MINFLUX or STED-MINFLUX microscope with increased temporal-spatial resolution and corresponding method

Abstract

To improve the spatial-temporal resolution of the localization of individual emitters, especially individual fluorophores, achieved when performing MINFLUX or STED-MINFLUX methods using a suitable microscope (1), an improved method and an improved microscope (1) are proposed. A measuring device (13) of the microscope (1) determines the current position or direction of the reference light beam (R) deflected or displaced by a beam scanner (10, 18), and a signal (S) is output depending on the current position or direction, which is indicative of the current position of the focal point formed by an illumination light (B) in the sample.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 into account the measured focus positions when evaluating MINFLUX or STED-MINFLUX measurement data.

Description

Technical field of the invention

[0001] The invention relates to a microscope and to MINFLUX or STED-MINFLUX methods to be performed with the microscope for the high-resolution temporal and spatial determination of the location of individual emitters or for the high-resolution temporal and spatial tracking of emitters. The emitters in question can, in particular, be individual fluorophores. State of the art

[0002] A relatively new microscopy technique 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 either scattered by the emitters or excites them to luminescence, especially fluorescence, and in this general sense is an excitation light.The fundamental feature of MINFLUX nanoscopy is that the illumination of the emitters, particularly the excitation of the fluorophores, is such that an emitter to be located, or an emitter whose movement within a sample is to be tracked, is positioned at various locations near a minimum of the excitation light distribution, ideally a zero point. The excitation light distribution exhibits a region of increasing intensity adjacent to the minimum. This ensures that the number of photons scattered by the emitter, the fluorescence photons, or each individual photon contains a particularly rich amount of information about the emitter's instantaneous position. This is especially relevant for applications where 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.

[0003] Based on this foundation, a number of refinements for information acquisition have been developed, enabling the localization of fluorophores with an uncertainty below 2 nm and the tracking of the movement of individual fluorophores both within a range of a few nanometers and within a range of a few micrometers. A detailed, but not exhaustive, description of MINFLUX nanoscopy can be found in 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). This publication experimentally demonstrates non-iterative MINFLUX localization and the tracking of individual fluorophores using MINFLUX. Furthermore, the concept of iterative MINFLUX is presented.In order to locate a fluorophore using MINFLUX nanoscopy as described in the aforementioned publication, the intensity minimum or zero point must be positioned at a multiple of locations relative to the fluorophore's position. As a preliminary step, the current position of the fluorophore must be estimated or known with a lower degree of accuracy. 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 such that the estimated position of the fluorophore is close to the minimum of the intensity distribution. The fluorescence response of the fluorophore is then measured.The same procedure is typically performed for several additional positions in the intensity distribution located around the estimated location, and optionally also for the position corresponding to the estimated location itself. The set of all such sampling positions constitutes a set of sampling positions. Using a ratiometric evaluation of the intensity ratios, the location of the fluorophore is determined with greater accuracy. This more precisely determined location can then be used as the starting point for repeating the sequence of the aforementioned steps, whereby, in the case of localizing a fluorophore, the positions of the minimum intensity distribution of the excitation light can be placed closer to the estimated location of the fluorophore. This procedure can be continued iteratively.To track the movement of an emitter, it can be advantageous to maintain the constant distance between the positions where the intensity minimum is placed and the position estimated in the previous step. The method, as described in the aforementioned publication, is performed using a microscope that incorporates both a piezoelectric scanner and electro-optical deflectors (EODs). This combined scanning setup allows for addressing an image field of approximately 20 µm × 20 µm. Using the EODs alone, an image field of approximately 2 µm × 2 µm can be addressed, although high-speed scanning is only possible within a field of approximately 300 nm × 300 nm. Both in localization and particle tracking, the control of the scanning positions within a set of positions is achieved exclusively using the high-speed EOD scanning.A combination of piezoelectric 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, an area covered by a set of scanning positions—can be achieved using a fixed set of scanning points.

[0004] European patent EP 3 372 990 B1 discloses a method in which a set or sets of scanning positions with a local zero point of an intensity distribution of fluorescence-preventing light, superimposed with, for example, a Gaussian intensity distribution of excitation light, are scanned. The determination of the fluorophore's position can then be carried out in essentially the same way as in MINFLUX methods, except that the dependence of the fluorescence intensity on the distance of the fluorophore with respect to the zero point 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 subsequently referred to in this application as STED-MINFLUX methods.

[0005] Patent DE 10 2019 008 304 B3 discloses a fluorescence microscope optimized for the stability of the detection beam path's alignment relative to the illumination beam paths. The fluorescence microscope can be configured as a MINFLUX microscope. Stabilization of the alignment is achieved by designing a beam path using a telescope such that the effective beam diameter is small at the point where the detection and illumination beam paths diverge.

[0006] German patent application DE 10 2021 107 704 A1 discloses a MINFLUX device for carrying out a corresponding MINFLUX process in which the drift of the sample is quantitatively detected and compensated. The drift is detected by determining the position of at least one reference marker using a MINFLUX method.

[0007] Document WO 2023 / 062099 A1 discloses a device for the rapid axial displacement of an illumination focus. The displacement is achieved by at least one polarization switch placed in the illumination path, which switches the linear polarization of the illumination light between two directions depending on a switching signal. A focusing element is arranged downstream of the switch, which influences the focusing of the illumination light depending on the polarization. Various focusing elements are proposed. A MINFLUX microscope with such a device is described.

[0008] The publication EP 4 167 011 A1 discloses a device for modifying the shape of an illumination focus, in particular for rapidly switching the shape of an illumination focus between different intensity profiles. Switching is achieved by a polarization switch placed in the illumination path, which, depending on a switching signal, reverses the linear polarization of the illumination light between two directions. Downstream of the switch are arranged a first surface of a wavefront modulator, which modulates light of a specific polarization direction, an element that rotates the polarization of the illumination light, and a second surface of a corresponding wavefront modulator, wherein the first and second surfaces of the wavefront modulator modulate light of the specific polarization differently.The polarization switch determines which of the modulator surfaces—the one positioned in the beam path before or the one after the additional polarization rotation element—modulates the light. A MINFLUX microscope equipped with such a device is described.

[0009] The publication DE 199 59 228 A1 discloses a laser scanning microscope in which the focus position is corrected based on a temperature measurement, using, for example, tables or curves stored in software that contain corresponding reference values.

[0010] International Patent Application WO 2021 / 122407 A1 discloses a method for correcting, in particular, short-term, transient disturbances in a laser scanning microscope and a laser scanning microscope with a device for detecting a disturbance, which is configured to perform a disturbance correction method. The microscope may include a scanner system with end-of-detection optical distortion (EOD) and a scanner that serves for comparatively slow, but coarse positioning of the focused excitation light over a large image field. The scanner may be implemented in a quad configuration. In particular, a method for correcting disturbances in image acquisition according to the MINFLUX principle is disclosed.The invention disclosed in the application solves the problem of achieving high localization accuracy even when interference cannot be avoided during data acquisition using the MINFLUX method. This is achieved by determining the magnitude of any interference during data acquisition for a given localization. A weighting factor is then derived from this measurement and taken into account when determining the location of a dye molecule. Alternatively, data acquisition can be interrupted and resumed 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 guiding 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 suppression light, and finally onto a position-sensitive detector.In this arrangement, disturbances that are transmitted to the beam-guiding elements, for example, of the excitation light, and that 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 by, in particular, applying a corrected control signal to electro-optical deflectors of a beam deflection device, so that a sampling position of an intensity minimum 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 coupled out again at another point by means of a further dichroic beam splitter, wherein in the section between the beam splitters the measuring light beam travels in the beam path of the excitation light. The excitation light beam travels in the beam direction before reaching the first dichroic beam splitter through an electro-optical deflection device, which has two electro-optical deflectors connected in series to deflect the excitation light beam in the horizontal and vertical directions, respectively, and after passing the further dichroic beam splitter through another scanner, for example in a quad configuration, i.e., a special arrangement with four tilting mirrors.In contrast, the beam path section common to the measuring light beam and the excitation light beam does not include any deflection device or scanner.

[0011] 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 incorporates an electro-optical deflection device and a quad scanner in the excitation light beam path. In this microscope, the excitation beam path is simultaneously the beam path of a depletion or STED light, which emerges from an optical fiber along with the excitation light. Furthermore, the electro-optical deflection device consists solely of an electro-optical deflector (EOD) operated in resonance and designed to deflect the excitation beam only in one direction, along the fast axis.The publication states that both EOD and mechanical scanners exhibit jitter, defined as a time-varying deviation of the actual position from a target position. The magnitude of the jitter depends on the scan speed. With a mechanical scanner, inertia causes disturbances during fast scanning, while slow scanning can lead to various drifts. Experimental determination of the respective jitters reveals that, with the given configuration, the jitter of both the EOD and the mechanical scanner is of the same order of magnitude, namely in the single-digit nanometer range, and can be neglected even in the context of high-resolution STED measurements.

[0012] Publication US 2002 / 179828 A1 describes a device for scanning a sample that can generate largely error-free image data even during rapid scanning. The device includes, among other things, a mechanism for determining the actual signal for each scan point from the scanning unit's settings. It also includes a processing unit for calculating a display signal and a pixel position from the actual signal and / or the target signal provided by a control unit of the scanning unit and the detection signal from a detection device, as well as a processing unit for assigning the display signal to the pixel position.

[0013] German patent DE 10 2007 008 009 B3 describes a scanning microscope comprising a beam deflection device and a detection device, wherein the detection device acquires at least one piece of information about a frequency and / or a phase and / or an amplitude of the beam deflection device. The patent relates to a method in which the measurement signal from the detection device is processed and, after processing, used either to control the deflection device or to assign image data to positions in the sample. A direct digital synthesis method is used for the processing; according to the standard definition in the field, this 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 drive signal of a resonant scanner and the scanning process, depending on the drive frequency and the quality factor of the resonator. The detection device can be 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 onto a position-sensitive detector, the signal of which constitutes the measurement signal.

[0014] 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 laser beam is collimated and directed onto a mirror of a resonant galvo scanner. The mirror then directs the beam through a focusing lens toward a photodiode. A slit is positioned in front of the photodiode at the lens's focus, so that the photodiode detects light and outputs a signal at regular zero crossings of the scanner, where 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 onto a further detection device consisting of a photodiode and a slit. The reference laser is switched such that a reference signal is received only during a single zero crossing of an oscillation cycle. This reference signal is fed back to the control signal of the resonant scanner.

[0015] Publication US 2021 / 239452 A1 describes 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 accuracy that angular changes, which, after focusing through an objective lens, 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 includes a laser whose light is collimated and directed to a beam splitter. Part of the light passes through the beam splitter and is focused into a sample by an objective lens; 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.

[0016] From the publication “Pixel hopping enables fast STED nanoscopy at low light dose” (Britta Vinçon, Opt. Express 28, 4516-4528 (2020), DOI: https: / / doi.org / 10.1364 / OE.385174) a STED microscope with a scanning unit comprising both galvo scanners and two electro-optic deflectors (EODs) is known. The EODs 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 EODs and the galvo scanners. The galvo scanners enable the scanning of a large field of view, while the EODs allow for rapid fine-tuning.To enable fast and accurate scanning at high speed within a large scan field, the combined scanning system is controlled as follows: A control unit sends a control signal to the scanning unit, splitting the signal so that the galvo scanners receive a target signal; in addition, an actual signal is tapped from the galvo scanners at a monitoring output, provided by the factory-implemented position sensors; the target signal is not only output to the galvo scanners but also to comparators, which receive both the target signal and the actual signal tapped from the monitoring output; the difference between the target signal and the actual signal is fed to the EOD as a correction signal, so that, in conjunction with the deflection provided by the EOD, the target scanning point is reached quickly and accurately, regardless of the inertia-related delay in setting the galvo scanners.This feature is used to vary the scanning speed, i.e., the duration for which a pixel is addressed or swept, significantly in a local area during a raster scan.

[0017] 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 / s41467-021-21652-z) describes the performance of MINFLUX techniques using a commercially available microscope. In addition to end-of-deflection optical (EOD) scanning, the microscope features a quad scanner consisting of four galvo scanners (two galvo scanners per deflection direction). With the quad scanner, in conjunction with a 100x magnification objective, an image field of approximately 80 µm × 80 µm can be addressed, making the microscope suitable for acquiring confocal or STED microscopy images. When performing a MINFLUX procedure, the sampling points of a single set of sampling points are addressed using the EOD. The adjustment of the center position of each set is then performed using the galvo scanners.Using a microscope, biomolecules are tracked for a period of seconds with a temporal resolution of 100 microseconds. The publication mentions that the accuracy of position determination during particle tracking is reduced because the particle shifts during the measurement of individual positions.

[0018] Investigations by the applicant have further shown that position determination when using beam scanners is additionally reduced because, during the measurement of fluorescence to a set of scan points, rapid irregular or seemingly irregular changes in the state of the beam scanner induce rapid irregular or at least seemingly irregular movement of the illumination light distribution in the sample. Both these rapid irregular changes in the state of the beam scanner and the rapid irregular movements of the illumination light distribution in the sample are referred to in this application as jitter. The jitter can superimpose itself on a controlled or regulated displacement of the illumination light and can also occur when the beam scanner is nominally aimed at a fixed position.It can occur particularly when using a beam deflection device that includes galvo scanners for addressing a large image field and EOD for addressing the set of scanning points for MINFLUX localization. In this case, rapid, irregular, or seemingly irregular movements of the galvo scanner mirrors, which are nominally in a stationary position during MINFLUX scanning, can induce irregular or seemingly irregular movement of the illumination light. In principle, such jitter can also occur with other beam scanner systems, especially if they are designed to scan a large image field in the microscope in question. Such large-field scanners typically have moving components, for example, movable mirrors.The effect of jitter is greater the shorter the effective measurement time at each point in the set of sampling points. Consequently, jitter limits the spatial-temporal resolution in microscopes known from the prior art. Object of the invention

[0019] The invention is therefore based on the objective of providing solutions that enable improved spatial-temporal resolution of the localization of individual emitters, in particular individual fluorophores, when using a microscope with a beam scanner in carrying out MINFLUX methods or STED-MINFLUX methods. Solution

[0020] 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. Definitions

[0021] In this application, emitters are understood to be objects which, 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.In this context, the maximum time delay, when tracking emitter movements, is related to the temporal resolution required to track the movements of the light-emitting particles or units, and to the speed at which the particles or units move within the sample. Time delays can be up to approximately 10 µs, but are typically in the range of up to several tens of nanoseconds, often in the range of 1 to 10 ns, and zero if the emission is scattered light. In this application, the term "fluorescence emitter" refers to 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 the application, excitation light is understood not only as fluorescence excitation light, but generally as any light that causes an emitter to emit light.

[0022] In the context of the application, an emitter is considered isolated if the distance to a nearest emitter, from which it is not optically distinguishable, is at least as large as the distance corresponding to the resolving power of the optical arrangement used to detect the emissions during the procedure.

[0023] In this application, emission suppression light is understood to mean light that prevents, reduces, or completely suppresses the emission of an emitter. In particular, emission suppression light can be stimulation light or STED light.

[0024] In this application, STED light is understood, as is generally the case, to mean light that suppresses fluorescence emission by exciting stimulated emission. Within the scope of this application, STED light is a specific form of emission suppression light.

[0025] In this application, the term MINFLUX encompasses localization and tracking methods for isolated emitters, in which light distributions of illumination light, which 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 light distributions in question or for different such light distributions in a near-area of ​​the emitter, wherein the emitter emits less light the smaller the distance between the emitter and the minimum of the light distribution.

[0026] In this application, STED-MINFLUX methods are understood to be methods corresponding to the MINFLUX methods as defined above, with the exception that instead of the intensity distribution of excitation light, an intensity distribution of emission suppression light, which overlaps with excitation light, is used.The fact that the intensity distribution of emission suppression light overlaps with excitation light means that the sample, in the region of the intensity minimum and the adjacent sections of the intensity increase regions of the emission suppression light, within which an emitter lies, is exposed to excitation light in such a way that emission from 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 from an emitter located within the intensity distribution of the emission suppression light decreases with increasing distance from the intensity minimum.If the emission suppression light is STED light and the emitter is a fluorescent emitter, this means that the spontaneous emission of the fluorescent 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 typical in STED microscopy; it is blocked, for example, by a filter. The fact that the intensity distribution of emission suppression light overlaps with excitation light does not mean that the excitation light and the emission suppression light are introduced into the sample strictly simultaneously. For example, as is common in STED microscopy, a short pulse of excitation light is applied, immediately followed by a pulse of STED light as emission suppression light.The excitation light, with which the intensity distribution of the emission suppression light overlaps, can, for example, exhibit an intensity distribution such as is common in confocal microscopy, thus corresponding at least approximately to an Airy or Gaussian function, where its central maximum can coincide with the minimum of the emission suppression light. It can also correspond to a tophat function. Constant wide-field excitation is also possible.

[0027] The term STED-MINFLUX is used in a generalized sense; it should by no means be understood to mean that the emission suppression light must be STED light.

[0028] In MINFLUX and STED-MINFLUX microscopy, the sample is illuminated with focused light. This light forms an intensity distribution around a geometric focus within the sample, characterized by a central intensity minimum, or zero. The position of a single emitter relative to this central minimum determines the intensity or photon emission rate of the emitter's light, which is used for position determination. Intensity distributions with a central minimum can be generated, in particular, by phase modulation of the illumination light using a phase filter or a spatial light modulator (SLM) in the illumination beam path, focusing the illumination light with an objective lens, and optionally, circular polarization of 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 sampling 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 located at a sampling position.

[0030] In this application, a donut-shaped intensity distribution is understood to be a special form of intensity distribution with a central minimum, which has a minimum that is surrounded in all spatial directions within a given plane by areas of increasing intensity.

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

[0032] In this application, a 3D minimum is understood to be a minimum surrounded by areas of increasing intensity in all spatial directions. A 3D minimum can be obtained, for example, by superimposing two light beams, whereby the wavefront of one beam is phase-modulated in each pupil using an annular phase plate and the wavefront of the other 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 for determining the position of a single emitter in a sample. In contrast to conventional light microscopy methods, this method does not require optical imaging of the sample. Instead, the MINFLUX localization method according to the invention detects light emissions from the emitter at various positions within the intensity distribution of the illumination light, and the emitter's position is determined from these detections, for example, by calculating 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 microscopic image.

[0034] Both localizations performed using a MINFLUX method and those performed using a STED-MINFLUX method are referred to, for the sake of simplicity, as MINFLUX localizations within the scope of this application.

[0035] Tracking an emitter is the process of determining the trajectory of an emitter moving within the sample, particularly through multiple, successive localizations.

[0036] In the context of this specification, a beam scanner is a device that variably deflects or displaces a light beam. Displacement 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 configured to displace a beam of illumination light, i.e., an illumination light beam, by means of one or more optical components, such that the focus of the illumination light on or in the sample is shifted in at least one spatial direction. The shift of the focus can, in particular, occur in two spatial directions, and furthermore, in a focal plane that is perpendicular to an optical axis of a lens. The spatial directions can be defined in various coordinate systems, e.g., in Cartesian coordinates (e.g.,x- and y-directions), in polar coordinates or in spherical coordinates (e.g., radial and azimuthal directions). The beam scanner can include, for example, mechanical scanners, in particular scanners based on tilting mirrors connected to a galvomotor, electro-optic deflectors, or acousto-optic deflectors.

[0037] For the purposes of this application, a galvo scanner is understood to be a module comprising a galvo drive and a tilting mirror connected to and movable by the galvo drive. The drive can be configured such that the angular positions of the tilting mirror are adjustable, or such that the tilting mirror performs a periodic rotational movement. Thus, for the purposes of this application, the term galvo scanner encompasses resonant galvo scanners and galvo scanners in which the angular positions of the tilting mirror are adjustable; the latter are referred to in this application as adjustable galvo scanners. The tilting mirror can be flat or curved, for example, concave. A galvo scanner may also include elements other than the galvo motor and the tilting mirror.

[0038] In this application, a galvo scanner system is defined as a scanning unit comprising one or more galvo scanners, configured as a whole to displace and / or deflect a light beam in one or more spatial directions. Similarly, in 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 configured as a whole to displace and / or deflect a light beam in one or more spatial directions.

[0039] In the context of this application, a mechanical scanner is a scanner that variably deflects or displaces an illumination light, wherein the variation of the deflection or displacement is achieved by moving at least one optical component, e.g., a mirror. The movement can, in particular, be a rotation, especially 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.

[0040] In the context of this application, the term deflection device refers to the entirety of all beam scanners in the beam path of the illumination light. Description of the invention

[0041] The microscope according to the invention is configured to perform a MINFLUX method and / or a STED-MINFLUX method. In the MINFLUX method and / or the STED-MINFLUX method according to the invention, the position of a single emitter in a sample is determined and / or an emitter is tracked.

[0042] The microscope according to the invention for carrying out a MINFLUX process and / or a STED-MINFLUX process comprises a light source for generating illumination. It further comprises optical elements that form an illumination beam path through which the illumination is guided as an illumination beam from the light source into a sample area. Within the illumination beam path, it includes optical means for focusing the illumination into a focal region within the sample such that, within the focal region, at least one focal point is bordered on both sides by regions of increasing intensity. The illumination beam path also includes a deflection device comprising a first beam scanner.The deflection device is designed to position the focal point in the sample at multiple locations. The microscope includes a detection device for recording emissions from the focal area of ​​the illumination light. The microscope according to the invention differs from prior art microscopes for performing MINFLUX or STED-MINFLUX methods by a measuring device for recording the position and / or direction of a reference light beam that is deflected and / or displaced by the first beam scanner. This 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 is indicative of the 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 include a lens.

[0044] The fact that, within the focus area of ​​the illumination light, intensity minimums at a focal point are bordered on both sides in at least one direction by areas of increasing intensity, includes in particular the cases where a 2D minimum is formed in the focus area of ​​the sample, where a 3D minimum is formed, and where, in a plane containing the optical axis and thus the focal point, a leaf-shaped intensity minimum, ideally a zero point, with adjacent areas of increasing intensity is formed. The optical means for focusing the illumination light into a focus area in a sample comprise one or more elements that cause, within the focus area of ​​the illumination light, intensity minimums at a focal point to be bordered on both sides in at least one direction by areas of increasing intensity.For example, such an element can comprise a vortex phase plate to create a 2D minimum, an annular phase plate to create a 3D minimum, or a phase plate with a 180° phase shift along a diameter to create a 1D minimum. Corresponding phase-influencing elements can also be implemented as adjustable spatial light modulators. Adjustable amplitude modulators, for example, can also be used to generate appropriate intensity distributions in the focal region.

[0045] As explained above, the microscope according to the invention has a deflection device in the beam path of the illumination light, which includes a first beam scanner. This first beam scanner is configured to position the focal point in the sample. The first beam scanner can include several individual beam scanners, that is, several individual deflection units or displacement units, for example, a galvo scanner system as defined above. The first beam scanner can also be the only beam scanner that the deflection device of the microscope has. The designation "the first beam scanner" serves to simplify the distinction in cases where 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. Rather, it means that the current position of the focal point in the sample is at least partially 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 implies that the current setting of the first beam scanner changes randomly, so that the current position of the focal point in the sample changes accordingly.

[0046] The first beam scanner can include a mechanical scanner or be designed entirely as a mechanical scanner. The mechanical scanner of the first beam scanner can, in particular, include a deflection unit with at least one continuous mirror that deflects all of the illumination light. Alternatively or additionally, this mechanical scanner can include one or more movable, in particular rotatable, elements through which the illumination light passes, such as a prism or 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, include or be configured as a MEMS scanner (MEMS: Micro-Electro-Mechanical Systems), piezo scanner, or galvo scanner system. Piezo scanners and MEMS scanners can be configured such that a mirror can be tilted about two mutually orthogonal axes. The first beam scanner can, in particular, include or be configured as resonant mechanical scanners with at least one continuous mirror deflecting all the illumination light, for example, resonant or adjustable galvo scanners or resonant or adjustable MEMS scanners. For the purposes of this application, adjustable scanners are understood to be those scanners that allow targeted addressing of focus positions, that is, in which the displacement and / or deflection they cause is directly adjustable.

[0047] The first beam scanner can comprise electro-optic deflectors (EOD) or acousto-optic deflectors (AOD), or be composed entirely of EOD or AOD, or of both. In preferred embodiments, the first beam scanner is composed of mechanical scanners, in particular tunable galvo scanners, piezo scanners, and / or MEMS scanners, and non-mechanical scanners, in particular AOD or EOD.

[0048] In order to perform a MINFLUX or STED-MINFLUX procedure using a lens through which the illumination light is directed into the sample, it is important that the illumination light beam is only tilted by the deflection device, i.e., by the first beam scanner or by the first beam scanner and further beam scanners, in the illumination light beam at the pupil of the lens and is not, or at least as little as possible, displaced, since a displacement of the illumination light beam would change the shape of the intensity distribution of the illumination light in the focus area and thus indirectly also the position of the focal point, i.e., the local minimum of the intensity distribution.Beam scanners that deflect the illumination beam in such a way as to satisfy the aforementioned condition can, for example, have a mirror tiltable about two axes, located in a pupil (i.e., in a plane into which the pupil of the objective lens is imaged), or they can have two mirrors, each tiltable about one axis and each located 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, e.g., WO 2021 / 122407 A1) as a quad-configuration scanner or 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. This is because, compared to other scanner types, their characteristics, such as their usable beam diameter and maximum deflection angle, typically allow for a larger usable scan area. Another preferred embodiment with two separate tiltable mirrors for shifting the focal point in the sample in one direction comprises two MEMS scanners, each with mirrors that can be tilted about two mutually orthogonal axes. Compared to a quad scanner, this embodiment requires fewer reflective surfaces.

[0049] In preferred embodiments, the microscope's deflection device can include additional beam scanners besides the first. All beam scanners, the first and the additional ones, can be designed as mechanical scanners. In preferred embodiments, the microscope can have an additional beam scanner in the illumination path, 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 paths by combining controlled deflections by the first beam scanner and controlled deflections by the additional beam scanner, or to address different scanning positions or scan different paths.This is achieved by only controlled deflections by the secondary beam scanner, while the primary beam scanner is controlled in such a way that it remains in a static position, as far as possible. In the case of a mechanical scanner, this means that it is in a rest position, or that it is controlled to perform very small adjustments, or in the case of a mechanical scanner, very small movements. These movements, if the secondary beam scanner's position remained unchanged, would cause the focal point of the illumination light in the sample to shift around a fixed position. Such control of very small movements can have the advantage of reducing irregularities in the shift of the focal point in the sample compared to irregularities that occur when a static setting of the primary beam scanner is maintained. This is particularly relevant if...if the first beam scanner includes adjustable galvo scanners and / or adjustable MEMS scanners.

[0050] Preferably, the deflection device is configured such that the focal point can be rapidly shifted within a short-range area with dimensions approximately corresponding to the extent of a diffraction-limited illumination spot. The ability to rapidly shift the focal point also includes cases where the rapid shift of the focal point in the sample is induced by rapid, irregular, or seemingly irregular 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 µm, 20 µm, 100 µm, or 500 µm.In preferred embodiments, the deflection unit therefore comprises several beam scanners, wherein one of the beam scanners, preferably the further beam scanner, is configured to rapidly 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 swept in less than 1 ms, and another of the beam scanners, preferably the first beam scanner, is configured to shift the focal point of the illumination light in the sample over larger distances, for example over 10 µm or 20 µm or 100 µm or 500 µm.

[0051] In preferred embodiments, the further beam scanner, that is, a different beam scanner than the first beam scanner, is an adjustable beam scanner. More preferably, the further beam scanner has a high bandwidth and is configured to rapidly shift the focal point of the illumination light across a set of scanning positions for minflux localization. In further preferred embodiments, the bandwidth of the further beam scanner is greater than that 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, that is, the time required for a scanner to move from one angular position to another, are preferably shorter than those of the first beam scanner, for example, only 50%, 20%, 10%, 1%, or less than 1% of that of the first beam scanner.

[0052] If the deflection device comprises a first beam scanner and a second beam scanner, in preferred embodiments the second beam scanner has one or more AODs or EODs, or is comprised of one or more AODs or EODs. AODs and EODs offer the advantage that the deflection angle adjustment is not limited by inertia. Both types of deflectors thus enable extremely rapid repositioning to any point within the addressable range of the second beam scanner. The bandwidth of an EOD, in conjunction with suitable control electronics, can be, for example, 1 MHz or 5 MHz or higher, thereby enabling repositioning of the focal point in less than 1 µs.

[0053] The microscope's secondary beam scanner can also include a mechanical scanner or be designed entirely as a mechanical scanner. The mechanical scanner of the secondary beam scanner can, in particular, include a deflection unit with at least one continuous mirror that deflects all of the illumination light. The mechanical scanner of the secondary beam scanner can, for example, contain or be configured as a MEMS scanner, piezo scanner, or galvo scanner system. In particular, piezo scanners and MEMS scanners can be configured such that a mirror can be tilted about two mutually orthogonal axes.

[0054] If the microscope's secondary beam scanner includes mechanical scanners with a deflection unit comprising at least one continuous mirror that deflects all of the illumination light, mechanical scanners with a small aperture are preferably selected for this purpose. More preferably, the microscope then includes an objective lens. The continuous mirror that deflects all of the illumination light can preferably be located in a pupil. The microscope can then include a dilator in the illumination beam following the secondary beam scanner, with which the illumination beam is adapted to the aperture of an objective lens.A mirror in a small-aperture mechanical scanner can be small and therefore have a low mass and consequently a low moment of inertia, allowing the bandwidth of the mechanical scanner system of the second beam scanner to be much larger than the bandwidth of the first beam scanner, and the response time of the second beam scanner to be correspondingly much smaller. If the mechanical scanners of the second beam scanner are resonant scanners, or if the mechanical scanner of the second beam scanner is a resonant scanner, it preferably has a high resonant frequency. Such resonant scanners, by their very nature, allow for significantly higher displacement speeds than adjustable scanners at the same usable aperture, but only along specific paths.For example, using two resonant scanners of identical frequency with axes perpendicular to each other when imaging into the sample, the focal point can be shifted onto circular paths; if the frequencies differ, other paths result, for example, Lissajous paths with integer frequency ratios. Instead of two resonant scanners, the microscope can also have a resonant scanner with two perpendicular tilting axes, in particular such a MEMS scanner.

[0055] In a further preferred embodiment, the additional beam scanner comprises a resonant mechanical scanner, which may be positioned in a pupil and which displaces the illumination beam in the sample in a first direction, and an adjustable mechanical scanner that displaces the illumination beam in the sample in a second direction orthogonal to the first direction. In a particularly preferred embodiment, the microscope additionally includes a dilation unit as described above. With such an embodiment, MINFLUX localization can be performed particularly quickly along a path that corresponds to a scanning path of a raster image acquisition.

[0056] In embodiments of the microscope comprising a first beam scanner, a further beam scanner, a lens and an expansion unit, the aperture of the further beam scanner and the expansion unit and the aperture of the lens are preferably matched to each other, and further preferably matched to each other in such a way.

[0057] In other preferred embodiments, the additional beam scanner may be or include a resonantly operated EOD.

[0058] With a resonant beam scanner, the focal point of the illumination light can be shifted particularly quickly.

[0059] Furthermore, the microscope according to the invention comprises a detection device for detecting light emitted from the focus region of the illumination light, i.e., for detecting emissions from the focus 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 specific position of the focal point in the sample. Preferably, the detection device comprises a detector capable of detecting 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.

[0060] Compared to microscopes known from the prior art, the microscope according to the invention differs, as already explained above, in that the microscope according to the invention has a measuring device for detecting the position and / or direction of a reference light beam that is deflected and / or displaced by the first beam scanner, wherein the measuring device is configured to output a signal at a signal output, depending on the position and / or direction, which is indicative of the position of the focal point in the sample. The reference light beam can be a light beam that has been split off from the light beam emitted by the illumination light source, or it can originate from a reference light source.The fact that the reference light beam is deflected and / or displaced by the first beam scanner means that the deflection and / or displacement of the reference light beam is caused 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 displaced by the same mirror surface(s). The fact that the signal is indicative of a focal point location in the sample means that the focal point location 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, corresponding 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, taking into account the imaging properties of the microscope in a manner known to those skilled in the art. Alternatively, the signal can directly be a measure of a position of the mechanical scanner that 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 to the fast, adjustable scanner, in particular an EOD scanner, which can be part of the first beam scanner or which forms the second 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.

[0061] The measuring device preferably has a position-sensitive or spatially resolved detector at one end of the reference light beam path. A further preferred 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 high temporal resolution, thus enabling fast data acquisition and rapid control, for example of an EOD scanner of the deflection device, at a frequency of several hundred kilohertz.

[0062] In a preferred embodiment, the measuring device is configured to detect the direction of the reference light beam deflected by the first beam scanner by focusing the reference light beam, or a portion thereof, onto the detector. For example, if the reference light beam is deflected by the first beam scanner—that is, if the reference light beam is angled by the first beam scanner to an optical axis of the beam scanner in its zero position—then, in particular, the angle imposed 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 convergently, as is the case, for example, with a pupil scanner where the scan lens is an integral part of the scanner, and when the reference light beam exits the first beam scanner collimated and is subsequently focused outside the illumination beam path.

[0063] In a preferred embodiment, the measuring device is configured to detect the position of the reference light beam deflected and / or displaced by the first beam scanner by collimating the reference light beam or a portion of the reference light beam and directing it onto the or a further position-sensitive or spatially resolving detector.

[0064] Further characteristics of the measuring device can be found in the descriptions of the procedure below, as well as in the figure descriptions.

[0065] Preferably, the reference light beam propagates along a reference light beam path, wherein the reference light beam path includes the first beam scanner.

[0066] In a preferred embodiment, the microscope includes a reference light source for generating the reference light beam. In preferred embodiments, the reference light source is a laser. Further characteristics of the reference light source will become apparent from the description of the method according to the invention given below.

[0067] 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 in the sample is positioned, for example, at scanning positions of a set of scanning points of a MINFLUX localization or along a scanning path for a MINFLUX localization. The first beam scanner can receive control signals, in particular electrical signals, from the control unit, which influence a state of the at least one deflection unit in order to shift or position the illumination 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 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 shifted within a larger image field, for example, to capture a confocal raster image. The control unit can, in particular, be an integral part of a computing and control unit. The computing and control unit is preferably also configured to receive, 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 configured to determine control signals for the deflection device based on the signals from the measuring device and to output them to the deflection device.

[0068] In the MINFLUX method and 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 illumination light is focused into the sample such that, in the focus area of ​​the illumination light, at least one intensity minimum is bordered on both sides of a focal point in at least one direction by areas of increasing intensity. The focal point is positioned at several positions in the sample by means of a deflection device which has a first beam scanner. An emitter in the focus area of ​​the illumination light is excited by the illumination light or by another light to emit luminescence or scattered light. The light emitted from the focus area of ​​the illumination light is detected by a detection device, whereby measured values ​​of the detection device are assigned to the scanning positions, and from the measured values ​​and the assigned scanning positions, a position or trajectory of an emitter is determined.

[0069] The MINFLUX method 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 and 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 direction of the deflected and / or displaced reference light beam and, depending on the position and / or direction, outputs a signal at a signal output that is indicative of a position of the focal point in the sample.

[0070] Positioning the focal point in the sample can be achieved by sequentially addressing different positions within 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, with the sequential positioning to different actual scanning positions resulting from random changes in the settings of the first beam scanner.In both cases, the positioning of the focal point is sequentially performed at various actual scanning positions. However, each actual scanning position, i.e., each position of the focal point, does not correspond exactly to the addressed scanning position, but deviates from it by an amount that depends on the difference between the respective actual setting of the first beam scanner and the respective target setting, i.e., the setting of the first beam scanner corresponding to the addressed scanning position. According to the invention, a measuring device detects the position and / or direction of the reference light beam deflected and / or displaced by the first beam scanner; 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.

[0071] In a preferred embodiment of the invention, the focal point in the sample is sequentially positioned at different scanning positions by means of a deflection device comprising a first beam scanner. The set of addressed scanning positions can form or contain a continuous path of scanning positions, for example, a circular path around the presumed position of the emitter; the path can also be raster-like or form a Lissajous path, a rosette, or a loop.

[0072] As described above, in the method according to the invention, an emitter is excited by the illumination light or by another light source in the focus region of the illumination light to emit luminescence or scattered light. If the excitation is caused by the illumination light, the illumination light can be the only light that is shone into the sample during the execution of the method. With such a method, both the position of an emitter by which illumination light is elastically scattered, such as a gold bead, and of a luminescent particle, such as a fluorescent bead or, in particular, a fluorophore, can be determined, and a corresponding emitter can be tracked.

[0073] In addition to the illumination light, further light can also be shone into the sample. In one embodiment, this further light can be a fluorescence suppression light, in particular a STED light, which suppresses the emission of free, i.e., unstimulated, fluorescence from an area adjacent to the focus area of ​​the illumination light and / or from outer areas of the focus area, where it is known from previous position determinations or estimates that the fluorophore whose position is to be determined is not located. In this case, the fluorescence suppression 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, generated, for example, by a vortex phase plate with a multiple increase in the wavefront phase modulation from 0 to 2π over one revolution along the circumference. It can also form a 3D donut, which can be generated, for example, with an annular phase plate. In this case, the additional light can be stationary within 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, though not necessarily in the same way.

[0074] In another embodiment of the method according to the invention, the illumination light can be a luminescence-prevention light, in particular a STED light; that is, the method according to the invention can be implemented as a STED-MINFLUX method according to the definition given in the relevant section. In STED-MINFLUX methods, the emitter, whose position is to be determined or which is to be tracked, is excited by additional light as excitation light. This additional light can be light focused together with the illumination light through a lens, wherein the shape of the intensity distribution can correspond at least approximately to an Airy function, a Gaussian function, or a top-hat function. Preferably, the intensity distribution has a broad region at its center—broad compared to the distances between the sampling positions of the illumination light to which the illumination light is shifted—in which the intensity does not change or changes only slightly.This condition can be met, for example, by focusing the additional light using only part of the objective aperture, i.e., not the full aperture. In this embodiment, the additional light can also be stationary within 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, though not necessarily in the same way.

[0075] The emitter's light emissions are detected by a detection device. This device can, for example, include a point detector such as an avalanche photodiode (APD), a photomultiplier, or a hybrid detector, or a spatially resolved detector such as a camera or an APD array. In particular, the detection device also includes evaluation electronics. The detection device is specifically designed to capture individual photons emitted by the emitter and register them in such a way that corresponding sampling positions can be assigned to the light emissions.

[0076] A specific relationship exists between the position and / or direction of the reference light beam deflected and / or displaced by the first beam scanner and the position and / or direction of the illumination light beam after the scanner. This relationship must take into account how the directions and / or positions of the reference light beam and the illumination light beam are related to each other. Furthermore, a specific relationship exists between the position and / or direction of the illumination light beam after the scanner and the position of the focal point in the sample. Determining the position and / or direction of the reference light beam therefore allows the determination of the respective actual scanning position, that is, 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 in particular, which can also be combined, to increase the spatial-temporal resolution.

[0077] 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. This correction value is then used to adjust the control signal of the deflection device or at least one beam scanner of the deflection device. For example, after appropriate amplification, the signal from the measuring device can 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, such as an operational amplifier tailored to the application. The reference light beam and the measuring device are thus used to control the deflection device. This control can be implemented using control strategies known in the art, for example, in the form of a PID controller (PID: Proportional-Integral-Differential).Using the measurement signal for control can be particularly advantageous when the deflection device has an additional adjustable beam scanner that allows random access to scanning points and has shorter, especially much shorter, response times than the first beam scanner—for example, only 20%, 10%, 1%, or less than 1%—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 this case, the deflection device is controlled by feeding the measurement signal, or a signal derived from the measurement signal, back to the fast unit(s).Accordingly, the control is preferably implemented such that rapid corrections to the position of the focal point in the sample are made by the second, faster beam scanner or by the fast unit(s) of the first beam scanner. This does not preclude slower corrections from also being made using, or in conjunction with, the first beam scanner or the slow unit(s). However, it is particularly simple to control the first beam scanner or the slow units of the first beam scanner and to regulate only the second beam scanner or the fast unit(s) of the first beam scanner. In this case, the detection of the position and / or direction of the reference light beam can be performed with a bandwidth or temporal resolution that is adapted to the bandwidth or response time of the second beam scanner.The bandwidth used to detect the position and / or direction of the reference light beam can be higher than the bandwidth of the second 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 relative to the bandwidth used to detect the position and / or direction. Particularly if the bandwidth of the second 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 very simply as proportional control of the deflection unit.

[0078] The other method involves determining actual sampling positions from the signal of the measuring device. For this purpose, the signal from 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 from the measuring device, is preferably stored in a storage device in such a way that it can be assigned to a measurement signal from the detection device for detecting the light emitted from the focus area. This can be achieved, for example, by storing both the signal from the measuring device or the signal derived from it, as well as the signal from the detection device, each with a timestamp.

[0079] 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 further beam scanner.

[0080] Determining the emitter's position or tracking it by defining a trajectory can now be performed using methods typically employed in MINFLUX or STED-MINFLUX procedures. This also applies to selecting the sampling positions where the focus point should be placed.

[0081] In the case where 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 indeed scanned with higher accuracy. This is particularly true if the first beam scanner is controlled during a sequential shift of the focal point in the sample in such a way that the focal point would not change its position if the first beam scanner were perfect. Investigations by the applicant using a typical microscope configuration with a quad scanner have shown that the jitter of all individual galvo scanners of the nominally stationary quad scanner can result in shifts 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 at several kilohertz.This can lead to an additional uncertainty of several nanometers in determining the instantaneous position of the emitter, particularly when tracking emitters, even independent of the uncertainty caused by the emitter's movement. Since measurements with uncertainties in the range of 1 nm are fundamentally possible using MINFLUX methods, this uncertainty due to jitter is therefore significant. Using an additional, particularly fast, adjustable beam scanner, such as an AOD or, especially, an EOD, the position of the focal point can be corrected within typical bandwidths, for example, within approximately 1 µs or less, provided the bandwidth of the position measurement is sufficiently large. This measurement with a correspondingly high bandwidth or temporal resolution can be achieved using methods known in the prior art, as will be explained below.With a smaller 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 to address a scanning position with an uncertainty of less than 1 nm. This significantly reduces the uncertainty of position determination during fast scanning, or conversely, enables high accuracy even during very fast measurements, such as those required when tracking a rapidly moving emitter.

[0082] It is also possible that the first beam scanner comprises several, particularly different, scanners, for example, a galvo scanner system and an EOD system. In these cases, the signal from the measuring device for detecting the position and / or direction of the reference light beam can be used to control the deflection device in such a way that only one or some of the scanners of the first beam scanner are controlled, while the control signal of other scanners is independent of the signal from the measuring device. In the specific example where 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 still fed back to the EOD system.

[0083] The measurement signal of the measuring device for determining the location and / or position, or the signal derived from it, can alternatively or additionally be used in another way to increase the spatial-temporal resolution, namely by taking into account, when determining the location or trajectory of the emitter from the emissions detected by the detection device, the actual focal points, i.e., those determined on the basis of the measurement of the location and / or direction of the reference light beam, at the time of excitation of the emission.

[0084] If only this alternative method of accuracy improvement is used, it is particularly suitable for evaluating previously acquired data. During data acquisition, the coordinates of the scanning positions can be based on the values ​​corresponding to the target positions, as is done in the prior art. For example, using methods known from the prior art, the position of the emitter can then be determined in real time, from which subsequent scanning positions can be determined. The focal point is then moved to these positions during the further course of the measurement 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.From the measured position of the reference light beam, a scanning position can now be determined with higher accuracy than from the data of the deflection device's control. If the actual scanning positions determined from the measurement—that is, the positions of the focal point in the sample—are used instead of the target positions to determine the emitter's position, then the emitter's position will also be determined with higher accuracy than if the target positions are used.

[0085] In principle, the actual positions of the focal point in the sample at the time of emission excitation—that is, the positions determined based on measurements of the position and / or direction of the reference light beam—can also be taken into account during data acquisition. This allows for real-time determination of the emitter's position or tracking of the emitter. For this purpose, the real-time data evaluation routine can be modified accordingly, and suitable hardware such as a high-performance Field Programmable Gate Array (FPGA) can be used. The actual sampling positions can be determined from the measurement signal of the measuring device, for example, in a separate unit and then sent to an FPGA to control the deflection unit. Alternatively, the signal from the measuring device can be sent directly to an FPGA to control the deflection unit and evaluated there in its entirety.

[0086] A combination of both uses of the measuring device's signal—namely, its use for controlling the deflection device and for determining the sampling positions, i.e., the positions of the focal point in the sample—is also advantageously possible. This is particularly true 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's signal, a deviation remains between the actual sampling position and the target position detected by the measuring device. This deviation is reduced compared to the deviation that would occur without 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 sampling positions, either by using the target positions as a basis for determining the position of the emitter or by taking into account the actual focus points at the time of excitation of the emission, i.e., those determined on the basis of the measurement of the position and / or direction of the reference light beam, during data acquisition.

[0087] In preferred embodiments of the method, a reference light beam is generated by means of a reference light source and this beam is directed over the first beam scanner.

[0088] 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, then generating the reference light beam may involve filtering the wavelengths.

[0089] In some embodiments, it is preferred that the light of the reference light beam does not include a wavelength range that contains wavelengths which are used in the microscope belonging to the method according to the invention for the microscopic images of the samples to be examined.In a preferred embodiment, the wavelength range of the reference light beam can, for example, consist exclusively of wavelengths above the longest wavelength used in carrying out microscopy procedures with the microscope, such as the longest wavelength of a STED laser or the longest wavelength of fluorescence to be detected. Alternatively, the wavelength range of the reference light source can consist exclusively of wavelengths below the shortest wavelength used in carrying out microscopy procedures, which, for example, can be the shortest 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.

[0090] 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 overall beam path. If the first beam scanner includes AOD and / or EOD, boundary conditions arise for the properties of the reference light beam. For example, the deflection angle imposed on a light beam by the first beam scanner can depend on polarization, as is typically the case with a single EOD, or it can depend on wavelength, as is typically the case with a single AOD.Accordingly, if the deflection unit has an EOD (extra-objective diffraction) and the reference light beam is guided through the deflection unit, it is preferred that the reference light beam guided through or over the first beam scanner has the same polarization as the illumination light beam, but preferably a different wavelength, thereby enabling the 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 an 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.

[0091] To guide the reference light beam across the first beam scanner, it can be introduced into the illumination beam path at a shallow angle on one side of the first beam scanner and, correspondingly, exited from the illumination beam path at a shallow angle on the other side. In this embodiment, the separation of the reference light beam occurs completely independently of differences in internal properties such as wavelength or polarization. The reference light beam undergoes essentially the same deflections as the illumination beam, insofar as these are caused by the first beam scanner.The angles at which the reference light beam can be introduced so that it is guided over the first beam scanner result from the parameters of the first beam scanner, for example from the dimensions of mirrors and the design of the beam path in the area of ​​the first beam scanner, which may, for example, have relay optics.

[0092] In another embodiment, the reference light beam can be coupled, for example, by means of a mirror, into a peripheral region of the illumination light's beam path parallel to the optical axis of the illumination light's beam path, which exists when the deflection device is in its neutral position. This peripheral region of the beam path cannot then be used by the illumination light.

[0093] In a preferred embodiment, the reference light beam is coupled into the illumination beam path 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 beam do not overlap, and is especially advantageous when the reference light beam does not have a wavelength used for the actual microscopic imaging. With this type of coupling, the reference light beam is preferably coupled parallel to the optical axis of the illumination beam path, which is present when the deflection device is in its neutral position. More preferably, the coupling occurs on this optical axis.

[0094] 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., that the illumination light source and the reference light source are on the same side of the scanner, or that the illumination light beam and the reference light beam pass through the first beam scanner in opposite directions, i.e., that the illumination light source and the reference light source are on opposite sides of the first beam scanner.

[0095] The extraction or decoupling process can be performed in the same way as the coupling process. It is not necessary for extraction to use the same method as coupling; combinations of different methods are also possible. For example, coupling can be achieved using a mirror in a peripheral region, and extraction using a beam splitter. Preferably, extraction occurs when the reference light beam is introduced at an angle, also without the use of any further optical element that would be touched by the illumination light. If the reference light is coupled onto the optical axis using a beam splitter, extraction also occurs 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 on the optical axis by means of a dichroic beam splitter and is also coupled out again by means of a dichroic beam splitter.

[0096] If a reference light beam is used which, due to its properties, for example its polarization, cannot be deflected by another beam scanner or several other beam scanners of the deflection device, the reference light beam can also be coupled or introduced into the beam path of the illumination light beam in such a way that the reference light beam path includes not only the first beam scanner but also the relevant additional beam scanner(s).

[0097] 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, in wavelength or polarization, or that the components are coupled in separately. "Separately" can mean, for example, spatially separated and parallel or at different angles, or temporally separated, e.g., by coupling intermittently short pulses.When using multiple different components, it is essential that the components can be detected separately after scanning.

[0098] The reference light beam can be coupled or introduced in such a way that, in the beam path section it shares with the illumination light beam, it has a state corresponding to that of the illumination light beam with regard to its convergence or divergence, or in particular with regard to the position of a focus within this beam path section.

[0099] In a preferred embodiment, the reference light, or a portion thereof, is coupled onto the optical axis by means of a beam splitter and, after the first beam scanner, is coupled out again at a location where the reference light beam is also collimated. The location of such locations can be easily determined by a person skilled in the art within a given beam path.

[0100] Within a quad scanner, such as that used in the applicant's microscopes, there are no imaging optical elements between the first and last tilting mirrors of the quad scanner. Accordingly, when using a quad scanner, collimated coupling or introduction can occur adjacent to one of the mirrors on the outer edge of the beam path, and coupling or output can occur adjacent to the mirror on the opposite edge of the beam path. According to the operating principle of a quad scanner (see, for example, DE 103 39 134 A1 or WO 2010 / 069 987 A1), in a microscope with an objective lens, a quad scanner deflects an illumination beam in such a way that, for each deflection angle, the axis of the illumination beam intersects the optical axis of the objective lens at the pupil, i.e., the posterior focal plane of the objective lens.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 before 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 focusing, so that it enters the objective lens collimated, whereby the angle of the illumination light beam to the optical axis of the objective lens, and thus the position of the focus of the illumination light in the sample, depends on the position of the quad scanner.If the objective lens and the second optical element form a telecentric system, the condition that the axis of the illumination beam intersects the optical axis of the objective lens for all scanner settings at the posterior focal plane or the pupil is fulfilled if, for all different scanner settings, the illumination beams after the last mirror of the scanner and in front of the second optical element are parallel to each other and to the optical axis; that is, if the scanner only displaces, but does not tilt, the illumination beam. The change in the position of the focal point of the illumination beam in the sample caused by the scanner is then directly dependent on the change in the displacement of the illumination beam caused by the scanner. This same change in displacement is then imposed on a reference light beam.If the reference light beam is coupled into the beam path of the illumination light in such a way, or introduced at a shallow angle, that it passes through the entire scanner as a collimated beam, the offset of the reference light beam, and thus the change in the offset of the illumination light beam, can be determined in a simple manner by directing or coupling 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 illumination light beam or against it, and collimating it onto a position-sensitive or spatially resolving detector, which is an essential element of the measuring device for detecting – here – the position of the reference light beam after the scanner.Alternatively or additionally, the reference light beam can be directed or coupled out 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 becomes an essential element of the measuring device for detecting the direction of the reference light beam. The position of the focus would not change under ideal scanner conditions. 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 posterior focal plane, i.e., the pupil of the lens.Detecting this change in position makes it possible to infer the change in the quality of the excitation light distribution and a shift in the focal point in the sample resulting from the change in position in the pupil.

[0101] Advantageous developments of the invention are set forth in the patent claims, the description, the drawings, and the accompanying explanations. The described advantages of features and / or combinations of features of the invention are merely examples and can have an effect 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 depicted relative arrangements and functional connections.

[0102] The combination of features from different embodiments of the invention or from features of different claims is also possible, deviating from the chosen cross-references in the claims, and is hereby encouraged. This also applies to features that are illustrated in separate drawings or mentioned in their description. These features can also be combined with features from different claims. Likewise, features listed in the claims can be omitted for further embodiments of the invention, but this does not apply to the independent claims of the granted patent.

[0103] The reference numerals contained in the patent claims do not constitute a limitation of the scope of the subject matter protected by the patent claims. They merely serve the purpose of making the patent claims easier to understand. Brief description of the characters Fig. Figure 1 shows an embodiment of a microscope according to the invention with a reference light source. Fig. Figure 2 shows an embodiment of a microscope according to the invention, in which a reference light beam is separated from light from an illumination light source. Fig. Figure 3 shows an embodiment of a microscope according to the invention, in which a reference light beam from a reference light source runs in the opposite direction to the illumination light beam. Fig. Figure 4 shows an embodiment of a microscope according to the invention, which has a measuring device whose signal is fed back analogously to a beam scanner. Fig. Figure 5 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a tilt. Fig. Figure 6 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a tilt with an enlarged measuring range. Fig. Figure 7 shows a section of an embodiment of a microscope according to the invention with a measuring device for determining a displacement. Description of the characters

[0104] In Fig. 1, Fig. 2 and in Fig. Figure 3 shows an embodiment of the microscope 1 according to the invention. In the illustrated embodiments, the microscope has a light source 2 for generating illumination. The illumination emanating from this light source 2 forms an illumination beam B, which is guided by optical elements forming an illumination beam path into a sample area 7, so that a focus is formed in a sample 8 placed in the sample area 7 such that, in the focus area of ​​the illumination, at least one focal point is bordered on both sides by areas of increased intensity, which can specifically mean that a 2D donut or a 3D donut is formed in the sample. The sample area 7 is in each case formed on an adjustable sample stage 20.The embodiments of the microscope shown in these figures each have a fast, adjustable scanner, specifically an EOD scanner 11, and they all have a mechanical beam scanner 10. In all the embodiments shown, the mechanical scanner 10 and the EOD scanner 11 form a deflection unit 9. All embodiments of the microscope 1 shown in these figures have a light modulator, 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. In particular, adjustable phase modulators can be reflective elements. For the sake of simplicity, a transmissive phase filter 3 is shown in all figures.The embodiments of the microscope 1 shown in the figures above each have a main beam splitter 4 that allows the illumination light to pass through, but deflects emissions 19 originating from the sample 8 onto a detection device 12. The embodiments of the microscope 1 shown in the figures above each further have a mechanical scanner 10 that deflects the illumination light beam, a lambda / 4 plate 5 for adjusting the polarization of the illumination light with a view to creating a minimum intensity of the lowest possible intensity, and an objective 6 that focuses the illumination light beam into the sample 8 in the sample area 7.

[0105] The ones mentioned Fig. In the embodiments of the microscope 1 shown in Figures 1 to 3, each further comprises a measuring device 13 configured to detect the position and / or direction of a reference light beam R deflected and / or displaced by a first beam scanner 18. In each of the illustrated embodiments of the microscope 1, the measuring device 13 has a signal output 13a, through which a signal S, indicative of the 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 illustration. The computing and control unit 17 can, for example, comprise FPGAs (Field Programmable Gate Arrays) and a storage device, neither of which are shown separately.In particular, the computing and control unit 17 is configured such that position values ​​corresponding to the signal S, or to a position of the focal point determined from the signal, or to the deviation of the position of the focal point from a corresponding target position, can be stored in the storage device, wherein the position values ​​can be assigned to time or assigned 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, the computing and control unit 17 receives measured values ​​from the detection device 12, which in the . Fig. 1 to 3 are each symbolized by a dotted line. Another dotted line indicates that a control signal, generated in the computing and control unit 17, is transmitted to the EOD scanner 11. The control signal is selected such that a scan is performed for carrying out a MINFLUX or STED-MINFLUX procedure. A correction is determined based on the signal S; that is, feedback or control of the deflection device 9, specifically the EOD scanner 11, is performed. This feedback and control is not necessary in all embodiments of the invention, but it is advantageous for improving the quality of the data acquisition when carrying out a MINFLUX or STED-MINFLUX procedure.The signal S can be used not only to improve data acquisition, which is usually linked to an initial data analysis performed in parallel, but also to improve data analysis performed after data acquisition. This is achieved by using the actual scanning positions—that is, the actual positions of the focal point in the sample—when determining the position of an emitter. A further 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 time-varying during localization using a MINFLUX or 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.

[0106] By means of the adjustable sample stage 20 shown in the figures, it can be ensured that the deflection device can be operated during a MINFLUX or STED-MINFLUX process with settings close to zero, in particular close to the zero position of the mechanical scanner 10, by regularly moving the sample into the center of the area scannable by the deflection device using the sample stage. This reduces the requirements for the measuring device 13, since the measuring range to be captured with respect to the position and / or direction of the reference light beam R is reduced. Accordingly, the method according to the invention can advantageously be implemented accordingly.

[0107] At the in Fig. In the embodiment of microscope 1 shown in Figure 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, from which the reference light beam R is deflected and / or displaced. In this embodiment, microscope 1 has a reference light source 16. The reference light beam R is coupled onto an optical axis of the microscope by means of a beam splitter, which is subsequently referred to as the second beam splitter 15. The illumination light beam propagates along this axis when the deflection device 9 is set to a zero position. For clarity, the reference light beam R is shown slightly offset parallel to the optical axis.The reference light beam R propagates along the illumination beam path after the beam splitter and is deflected and / or displaced in the same manner as the illumination light beam, according to 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 separates the reference light beam R from the illumination light beam and directs it towards 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, for example, different polarizations or different wavelengths.

[0108] At the in Fig. In the embodiment of microscope 1 shown in Figure 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, constitutes 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, from which the reference light beam R is deflected and / or displaced. The Fig. Figure 2 shows that microscope 1 has a first beam splitter 14 in the illumination beam path after the mechanical scanner 10. This beam splitter separates the reference light beam R from the illumination light beam and directs it towards the measuring device 13. In the specific embodiment shown, the reference light beam R is generated by the light source 2 for the illumination light and passes through all optical elements together with the illumination light beam as a single light beam up to the first beam splitter 14. The reference light beam R thus undergoes identical deflections and / or displacements as the illumination light beam up to the first beam splitter. The beam splitter then divides off a portion, for example, half, of the light beam emitted by the light source.Since the reference light beam R deflected and / or displaced by the first beam scanner 18 has undergone phase modulation in this embodiment of the microscope 1, a measuring device 13 adapted for this purpose is preferably used in this embodiment of the microscope 1.

[0109] At the in Fig. The embodiment of microscope 1 shown in 3 forms, as in the one described in Fig. In the embodiment of microscope 1 shown in Figure 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 microscope 1 has a reference light source 16. In contrast to the embodiment shown in Figure 1, the first beam scanner 18, together with the EOD scanner 11, forms a deflection device 9. Fig. In the illustrated embodiment of the microscope, the reference light beam R, emanating from the reference light source 16, is not coupled into the illumination light beam path 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 true to angle in the figure for the sake of simplicity. This angle causes the reference light beam R and the illumination light beam to be deflected and / or displaced by the first beam scanner 18, which is identical here to the mechanical beam scanner 10, in a corresponding manner. Accordingly, the reference light beam R is exited from the illumination light beam path at a shallow angle on the other side of the first beam scanner.Even in the zero position of the mechanical scanner, the angle at which the reference light beam is introduced need not be identical to the angle at which the reference light beam is emitted on the other side. Rather, the relationship between these two angles depends on which imaging elements the first beam scanner comprises. Another difference from that in... Fig. In the embodiment of microscope 1 shown in Figure 1, the reference light beam is passed through the first beam scanner 18 in the opposite direction to the illumination light beam.

[0110] The properties of the embodiments of the Fig. 1 and the Fig. Three methods could also be combined. For example, the reference light beam R could be introduced at a shallow angle but in the direction of the illumination light beam, or conversely, it could be coupled in using a beam splitter in the opposite direction to the illumination light.

[0111] The in Fig. The embodiment shown in Figure 4 corresponds in most respects to the one shown in Figure 4. Fig. Figure 3. The reference light source 16 and the measuring device 13 are shown in slightly different positions. Unlike the one in Fig. The embodiment shown in 3 has the Fig. Figure 4 shows 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 part of the measuring device 13 and is therefore not shown separately. The measuring device can, in particular, be based on a position-sensitive diode.

[0112] The Fig. Figures 1 to 4 are schematic representations in which not all elements that are typically present, such as lenses for focusing or collimating the illumination light beam or the reference light beam R, are shown.

[0113] In Fig. Figure 5 shows a section of an embodiment of the microscope 1. The section includes 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 includes the lambda / 4 plate 5, the objective 6, and 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 Figure 5. Fig. 1, to which reference is hereby made. In contrast to the representation of the Fig. Figure 1 shows an entrance lens 22 and an exit lens 23, which define the boundaries of the mechanical scanner 10 and are to be understood here as elements of the mechanical scanner; this does not mean that the lenses 22 and 23 form a common component, but merely 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, i.e., in particular no lens, is shown between the exit lens 23 and the objective 6. While the Fig. Figures 1 to 4 are schematic representations in which not all elements that are typically present, such as lenses for focusing or collimating the illumination light beam or the reference light beam R, are also shown. Fig. Paragraph 5 is to be understood as meaning that, in the illustrated embodiment of the microscope 1, there is actually no beam-shaping element, i.e., 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. The reference light beam R is coupled into the illumination beam path by means of the second beam splitter 15. The reference light beam 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 at the entrance lens 22 in collimated form and exit the first beam scanner 18 at the exit lens 23, also in collimated form. 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 beam B, in particular, can enter at an angle relative to the optical axis of another beam scanner (not shown here), such as an EOD scanner, depending on the setting of that second beam scanner, which has its zero position. The fact that a beam is tilted does not preclude it from also being displaced. In the example shown, a displacement would cause the axis of the illumination beam B to pass through the rear aperture of the lens 6 at different points, depending on the displacement. After the first beam splitter, the illumination beam B passes through the lambda / 4 plate 5 and is focused by the lens 6 into the sample 8 in sample area 7.The position of the focal point in the sample is determined by the overall tilt imparted to the illumination beam, which results additively from the tilt with which the illumination beam B entered the first beam scanner 18 and the tilt imparted by the first beam scanner 18, and by the properties of the lens 6, in particular its focal length. The reference beam R is coupled out of the illumination beam by the first beam splitter 14 and directed towards an embodiment of the measuring device 13. The measuring device 13 has a lens 25 that focuses the reference beam R, collimated in front of the lens 25, onto a detector of the measuring device, specifically a position-sensitive diode 24.The position of the focal point of the reference light beam 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 either high sensitivity for determining the tilt and a smaller measuring range in which tilts can be measured, or lower sensitivity and a larger measuring range.

[0114] In Fig. Figure 6 shows a section of a further embodiment of the microscope, which differs with regard to the guidance of the illumination light beam B and the reference light beam up to the first beam splitter 14 of the Fig. This corresponds to the configuration shown in Figure 5. To achieve high sensitivity for determining the tilt while simultaneously covering a larger measurement range of tilts, the measuring device 13 comprises, on the one hand, a diffractive optical element (DOE) 26 and, on the other hand, several position-sensitive diodes 24 arranged side by side. The DOE 26 splits the incident reference light beam R into several partial beams. Each of the partial beams is imprinted with an identical tilt by the first scanner 18, so that, in principle, each of the partial beams is suitable for measuring the imprinted tilt. In the situation shown, the reference light beam R is not imprinted with a tilt by the first beam scanner 18, so that the partial beam of the 0th order falls centrally onto the center of the position-sensitive diodes 13.With a greater tilt, the zeroth order beam now drifts out of the area of ​​the central position-sensitive diode 13, while, depending on the direction of the tilt, either the first-order or the -1st-order partial beam drifts into the area of ​​the central position-sensitive diode 13. In general, the splitting and detection with multiple position-sensitive diodes 13 ensures that even small changes in the tilt of the reference light beam R can be measured, even with large tilts. 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 in one spatial direction is shown.In fact, the DOE 26 can preferably be a 2D DOE, in which case the position-sensitive diodes 13 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 in two spatial directions, can be determined by means of the measuring devices. At least three position-sensitive diodes 13 are required for a two-dimensional arrangement. However, more than three position-sensitive diodes 13 can preferably also be arranged, for example on a Cartesian or a hexagonal grid.

[0115] A corresponding division as in Fig. Figure 6 shows that it can also be used in a corresponding way to determine displacements of the reference light beam R.

[0116] In Fig. Figure 7 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 by the first scanner 18. The arrangement has essentially the same components as the one in Figure 7. Fig. Arrangement shown in section 5. In contrast to the arrangement according to Fig. 5 is in Fig.7. The lens 23 is not part of the first scanner, but 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 beam path. The lens 22, which for the purposes of this application is functionally an element of the first beam scanner 18, focuses the reference light beam R. The convergent reference light beam is coupled out of the illumination beam path by means of the first beam splitter 14. A focus of the reference light beam F 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 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 is collimated by the lens 25 of the measuring device 13. The collimated reference light beam then travels at an angle to the aforementioned optical axis, the angle depending on the displacement of the focus F of the reference light beam. The detector of the measuring device 13, the position-sensitive diode 13, is located at a distance that is very large in relation to the focal length of the lens 25, so that the point of impact of the collimated reference light beam is sensitive to the position of the focus relative to the optical axis, and thus to the displacement to be measured. Reference symbol list 1 microscope 2 light sources 3-phase filter 4 main beam splitters 5 lambda / 4 plate 6 lens 7 Sample area 8 Sample 9. Deflection device 10 mechanical beam scanners 11 EOD scanners 12 Detection device 13 Measuring device 13a Signal output 14 first beam splitter 15 second beam splitter 16 Reference light source 17 Calculation and control unit 18 first beam scanner 19 emissions 20 adjustable sample table 21 Analog line 22 Entrance lens 23 Exit lens 24 position-sensitive diodes 25 lens 26 diffractive optical element (DOE) B Illumination light beam R Reference light beam S Signal F Focus of the reference light beam

Claims

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

Images