METHOD AND DEVICE FOR DETERMINING POSITIONS OF MOLECULES IN A SAMPLE

Abstract

The invention relates to a method for determining positions of mutually spaced molecules (M) in a sample (20), having the steps of generating (101) a plurality of light distributions, each light distribution having a local intensity minimum (110, 310) and adjacent regions (120, 320) of increasing intensity, comprising an excitation light distribution (100) and a deactivation light distribution (300); illuminating (102) the sample (20) with the excitation light distribution (100) and the deactivation light distribution (300); detecting (103) photons emitted by the molecule (M) for different positions of the excitation light distribution (100); and deriving (104) the position of the molecule (M) on the basis of the photons detected for the different positions of the excitation light distribution (100), wherein the local minimum (110) of the excitation light distribution (100) is arranged at a plurality of scanning positions (201) one after the other within a scanning region (200), and the light intensity of the deactivation light in a catching region (210), which is paired with the scanning region (200) and in which the position of the molecule (M) can be unambiguously derived from the scanning positions (201) and the paired detected photons, corresponds maximally to three times the saturation intensity of the deactivation light. The invention further relates to a device for carrying out said method.

Claims

1.-15. (canceled)

16. A method for determining positions of molecules spaced apart from one another in one or more spatial directions in a sample comprising the steps of: generating a plurality of light distributions, each light distribution comprising a local intensity minimum and intensity increasing regions adjacent thereto, the light distributions comprising an excitation light distribution and a deactivation light distribution, particularly a STED light distribution, illuminating the sample with the excitation light distribution and the deactivation light distribution, detecting photons emitted by the molecule for different positionings of the excitation light distribution, and deriving the position of the molecule based on the photons detected for the different positionings of the excitation light distribution, wherein the local minimum of the excitation light distribution is successively arranged at a plurality of scanning positions within a scanning region, wherein the light intensity of the deactivation light in a catch region assigned to the scanning region, in which the position of the molecule can be unambiguously derived from the scanning positions and the associated detected photons, corresponds at most to three times a saturation intensity.

17. The method according to claim 16, wherein the light intensity of the deactivation light in the catch region corresponds, at most to twice the saturation intensity.

18. The method according to claim 16, wherein the light intensity of the deactivation light in the catch region corresponds at most the saturation intensity.

19. The method according to claim 16, wherein the excitation light distribution is repositioned while the deactivation light distribution is left stationary so that the local minimum of the excitation light distribution is positioned differently more than once within a vicinity of the local intensity minimum of the deactivation light distribution.

20. The method according to claim 16, wherein the excitation light distribution and the deactivation light distribution are repositioned together.

21. The method according to claim 16, wherein a region between two local maxima of the deactivation light distribution adjacent to the local intensity minimum of the deactivation light distribution is extended further, particularly at least 10% further, at least in one spatial direction than a corresponding region between two local maxima of the excitation light distribution adjacent to the local minimum of the excitation light distribution.

22. The method according to claim 16, wherein a plurality of scanning steps is performed, wherein the local minimum of the excitation light distribution in each of the scanning steps is positioned at a plurality of the scanning positions of a respective scanning region, wherein the respective scanning region of each scanning step comprises a smaller area or volume than the scanning regions of the preceding scanning steps, and the deactivation light distribution is adjusted in at least a part of the scanning steps depending on the area or volume of the respective scanning area, wherein a total intensity and/or a shape of the deactivation light distribution is adjusted depending on the area or volume of the respective scanning area.

23. The method according to claim 16, wherein the deactivation light distribution remains constant during repositioning of the excitation light distribution.

24. The method according claim 16, wherein the deactivation light distribution is formed as a 2D donut or a 3D donut.

25. The method according to claim 24, wherein the 2D donut is generated by phase modulation of the deactivation light with a first phase pattern, the first phase pattern comprising a phase increasing in a circumferential direction with respect to an optical axis, particularly continuously, from 0 to 2π.Math.n, wherein n is a natural number greater than 1.

26. The method according to claim 16, wherein the deactivation light distribution is formed by irradiating a deactivation light beam onto the sample along an optical axis, wherein the deactivation light beam is focused into the sample by means of an objective, and wherein a beam cross-section of the deactivation light beam is adjusted such that a pupil of the objective lens is under-illuminated so that the deactivation light distribution is stretched in the direction of the optical axis.

27. The method according to claim 26, wherein the beam cross-section of the deactivation light beam is adjusted by adapting an active surface of a beam shaping device, particularly a light modulator.

28. A method according to claim 27, wherein an orientation of a blazed grating in an outer region of the active surface of the beam shaping device is adjusted

29. The method according to claim 27, wherein a second phase pattern is generated on the active surface of the beam shaping device.

30. The method according to claim 29, wherein the second phase pattern is formed as a ring extending in a circumferential direction to the optical axis.

31. The method according to claim 30, wherein the ring comprises a plurality of segments, wherein adjacent segments respectively comprise a phase difference of π to each other.

32. The method according to claim 26, wherein the deactivation light beam is formed at least approximately as a Bessel beam, particularly by means of an axicon or a light modulator.

33. A device, particularly a microscope, for determining positions of molecules spaced apart from one another in one or more spatial directions in a sample comprising a. at least one light source for generating an excitation light beam and a deactivation light beam, particularly a STED beam, b. at least one beam shaping device for forming an excitation light distribution from the excitation light beam and a deactivation light distribution from the deactivation light beam, wherein the excitation light distribution and the deactivation light distribution each comprise a local intensity minimum and intensity increasing regions adjacent thereto, c. an optical arrangement for illuminating the sample with the excitation light distribution and the deactivation light distribution, d. at least one detector for detecting photons emitted by the molecule for different positionings of the excitation light distribution, and e. a computing unit for deriving the position of the molecule based on the photons detected for the different positionings of the excitation light distribution, wherein the device comprises at least one beam deflection device configured to successively arrange the local minimum of the excitation light distribution at a plurality of scanning positions within a scanning region, wherein the device comprises a control unit configured to adjust the light intensity of the deactivation light in a catch region assigned to the scanning region, in which the position of the molecule can be unambiguously derived from the scanning positions and the associated detected photons, to at most three times a saturation intensity.

34. The device according to claim 33, wherein the at least one beam deflection device is configured to reposition the excitation light distribution relative to the deactivation light distribution and to position the local minimum of the excitation light distribution differently more than once within a vicinity of the local intensity minimum of the deactivation light distribution.

35. The device according to claim 33, wherein the device comprises a first beam shaping device for generating the excitation light distribution and a second beam shaping device for generating the deactivation light distribution independently of the generation of the excitation light distribution.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0140] In the following, the invention is further explained and described with reference to preferred embodiments shown in the figures.

[0141] FIG. 1 shows schematically the sequence of the method according to the invention;

[0142] FIG. 2 schematically shows several steps of a MINFLUX localization method;

[0143] FIG. 3 shows a device for determining positions of a molecule according to a first embodiment of the present invention;

[0144] FIG. 4 shows a device for determining positions of a molecule according to a second embodiment of the present invention;

[0145] FIG. 5 shows a beam shaping device as part of a device according to an embodiment of the invention;

[0146] FIG. 6A-D show different phase patterns for generating deactivation light distributions and corresponding contour plots generated by simulations;

[0147] FIG. 7A-D shows additional phase patterns for generating deactivation light distributions and corresponding simulated contour plots;

[0148] FIGS. 8A-D show plots of an excitation light distribution and various deactivation light distributions along a direction in the focal plane;

[0149] FIGS. 9A-D show a further phase pattern for generating a deactivation light distribution as well as different plots of the deactivation light distribution generated by the phase pattern and an excitation light distribution;

[0150] FIG. 10A-E show a further phase pattern for generating a deactivation light distribution, and various plots of the deactivation light distribution generated by the phase pattern compared to a bottle beam distribution.

FIGURE DESCRIPTION

[0151] FIG. 1 illustrates the sequence of the method according to the invention as a block diagram. The method is in particular a MINFLUX localization with additional deactivation (e.g., STED) light.

[0152] In step 101, an excitation light distribution 100 and a deactivation light distribution 300, in particular a STED light distribution, are generated. Both light distributions have a local intensity minimum 110, 310 and intensity increasing areas 120, 320 adjacent thereto (see also FIG. 8). Around the respective local intensity minimum 110, 310, the light distributions each have a region of relatively low light intensity. Therein, this region of the deactivation light distribution 300 is in particular wider than the corresponding region of the excitation light distribution 100. In particular, the deactivation light distribution 300 forms a jacket around a region of the excitation light distribution 100.

[0153] According to step 102, the sample 20 is illuminated with the excitation light distribution 100 and the deactivation light distribution 300 so that a molecule M to be localized in the sample 20 is excited by the excitation light and accordingly emits photons, while the deactivation light suppresses the emission of background photons from other molecules M in the sample 20.

[0154] In step 103, photons emitted by the molecule are detected for different positionings of the excitation light distribution 100 using a detector, such as a point detector confocal to the focus.

[0155] Finally, in step 104, the position of the molecule M is determined based on the photons detected for the different positionings of the excitation light distribution 100, e.g., using a maximum likelihood estimator. In particular, the method is performed such that the excitation light distribution 100 and the deactivation light distribution 300 during the different positionings of the excitation light distribution 100 are such that an effective detection PSF, in particular a width of the effective emission point spread function, is unaffected by the deactivation light distribution 300, i.e., is only affected by the excitation light distribution. This improves localization while suppressing background emission. Of course, the representation of FIG. 1 as a block diagram does not mean that the individual steps of the method are necessarily carried out one after the other.

[0156] FIG. 2 shows an embodiment of the method according to the invention, which is a MINFLUX localization, in particular performed in several iterations, wherein the minimum 110 of the excitation light distribution 100 is arranged in each iteration at a plurality of scanning positions 201 in a scanning region 200 around a previously determined estimated position of the molecule M. The scanning region 200 is defined as a circle with a radius L around an estimated position of the molecule to be localized. The position estimate selected as the center of the scanning region 200 in the initial step can be determined by an independent method (e.g., PALM/STORM microscopy, confocal scan with Gaussian excitation light distribution, or pinhole orbit scan). Thereafter, in particular, the position estimate determined in the previous MINFLUX iteration is used as the center of the circle in each case.

[0157] The scanning positions 201 of an iteration form a so-called set of targeted coordinates (STC, or target coordinate pattern, TCP). In particular, the individual scanning positions 201 may be located on a circle with radius L around the estimated position of the molecule M, wherein the scanning positions 201 are particularly arranged symmetrically around the center. Further, in particular, the estimated position of the molecule M is also used as a scanning position 201. In analogy to the example shown in two dimensions, the sample 20 can be scanned in three dimensions with scanning positions 201 located on or within a sphere of radius L to perform a three-dimensional localization of the molecule M (3D MINFLUX). To shift the excitation light distribution 100 (and in particular also the deactivation light distribution 300) in the z-direction (along the optical axis), a deformable mirror can be used, for example. In particular, a catch region 210 is located within the scanning region 200, wherein the molecule M can be unambiguously localized by means of the method according to the invention, provided that it is located within the catch region 210.

[0158] As shown in FIG. 2, the radius L can become smaller in each step, so that the accuracy of the position estimate also increases in each step, at least as long as the molecule M is located in the corresponding reduced scanning region 200. The procedure is typically carried out until the molecule M loses its emissivity (e.g., due to bleaching or a transition to a dark state in the case of fluorophores) or, if possible, until the accuracy of the position estimate approaches a threshold value given by the signal-to-noise ratio. In particular, the whole procedure is then repeated with several further molecules M. A high-resolution image of the sample comprising the multiple localized molecules M can then be calculated from the multiple localizations. The described reduction of the scanning region 200 also results in a smaller catch region 210 in each case than in the previous iteration step.

[0159] In FIG. 2, the deactivation light distribution 300 is further schematically shown as a circle concentrically arranged around the scanning region 200. At the position marked by the circle, the intensity of the deactivation light may correspond to, for example, the saturation intensity or a lower value.

[0160] As shown in FIG. 2, the deactivation light distribution 300 may remain stationary during at least one MINFLUX iteration, and in particular across multiple MINFLUX iterations, while the excitation light distribution 100 is moved around the local minimum 310 of the deactivation light distribution 300 within a region of lower deactivation light intensity to head for the the scanning positions 201. This has the advantage that fast beam deflection devices such as EODs need only be provided for the excitation light beam, which reduces the complexity of the device and allows for a more compact design. In particular, the deactivation light distribution 300 is then arranged in the center of a projection of a detection pinhole aperture into the focal plane in a corresponding sample region at the beginning of each new localization of a molecule M, in particular by a galvanometric scanner (slower compared to EODs but present anyway). In particular, the deactivation light distribution 300 can be adjusted between iteration steps such that an area where, for example, the saturation intensity of the deactivation light or a lower light intensity prevails, becomes smaller in each iteration step as the scanning region 200 is reduced (see FIG. 2). This can be achieved, in particular, by controlling the total intensity of the deactivation light or by varying the beam shaping (e.g., different phase patterns on an SLM).

[0161] Alternatively, the excitation light distribution 100 may be moved together with the deactivation light distribution 300, so that in each iteration the local minima of the two light distributions are arranged together in succession at the scanning positions 201. Due to the required scanning speed, this is performed in particular by means of a common beam deflection with electro-optical or acousto-optical deflectors.

[0162] FIGS. 3 and 4 show embodiments of a device 1 according to the invention for determining positions of a molecule M in the form of a MINFLUX microscope with an additional deactivation (in particular STED) light source. The basic principle of the device is first explained with reference to FIG. 3. Subsequently, differences of the embodiment according to FIG. 4 compared to the embodiment according to FIG. 3 are discussed.

[0163] FIG. 3 shows a device 1 with a first light source 11, in particular a laser source, for generating a coherent excitation light beam A and a second light source 12, in particular a laser source, for generating a coherent deactivation light beam D. Therein, the light from the first light source 11 has a suitable wavelength for exciting the molecules M in the sample, and the second light source 12 generates light of a wavelength suitable for deactivating the molecules M so that the molecules can no longer emit photons when the excitation light is irradiated (e.g. depletion by stimulated emission, i.e. STED). The wavelengths of the excitation light and the deactivation light are particularly different from each other. As an alternative to two separate light sources 11, 12, the device 1 may also comprise only one light source, in particular a laser source, which generates both the excitation light and the deactivation light. For this purpose, the light source may, e.g., generate a continuum of wavelengths from which excitation light and deactivation light of suitable wavelengths are then obtained, for example by means of optical filters.

[0164] In the example shown in FIG. 3, the excitation light beam A and the deactivation light beam D are coupled into a common beam path at a first, particularly dichroic, beam splitter 13a. The light beams then pass together through a lateral beam deflection device 15, which consists of two beam deflection units 15a,15b arranged in series in the beam path. The beam deflection units 15a,15b may be electro-optical deflectors, e.g., the first beam deflection unit 15a being designed to deflect the light beams passing through it in a first direction x perpendicular to the optical axis OA, and the second beam deflection unit 15b being designed to deflect the light beams passing through it in a second direction y perpendicular to the optical axis OA and perpendicular to the first direction. In a manner known from the prior art, the lateral beam deflection device 15 particularly comprises further optical components, in particular lenses for focusing the light beams, which are not shown here for the sake of clarity.

[0165] At a second, particularly dichroic, beam splitter 13b, the excitation light beam A and the deactivation light beam D are then split into two parallel beam paths. The excitation light beam A impinges on a first beam shaping device 16a, particularly a first light modulator, further in particular a phase modulating spatial light modulator SLM with controllable pixels. By diffraction of the excitation light beam A at a blazed grating of an active surface of the light modulator, a phase pattern set by means of the controllable pixels is impressed on the excitation light beam A, resulting in formation of the excitation light distribution 100 with the local minimum 110 in the focal plane in the sample 20 by interference. The phase-modulated excitation light beam A is coupled into the main beam path via a first mirror 14a and a second mirror 14b.

[0166] In an analogous manner, the deactivation light beam D is directed via a third mirror 14c onto a second beam shaping device 16b, particularly a second light modulator, which imparts to it a preset phase pattern which may differ in particular from the phase pattern of the first beam shaping device 16a. The phase-modulated deactivation light beam D is then deflected via a fourth mirror 14d and then combined with the phase-modulated excitation light beam A via a third, particularly dichroic, beam splitter 13c. Of course, a configuration is also possible in which the deactivation light beam D is coupled into the main beam path via mirrors and the excitation light beam A is combined with the deactivation light beam D via a beam splitter.

[0167] Alternatively to the first light modulator 16a and the second light modulator 16b, phase plates with fixed predetermined phase patterns can be used, e.g., which are then typically traversed by the respective light beams (instead of diffraction at a grating of the active surface of the light modulator) in order to impress the respective phase patterns thereon. In addition, optical elements can be arranged in front of the beam shaping devices 16a,16b which influence the polarization direction of the respective light beams, e.g., λ/2 plates (not shown). Furthermore, especially when imposing a vortex phase pattern to generate a 2D donut, a circular polarizing element (e.g., an λ/4 plate) can be provided in the beam path behind the beam shaping devices 16a,16b to achieve the desired light distribution in the focus (not shown).

[0168] In particular, the beam shaping devices 16a,16b are each arranged in a plane perpendicular to the optical axis OA, which is conjugate to a pupil of the lens 19. Particularly in the case of light modulators, a slight deviation from this position can also be corrected by adjusting the phase pattern accordingly.

[0169] By means of the beam shaping devices 16a, 16b, in particular different phase patterns are imposed on the excitation light beam A and the deactivation light beam D, in particular so that a broader low intensity region (e.g., below the saturation intensity) around the local minimum 310 results in the focal plane for the deactivation light distribution 300 than for the excitation distribution 100. For example, for a 2D MINFLUX localization, the excitation light distribution 100 could be formed as a 2D donut generated by a vortex phase distribution between 0 and 2π, while the deactivation light distribution is formed as a wider 2D donut generated by a vortex phase distribution between 0 and 4π, 6π, 8λ, or 10λ. Similarly, it is possible, for example, that for 3D MINFLUX localization, an excitation light distribution 100 and a deactivation light distribution 300 are each in the form of a 3D donut, with the 3D donut of the deactivation light additionally being axially stretched.

[0170] In the common beam path of the excitation light beam A and the deactivation light beam D there is furthermore an axial beam deflection device 17, e.g., comprising a deformable mirror. With the aid of the axial beam deflection device 17, the excitation light beam A and the deactivation light beam D can be displaced together in the z-direction, i.e., parallel to the optical axis OA, e.g., in order to head for scanning positions 201 in the sample in the z-direction for a 3D localization of the molecule M.

[0171] Finally, a scanning device 18, e.g., a galvanometric scanner, is arranged in the beam path to scan the excitation light beam A and the deactivation light beam D together across the sample 20. The excitation light beam A and the deactivation light beam D are focused by an objective 19 having a pupil 19a into a sample 20 containing molecules M to be localized, which are spaced apart from each other. At least the objective 19 and the scanning device 18 thereby particularly form an optical arrangement 2 for illuminating the sample 20 with the excitation light distribution 100 and the deactivation light distribution 300. The excitation light beam A and the deactivation light beam D enter the objective 19 along an optical axis.

[0172] Due to the excitation with the excitation light, the molecules emit photons (e.g., fluorescent light). The light E emitted by the sample 20 is descanned by the scanning device 18 and coupled into a detection beam path via a fourth, particularly dichroic, beam splitter 13d. In this detection beam path, there is in particular a pinhole 21 which is arranged confocal to the focus of the excitation and deactivation light beam in the sample 20. Behind the pinhole 21 a detector 22, in the example shown here a point detector, e.g., an avalanche photodiode or a photomultiplier, is arranged, which detects and in particular counts the photons emitted by the molecule M.

[0173] Furthermore, the device 1 comprises a computing unit 23 which is in electrical connection or data connection with the detector 22 and is configured to determine the position of the molecule M from the photons detected by the detector 22 and the associated positions of the local minimum 110 of the excitation light distribution 100, for example by means of a maximum likelihood estimator implemented on the software or hardware side on the computing unit 23. In order to obtain the positions of the excitation light distribution 100 belonging to the detected photon numbers or photon count rates, the computing unit 23 particularly receives a signal from the control unit 24 or, e.g., directly from the lateral beam deflection device 15, the axial beam deflection device 17 and/or the scanning device 18.

[0174] The control unit 24 is further configured to control the lateral beam deflection device 15, the axial beam deflection device 17, the scanning device 18, and the beam shaping devices 16a and 16b.

[0175] With the device 1 shown in FIG. 3, the method according to the invention is carried out in particular in such a way that the excitation light beam A and the deactivation light beam D are moved together relative to the sample 20 in a plurality of MINFLUX iterations by means of the lateral beam deflection device 15 (and optionally also by means of the axial beam deflection device 17). In this process, the scanning positions 201 (see FIG. 2) are scanned with the local minimum 110 of the excitation light distribution 100, and by means of the detector 22 the photons emitted by the molecule M are detected for each scanning position 201. Then, for each MINFLUX iteration, an estimated position of the molecule M is determined by means of the computing unit 23 from the detected photons and the corresponding positions of the excitation light distribution 100.

[0176] FIG. 4 shows a device 1 according to the invention, which differs from the device 1 shown in FIG. 3 in that the lateral beam deflection device 15 and the axial beam deflection device 17 are arranged in a partial beam path via which the excitation light beam A is directed onto the sample 20. The deactivation light beam D, on the other hand, is guided on a parallel partial beam path without corresponding beam deflection devices. The light beams A, D are split between the partial beam paths by means of the second, particularly dichroic, beam splitter 13b and are recombined via the third, particularly dichroic, beam splitter 13c. The beam shaping devices 16a,16b described in connection with FIG. 3 are also located in the corresponding partial beam paths.

[0177] When using the embodiment of the device 1 shown in FIG. 4, in particular prior to the localization of a molecule M, the excitation light beam A and the deactivation light beam D are jointly directed by means of the scanning device 18 onto a region of the sample 20 in which the molecule M to be localized is suspected according to a pre-localization. Then, the excitation light beam A is moved over the sample by means of the lateral beam deflection device 15 and optionally also by means of the axial beam deflection device 17 to target the scanning positions 201 with the local minimum 110 of the excitation light distribution 100. Meanwhile, the deactivation light distribution 300 particularly remains stationary.

[0178] FIG. 5 shows a schematic cross-sectional view of a second beam shaping device 16b for phase modulating the deactivation light beam D. The second beam shaping device has an active surface 160 with a blazed grating, which diffracts the incident deactivation light beam D in a desired order and thus couples it into the further beam path so that the deactivation light beam is focused by the objective 19 into the sample 20. A phase pattern can further be superimposed on the active surface with the blazed grating, so that a corresponding phase distribution is imposed on the deactivation light beam D, which forms the deactivation light distribution 300 at the focus by interference. In particular, the active surface 160 is a plane conjugate to the pupil 19a of the objective 19 (see FIGS. 3-4). The phase pattern can be generated, in particular, in the form of controllable pixels, wherein a phase value can be specified for each pixel. The phase pattern and in particular also the orientation of the blazed grating are controllable by means of the control unit 24.

[0179] FIG. 5 further schematically illustrates an outer region 161 of the active surface 160 and an inner region 162 of the active surface 160 enclosed by the outer region 161.

[0180] By means of the control unit 24, the orientation of the blazed grating can be changed in the outer region 161 such that the deactivation light beam D is not coupled into the beam path but, for example, into a beam trap (not shown). In this way, the beam cross-section of the deactivation light beam D can be reduced such that the pupil 19a of the lens 19 is under-illuminated and thus the deactivation light distribution 300 is stretched in the direction of the optical axis OA (i.e., axially).

[0181] In another embodiment, the inner region 162 may represent the first phase pattern for generating the deactivation light distribution 300 and the outer region 161 may represent a second phase pattern that effectively results in reducing the beam cross-section of the deactivation light beam D and thus axially stretching the deactivation light distribution 300.

[0182] FIGS. 6 to 7 and 9 to 10 show phase patterns (FIGS. 6A-D, above) that can be generated, for example, on an active surface 160 of a beam shaping device 16a,16b to modulate the phase distribution of the excitation light beam A or the deactivation light beam D, in particular the deactivation light beam D, so that a corresponding light intensity distribution with a local minimum is produced at the focus of the respective light beam in the sample 20 by interference. Below the phase patterns, contour plots of corresponding simulated light distributions are shown, respectively as a section through the focal plane (x-y plane, perpendicular to the optical axis, where the z coordinate is zero, FIG. 6A-D, center) and as a section in the y-z plane, where the x coordinate is zero (FIG. 6A-D, bottom). The location coordinates in FIGS. 6A-D, center and bottom, are each in units of micrometers. In particular, the phase patterns are generated in a plane conjugate to the plane of the pupil 19a of the objective 19.

[0183] FIG. 6A (top) shows a first phase pattern 610 consisting of a first ring 602 arranged concentrically around a circular disk 601, wherein the first ring 602 and the circular disk 601 have a phase difference of π with respect to each other. With such a phase pattern, a 3D donut (also referred to as a bottle beam) with a central local minimum 310 can be generated at the focus of the phase-modulated light beam.

[0184] Also shown in each of FIGS. 6B to 6D (top) is a first phase pattern 610 having a first ring 602 arranged concentrically around a circular disk 601, wherein the circular disk 601 and the first ring 602 have a phase shift of π. However, the radius of the first ring 602 and the circular disk 601 are each smaller than in the first phase pattern 610 shown in FIG. 6A (above). In addition, FIGS. 6B-6D show another second phase pattern 611 comprising a second ring 603 concentrically arranged around the first ring 602 of the first phase pattern 610. This second ring 603 comprises a plurality of segments 604 having alternating phase values corresponding to the phase value of the first ring 602 and the phase value of the circular disk 601. Accordingly, the segments 604 adjacent on the second ring 603 comprise phase jumps of π with respect to each other. In the example shown here, all segments 604 have identical dimensions and are thus symmetrically arranged around the first ring 602.

[0185] As can be seen from the comparison between the contour line diagrams in FIG. 6A (bottom) and FIG. 6B (bottom), the additional second phase pattern 611 with the segmented second ring 603 results in a stretching of the 3D donut in the axial direction (z-direction). In contrast, there is only a slight change in the light intensity distribution in the focal plane (FIGS. 6A and 6B, center). Stretching a 3D donut-shaped deactivation light distribution 300 in the z-direction is advantageous for the method according to the invention, since it can effectively suppress background emission from regions lying above and below the focus in the z-direction.

[0186] Furthermore, it can be seen from FIGS. 6C and 6D that by widening the second ring 603 radially inward while simultaneously reducing the size of the circular disk 601, an even further stretching in the z-direction can be achieved than for the case shown in FIG. 6A. In the configuration shown in FIG. 6D, a broadening of the central local minimum 310 in the focal plane is additionally visible (FIG. 6D, center), which can also have a beneficial effect on the suppression of background fluorescence.

[0187] FIGS. 7A and 7B show additional phase patterns for phase modulation of the deactivation light beam. FIGS. 7C-D show corresponding contour plots of the light intensity distribution in the XZ plane obtained at the focus, with FIG. 7C corresponding to the phase pattern shown in FIG. 7A and FIG. 7D corresponding to the phase pattern shown in FIG. 7B. FIG. 7A, like FIG. 6A, shows a first phase pattern formed by a circular disk 601 and a first ring 602 concentrically arranged around the circular disk 601, with a phase difference of π. Accordingly, FIG. 7C shows the contour line diagram of a 3D donut.

[0188] According to FIG. 7B, in addition to the circular disk 601 and the first ring 602, a second ring 603 and a third ring 605 are provided, with adjacent rings each having a phase difference of π from one another. This advantageously results in an intensity distribution stretched in the z-direction at the focus (FIG. 7D). The scales are given in the unit μm.

[0189] FIGS. 8A-D show plots along a direction in the focal plane (e.g., along the x-coordinate) of a simulated 2D donut-shaped excitation light distribution 100 with a wavelength of 642 nm each superimposed on simulated 2D donut-shaped deactivation light distributions 300 of different shapes and widths with a wavelength of 775 nm. The scale of the x-axis is in units of micrometers. The excitation light distribution 100 has a central local minimum 110 and the deactivation light distributions 300 each have at least one local minimum 310. Adjacent to the local minima 110, 310, the distributions 100, 300 have intensity increasing areas 120, 320 and at least two local maxima 130, 330 each. The excitation light distribution 100 was generated by phase modulating the excitation light beam A with a vortex-shaped phase pattern in a plane conjugate to the objective pupil, which has phase values gradually increasing from 0 to 2π in a clockwise circumferential direction with respect to the optical axis. In addition, the excitation light beam A has been left circularly polarized so that the excitation light distribution 100 is obtained in the focal plane by interference.

[0190] The deactivation light distribution 300 shown in FIG. 8A was generated by phase modulating the deactivation light beam 100 with the same vortex-shaped phase pattern, also with left circular polarization. The local minima 110, 310 of the distributions 100, 300 are here at the same position. The deactivation light distribution 300 according to FIG. 8A is slightly wider than the excitation light distribution 100 due to the higher wavelength.

[0191] In contrast, the deactivation light distributions 300 shown in FIGS. 8B to 8D were generated with vortex-shaped phase patterns that have gradually increasing first phase patterns in the circumferential direction between 0 and 4π (FIG. 8B), 0 and 6π (FIG. 8C), and 0 and 10p (FIG. 8D), respectively. As a result, as the maximum phase value increases, the deactivation light distributions 300 exhibit an increasing width, i.e., a greater distance between the local maxima 130, 330 adjacent to the local minimum 110, 310 and a wider region of relatively low light intensity between the local maxima 130, 330. This region is particularly wide for a maximum of 10π. Advantageously, the deactivation light of these distributions reduces the background fluorescence but does not significantly affect the detection PSF of the emission light.

[0192] The deactivation light distribution 300 shown in FIG. 8B comprises a further local maximum 330 at the position of the minimum of the excitation light distribution 100. This effect results from the polarization direction of the light in this particular phase pattern (vortex pattern from 0 to 4π).

[0193] FIG. 9A shows a first phase pattern 610 for generating the deactivation light distribution 300 shown in FIGS. 9B and 9D, which is overlaid with a third phase pattern 612 for generating secondary maxima. The first phase pattern 610 is vortex-shaped with phase increasing in the circumferential direction from 0 to 10π. This pattern is superimposed with a third phase pattern 612 in the form of a circular disk 601 divided into segments 604, the segments 604 having a phase difference relative to the radially adjacent region of the first ring 602 of

[00002]$+\frac{\pi }{2}\mathrm{or}-\frac{\pi }{2}.$

The outer region of the superimposed phase pattern, on which the multiple vortex phase pattern is best seen, forms a first ring 602 concentrically arranged around the circular disk 601. The segments 604 with positive and negative phase jump to the first ring 602 are thereby arranged alternately. Accordingly, segments 604 adjacent to each other in the circumferential direction each have a phase difference of π.

[0194] The phase pattern shown can be used to create a 2D donut-shaped deactivation light distribution 300 with a wide range of low intensity around the central local minimum 310 (see FIG. 8D) and additional secondary maxima of light intensity. These secondary maxima advantageously suppress background emission in areas further away from the focus.

[0195] FIG. 9B shows a plot of the deactivation light distribution 300 along the x coordinate. FIG. 9D shows a 2D plot of the deactivation light distribution 300 in the xy plane, and FIG. 9C shows an xy plot of a 2D donut of the deactivation light distribution 300 shown in FIG. 8A for comparison.

[0196] FIG. 10A shows another first phase pattern 610 for phase modulating the deactivation light, which is superimposed with a second phase pattern 611 for stretching the deactivation light distribution 300 in the z-direction and with a third phase pattern 612 for generating secondary maxima. The first phase pattern 610 comprises a first ring 602 and a second ring 603 arranged concentrically around the first ring 602, the first ring 602 and the second ring 603 having a phase difference of π with respect to each other so that a 3D donut is formed at the focus. The second phase pattern 611 is formed by a third ring 605 arranged concentrically around the second ring 603, the third ring 605 being divided into alternating segments 604, the segments 604 having alternating phase differences of 0 or π relative to the second ring 603. The third phase pattern 612 forms a circular disk 601 arranged concentrically within the first ring 602, which is divided into segments 604 having alternating phase differences of 0 or π relative to the first ring 602.

[0197] FIGS. 10B and 10C show a corresponding deactivation light distribution 300 in the focus generated by the phase pattern shown in FIG. 10A, wherein FIG. 10B is an x-y section (focal plane) and FIG. 10C is an x-z section. FIGS. 10D and 10E show sections through a regular 3D donut, i.e., bottle beam (see phase patterns FIGS. 6A and 7A) for comparison. All scales have the unit μm.

[0198] In particular, as can be seen from FIG. 10B, the third phase pattern 612 creates secondary maxima that have a beneficial effect on the suppression of background emission by the deactivation light. Additionally, the deactivation light distribution 300 is stretched in the z-direction by the second phase pattern 611, as can be seen from FIG. 10C.

LIST OF REFERENCE SIGNS

[0199] 1 Device for determining positions of a molecule

[0200] 2 Optical arrangement

[0201] 11 Excitation light source

[0202] 12 deactivation light source

[0203] 13a First beam splitter

[0204] 13b Second beam splitter

[0205] 13c Third beam splitter

[0206] 13d Fourth beam splitter

[0207] 14a First mirror

[0208] 14b Second mirror

[0209] 14c Third mirror

[0210] 14d Fourth mirror

[0211] 15 Lateral beam deflection device

[0212] 15a First beam deflection unit

[0213] 15b Second beam deflection unit

[0214] 16a First beam shaping device

[0215] 16b Second beam shaping device

[0216] 17 Axial beam deflection device

[0217] 18 Scanning device

[0218] 19 Objective

[0219] 19a Pupil

[0220] 20 Sample

[0221] 21 Pinhole

[0222] 22 Detector

[0223] 23 Computing unit

[0224] 24 Control unit

[0225] 100 Excitation light distribution

[0226] 101 Generating a plurality of light distributions

[0227] 102 Illuminating with the excitation light distribution

[0228] 103 Detecting emitted photons

[0229] 104 Deriving the position of the molecule

[0230] 110 Local minimum of the excitation light distribution

[0231] 120 Intensity increasing region of the excitation light distribution

[0232] 130 Local maximum of the excitation light distribution

[0233] 160 Active surface

[0234] 161 Outer region

[0235] 162 Inner region

[0236] 200 Scanning region

[0237] 201 Scanning position

[0238] 210 Catch region

[0239] 300 Deactivation light distribution

[0240] 310 Local minimum of the deactivation light distribution

[0241] 320 Intensity increasing region of the deactivation light distribution

[0242] 330 Local maximum of the deactivation light distribution

[0243] 601 Circular disk

[0244] 602 First ring

[0245] 603 Second ring

[0246] 604 Segment

[0247] 605 Third ring

[0248] 610 First phase pattern

[0249] 611 Second phase pattern

[0250] 612 Third phase pattern

[0251] A Excitation light beam

[0252] D Deactivation light beam

[0253] E Emitted light

[0254] L Radius

[0255] M Molecule

[0256] OA Optical axis