribution in the sample. Both these rapid, irregular changes in the state of the beam scanner and the rapid, irregular movements in the illumination light distribution in the sample are referred to in this application as jitter. Jitter can overlay a controlled or regulated displacement of the illumination light and can occur even when the beam scanner is nominally aimed at a fixed position.It can occur particularly when using a beam deflection device that has a galvo scanner for addressing a large image field and an EOD for addressing the set of scanning points for MINFLUX localization. In this case, rapid, irregular or irregular-appearing movements of the galvo scanner mirrors, which are nominally in a rest position during MINFLUX scanning, can induce the irregular or irregular-appearing movement of the illumination light. In principle, such jitter can also occur in other beam scanner systems, particularly 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, such as movable mirrors.The impact of jitter is greater the shorter the effective measurement time at each point in the sample set. Consequently, jitter limits the spatial and temporal resolution in state-of-the-art microscopes.

[0016] Object of the invention

[0017] 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.

[0018] Solution

[0019] The object of the invention is achieved by a microscope having the features of independent claim 1 and by a method having the features of claim 20. Dependent claims 2 to 19 relate to preferred embodiments of the microscope, and dependent claims 21 and 22 relate to preferred embodiments of the method.

[0020] Definitions

[0021] In this application, emitters are understood to be objects that, when illuminated with excitation light, can be considered point light sources with regard to the measurements according to the invention. The light emitted by the object acting as a point light source can, for example, be scattered light resulting from elastic scattering such as Rayleigh scattering or inelastic scattering such as Raman scattering, or it can be luminescent light, in particular fluorescent light. It is essential for an emitter that it emits light immediately or with a short time delay in response to illumination.When emitter movements are to be tracked, the maximum time delay is related to the temporal resolution with which the movements of the light-emitting particles or light-emitting units are to be tracked and to the speed at which the particles or units move in the sample. The time delays can be up to around 10 ps, ​​but are usually in the range of up to a few tens of nanoseconds, frequently in the range of 1 to 10 ns and, if the emission is scattered light, zero. In this application, the term fluorescence emitter is used to describe emitters that act as fluorescent point light sources. Emitters can be, for example, metallic nanoparticles. The more specific term fluorescence emitter includes, for example, individual fluorescent dye molecules or their fluorescent chemical groups.Instead of dyes, other fluorescent units such as quantum dots or upconverting nanoparticles can also be used for labeling. Accordingly, in the context of this application, excitation light is understood not only to mean fluorescence excitation light, but generally to mean light that causes light to emanate from an emitter.

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

[0023] In this application, emission suppression light refers to 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.

[0024] In this application, STED light is understood, as is generally the case, to be light that suppresses fluorescence emission by exciting stimulated emission. STED light, in the context of this application, is a special form of emission-suppressing light.

[0025] In this application, MINFLUX methods are used to summarize localization and tracking methods for isolated emitters in which light distributions of illuminating light that excites light emissions from the emitter are generated at the focus in the sample, wherein the light distributions have a local minimum, and in which the position of an isolated emitter is determined by detecting light emissions from the emitter for different positions of the minima of the respective light distributions or for different such light distributions in a close range of the emitter, wherein the smaller the distance between the emitter and the minimum of the light distribution, the less light is emitted by the emitter.

[0026] In this application, STED-MINFLUX methods are understood to mean methods corresponding to the MINFLUX methods as defined above, with the difference that the intensity distribution of excitation light is replaced by an intensity distribution of emission suppression light that 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, is exposed to excitation light in such a way that an emission of the emitter is excited or would be excited in the absence of emission suppression light, so that the emission suppression light, according to its intensity distribution, affects the excited emission or the excitation of the emission in such a way that a measured emission of an emitter located within the intensity distribution of the emission suppression light decreases with increasing distance from the intensity minimum.If the emission quenching light is STED light and the emitter is a fluorescence emitter, this means that the spontaneous emission of the fluorescence emitter decreases with increasing distance from the intensity minimum of the STED light. The stimulated emission of the emitter, which has the same wavelength as the STED light, does not contribute to the measured emission, as is common in STED microscopy; it is blocked, for example, by a filter. The fact that the intensity distribution of the emission quenching light overlaps with the excitation light does not mean that the excitation light and the emission quenching light are introduced into the sample strictly simultaneously. For example, it is possible, as is common in STED microscopy, to apply a short pulse of excitation light, followed immediately by a pulse of STED light as emission quenching light.The excitation light overlapping the intensity distribution of the emission suppression light can, for example, have an intensity distribution as is common in confocal microscopy, i.e., at least approximately corresponding to an Airy function or a Gaussian function, whose central maximum may coincide with the minimum of the emission suppression light. It can also correspond to a top-hat function. Constant excitation in the wide field is also possible.

[0027] The term STED-MINFLUX is used in a generalized sense; it should not be understood that the emission suppression light must be STED light.

[0028] In MINFLUX microscopy and STED-MINFLUX microscopy, the sample is illuminated with focused illumination light. The illumination light forms an intensity distribution with a central intensity minimum, specifically an intensity zero, around a geometric focus in the sample. The position of an individual emitter relative to the central minimum then determines the intensity or photon emission rate of the emitter's light emissions, which are used to determine the position. Intensity distributions with a central minimum can be generated, in particular, by phase modulating the illumination light using a phase filter or a spatial light modulator (SLM) in the illumination beam path, focusing the illumination light using an objective lens, and, if necessary, additionally circularly polarizing the illumination light. The intensity distribution then arises at the geometric focus through interference.

[0029] When the present application refers to the fact that the intensity distribution of the illumination light is shifted at scanning positions, this means that the intensity distribution of the illumination light is shifted relative to the sample in such a way that the focal point in which the central minimum is formed is arranged at a respective scanning position.

[0030] 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.

[0031] If this minimum extends along an axis perpendicular to this plane, this minimum is referred to in this application as a 2D minimum. A 2D minimum can be obtained, for example, using a vortex phase plate in the beam path, whereby the minimum can then extend, for example, along the optical axis of a lens.

[0032] In this application, a 3D minimum is understood to be a minimum surrounded by regions of increasing intensity in all spatial directions. A 3D minimum can be obtained, for example, by superimposing two light beams, with the wavefront of one of the two being phase-modulated in a pupil using an annular phase plate and the wavefront of the other being phase-modulated using a vortex phase plate. A 3D minimum can also exhibit a donut-shaped intensity distribution in a given plane.

[0033] In this application, the term "localization" describes a method in which the position of an individual emitter in a sample is determined. In contrast to conventional light microscopy methods, this method does not require optical imaging of the sample. Rather, the MINFLUX localization method according to the invention detects light emissions from the emitter for various positions of the intensity distribution of the illumination light, and from these, the position of the emitter is determined, for example, calculated using a position estimator. This process can be performed sequentially for several individual emitters, and the determined positions can be displayed in a localization map, which can produce a high-resolution light microscopy image.Both localizations performed using a MINFLUX method and those performed using a STED-MINFLUX method are referred to as MINFLUX localizations for the purposes of this application.

[0034] Tracking an emitter means determining the trajectory of an emitter moving in the sample, particularly by repeatedly locating it in succession.

[0035] In the context of the present specification, a beam scanner is a device that variably deflects or shifts a light beam. A shift of 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 illumination light, i.e., an illumination light beam, by means of one 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 shift of the focus can, in particular, take place in two spatial directions, furthermore, in particular, in a focal plane that is perpendicular to an optical axis of an objective. The spatial directions can be defined in a wide variety of coordinate systems, e.g., in Cartesian coordinates (e.g.,x- and y-direction), 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 connected to a galvo motor, electro-optical deflectors, or acousto-optical deflectors.

[0036] For the purposes of this application, a galvo scanner is understood to be a module with a galvo drive and a tilting mirror connected to it and movable by the galvo drive. The drive can be designed such that the angular positions of the tilting mirror are adjustable, or such that the tilting mirror performs a periodic rotary motion. For the purposes of this application, the term galvo scanner therefore includes resonant galvo scanners and galvo scanners in which the angular positions of the tilting mirror are adjustable; these are referred to as adjustable galvo scanners for the purposes 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.

[0037] For the purposes of this application, a galvoscanner system is defined as a scanning unit comprising one or more galvoscanners, which as a whole is configured to shift and / or deflect a light beam in one or more spatial directions. Similarly, for the purposes of this application, a MEMS scanner system is defined as a scanning unit comprising one or more MEMS scanners, and a piezo scanner system is defined as a scanning unit comprising one or more piezo scanners, each of which as a whole is configured to shift and / or deflect a light beam in one or more spatial directions.

[0038] 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.

[0039] 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.

[0040] Description of the invention

[0041] The microscope according to the invention is designed to perform 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.

[0042] The microscope according to the invention for carrying out a MINFLUX method and / or a STED-MINFLUX method comprises a light source for generating an illuminating light. It further comprises optical elements that form an illuminating light beam path through which the illuminating light is guided as an illuminating light beam from the light source into a sample region. In the illuminating light beam path, it comprises optical means for focusing the illuminating light into a focus region in a sample in the sample region such that, in the focus region of the illuminating light, an intensity minimum of the illuminating light at a focal point is bordered on both sides in at least one direction by regions of increasing intensity. Furthermore, in the illuminating light beam path, it comprises a deflection device that includes a first beam scanner.The deflection device is designed to position the focal point in the sample at multiple positions within the sample. The microscope has a detection device for detecting emissions from the focal region of the illumination light. The microscope according to the invention differs from microscopes known from the prior art for carrying out MINFLUX methods or STED-MINFLUX methods in that it includes 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. The measuring device is designed to output a signal at a signal output depending on the position and / or direction of the reference light beam, which signal is indicative of a position of the focal point in the sample.

[0043] The optical means for focusing the illumination light into a focus area in a sample may in particular comprise an objective lens.

[0044] The fact that in the focus region of the illumination light, regions of intensity increase border on both sides in at least one direction an intensity minimum of the illumination light at a focus point includes in particular the cases where a 2D minimum is formed in the sample in the focus region, where a 3D minimum is formed, and where a sheet-shaped intensity minimum, ideally a zero point, with adjacent intensity increase regions is formed in a plane containing the optical axis and thus the focus point. For this purpose, the optical means for focusing the illumination light into a focus region in a sample comprise one or more elements which cause or cause an intensity increase region to border on both sides in at least one direction an intensity minimum of the illumination light at a focus point in the focus region of the illumination light.For example, such an element can comprise a vortex phase plate to form a 2D minimum, an annular phase plate to form a 3D minimum, or a phase plate with a 180° phase shift along a diameter to form a 1D minimum. Corresponding elements that influence 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 focal area.

[0045] 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 focal 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 as defined further 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 simplify differentiation in cases in which the deflection device of the microscope has several beam scanners.The fact that the first beam scanner is configured to position the focal point in the sample does not mean that the positioning of the focal point in the sample is carried out exclusively by the first beam scanner, but rather that the current position of the focal point in the sample is at least partly determined by the current setting of the first beam scanner. The fact that the focal 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 focal point in the sample changes according to the random change in the setting of the first beam scanner.

[0046] The first beam scanner may comprise a mechanical scanner or be designed entirely as a mechanical scanner. The mechanical scanner of the first beam scanner may, in particular, comprise a deflection unit with at least one continuous mirror that deflects the entire illumination light. Alternatively or additionally, this mechanical scanner may comprise one or more movable, in particular rotatable, elements through which the illumination light passes, such as one or more 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, comprise or be formed from resonant mechanical scanners with at least one connected mirror that deflects the entire 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.

[0047] The first beam scanner may comprise electro-optical deflectors (EOD) or acousto-optical deflectors (AOD), or may 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 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 illuminating light is directed into the sample, it is important that the illuminating light beam is only tilted in the illuminating light beam in the pupil of the objective by the deflection device, i.e. by the first beam scanner or by the first beam scanner and further beam scanners, and is not displaced or at least displaced as little as possible, since a displacement of the illuminating light beam would change the shape of the intensity distribution of the illuminating 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 into 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 focal 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.Galvo scanner-based mechanical scanners are preferred when large image fields need to be addressed, for example, to acquire a confocal overview image or to shift the focal point of the illumination light to different areas within the larger image field. Among other things, they typically enable a larger usable scanning area compared to other scanner types due to their properties such as their usable beam diameter and 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, with the mirrors of the MEMS scanners each being tiltable about two mutually orthogonal axes. Compared to a quad scanner, this embodiment requires fewer reflective surfaces.

[0048] In preferred embodiments, the deflection device of the microscope can comprise additional beam scanners in addition to the first beam scanner. All beam scanners, the first beam scanner and the additional beam scanners, can be designed as mechanical scanners. In preferred embodiments, the microscope can comprise an additional 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 displacement by the first beam scanner. It is possible to address different scanning positions or scan different scanning paths by combining controlled deflections by the first beam scanner and controlled deflections by the additional beam scanner, as well as to address different scanning positions or scan different scanning paths.by only controlled deflections being carried out by the additional beam scanner, while the first beam scanner is controlled in such a way that it is, as far as achievable, in a static position, i.e., 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 adjustment changes, in the case of a mechanical scanner, very small movements, which, if the adjustment of the additional beam scanner were not changed, 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 focal point in the sample compared to irregularities that occur when a static adjustment of the first beam scanner is desired. This applies in particular,if the first beam scanner comprises adjustable galvo scanners and / or adjustable MEMS scanners.

[0049] Preferably, the deflection device is configured such that the focal point can be rapidly shifted within a close range with dimensions that approximately correspond to the extent of a diffraction-limited illumination spot. The ability to rapidly shift the focal point in the sample also includes cases in which the rapid shift of the focal point in the sample is induced by rapid, irregular, or irregular-appearing changes in the state of the deflection device, for example, the first beam scanner. Furthermore, the deflection device is preferably configured to shift the focal point of the illumination light in the sample over larger distances, for example, over 10 pm, 20 pm, 100 pm, or 500 pm.In preferred embodiments, the deflection unit therefore comprises 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 scanned 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 greater distances, for example over 10 pm or 20 pm or 100 pm or 500 pm.

[0050] In preferred embodiments, the additional beam scanner, i.e., a different beam scanner than the first beam scanner, is an adjustable beam scanner. Further preferably, the additional beam scanner has a high bandwidth and is configured to quickly shift the focal point of the illumination light to a set of scanning positions for MINFLUX localization. In further preferred embodiments, the bandwidth of the additional 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 one hundred or more than one hundred times as large. Accordingly, the response times of the deflection unit, i.e., the time required for a scanner to be moved from one angular position to another, are preferably shorter than those of the first beam scanner, for example, they are only 50%, 20%, 10%, 1%, or less than 1% of those of the first beam scanner.

[0051] If the deflection device comprises a first beam scanner and a further beam scanner, in preferred embodiments the further beam scanner comprises 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 mass inertia. Both types of deflectors thus enable extremely fast relocation to any point within the range addressable 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, thus enabling a relocation of the focal point within less than 1 ps.

[0052] The additional beam scanner of the microscope can also comprise a mechanical scanner or be designed entirely as a mechanical scanner. The mechanical scanner of the additional beam scanner can, in particular, comprise a deflection unit with at least one continuous mirror that deflects the entire illumination light. The mechanical scanner of the additional beam scanner can, for example, contain a MEMS scanner, piezo scanner, or galvo scanner, or can be designed as a MEMS scanner system, piezo scanner system, or galvo scanner system. In particular, piezo scanners and MEMS scanners can be designed such that a mirror can be tilted about two mutually orthogonal axes.

[0053] If the microscope's additional beam scanner comprises mechanical scanners with a deflection unit including at least one continuous mirror deflecting the entire illumination light, mechanical scanners with a small aperture are preferably selected for this purpose. The microscope then preferably comprises an objective lens. The continuous mirror deflecting the entire illumination light can preferably be arranged in a pupil. Then, the microscope can comprise an expansion unit in the illumination light beam downstream of the additional beam scanner, with which the illumination light beam is adapted to the aperture of an objective lens.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 correspondingly 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 resonant 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 specific paths.For example, using two resonant scanners of identical frequencies with mutually perpendicular axes after imaging into the sample, the focal point can be shifted to circular orbits. If the frequencies differ, different orbits result, for example, Lissajous orbits 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 a MEMS scanner.

[0054] In a further preferred embodiment, the additional beam scanner comprises a resonant mechanical scanner, which can be placed in a pupil in particular and which shifts the illumination light beam in the sample in a first direction, and an adjustable mechanical scanner which shifts 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 acquisition.

[0055] 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 matched to one another, more preferably matched to one another in such a way.

[0056] In other preferred embodiments, the further beam scanner may be or comprise a resonantly operated EOD.

[0057] With a resonantly operated beam scanner, the focal point of the illumination light can be shifted particularly quickly. Furthermore, the microscope according to the invention has a detection device for detecting light emitted from the focal region of the illumination light, i.e., for detecting emissions from the focal region of the illumination light. The detection device is configured to detect emissions in such a way that each emission can be assigned to a position of the focal 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 focal point in the sample, but also to a location in the detection plane.

[0058] 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 direction of a reference light beam that is deflected and / or displaced by the first beam scanner. The measuring device is designed to output a signal at a signal output as a function of the position and / or direction, which signal is indicative of a position of the focal point in the sample. The reference light beam can be a light beam that has been split 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 reference light beam is deflected and / or shifted by the same deflecting element(s) 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, this means that the reference light beam and the illumination light beam are deflected and / or shifted by the same mirror surface(s). 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, which corresponds to a target position of the focal point in the sample. The position of the focal point in the sample can then 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 those skilled in the art. However, the signal can also directly be a 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 a component of the first beam scanner or which forms the additional beam scanner.Then, after appropriate amplification, the signal from the measuring device is fed back to the fast beam scanner as an analog control signal.

[0059] The measuring device preferably has a position-sensitive or spatially resolving detector at one end of the reference light beam path. Further preferably, a detector of the measuring device is a position-sensitive diode, in particular a diode that is position-sensitive in two spatial directions. Position-sensitive diodes have the particular advantage of having high temporal resolution and thus allowing fast data acquisition and fast control, for example, of an EOD scanner of the deflection device, for example at a frequency of several hundred kilohertz.

[0060] In a preferred embodiment, the measuring device is configured 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 onto the detector. For example, if 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 relative to an optical axis of the beam scanner, which axis is in a zero position, the imposed 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 exits the first beam scanner in a convergent manner, 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 exits the first beam scanner in a collimated manner and is subsequently focused outside the illumination beam path.

[0061] In a preferred embodiment, the measuring device is configured 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 onto the or a further position-sensitive or spatially resolving detector.

[0062] Further properties of the measuring device are described in the method descriptions below, as well as in the descriptions of the figures. Preferably, the reference light beam propagates along a reference light beam path, which includes the first beam scanner.

[0063] 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 will become apparent from the description of the method according to the invention given below.

[0064] The microscope according to the invention preferably has a control unit configured to control the first beam scanner or the further beam scanner(s) or the first beam scanner and the further beam scanner(s) such 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, in particular electrical signals, from the control unit that 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 configured, in particular, to control the first beam scanner such that the focal point in the sample is positioned and / or displaced within a larger image field, for example, such that 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 configured, 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, whereby the connection can be direct or indirect, mediated by further elements of the computing and control unit, of which it can be an integral part. In a further preferred embodiment, the computing and control unit is designed to determine control and regulation signals for the deflection device based on the signals from the measuring device and to output them to the deflection device.

[0065] 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 is determined.For this purpose, an illuminating light is focused into the sample in such a way that, in the focus area of ​​the illuminating light, areas of increasing intensity border on both sides of an intensity minimum of the illuminating light at a focus point 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 illuminating light is excited by the illuminating light or by another light to emit luminescence or scattered light. The light emitted from the focus area of ​​the illuminating light is detected by a detection device, measurement values ​​from the detection device being assigned to the scanning positions, and a position or trajectory of an emitter is determined from the measurement values ​​and the assigned scanning positions.

[0066] 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 which is indicative of a position of the focal point in the sample.

[0067] 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 be raster-like 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 adjustments of the first beam scanner.In both cases, the positioning of the focal point is then carried out sequentially at different actual scanning positions, whereby each actual scanning position, i.e. each position of the focal point, does not correspond exactly to the respectively 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. on 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 measurement signal, which is indicative of a respective position of the focal point in the sample, i.e. from which the respective position of the focal point in the sample can be determined, is output at a signal output.

[0068] In a preferred embodiment of the invention, the focal point in the sample is sequentially positioned at various 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 be raster-like or form a Lissajous path, a rosette, or a loop.

[0069] As stated above, in the method according to the invention, an emitter is excited by the illumination light or by another light in the focal region of the illumination light to emit luminescent or scattered light. If the excitation occurs by the illumination light, the illumination light can be the only light irradiated into the sample during the method. With such a method, the position of an emitter at which illumination light is elastically scattered, such as a gold bead, or of a luminescent particle, such as a fluorescent bead or, in particular, a fluorophore, can be determined, as well as the corresponding emitter can be tracked.

[0070] In addition to the illumination light, additional light can also be irradiated into the sample. In one embodiment, the additional light can be a fluorescence-preventing light, in particular a STED light, which suppresses the emission of free, i.e., unstimulated, fluorescence from an area adjacent to the focal area of ​​the illumination light and / or from outer areas of the focal area, for which it is known from previous position determinations or position estimations that the fluorophore whose position is to be determined is not located. In this case, the fluorescence-preventing light can therefore serve, in particular, to suppress background light, which would impair the quality of the position determination.For this purpose, the fluorescence-preventing light can form a wide STED donut, which is generated, for example, using a vortex phase plate with a multiple increase in the modulation of the wavefront phase from 0 to 2TT over one revolution along the circumferential direction. It can also form a 3D donut, which can be generated, for example, using an annular phase plate. In this case, the additional light can be stationary in the sample, so that the illumination light is shifted relative to the additional light, or the additional light can be shifted 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-preventing 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 mentioned in the relevant section.In STED-MINFLUX methods, the emitter, whose position is to be determined or tracked, is excited with additional light as excitation light. The additional light can be light focused together with the illumination light through an objective, whereby the shape of the intensity distribution can at least approximately correspond to an Airy function, a Gaussian function, or a top-hat function. Preferably, the intensity distribution has a broad range at its center—wide compared to the distances between the scanning positions of the illumination light to which the illumination light is shifted—in which the intensity changes little or not. This condition can be met, for example, by focusing the additional light using part of the objective's aperture, 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.

[0071] The light emissions from the emitter are detected by a detection device. This can, for example, comprise 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 further comprises evaluation electronics. The detection device is designed, in particular, to detect individual photons emitted by the emitter and to register them in such a way that the scanning positions associated with the light emissions can be assigned.

[0072] There is 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 downstream of the scanner, whereby the relationship between the directions and / or positions of the reference light beam and the illumination light beam must be taken into account. Furthermore, there is a specific relationship between the position and / or direction of the illumination light beam downstream of the scanner and the position of the focal point in the sample. Detecting 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 from the measuring device, can be used in two ways, which can also be combined, to increase the spatial-temporal resolution.

[0073] One approach involves acquiring 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 thus 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-derivative).The use of the measurement signal for control can be particularly advantageous when the deflection device has another adjustable beam scanner that allows random access to scanning points and has shorter, in particular much shorter, response times than the first beam scanner, for example, only 20%, 10%, 1%, or less than 1% of that of 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 is achieved 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 rapid corrections to the position of the focal point in the sample are effected by the additional, fast beam scanner or by the fast unit(s) of the first beam scanner. This does not preclude the possibility of slower corrections being made using or jointly using the first beam scanner or the slow unit(s) for the correction. However, it is particularly simple to control the first beam scanner or the slow units of the first beam scanner and to regulate exclusively the additional beam scanner or the fast unit(s) of the first beam scanner. In this case, 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 additional 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 additional 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 position and / or direction detection. 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.

[0074] 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 direction or a signal derived from a control signal of the deflection device and the signal of the measuring device is preferably stored in a memory 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.

[0075] 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.

[0076] Determining the position of the emitter or tracking the emitter by determining a trajectory can now be performed using the methods commonly used for MINFLUX or STED-MINFLUX methods. This also applies to the selection of the sampling positions at which the focus point is to be positioned.

[0077] If the deflection device is controlled based on the signal from the measuring device, an advantage arises simply 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 displacement of the focal point in the sample in such a way that the focal point would not change its position, the first beam scanner would be 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 displacements of the focal point in the sample with a displacement of several nanometers, with the noise power spectrum exhibiting large values ​​in the frequency range of several kilohertz.This can result in an (additional) uncertainty of several nanometers when determining the instantaneous 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 fundamentally possible using the MINFLUX method, this uncertainty due to the jitter is therefore significant. Using an additional, 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 approximately 1 ps or less, provided the bandwidth of the measurement of the position and / or orientation is sufficiently large. This measurement with a correspondingly high bandwidth or time resolution can be realized using means known in the art, as will be explained below.With a narrower measurement bandwidth, this limits the time constant of the correction. Using the measuring device and, for example, the EOD of the deflection device, it is possible, for example, to address a scanning position with an uncertainty of less than 1 nm. This significantly reduces the uncertainty of determining the position during fast scanning or, conversely, enables high accuracy even during very fast measurements, as is particularly necessary when tracking a fast-moving emitter.

[0078] It is also possible for the first beam scanner to comprise multiple, 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 such 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. However, in this example, the measurement signal is nevertheless fed back to the EOD system.

[0079] The measurement signal from the measuring device for determining the orientation and / or position, or the signal derived from it, can alternatively or additionally be used in another way to increase the spatial and temporal resolution. This is to say, when determining the orientation or trajectory of the emitter from the emissions detected by the detection device, the actual focal points, i.e., the focal points determined on the basis of the measurement of the orientation and / or direction of the reference light beam, are taken into account at the time of excitation of the emission. 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 ​​corresponding 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 state of the art, the position of the emitter can be determined in real time. Based on this position, subsequent scanning positions are determined, to which the focal point is shifted during 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 directly affect the rest of the measurement process. However, from the measured position and / or position of the reference light beam, a scanning position can now be determined with greater accuracy than from the data from the control of 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.

[0080] In principle, however, the actual positions of the focal point in the sample at the time of emission excitation can also be taken into account during data acquisition, i.e. the positions determined based on the measurement of the position and / or direction of the reference light beam, 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 customized 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 passed from there to an FPGA for controlling the deflection unit, or the signal from the measuring device can be passed to an FPGA for controlling the deflection unit and fully evaluated there.

[0081] A combination of both types of use of the measuring device signal—that is, the use for controlling the deflection device and the use 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, even when the deflection unit is controlled by the measuring device signal, a deviation remains between the actual scanning position and the target position detected by the measuring device. This deviation is reduced compared to the deviation in the absence of control.The evaluation of the data in real time can now be carried out in the same way as when using only the signal from the measuring device 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 the direction of the reference light beam at the time of excitation of the emission, during the data acquisition.

[0082] 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.

[0083] The reference light source can be, for example, a laser, a light-emitting diode, or a laser diode. Particularly if the reference light source is a broadband light source, generating the reference light beam can involve filtering the wavelengths.

[0084] 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 longest wavelength used when performing microscopy methods with the microscope, for example, a longest STED laser wavelength or a longest fluorescence wavelength to be detected; alternatively, the wavelength range of the reference light source can exclusively comprise wavelengths below the smallest wavelength used for performing 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.

[0085] 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 overall beam path. If the first beam scanner includes an AOD and / or an EOD, boundary conditions for the properties of the reference light beam arise. For example, the deflection angle that the first beam scanner imposes on a light beam can be dependent on polarization, as is often the case with a single EOD, or it can be dependent on wavelength, as is often the case with a single AOD.Accordingly, if the deflection unit has EOD and the reference light beam is guided through the deflection unit, it is preferred that the reference light beam guided through or via the first beam scanner has the same polarization as the illumination light beam, but preferably a different wavelength, thereby enabling separation of the reference light beam 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.

[0086] 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, can be exited from 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 its internal properties, such as wavelength or polarization. The reference light beam essentially undergoes 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 are determined by the parameters of the first beam scanner, for example the dimensions of mirrors and the structure of the beam path in the area of ​​the first beam scanner, which can, for example, have a relay optics.

[0087] 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 axis 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.

[0088] 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, a dichroic beam splitter. This is particularly possible when the wavelength ranges of the reference light beam and the illumination light beam do not overlap, and is particularly advantageous when 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 coupled 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. More preferably, the coupling takes place on this optical axis.

[0089] 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, i.e., the illumination light source and the reference light source are located on the same side of the scanner, or such that the illumination light beam and the reference light beam pass through the first beam scanner in opposite directions, i.e., the illumination light source and the reference light source are located on opposite sides of the first beam scanner.

[0090] The output or decoupling can be carried out in the same way as the input. The output does not necessarily have to be carried out using the same method as the input; combinations of different methods are also possible. For example, the input can be carried out using a mirror in an edge region and the output using a beam splitter. Preferably, the output takes place when the reference light beam was introduced at an angle, likewise without the use of an additional 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 output 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 in by means of a dichroic beam splitter on the optical axis and is also coupled out again using a dichroic beam splitter.

[0091] 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 illuminating 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.

[0092] 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, in particular, have two different components upon coupling or introduction, for example a collimated and a convergent component. If the reference light has such different components, it is preferred that it additionally differs from the illumination light either in another property, for example the 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 intermittently coupling in short pulses.When using several different components, it is essential that the components can be detected separately after the scanner.

[0093] The reference light beam can further be coupled or introduced in such a way that, in the beam path section which it has in common with the illuminating light beam, it has a state corresponding to that of the illuminating 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.

[0094] 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. After the first beam scanner, it is coupled out again at a location where the reference light beam is also collimated. The position of such locations in a given beam path can be easily determined by a person skilled in the art.

[0095] There are no imaging optical elements within a quad scanner, such as those 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, collimated coupling or introduction can occur adjacent to one of the outer mirrors along the beam path and coupling or exit can occur adjacent to the outer mirror opposite the beam path. According to the functional principle of a quad scanner (see, for example, 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 objective in a collimated state, whereby an angle of the illumination light beam to the optical axis of the objective, and thus the position of the focus of the illumination light in the sample, depends on the position of the quad scanner.If the objective lens and the second optical element form a telecentric system, the condition that the axis of the illuminating light beam intersects the optical axis of the objective lens in the rear focal plane or pupil for all scanner settings is met if, for all different scanner settings, the illuminating light beams after the last mirror of the scanner and before the second optical element are parallel to each other and to the optical axis, i.e., if the scanner only offsets the illuminating light beam but does not tilt it. The change in the position of the focal point of the illuminating light in the sample caused by the scanner is then directly dependent on the change in the offset of the illuminating light caused by the scanner. The same change in offset is now imposed on a reference light beam.If the reference light beam is coupled into the beam path of the illuminating light or introduced at a shallow angle such that it passes the entire scanner as a collimated beam, the offset of the reference light beam and thus the change in the offset of the illuminating light beam can be determined in a simple manner by directing or decoupled the reference light beam in the direction of the reference light beam after the scanner and before the first or the second optical element, depending on whether the reference light beam propagates in the direction of the illuminating 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 diverted 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 then forms an essential element of the measuring device for detecting the – in this case – 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 angular change that the scanner imposes on the reference light beam and thus also on the illumination light beam. This angular change, which is detected by 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.The detection of this change in position makes it possible to draw conclusions about the change in the quality of the excitation light distribution and a shift of the focal point in the sample resulting from the change in position in the pupil.

[0096] Advantageous developments of the invention emerge from the patent claims, the description, the drawings, and the associated explanations of the drawings. The described advantages of features and / or combinations of features of the invention are merely exemplary and may be effective alternatively or cumulatively. Regarding the disclosure content (but not the scope of protection) of the original application documents and the patent, the following applies: Further features can be found in the drawings—in particular, the relative arrangements and operative connections shown.

[0097] The combination of features of different embodiments of the invention or of features of different patent claims is also possible, deviating from the chosen references of the patent claims, and is hereby encouraged. This also applies to features shown in separate drawings or mentioned in their description. These features can also be combined with features of different patent claims. Likewise, features listed in the patent claims can be omitted for further embodiments of the invention; however, this does not apply to the independent claims of the granted patent.

[0098] The reference signs contained in the patent claims do not represent a limitation of the scope of the subject-matter protected by the patent claims. They serve solely to make the patent claims easier to understand.

[0099] Short description of the characters

[0100] Fig. 1 shows an embodiment of a microscope according to the invention with a reference light source.

[0101] Fig. 2 shows an embodiment of a microscope according to the invention, in which a

[0102] Reference light beam is split from light from an illuminating light source.

[0103] Fig. 3 shows an embodiment of a microscope according to the invention, in which a

[0104] Reference light beam from a reference light source runs in the opposite direction to the illumination light beam.

[0105] 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.

[0106] Fig. 5 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a tilt.

[0107] Fig. 6 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining tilt with an enlarged measuring range. Fig. 7 shows a section of another embodiment of a microscope according to the invention with a measuring device for determining tilt with an enlarged measuring range and improved sensitivity.

[0108] Fig. 8 shows a part of the measuring device of the microscope shown in Fig. 7 in detail.

[0109] Fig. 9 shows exemplary measurement data that can be recorded with the measuring device of the microscope shown in Fig. 7.

[0110] Fig. 10 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a displacement.

[0111] Description of the characters

[0112] Fig. 1, Fig. 2 and Fig. 3 each show an embodiment of the microscope 1 according to the invention. 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 at a focus point is bordered on both sides in at least one direction by regions of intensity increase, which in concrete terms can mean that a 2D donut or a 3D donut is formed in the sample. The sample region 7 is formed in each case on an adjustable sample stage 20.The embodiments of the microscope 1 illustrated in these figures each have a fast, adjustable scanner, which here is specifically an EOD scanner 11, and they all have a mechanical beam scanner 10. In all illustrated embodiments, the mechanical scanner 10 and the EOD scanner 11 form a deflection unit 9. All embodiments of the microscope 1 illustrated in these figures have a light modulator, which here is specifically designed as a phase filter 3. 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 illustrated in all figures.The embodiments of the microscope 1 illustrated in the figures above 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 illustrated in the figures above each further have a mechanical scanner 10, which deflects the illumination light beam B, a 4-wave plate 5 for adjusting the 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.

[0113] The embodiments of the microscope 1 illustrated in Figures 1 to 3 each further comprise a measuring device 13 configured to detect a position and / or a direction of a reference light beam R deflected and / or shifted by a first beam scanner 18. In the illustrated embodiments of the microscope 1, 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 is preferably 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 respectively associated target position can be stored in the memory 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 illustrated embodiments of the microscope 1, 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 transferred to the EOD scanner 11.The control signal is selected such that a scan is performed for performing a MINFLUX method or a STED-MINFLUX method. A correction is determined based on the signal S, i.e., feedback or control of the deflection device 9, specifically in particular of the EOD scanner 11, is provided. This feedback and control is not necessary in all embodiments of the invention, but it is advantageous for improving the quality of data acquisition when performing a MINFLUX method or a STED-MINFLUX method.However, the signal S can be used not only to improve the data acquisition, which is usually associated with an initial data analysis performed in parallel with the data acquisition, but also to improve the data analysis performed after the data acquisition has been completed by determining the position of an emitter based on the actual scanning positions, i.e., the respective actual positions of the focal point in the sample. Another dotted line indicates that a control signal, generated in the computing and control unit 17, is transmitted to the mechanical beam scanner 10. This control signal can be static or temporally variable during the execution of a localization using a MINFLUX method or a STED-MINFLUX method.When determining the control signal for the mechanical scanner 10, the signal S of the measuring device 13 can also be taken into account.

[0114] By means of the adjustable sample table 20 shown in the figures, it can be ensured that the deflection device 9 can be operated with settings close to a zero position, in particular close to a zero position of the mechanical scanner 10, during the implementation of a MINFLUX method or a STED-MINFLUX method, by regularly pushing the sample 8 into the center of the area scanned by the deflection device 9 using the sample table 20. This reduces the demands on the measuring device 13, since the measurement 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 implemented accordingly.

[0115] 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, together with the EOD scanner 11, forms a deflection device 9. 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 slightly offset in parallel for reasons of clarity.The reference light beam R propagates downstream of 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, depending on the setting of the first beam scanner 18, which is formed by the mechanical scanner 10. Downstream of the first beam scanner 18 is another beam splitter, 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 toward the measuring device 13. Both the first beam splitter 14 and the second beam splitter 15 are tuned to a difference between the reference light beam R and the illumination light beam B, for example, to different polarizations or different wavelengths.

[0116] 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, here forms the entire deflection device 9. 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 downstream of 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 specifically illustrated embodiment, 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 deflections and / or displacements identical to those of the illumination light beam B up to the first beam splitter 14. The beam splitter 14 then 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 shifted by the first beam scanner 18 has undergone phase modulation in this embodiment of the microscope 1, a corresponding embodiment of the measuring device 13 is preferably used in this embodiment of the microscope 1.

[0117] 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, together with the EOD scanner 11, forms a deflection device 9. The illustrated embodiment of the microscope 1 has a reference light source 16. In contrast to the embodiment shown in Fig.In the embodiment of the microscope 1 shown in Figure 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. Instead, the reference light beam R is introduced into the illumination light beam path at a shallow angle, which is not shown at a true 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 here is identical to the mechanical beam scanner 10. Accordingly, the reference light beam R is correspondingly output from the beam path of the illumination light at a shallow 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 does not have to be identical to the angle at which the reference light beam R is output 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 transmitted through the first beam scanner 18 in the opposite direction to the illumination light beam B.

[0118] The properties of the embodiments of Fig. 1 and Fig. 3 could also be combined. Thus, the reference light beam R could be introduced at a shallow 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.

[0119] 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, generated by the measuring device 13 in conjunction with an analog amplifier, is fed back to the EOD scanner 11 as a control signal. The analog amplifier can also be considered 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.

[0120] 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.

[0121] 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 Å / 4 plate 5, the objective 6, the sample area 7 with the sample 8, which is placed on the adjustable sample stage 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 shows an entrance lens 22 and an exit lens 23, 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 merely 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-shaping element, that is to say 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 shown, Fig.5 is to be understood to mean that in the illustrated embodiment of the microscope 1, there is actually no beam-shaping element, that is to say in particular no lens, between the exit lens 23 and the objective 6. In this embodiment, the microscope 1 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 in a collimated state at the entrance lens 22 and exit the first beam scanner 18 in a collimated state at the exit lens 23. Upon 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 relative to the optical axis of the additional beam scanner, which is in a zero position, depending on the 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 shifted. In the example shown, a shift 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 shift. 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 region 7.The position of the focal point in the sample 8 is determined by the total tilt imposed on the illumination light beam B, which results from the additive 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 its focal length. The reference light beam R is decoupled from the illumination beam path by the first beam splitter 14 and directed toward an embodiment of the measuring device 13. The measuring device 13 has a lens 25 that 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 tilt imparted to the reference light beam R by the first beam scanner 18 and on the properties of the lens 25, in particular 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 for determining the tilt and a smaller measuring range in which tilts can be measured, or a lower sensitivity and a larger measuring range.

[0122] Fig. 6 shows a section of a further 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 measurement range of the tilts while maintaining high sensitivity for determining the tilt, the measuring device 13 has, on the one hand, a diffractive optical element (DOE) 26 and, on the other hand, several position-sensitive diodes 24 arranged next to one another. The DOE 26 splits the incident reference light beam R into several partial beams. An identical tilt is impressed on each of the partial beams 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 subjected to any tilt by the first beam scanner 18, so that the 0th-order partial beam falls centrally onto the central one of the position-sensitive diodes 24. With a greater tilt, the 0th-order beam now moves out of the area of ​​the central position-sensitive diode 24, while either the 1st-order partial beam or the -1st-order partial beam moves into the area of ​​the central position-sensitive diode 24, depending on the direction of the tilt. In general, the splitting and detection with multiple position-sensitive diodes 24 ensures that even with large tilts of the reference light beam R, small changes in the tilt can be measured. For this purpose, the individual signals from the multiple diodes 13 can also be combined.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. For a two-dimensional arrangement, at least three position-sensitive diodes 24 are required. 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.

[0123] Fig. 7 shows a section of a further 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 with simultaneously improved 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, for example, as photodiodes.7 shows the arrangement in cross section in the plane of the optical axis of the incident reference light beam R, where the optical axis is defined by the zero position of the reference light beam R.

[0124] 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 behind the first transmission grating 30a in the light path of the reference light beam R, and the second measuring sensor 31b is arranged behind the second transmission grating 30b in the light path of the reference light beam R. The reference light beam R is incident 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 also shows a deflection direction A in which the reference light beam R migrates on the arrangement 29 when the tilt or displacement of the reference light beam R changes.

[0125] Fig. 9 shows first measurement data 32 of the position-sensitive diode 24 of the measuring device 13 shown in Fig. 7, as well as 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 measuring range shown, but has a comparatively low sensitivity. The second measurement data 33 consists of two phase-shifted sinusoidal signals from 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.Sensitivity to small deflections / tilts can be determined more precisely 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, however, the position signal of the arrangement is not unambiguous over the entire measuring range, i.e. 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.

[0126] 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 when, 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 tilts and / or displacements of the reference light beam R are required. These relationships also apply to the embodiment shown in Fig. 6.

[0127] The measuring device 13 shown in Fig. 7 can be extended to a position determination in two spatial directions, for example by 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).

[0128] Fig. 10 shows an arrangement in which a reference light beam R is collimated and directed onto 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 rather is located exclusively in the illumination beam path, functioning as a tube lens. The reference light beam R is collimated and directed onto 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 within the meaning of this application, focuses the reference light beam R.The convergent reference light beam R is decoupled from 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, wherein the angle depends on the shift 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.

[0129] List of reference symbols

[0130] 1 microscope

[0131] 2 light source

[0132] 3 phase filters

[0133] 4 main beam splitters

[0134] 5 lambda / 4 plate

[0135] 6 Lens

[0136] 7 Sample area

[0137] 8 Sample

[0138] 9 Deflection device

[0139] 10 mechanical beam scanners

[0140] 11 EOD scanners

[0141] 12 Detection device

[0142] 13 Measuring device

[0143] 13a Signal output

[0144] 14 first beam splitter

[0145] 15 second beam splitter

[0146] 16 Reference light source

[0147] 17 Computing and control unit

[0148] 18 first beam scanner

[0149] 19 emissions

[0150] 20 adjustable sample table

[0151] 21 Analog line

[0152] 22 entrance lens

[0153] 23 Exit lens

[0154] 24 position-sensitive diodes

[0155] 25 lenses

[0156] 26 diffractive optical elements (DOE)

[0157] 27 third beam splitter

[0158] 28 mirrors

[0159] 29 Arrangement

[0160] 30a, 30b transmission grating

[0161] 31a, 31b measuring sensor

[0162] 32 First measurement data 33 Second measurement data

[0163] A Deflection direction

[0164] B Illumination light beam

[0165] R Reference light beam

[0166] S Signal

[0167] 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 illuminating light, - optical elements forming an illuminating light beam path through which the illuminating light is guided as an illuminating light beam (B) from the light source (2) into a sample area (7), - in the illuminating light beam path, optical means for focusing the illuminating light into a focus region in a sample (8) in the sample region (7) such that, in the focus region of the illuminating light, an intensity minimum of the illuminating light at a focus point is bordered on both sides in at least one direction by intensity increase regions, - a deflection device (9) comprising a first beam scanner (18) in the beam path of the illuminating light, which is designed to position the focal point in the sample (8) at a plurality of positions in the sample (8), and - a detection device (12) for detecting emissions (19) from the focus area of ​​the illuminating 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, in addition to the first beam scanner (18), a further beam scanner, 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 directed at an angle to the illuminating light beam onto the first beam scanner (18), in particular is introduced into the illuminating light beam path in front of the first beam scanner (18) at a shallow angle, such that the reference light beam (R) and the illuminating light beam are deflected and / or displaced by the first beam scanner (18) in a mutually corresponding manner.

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 memory 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 desired position can be stored in the memory device.

15. Microscope (1) according to claim 14, characterized in that the position values ​​in the storage device can be assigned in time or can be stored in a time-associated manner to measured values ​​of the detection device (12) for detecting emissions (19) from the focus area of ​​the illuminating 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 arranged 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) onto the detector.

19. Microscope (1) according to claim 3 or 4, characterized in that the measuring device (13) is designed 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 onto 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 illuminating light is focused into the sample (8) in such a way that, in the focus area of ​​the illuminating light, an intensity minimum of the illuminating light at a focus point is bordered on both sides in at least one direction by intensity increase areas, - the focal 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 excited by the illuminating light or by another light in the focus area of ​​the illuminating light to emit emissions (19), - the emissions (19) from the focus area of ​​the illuminating 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 derived from the signal and a control signal of the deflection device (9) is stored in a memory device in such a way that it can be assigned to a respective measurement signal of the detection device (12) for detecting the light emitted from the focus area.

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